Publication from Kathrin Göritzer (Schrödinger fellowship)
Stability Engineering of Recombinant Secretory IgA
Abstract Secretory IgA (SIgA) presents a promising avenue for mucosal immunotherapy yet faces challenges in expression, purification, and stability. IgA exists in two primary isotypes, IgA1 and IgA2, with IgA2 further subdivided into two common allotypes: IgA2m(1) and IgA2m(2). The major differences between IgA1 and IgA2 are located in the hinge region, with IgA1 featuring a 13-amino acid elongation that includes up to six O-glycosylation sites. Furthermore, the IgA2m(1) allotype lacks a covalent disulfide bond between heavy and light chains, which is present in IgA1 and IgA2m(2). While IgA1 demonstrates superior epitope binding and pathogen neutralization, IgA2 exhibits enhanced effector functions and stability against mucosal bacterial degradation. However, the noncovalent linkage in the IgA2m(1) allotype raises production and stability challenges. The introduction of distinct single mutations aims to facilitate an alternate disulfide bond formation to mitigate these challenges. We compare four different IgA2 versions with IgA1 to further develop secretory IgA antibodies against SARS-CoV-2 for topical delivery to mucosal surfaces. Our results indicate significantly improved expression levels and assembly efficacy of SIgA2 (P221R) in Nicotianabenthamiana. Moreover, engineered SIgA2 displays heightened thermal stability under physiological as well as acidic conditions and can be aerosolized using a mesh nebulizer. In summary, our study elucidates the benefits of stability-enhancing mutations in overcoming hurdles associated with SIgA expression and stability.
A synchronized, large-scale field experiment using Arabidopsis thaliana reveals the significance of the UV-B photoreceptor UVR8 under natural conditions
Abstract This study determines the functional role of the plant ultraviolet-B radiation (UV-B) photoreceptor, UV RESISTANCE LOCUS 8 (UVR8) under natural conditions using a large-scale 'synchronized-genetic-perturbation-field-experiment'. Laboratory experiments have demonstrated a role for UVR8 in UV-B responses but do not reflect the complexity of outdoor conditions where 'genotype × environment' interactions can mask laboratory-observed responses. Arabidopsisthaliana knockout mutant, uvr8-7, and the corresponding Wassilewskija wild type, were sown outdoors on the same date at 21 locations across Europe, ranging from 39°N to 67°N latitude. Growth and climatic data were monitored until bolting. At the onset of bolting, rosette size, dry weight, and phenolics and glucosinolates were quantified. The uvr8-7 mutant developed a larger rosette and contained less kaempferol glycosides, quercetin glycosides and hydroxycinnamic acid derivatives than the wild type across all locations, demonstrating arole for UVR8 under field conditions. UV effects on rosette size and kaempferol glycoside content were UVR8 dependent, but independent of latitude. In contrast, differences between wild type and uvr8-7 in totalquercetin glycosides, and the quercetin-to-kaempferol ratio decreased with increasing latitude, that is, a more variable UV response. Thus, the large-scale synchronized approach applied demonstrates a location-dependent functional role of UVR8 under natural conditions.
Publication from Korbei Lab with contributions from Elsa Arcalis and the Luschnig Lab
Modulation of abscisic acid signaling via endosomal TOL proteins
Abstract The phytohormone abscisic acid (ABA) functions in the control of plant stress responses, particularly in drought stress. A significant mechanism in attenuating and terminating ABA signals involves regulated protein turnover, with certain ABA receptors, despite their main presence in the cytosol and nucleus, subjected to vacuolar degradation via the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Collectively our findings show that discrete TOM1-LIKE (TOL) proteins, which are functional ESCRT-0 complex substitutes in plants, affect the trafficking for degradation of core components of the ABA signaling and transport machinery. TOL2,3,5 and 6 modulate ABA signaling where they function additively in degradation of ubiquitinated ABA receptors and transporters. TOLs colocalize with their cargo in different endocytic compartments in the root epidermis and in guard cells of stomata, where they potentially function in ABA-controlled stomatal aperture. Although the tol2/3/5/6 quadruple mutant plant line is significantly more drought-tolerant and has a higher ABA sensitivity than control plant lines, it has no obvious growth or development phenotype under standard conditions, making the TOL genes ideal candidates for engineering to improved plant performance. Keywords: ABA receptors; ABA signaling pathway; ABA transporters; ESCRT; TOL; vacuolar de
Glycans, with their variable compositions and highly dynamic conformations, vastly expand the heterogeneity of whatever factor or cell they are attached to. These properties make them crucial contributors to biological function and organismal health and also very difficult to study. That may be changing as we look to the future of glycobiology.
Tamara Krska (AG Adam) was awarded with the "best poster" prize at the 45th Mycotoxin Workshop (Vienna, Jun 3-6, 2024) with her contribution "The ceramide synthase CER1 plays a role in self-protection against fumonisin B1".
Photo: (from left to right) Tamara Krska and organizers Prof. Doris Marko (UniVie) and Assoc. Prof. Elisabeth Varga (VetMedUni) in the ceremony hall of the University of Vienna.
Mechanism of Fumonisin Self-Resistance: Fusariumverticillioides Contains Four Fumonisin B1-Insensitive-Ceramide Synthases
Abstract Fusarium verticillioides produces fumonisins, which are mycotoxins inhibiting sphingolipid biosynthesis in humans, animals, and other eukaryotes. Fumonisins are presumed virulence factors of plant pathogens, but may also play a role in interactions between competing fungi. We observed higher resistance to added fumonisin B1 (FB1) in fumonisin-producing Fusariumverticillioides than in nonproducing F.graminearum, and likewise between isolates of Aspergillus and Alternaria differing in production of sphinganine-analog toxins. It has been reported that in F. verticillioides, ceramide synthase encoded in the fumonisin biosynthetic gene cluster is responsible for self-resistance. We reinvestigated the role of FUM17 and FUM18 by generating a double mutant strain in a fum1 background. Nearly unchanged resistance to added FB1 was observed compared to the parental fum1 strain. A recently developed fumonisin-sensitive baker’s yeast strain allowed for the testing of candidate ceramide synthases by heterologous expression. The overexpression of the yeast LAC1 gene, but not LAG1, increased fumonisin resistance. High-level resistance was conferred by FUM18, but not by FUM17. Likewise, strong resistance to FB1 was caused by overexpression of the presumed F. verticillioides “housekeeping” ceramide synthases CER1, CER2, and CER3, located outside the fumonisin cluster, indicating that F.verticillioides possesses a redundant set of insensitive targets as a self-resistance mechanism.
Cytosolic UDP-L-arabinose synthesis by bifunctional UDP-glucose 4-epimerases in Arabidopsis
SUMMARY L-Arabinose (L-Ara) is a plant-specific sugar found in cell wall polysaccharides, proteoglycans, glycoproteins, and small glycoconjugates, which play physiologically important roles in cell proliferation and other essential cellular processes. L-Ara is synthesized as UDP-L-arabinose (UDP-L-Ara) from UDP-xylose (UDP-Xyl) by UDP-Xyl 4-epimerases (UXEs), a type of de novo synthesis of L-Ara unique to plants. In Arabidopsis, the Golgi-localized UXE AtMUR4 is the main contributor to UDP-L-Ara synthesis. However, cytosolic bifunctional UDP-glucose 4-epimerases (UGEs) with UXE activity, AtUGE1, and AtUGE3 also catalyze this reaction. For the present study, we first examined the physiological importance of bifunctional UGEs in Arabidopsis. The uge1 and uge3 mutants enhanced the dwarf phenotype of mur4 and further reduced the L-Ara content in cell walls, suggesting that bifunctional UGEs contribute to UDP-L-Ara synthesis. Through the introduction of point mutations exchanging corresponding amino acid residues between AtUGE1 with high UXE activity and AtUGE2 with low UXE activity, two mutations that increase relative UXE activity of AtUGE2 were identified. The crystal structures of AtUGE2 in complex forms with NAD+ and NAD+/UDP revealed that the UDP-binding domain of AtUGE2 has a more closed conformation and smaller sugar-binding site than bacterial and mammalian UGEs, suggesting that plant UGEs have the appropriate size and shape for binding UDP-Xyl and UDP-L-Ara to exhibit UXE activity. The presented results suggest that the capacity for cytosolic synthesis of UDP-L-Ara was acquired by the small sugar-binding site and several mutations of UGEs, enabling diversified utilization of L-Ara in seed plants.
Publication from Anna Atanasoff-Kardjalieff - Studt Lab
Incorporation of the histone variant H2A.Z counteracts gene silencing mediated by H3K27 trimethylation in Fusariumfujikuroi
Abstract
Background Fusariumfujikuroi is a pathogen of rice causing diverse disease symptoms such as ‘bakanae’ or stunting, most likely due to the production of various natural products (NPs) during infection. Fusaria have the genetic potential to synthesize a plethora of these compounds with often diverse bioactivity. The capability to synthesize NPs exceeds the number of those being produced by far, implying a gene regulatory network decisive to induce production. One such regulatory layer is the chromatin structure and chromatin-based modifications associated with it. One prominent example is the exchange of histones against histone variants such as the H2A variant H2A.Z. Though H2A.Z already is well studied in several model organisms, its regulatory functions are not well understood. Here, we used F. fujikuroi as a model to explore the role of the prominent histone variant FfH2A.Z in gene expression within euchromatin and facultative heterochromatin.
Results Through the combination of diverse ‘-omics‘ methods, we show the global distribution of FfH2A.Z and analyze putative crosstalks between the histone variant and two prominent histone marks, i.e., H3K4me3 and H3K27me3, important for active gene transcription and silencing, respectively. We demonstrate that, if FfH2A.Z is positioned at the + 1-nucleosome, it poises chromatin for gene transcription, also within facultative heterochromatin. Lastly, functional characterization of FfH2A.Z overexpression and depletion mutants revealed that FfH2A.Z is important for wild type-like fungal development and secondary metabolism.
Conclusion In this study, we show that the histone variant FfH2A.Z is a mark of positive gene transcription and acts independently of the chromatin state most likely through the stabilization of the + 1-nucleosome. Furthermore, we demonstrate that FfH2A.Z depletion does not influence the establishment of both H3K27me3 and H3K4me3, thus indicating no crosstalk between FfH2A.Z and both histone marks. These results highlight the manifold functions of the histone variant FfH2A.Z in the phytopathogen F. fujikuroi, which are distinct regarding gene transcription and crosstalk with the two prominent histone marks H3K27me3 and H3K4me3, as proposed for other model organisms.
Publication from AG Strasser/Stöger in Frontiers in Bioengineering and Biotechnology
Implications of O-glycan modifications in the hinge region of a plant-produced SARS-CoV-2-IgA antibody on functionality
Introduction: Prolyl-4-hydroxylases (P4H) catalyse the irreversible conversion of proline to hydroxyproline, constituting a common posttranslational modification of proteins found in humans, plants, and microbes. Hydroxyproline residues can be further modified in plants to yield glycoproteins containing characteristic O-glycans. It is currently unknown how these plant endogenous modifications impact protein functionality and they cause considerable concerns for the recombinant production of therapeutic proteins in plants. In this study, we carried out host engineering to generate a therapeutic glycoprotein largely devoid of plant-endogenous O-glycans for functional characterization. Methods: Genome editing was used to inactivate two genes coding for enzymes of the P4H10 subfamily in the widely used expression host Nicotiana benthamiana. Using glycoengineering in plants and expression in human HEK293 cells we generated four variants of a potent, SARS-CoV-2 neutralizing antibody, COVA2-15 IgA1. The variants that differed in the number of modified proline residues and O-glycan compositions of their hinge region were assessed regarding their physicochemical properties and functionality. Results: We found that plant endogenous O-glycan formation was strongly reduced on IgA1 when transiently expressed in the P4H10 double mutant N. benthamiana plant line. The IgA1 glycoforms displayed differences in proteolytic stability and minor differences in receptor binding thus highlighting the importance of O-glycosylation in the hinge region of human IgA1. Discussion: This work reports the successful protein O-glycan engineering of an important plant host for recombinant protein expression. While the complete removal of endogenous hydroxyproline residues from the hinge region of plant-produced IgA1 is yet to be achieved, our engineered line is suitable for structure-function studies of O-glycosylated recombinant glycoproteins produced in plants.
Bacterial Lactonases ZenA with Noncanonical Structural Features Hydrolyze the Mycotoxin Zearalenone
Abstract Zearalenone (ZEN) is a mycoestrogenic polyketide produced by Fusariumgraminearum and other phytopathogenic members of the genus Fusarium. Contamination of cereals with ZEN is frequent, and hydrolytic detoxification with fungal lactonases has been explored. Here, we report the isolation of a bacterial strain, Rhodococcuserythropolis PFA D8–1, with ZEN hydrolyzing activity, cloning of the gene encoding α/β hydrolase ZenA encoded on the linear megaplasmid pSFRL1, and biochemical characterization of nine homologues. Furthermore, we report site-directed mutagenesis as well as structural analysis of the dimeric ZenARe of R.erythropolis and the more thermostable, tetrameric ZenAScfl of Streptomycescoelicoflavus with and without bound ligands. The X-ray crystal structures not only revealed canonical features of α/β hydrolases with a cap domain including a Ser-His-Asp catalytic triad but also unusual features including an uncommon oxyanion hole motif and a peripheral, short antiparallel β-sheet involved in tetramer interactions. Presteady-state kinetic analyses for ZenARe and ZenAScfl identified balanced rate-limiting steps of the reaction cycle, which can change depending on temperature. Some new bacterial ZEN lactonases have lower KM and higher kcat than the known fungal ZEN lactonases and may lend themselves to enzyme technology development for the degradation of ZEN in feed or food.
Paper of AG Adam in collaboration with Bimm and Department of Agrobiotechnology, IFA Tulln
Fusariumsporotrichioides Produces Two HT-2-α-Glucosides on Rice
Abstract Fusarium is a genus that mostly consists of plant pathogenic fungi which are able to produce a broad range of toxic secondary metabolites. In this study, we focus on a type A trichothecene-producing isolate (15-39) of Fusariumsporotrichioides from Lower Austria. We assessed the secondary metabolite profile and optimized the toxin production conditions on autoclaved rice and found that in addition to large amounts of T-2 and HT-2 toxins, this strain was able to produce HT-2-glucoside. The optimal conditions for the production of T-2 toxin, HT-2 toxin, and HT-2-glucoside on autoclaved rice were incubation at 12 °C under constant light for four weeks, darkness at 30 °C for two weeks, and constant light for three weeks at 20 °C, respectively. The HT-2-glucoside was purified, and the structure elucidation by NMR revealed a mixture of two alpha-glucosides, presumably HT-2-3-O-alpha-glucoside and HT-2-4-O-alpha-glucoside. The efforts to separate the two compounds by HPLC were unsuccessful. No hydrolysis was observed with two the alpha-glucosidases or with human salivary amylase and Saccharomycescerevisiae maltase. We propose that the two HT-2-alpha-glucosides are not formed by a glucosyltransferase as they are in plants, but by a trans-glycosylating alpha-glucosidase expressed by the fungus on the starch-containing rice medium.
Publication from Kathrin Göritzer (Schrödinger fellowship)
Recombinant neutralizing secretory IgA antibodies for preventing mucosal acquisition and transmission of SARS-CoV-2
Passive delivery of antibodies to mucosal sites may be a valuable adjunct to COVID-19 vaccination to prevent infection, treat viral carriage, or block transmission. Neutralizing monoclonal IgG antibodies are already approved for systemic delivery, and several clinical trials have been reported for delivery to mucosal sites where SARS-CoV-2 resides and replicates in early infection. However, secretory IgA may be preferred because the polymeric complex is adapted for the harsh, unstable external mucosal environment. Here, we investigated the feasibility of producing neutralizing monoclonal IgA antibodies against SARS-CoV-2. We engineered two class-switched mAbs that express well as monomeric and secretory IgA (SIgA) variants with high antigen-binding affinities and increased stability in mucosal secretions compared to their IgG counterparts. SIgAs had stronger virus neutralization activities than IgG mAbs and were protective against SARS-CoV-2 infection in an invivo murine model. Furthermore, SIgA1 can be aerosolized for topical delivery using a mesh nebulizer. Our findings provide a persuasive case for developing recombinant SIgAs for mucosal application as a new tool in the fight against COVID-19.
H3K27me3 is vital for fungal development and secondary metabolite gene silencing, and substitutes for the loss of H3K9me3 in the plant pathogen Fusarium proliferatum
H3K27me3 is a hallmark of facultative heterochromatin, and as such tightly connected with gene silencing. In the genus Fusarium, a large proportion of the genome is allocated with this histone mark. Not surprisingly its removal is detrimental and even lethal for some fusaria e.g., the plant pathogen Fusariumfujikuroi for still unknown reasons. Here, we show that Kmt6 is vital but not essential in the closely related Fusariumproliferatum, allowing for the first time a comprehensive analysis of Kmt6 loss-of-function in a member of the Fusariumfujikuroi species complex (FFSC). By an “omics”-oriented approach, we show the genome-wide distribution of activating and silencing histone marks in the wild-type as well as a Δfpkmt6 strain, mapped on a newly annotated genome assembled to near chromosome level. Loss of H3K27me3 and H3K9me3 involved in facultative- and constitutive heterochromatin, respectively, revealed a crucial crosstalk between both histone marks in this genus. These results may explain the subtle phenotype of KMT1 deletion exhibited in this but also in other fusaria, which stands in marked contrast to other fungal species.
Enzymatic synthesis of the modified mycotoxins 3-lactyl- and 3-propionyl-deoxynivalenol
The use of lactic acid bacteria as a low-cost sustainable management tool to prevent further build-up of Fusarium mycotoxins during grain storage is increasingly propagated. It has been reported that even deoxynivalenol contamination already formed in the field can be reduced by such treatment in unknown ways. An enigmatic deoxynivalenol derivative, 3-lactyl-deoxynivalenol, has been reported already in 1982 as the toxic principle of Fusarium-infected barley from China, but very little is known about this metabolite. Here, we show that the enzymatic machinery of Fusariumgraminearum is sufficient for its biosynthesis. Similarly, when challenged with propionic acid, F. graminearum can form a novel modified mycotoxin, 3-propionyl-deoxynivalenol. Lactic acid and propionic acid are first converted into lactyl-CoA and propionyl-CoA, respectively. These acyl-CoA derivatives can subsequently be used by the 3-O-acyltransferase encoded by TRI101. We expressed the respective genes in E. coli and utilized the affinity-purified proteins for enzymatic synthesis of the reference substances 3-lactyl- and 3-propionyl-deoxynivalenol. The structures of the purified compounds were confirmed by nuclear magnetic resonance spectroscopy. Preliminary toxicological assessment using invitro translation assays indicated residual toxicity, most likely due to reactivation of deoxynivalenol by de-acylation. In conclusion, this study reports a method to synthesize 3-lactyl- and 3-propionyl-deoxynivalenol reference substances, which will be highly useful to determine occurrences of these acylated deoxynivalenol-derivatives in cereal samples and to perform more detailed studies to evaluate their toxicological relevance.
Development of a fumonisin-sensitive Saccharomycescerevisiae indicator strain and utilization for activity testing of candidate detoxification genes
Importance Fumonisins can cause diseases in animals and humans consuming Fusarium-contaminated food or feed. The search for microbes capable of fumonisin degradation, or for enzymes that can detoxify fumonisins, currently relies primarily on chemical detection methods. Our constructed fumonisin B1-sensitive yeast strain can be used to phenotypically detect detoxification activity and should be useful in screening for novel fumonisin resistance genes and to elucidate fumonisin metabolism and resistance mechanisms in fungi and plants, and thereby, in the long term, help to mitigate the threat of fumonisins in feed and food.
Publication from David Aleksza and Marie-Theres Hauser
Phytosiderophore pathway response in barley exposed to iron, zinc or copper starvation
Highlights:
Efficient micronutrient acquisition is a critical factor in selecting micronutrient dense crops for human consumption.
In this study we investigated the phytosiderophore pathway response and other genes triggered under either Fe, Zn or Cu starvation in two barley lines differing in micronutrient efficiency by RNAseq.
Our results demonstrate that the PS pathway is not only triggered under Fe and Zn deficiency but also under Cu starvation with the response strength decreasing in the order Fe > Zn > Cu.
PS pathway gene expression, PS exudation, and micronutrient uptake were generally greater in the micronutrient efficient line. Interestingly the fold-change in gene expression and phytosiderophore release compared to the respective control was mostly greater in the inefficient line due to the low control levels. These findings suggest a potential link between a highly expressed PS pathway and high micronutrient accumulation.
Nina Valente was awarded a DOC scholarship by the Austrian Academy of Sciences (ÖAW) for her work on Mechanisms of Resistance to the Own Toxin in Trichothecene Producing Fungi. With the DOC programme, the Austrian Academy of Sciences supports highly qualified doctoral candidates from all fields of research who are completing their doctoral studies at an Austrian university. The certificate was presented to Nina Valente on 8 November 2023 by Heinz Faßmann (ÖAW President) and Ulrike Diebold (ÖAW Vice President).
Marie-Theres Hauser was involved in a childrens book initiated in the COST action PLANTSMETALS and written by Seçkin Eroğlu and Esra Eret. The book is called "Neal's adventures - the mysteries inside plants" and can be downloaded at https://plantmetals.eu/plantmetals-education.html
The key aim was to raise awareness about plants in kids in primary school. It is available in many languages.
Mit hochentwickelten Methoden bringt die Plattform Bioactive Microbial Metabolites (BiMM) Pilze dazu, im Labor neuartige Substanzen als Basis von Wirkstoffen für medizinische und agrarische Anwendungen zu bilden.
Genome analysis of Cephalotrichumgorgonifer and identifcation of the biosynthetic pathway for rasfonin, an inhibitor of KRAS dependent cancer
Background Fungi are important sources for bioactive compounds that find their applications in many important sectors like in the pharma-, food- or agricultural industries. In an environmental monitoring project for fungi involved in soil nitrogen cycling we also isolated Cephalotrichumgorgonifer (strain NG_p51). In the course of strain characterisation work we found that this strain is able to naturally produce high amounts of rasfonin, a polyketide inducing autophagy, apoptosis, necroptosis in human cell lines and showing anti-tumor activity in KRAS-dependent cancer cells.
Abstract Plants can be engineered to recombinantly produce high-quality proteins such as therapeutic proteins and vaccines, also known as molecular farming. Molecular farming can be established in various settings with minimal cold-chain requirements and could thus ensure rapid and global-scale deployment of biopharmaceuticals, promoting equitable access to pharmaceuticals. State of the art plant-based engineering relies on rationally assembled genetic circuits, engineered to enable the high-throughput and rapid expression of multimeric proteins with complex post-translational modifications. In this Review, we discuss the design of expression hosts and vectors, including Nicotianabenthamiana, viral elements and transient expression vectors, for the production of biopharmaceuticals in plants. We examine engineering of post-translational modifications and highlight the plant-based expression of monoclonal antibodies and nanoparticles, such as virus-like particles and protein bodies. Techno-economic analyses suggest a cost advantage of molecular farming compared with mammalian cell-based protein production systems. However, regulatory challenges remain to be addressed to enable the widespread translation of plant-based biopharmaceuticals.
With two articles published in PBJ march issue Herta Steinkellner and Roman Palt were awarded with the front cover image
The publications refer to a cooperation with Qiang Chen (Arizona State University, USA), who held a guest professorship at BOKU in SS2022.
Plant Biotechnology Journal (PBJ) Impact Factor 13 and CiteScore 16. The journal ranks third among plant science journals publishing original research, irrespective of evaluation criteria.
Identification of a UDP-glucosyltransferase conferring deoxynivalenol resistance in Aegilopstauschii and wheat
Summary: Aegilopstauschii is the diploid progenitor of the wheat D subgenome and a valuable resource for wheat breeding, yet, genetic analysis of resistance against Fusarium head blight (FHB) and the major Fusarium mycotoxin deoxynivalenol (DON) is lacking. We treated a panel of 147 Ae. tauschii accessions with either Fusariumgraminearum spores or DON solution and recorded the associated disease spread or toxin-induced bleaching. A k-mer-based association mapping pipeline dissected the genetic basis of resistance and identified candidate genes. After DON infiltration nine accessions revealed severe bleaching symptoms concomitant with lower conversion rates of DON into the non-toxic DON-3-O-glucoside. We identified the gene AET5Gv20385300 on chromosome 5D encoding a uridine diphosphate (UDP)-glucosyltransferase (UGT) as the causal variant and the mutant allele resulting in a truncated protein was only found in the nine susceptible accessions. This UGT is also polymorphic in hexaploid wheat and when expressed in Saccharomycescerevisiae only the full-length gene conferred resistance against DON. Analysing the D subgenome helped to elucidate the genetic control of FHB resistance and identified a UGT involved in DON detoxification in Ae.tauschii and hexaploid wheat. This resistance mechanism is highly conserved since the UGT is orthologous to the barley UGT HvUGT13248 indicating descent from a common ancestor of wheat and barley.
Publication from Anna Atanasoff-Kardjalieff and Lena Studt-Reinhold
Biosynthesis of the Isocoumarin Derivatives Fusamarins Is Mediated by the PKS8 Gene Cluster in Fusarium
Abstract Fusarium mangiferae causes the mango malformation disease (MMD) on young mango trees and seedlings resulting in economically significant crop losses. Next to this, F. mangiferae produces a vast array of secondary metabolites (SMs), including mycotoxins that may contaminate the harvest. Their production is tightly regulated at the transcriptional level. Here, we show that lack of the H3K9-specific histone methyltransferase, FmKmt1, influences the expression of the F. mangiferae polyketide synthase (PKS) 8 ( FmPKS8 ), a so far cryptic PKS. By a combination of reverse genetics, untargeted metabolomics, bioinformatics and chemical analyses including structural elucidation, we determined the FmPKS8 biosynthetic gene cluster (BGC) and linked its activity to the production of fusamarins (FMN), which can be structurally classified as dihydroisocoumarins. Functional characterization of the four FMN cluster genes shed light on the biosynthetic pathway. Cytotoxicity assays revealed moderate toxicities with IC 50 values between 1 and 50 µM depending on the compound.
Poster prizes for Benjamin Kogelmann and Lukas Eidenberger
Congratulations to the two doctoral students Benjamin Kogelmann and Lukas Eidenberger (group Steinkellner) for 2 poster prizes awarded during the International Society for Plant Molecular Farming Conference in Rome!
New paper from AG Adam (collaboration with Rainer Schuhmachers group, SFB Fusarium)
Gramiketides, Novel Polyketide Derivatives of FusariumGraminearum, Are Produced during the Infection of Wheat
Abstract The plant pathogen Fusarium graminearum is a proficient producer of mycotoxins and other in part still unknown secondary metabolites, some of which might act as virulence factors on wheat. The PKS15 gene is expressed only in planta, so far hampering the identification of an associated metabolite. Here we combined the activation of silent gene clusters by chromatin manipulation (kmt6) with blocking the metabolic flow into the competing biosynthesis of the two major mycotoxins deoxynivalenol and zearalenone. Using an untargeted metabolomics approach, two closely related metabolites were found in triple mutants (kmt6 tri5 pks4,13) deficient in production of the major mycotoxins deoxynivalenol and zearalenone, but not in strains with an additional deletion in PKS15 (kmt6 tri5 pks4,13 pks15). Characterization of the metabolites, by LC-HRMS/MS in combination with a stable isotope-assisted tracer approach, revealed that they are likely hybrid polyketides comprising a polyketide part consisting of malonate-derived acetate units and a structurally deviating part. We propose the names gramiketide A and B for the two metabolites. In a biological experiment, both gramiketides were formed during infection of wheat ears with wild-type but not with pks15 mutants. The formation of the two gramiketides during infection correlated with that of the well-known virulence factor deoxynivalenol, suggesting that they might play a role in virulence.
The KdmB-EcoA-RpdA-SntB chromatin complex binds regulatory genes and coordinates fungal development with mycotoxin synthesis
Abstract Chromatin complexes control a vast number of epigenetic developmental processes. Filamentous fungi present an important clade of microbes with poor understanding of underlying epigenetic mechanisms. Here, we describe a chromatin binding complex in the fungus Aspergillus nidulans composing of a H3K4 histone demethylase KdmB, a cohesin acetyltransferase (EcoA), a histone deacetylase (RpdA) and a histone reader/E3 ligase protein (SntB). In vitro and in vivo evidence demonstrate that this KERS complex is assembled from the EcoA-KdmB and SntB-RpdA heterodimers. KdmB and SntB play opposing roles in regulating the cellular levels and stability of EcoA, as KdmB prevents SntB-mediated degradation of EcoA. The KERS complex is recruited to transcription initiation start sites at active core promoters exerting promoter-specific transcriptional effects. Interestingly, deletion of any one of the KERS subunits results in a common negative effect on morphogenesis and production of secondary metabolites, molecules important for niche securement in filamentous fungi. Consequently, the entire mycotoxin sterigmatocystin gene cluster is downregulated and asexual development is reduced in the four KERS mutants. The elucidation of the recruitment of epigenetic regulators to chromatin via the KERS complex provides the first mechanistic, chromatin-based understanding of how development is connected with small molecule synthesis in fungi.
"How to Completely Squeeze a Fungus—Advanced Genome Mining Tools for Novel Bioactive Substances"
Abstract Fungal species have the capability of producing an overwhelming diversity of bioactive substances that can have beneficial but also detrimental effects on human health. These so-called secondary metabolites naturally serve as antimicrobial “weapon systems”, signaling molecules or developmental effectors for fungi and hence are produced only under very specific environmental conditions or stages in their life cycle. However, as these complex conditions are difficult or even impossible to mimic in laboratory settings, only a small fraction of the true chemical diversity of fungi is known so far. This also implies that a large space for potentially new pharmaceuticals remains unexplored. We here present an overview on current developments in advanced methods that can be used to explore this chemical space. We focus on genetic and genomic methods, how to detect genes that harbor the blueprints for the production of these compounds (i.e., biosynthetic gene clusters, BGCs), and ways to activate these silent chromosomal regions. We provide an in-depth view of the chromatin-level regulation of BGCs and of the potential to use the CRISPR/Cas technology as an activation tool.
Abstract Directionality in the intercellular transport of the plant hormone auxin is determined by polar plasma membrane localization of PIN-FORMED (PIN) auxin transport proteins. However, apart from PIN phosphorylation at conserved motifs, no further determinants explicitly controlling polar PIN sorting decisions have been identified. Here we present Arabidopsis WAVY GROWTH 3 (WAV3) and closely related RING-finger E3 ubiquitin ligases, whose loss-of-function mutants show a striking apical-to-basal polarity switch in PIN2 localization in root meristem cells. WAV3 E3 ligases function as essential determinants for PIN polarity, acting independently from PINOID/WAG-dependent PIN phosphorylation. They antagonize ectopic deposition of de novo synthesized PIN proteins already immediately following completion of cell division, presumably via preventing PIN sorting into basal, ARF GEF-mediated trafficking. Our findings reveal an involvement of E3 ligases in the selective targeting of apically localized PINs in higher plants.
"RimO (SrrB) is required for carbon starvation signaling and production of secondary metabolites in Aspergillusnidulans"
Abstract Depending on the prevailing environmental, developmental and nutritional conditions, fungi activate biosynthetic gene clusters (BGCs) to produce condition-specific secondary metabolites (SMs). For activation, global chromatin-based de-repression must be integrated with pathway-specific induction signals. Here we describe a new global regulator needed to activate starvation-induced SMs. In our transcriptome dataset, we found locus AN7572 strongly transcribed solely under conditions of starvation-induced SM production. The predicted AN7572 protein is most similar to the stress and nutritional regulator Rim15 of Saccharomycescerevisiae, and to STK-12 of Neurosporacrassa. Based on this similarity and on stress and nutritional response phenotypes of A. nidulans knock-out and overexpression strains, AN7572 is designated rimO. In relation to SM production, we found that RimO is required for the activation of starvation-induced BGCs, including the sterigmatocystin (ST) gene cluster. Here, RimO regulates the pathway-specific transcription factor AflR both at the transcriptional and post-translational level. At the transcriptional level, RimO mediates aflR induction following carbon starvation and at the post-translational level, RimO is required for nuclear accumulation of the AflR protein. Genome-wide transcriptional profiling showed that cells lacking rimO fail to adapt to carbon starvation that, in the wild type, leads to down-regulation of genes involved in basic metabolism, membrane biogenesis and growth. Consistently, strains overexpressing rimO are more resistant to oxidative and osmotic stress, largely insensitive to glucose repression and strongly overproduce several SMs. Our data indicate that RimO is a positive regulator within the SM and stress response network, but this requires nutrient depletion that triggers both, rimO gene transcription and activation of the RimO protein.
"Polaramycin B, and not physical interaction, is the signal that rewires fungal metabolism in the Streptomyces–Aspergillus interaction"
Abstract Co-culturing the bacterium Streptomycesrapamycinicus and the ascomycete Aspergillusnidulans has previously been shown to trigger the production of orsellinic acid (ORS) and its derivates in the fungal cells. Based on these studies it was assumed that direct physical contact is a prerequisite for the metabolic reaction that involves a fungal amino acid starvation response and activating chromatin modifications at the biosynthetic gene cluster (BGC). Here we show that not physical contact, but a guanidine containing macrolide, named polaramycin B, triggers the response. The substance is produced constitutively by the bacterium and above a certain concentration, provokes the production of ORS. In addition, several other secondary metabolites were induced by polaramycin B. Our genome-wide transcriptome analysis showed that polaramycin B treatment causes downregulation of fungal genes necessary for membrane stability, general metabolism and growth. A compensatory genetic response can be observed in the fungus that included upregulation of BGCs and genes necessary for ribosome biogenesis, translation and membrane stability. Our work discovered a novel chemical communication, in which the antifungal bacterial metabolite polaramycin B leads to the production of antibacterial defence chemicals and to the upregulation of genes necessary to compensate for the cellular damage caused by polaramycin B.
Publication from Elena Feraru with scientific contribution from Korbei Lab
"PILS proteins provide a homeostatic feedback on auxin signaling output"
Abstract Multiple internal and external signals modulate the metabolism, intercellular transport and signaling of the phytohormone auxin. Considering this complexity, it remains largely unknown how plant cells monitor and ensure the homeostasis of auxin responses. PIN-LIKES (PILS) intracellular auxin transport facilitators at the endoplasmic reticulum are suitable candidates to buffer cellular auxin responses because they limit nuclear abundance and signaling of auxin. We used forward genetics to identify gloomyandshinypils (gasp) mutants that define the PILS6 protein abundance in a post-translational manner. Here, we show that GASP1 encodes an uncharacterized RING/U-box superfamily protein that impacts on auxin signaling output. The low auxin signaling in gasp1 mutants correlates with reduced abundance of PILS5 and PILS6 proteins. Mechanistically, we show that high and low auxin conditions increase and reduce PILS6 protein levels, respectively. Accordingly, non-optimum auxin concentrations are buffered by alterations in PILS6 abundance, consequently leading to homeostatic auxin output regulation. We envision that this feedback mechanism provides robustness to auxin-dependent plant development.
"Identification and Functional Characterisation of Two Oat UDP-Glucosyltransferases Involved in Deoxynivalenol Detoxification"
Abstract Oat is susceptible to several Fusarium species that cause contamination with different trichothecene mycotoxins. The molecular mechanisms behind Fusarium resistance in oat have yet to be elucidated. In the present work, we identified and characterised two oat UDP-glucosyltransferases orthologous to barley HvUGT13248. Overexpression of the latter in wheat had been shown previously to increase resistance to deoxynivalenol (DON) and nivalenol (NIV) and to decrease disease the severity of both Fusarium head blight and Fusarium crown rot. Both oat genes are highly inducible by the application of DON and during infection with Fusariumgraminearum. Heterologous expression of these genes in a toxin-sensitive strain of Saccharomycescerevisiae conferred high levels of resistance to DON, NIV and HT-2 toxins, but not C4-acetylated trichothecenes (T-2, diacetoxyscirpenol). Recombinant enzymes AsUGT1 and AsUGT2 expressed in Escherichiacoli rapidly lost activity upon purification, but the treatment of whole cells with the toxin clearly demonstrated the ability to convert DON into DON-3-O-glucoside. The two UGTs could therefore play an important role in counteracting the Fusarium virulence factor DON in oat.
"The Hydrophilic Loop of Arabidopsis PIN1 Auxin Efflux Carrier Harbors Hallmarks of an Intrinsically Disordered Protein"
Abstract Much of plant development depends on cell-to-cell redistribution of the plant hormone auxin, which is facilitated by the plasma membrane (PM) localized PIN FORMED (PIN) proteins. Auxin export activity, developmental roles, subcellular trafficking, and polarity of PINs have been well studied, but their structure remains elusive besides a rough outline that they contain two groups of 5 alpha-helices connected by a large hydrophilic loop (HL). Here, we focus on the PIN1 HL as we could produce it in sufficient quantities for biochemical investigations to provide insights into its secondary structure. Circular dichroism (CD) studies revealed its nature as an intrinsically disordered protein (IDP), manifested by the increase of structure content upon thermal melting. Consistent with IDPs serving as interaction platforms, PIN1 loops homodimerize. PIN1 HL cytoplasmic overexpression in Arabidopsis disrupts early endocytic trafficking of PIN1 and PIN2 and causes defects in the cotyledon vasculature formation. In summary, we demonstrate that PIN1 HL has an intrinsically disordered nature, which must be considered to gain further structural insights. Some secondary structures may form transiently during pairing with known and yet-to-be-discovered interactors.
Der Manfred Schwanninger Preis wird für die Entwicklung von qualitativhochwertigenLehr-undLernmaterialien und dessen didaktischsinnvollenEinsatz verliehen.
Sabine Strauss-Goller und Claudia Puck haben den Preis für die Entwicklung einer 3D Virtuell Reality Experience erhalten, mit Hilfe derer man in das “Innenleben einer Pilzzelle“ blicken kann.
Die offiziellen Preisverleihung findet am 11.05.2022 um 16:30 statt.
"Ubiquitination of the ubiquitin-binding machinery: how early ESCRT components are controlled"
Abstract To be able to quickly and accurately respond to the environment, cells need to tightly control the amount and localization of plasma membrane proteins. The post-translation modification by the protein modifier ubiquitin is the key signal for guiding membrane-associated cargo to the lysosome/vacuole for their degradation. The machinery responsible for such sorting contains several subunits that function as ubiquitin receptors, many of which are themselves subjected to ubiquitination. This review will focus on what is currently known about the modulation of the machinery itself by ubiquitination and how this might affect its function with a special emphasis on current findings from the plant field.
Diese Tabakpflanze rettet Leben - FWF scilog mit Herta Steinkellner
Der Molekularbiologin Herta Steinkellner ist es gemeinsam mit ihrem Team gelungen, hochwirksame Antikörper gegen Covid-19 zu erzeugen. Das Erstaunliche an ihrer Methode: Die medizinischen Wunderwaffen werden von – humanisierten – Pflanzen produziert. Möglich ist das, weil auf genetischer Ebene die Grenzen zwischen den Lebewesen verschwimmen.
Qiang Chen Professor, Biodesign Center for Immunotherapy, Vaccines and Virotherapy Qiang.Chen.4@asu.edu
Guestprofessor at BOKU Host: Herta Steinkellner
Content: • Viral infection mechanism and immunological response • Induction of specific immune response (epitope profiling) • Biotechnology innovations in vaccine development (nucleic acid -,protein and vector based) • How to approach temperature stability, needle free delivery, and simplicity of manufacture • The long and winding road to approval
Learning outcomes: Students enrolled in this course should • gain a comprehensive understanding of the science (e.g. virology, immunology, vaccinology) behind various vaccines. • be able to critically evaluate current vaccines and vaccine platforms. • be able to propose innovative ideas of new vaccine candidates.
Paper from Anna Atanasoff-Kardjalieff and Lena Studt
"Secondary Metabolite Gene Regulation in Mycotoxigenic Fusarium Species: A Focus on Chromatin"
Abstract Fusarium is a species-rich group of mycotoxigenic plant pathogens that ranks as one of the most economically important fungal genera in the world. During growth and infection, they are able to produce a vast spectrum of low-molecular-weight compounds, so-called secondary metabolites (SMs). SMs often comprise toxic compounds (i.e., mycotoxins) that contaminate precious food and feed sources and cause adverse health effects in humans and livestock. In this context, understanding the regulation of their biosynthesis is crucial for the development of cropping strategies that aim at minimizing mycotoxin contamination in the field. Nevertheless, currently, only a fraction of SMs have been identified, and even fewer are considered for regular monitoring by regulatory authorities. Limitations to exploit their full chemical potential arise from the fact that the genes involved in their biosynthesis are often silent under standard laboratory conditions and only induced upon specific stimuli mimicking natural conditions in which biosynthesis of the respective SM becomes advantageous for the producer. This implies a complex regulatory network. Several components of these gene networks have been studied in the past, thereby greatly advancing the understanding of SM gene regulation and mycotoxin biosynthesis in general. This review aims at summarizing the latest advances in SM research in these notorious plant pathogens with a focus on chromatin structure.
Nature paper on COVID-19 with contribution from Herta Steinkellner
"Virus-induced senescence is a driver and therapeutic target in COVID-19"
Abstract Derailed cytokine and immune cell networks account for the organ damage and the clinical severity of COVID-19 (refs. 1,2,3,4). Here we show that SARS-CoV-2, like other viruses, evokes cellular senescence as a primary stress response in infected cells. Virus-induced senescence (VIS) is indistinguishable from other forms of cellular senescence and is accompanied by a senescence-associated secretory phenotype (SASP), which comprises pro-inflammatory cytokines, extracellular-matrix-active factors and pro-coagulatory mediators5,6,7. Patients with COVID-19 displayed markers of senescence in their airway mucosa in situ and increased serum levels of SASP factors. In vitro assays demonstrated macrophage activation with SASP-reminiscent secretion, complement lysis and SASP-amplifying secondary senescence of endothelial cells, which mirrored hallmark features of COVID-19 such as macrophage and neutrophil infiltration, endothelial damage and widespread thrombosis in affected lung tissue1,8,9. Moreover, supernatant from VIS cells, including SARS-CoV-2-induced senescence, induced neutrophil extracellular trap formation and activation of platelets and the clotting cascade. Senolytics such as navitoclax and a combination of dasatinib plus quercetin selectively eliminated VIS cells, mitigated COVID-19-reminiscent lung disease and reduced inflammation in SARS-CoV-2-infected hamsters and mice. Our findings mark VIS as a pathogenic trigger of COVID-19-related cytokine escalation and organ damage, and suggest that senolytic targeting of virus-infected cells is a treatment option against SARS-CoV-2 and perhaps other viral infections.
Publication from Izadi Shiva and Alexandra Castilho
"Reteplase Fc-fusions produced in N.benthamiana are able to dissolve blood clots exvivo"
Abstract Thrombolytic and fibrinolytic therapies are effective treatments to dissolve blood clots in stroke therapy. Thrombolytic drugs activate plasminogen to its cleaved form plasmin, a proteolytic enzyme that breaks the crosslinks between fibrin molecules. The FDA-approved human tissue plasminogen activator Reteplase (rPA) is a non-glycosylated protein produced in E. coli. rPA is a deletion mutant of the wild-type Alteplase that benefits from an extended plasma half-life, reduced fibrin specificity and the ability to better penetrate into blood clots. Different methods have been proposed to improve the production of rPA. Here we show for the first time the transient expression in Nicotianabenthamiana of rPA fused to the immunoglobulin fragment crystallizable (Fc) domain on an IgG1, a strategy commonly used to improve the stability of therapeutic proteins. Despite our success on the expression and purification of dimeric rPA-Fc fusions, protein instability results in high amounts of Fc-derived degradation products. We hypothesize that the “Y”- shape of dimeric Fc fusions cause steric hindrance between protein domains and leads to physical instability. Indeed, mutations of critical residues in the Fc dimerization interface allowed the expression of fully stable rPA monomeric Fc-fusions. The ability of rPA-Fc to convert plasminogen into plasmin was demonstrated by plasminogen zymography and clot lysis assay shows that rPA-Fc is able to dissolve blood clots exvivo. Finally, we addressed concerns with the plant-specific glycosylation by modulating rPA-Fc glycosylation towards serum-like structures including α2,6-sialylated and α1,6-core fucosylated N-glycans completely devoid of plant core fucose and xylose residues.
BiMM paper on a new bioactive molecule from a new fungus
"Luteapyrone, a Novel ƴ-Pyrone Isolated from the Filamentous Fungus Metapochonialutea"
Abstract In the process of screening for new bioactive microbial metabolites we found a novel ƴ-pyrone derivative for which we propose the trivial name luteapyrone, in a recently described microscopic filamentous fungus, Metapochonialutea BiMM-F96/DF4. The compound was isolated from the culture extract of the fungus grown on modified yeast extract sucrose medium by means of flash chromatography followed by preparative HPLC. The chemical structure was elucidated by NMR and LC-MS. The new compound was found to be non-cytotoxic against three mammalian cell lines (HEK 263, KB-3.1 and Caco-2). Similarly, no antimicrobial activity was observed in tested microorganisms (gram positive and negative bacteria, yeast and fungi).
"Highly active engineered IgG3 antibodies against SARS-CoV-2"
Abstract Monoclonal antibodies (mAbs) that efficiently neutralize SARS-CoV-2 have been developed at an unprecedented speed. Notwithstanding, there is a vague understanding of the various Ab functions induced beyond antigen binding by the heavy-chain constant domain. To explore the diverse roles of Abs in SARS-CoV-2 immunity, we expressed a SARS-CoV-2 spike protein (SP) binding mAb (H4) in the four IgG subclasses present in human serum (IgG1-4) using glyco-engineered Nicotianabenthamiana plants. All four subclasses, carrying the identical antigen-binding site, were fully assembled in planta and exhibited a largely homogeneous xylose- and fucose-free glycosylation profile. The Ab variants ligated to the SP with an up to fivefold increased binding activity of IgG3. Furthermore, all H4 subtypes were able to neutralize SARS-CoV-2. However, H4-IgG3 exhibited an up to 50-fold superior neutralization potency compared with the other subclasses. Our data point to a strong protective effect of IgG3 Abs in SARS-CoV-2 infection and suggest that superior neutralization might be a consequence of cross-linking the SP on the viral surface. This should be considered in therapy and vaccine development. In addition, we underscore the versatile use of plants for the rapid expression of complex proteins in emergency cases.
Paper in EMBO Reports with contribution of the Schüller/ BiMM lab
"A phosphatase-centric mechanism drives stress signaling response"
Abstract Changing environmental cues lead to the adjustment of cellular physiology by phosphorylation signaling networks that typically center around kinases as active effectors and phosphatases as antagonistic elements. Here, we report a signaling mechanism that reverses this principle. Using the hyperosmotic stress response in Saccharomycescerevisiae as a model system, we find that a phosphatase-driven mechanism causes induction of phosphorylation. The key activating step that triggers this phospho-proteomic response is the Endosulfine-mediated inhibition of protein phosphatase 2A-Cdc55 (PP2ACdc55), while we do not observe concurrent kinase activation. In fact, many of the stress-induced phosphorylation sites appear to be direct substrates of the phosphatase, rendering PP2ACdc55 the main downstream effector of a signaling response that operates in parallel and independent of the well-established kinase-centric stress signaling pathways. This response affects multiple cellular processes and is required for stress survival. Our results demonstrate how a phosphatase can assume the role of active downstream effectors during signaling and allow re-evaluating the impact of phosphatases on shaping the phosphorylome.
"Light regulates alternative splicing outcomes via the TOR kinase pathway"
Summary For plants, light is the source of energy and the most relevant regulator of growth and adaptations to the environment by inducing changes in gene expression at various levels, including alternative splicing. Light-triggered chloroplast retrograde signals control alternative splicing in Arabidopsisthaliana. Here, we provide evidence that light regulates the expression of a core set of splicing-related factors in roots. Alternative splicing responses in roots are not directly caused by light but are instead most likely triggered by photosynthesized sugars. The target of rapamycin (TOR) kinase plays a key role in this shoot-to-root signaling pathway. Knocking down TOR expression or pharmacologically inhibiting TOR activity disrupts the alternative splicing responses to light and exogenous sugars in roots. Consistently, splicing decisions are modulated by mitochondrial activity in roots. In conclusion, by activating the TOR pathway, sugars act as mobile signals to coordinate alternative splicing responses to light throughout the whole plant.
"Development and Validation of an LC-MS/MS Based Method for the Determination of Deoxynivalenol and Its Modified Forms in Maize"
Abstract The Fusarium mycotoxin deoxynivalenol (DON) is a common contaminant of cereals and is often co-occurring with its modified forms DON-3-glucoside (D3G), 3-acetyl-DON (3ADON) or 15-acetyl-DON (15ADON). A stable-isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) based method for their determination in cereals was developed and validated for maize. Therefore, 13C-labelled D3G was enzymatically produced using 13C-DON and [13C6Glc]-sucrose and used as an internal standard (IS) for D3G, while uniformly 13C labelled IS was used for the other mycotoxins. Baseline separation was achieved for the critical peak pair DON/D3G, while 3ADON/15ADON could not be fully baseline separated after testing various reversed phase, fluorinated phase and chiral LC columns. After grinding, weighing and extracting the cereal samples, the raw extract was centrifuged and a mixture of the four 13C-labelled ISs was added directly in a microinsert vial. The subsequent analytical run took 7 min, followed by negative electrospray ionization and selected reaction monitoring on a triple quadrupole MS. Maize was used as a complex cereal model matrix for validation. The use of the IS corrected the occurring matrix effects efficiently from 76 to 98% for D3G, from 86 to 103% for DON, from 68 to 100% for 15ADON and from 63 to 96% for 3ADON.
"Polyphasic Approach Utilized for the Identification of Two New Toxigenic Members of Penicillium Section Exilicaulis, P. krskae and P. silybi spp. nov."
Abstract Two new species, Penicilliumkrskae (isolated from the air as a lab contaminant in Tulln (Austria, EU)) and Penicilliumsilybi (isolated as an endophyte from asymptomatic milk thistle (Silybummarianum) stems from Josephine County (Oregon, USA)) are described. The new taxa are well supported by phenotypic (especially conidial ornamentation under SEM, production of red exudate and red pigments), physiological (growth at 37 °C, response to cycloheximide and CREA), chemotaxonomic (production of specific extrolites), and multilocus phylogenetic analysis using RNA-polymerase II second largest subunit (RPB2), partial tubulin (benA), and calmodulin (CaM). Both new taxa are resolved within the section Exilicaulis in series Restricta and show phylogenetic affiliation to P.restrictumsensustricto. They produce a large spectrum of toxic anthraquinoid pigments, namely, monomeric anthraquinones related to emodic and chloremodic acids and other interesting bioactive extrolites (i.e., endocrocin, paxilline, pestalotin, and 7-hydroxypestalotin). Of note, two bianthraquinones (i.e., skyrin and oxyskyrin) were detected in a culture extract of P.silybi. Two new chloroemodic acid derivatives (2-chloro-isorhodoptilometrin and 2-chloro-desmethyldermoquinone) isolated from the exudate of P.krskae ex-type culture were analyzed by nuclear magnetic resonance (NMR) and liquid chromatography–mass spectrometry (LC–MS).
"Identification and Functional Characterization of the Gene Cluster Responsible for Fusaproliferin Biosynthesis in Fusariumproliferatum"
Abstract The emerging mycotoxin fusaproliferin is produced by Fusariumproliferatum and other related Fusarium species. Several fungi from other taxonomic groups were also reported to produce fusaproliferin or the deacetylated derivative, known as siccanol or terpestacin. Here, we describe the identification and functional characterization of the Fusariumproliferatum genes encoding the fusaproliferin biosynthetic enzymes: a terpenoid synthase, two cytochrome P450s, a FAD-oxidase and an acetyltransferase. With the exception of one gene encoding a CYP450 (FUP2, FPRN_05484), knock-out mutants of the candidate genes could be generated, and the production of fusaproliferin and intermediates was tested by LC-MS/MS. Inactivation of the FUP1 (FPRN_05485) terpenoid synthase gene led to complete loss of fusaproliferin production. Disruption of a putative FAD-oxidase (FUP4, FPRN_05486) did not only affect oxidation of preterpestacin III to terpestacin, but also of new side products (11-oxo-preterpstacin and terpestacin aldehyde). In the knock-out strains lacking the predicted acetyltransferase (FUP5, FPRN_05487) fusaproliferin was no longer formed, but terpestacin was found at elevated levels. A model for the biosynthesis of fusaproliferin and of novel derivatives found in mutants is presented.
"Molecular systematics of Keratinophyton: the inclusion of species formerly referred to Chrysosporium and description of four new species"
Abstract Four new Keratinophyton species (Ascomycota, Pezizomycotina, Onygenales), K.gollerae, K.lemmensii, K.straussii, and K.wagneri, isolated from soil samples originating from Europe (Austria, Italy, and Slovakia) are described and illustrated. The new taxa are well supported by phylogenetic analysis of the internal transcribed spacer region (ITS) region, the combined data analysis of ITS and the nuclear large subunit (LSU) rDNA, and their phenotype. Based on ITS phylogeny, within the Keratinophyton clade, K.lemmensii is clustered with K.durum, K.hubeiense, K.submersum, and K.siglerae, while K.gollerae, K.straussii and K.wagneri are resolved in a separate terminal cluster. All four new species can be well distinguished from other species in the genus based on phenotype characteristics alone. Ten new combinations are proposed for Chrysosporium species which are resolved in the monophyletic Keratinophyton clade. A new key to the recognized species is provided herein.
"Biosynthesis of Fusapyrone Depends on the H3K9 Methyltransferase, FmKmt1, in Fusariummangiferae"
The phytopathogenic fungus Fusariummangiferae belongs to the Fusariumfujikuroi species complex (FFSC). Members of this group cause a wide spectrum of devastating diseases on diverse agricultural crops. F.mangiferae is the causal agent of the mango malformation disease (MMD) and as such detrimental for agriculture in the southern hemisphere. During plant infection, the fungus produces a plethora of bioactive secondary metabolites (SMs), which most often lead to severe adverse defects on plants health. Changes in chromatin structure achieved by posttranslational modifications (PTM) of histones play a key role in regulation of fungal SM biosynthesis. Posttranslational tri-methylation of histone 3 lysine 9 (H3K9me3) is considered a hallmark of heterochromatin and established by the SET-domain protein Kmt1. Here, we show that FmKmt1 is involved in H3K9me3 in F. mangiferae. Loss of FmKmt1 only slightly though significantly affected fungal hyphal growth and stress response and is required for wild type-like conidiation. While FmKmt1 is largely dispensable for the biosynthesis of most known SMs, removal of FmKMT1 resulted in an almost complete loss of fusapyrone and deoxyfusapyrone, γ-pyrones previously only known from Fusarium semitectum. Here, we identified the polyketide synthase (PKS) FmPKS40 to be involved in fusapyrone biosynthesis, delineate putative cluster borders by co-expression studies and provide insights into its regulation.
"The Instability of Dimeric Fc-Fusions Expressed in Plants Can Be Solved by Monomeric Fc Technology"
The potential therapeutic value of many proteins is ultimately limited by their rapid invivo clearance. One strategy to limit clearance by metabolism and excretion, and improving the stability of therapeutic proteins, is their fusion to the immunoglobulin fragment crystallizable region (Fc). The Fc region plays multiple roles in (i) dimerization for the formation of “Y”-shaped structure of Ig, (ii) Fc-mediated effector functions, (iii) extension of serum half-life, and (iv) a cost-effective purification tag. Plants and in particular Nicotianabenthamiana have proven to be suitable expression platforms for several recombinant therapeutic proteins. Despite the enormous success of their use for the production of full-length monoclonal antibodies, the expression of Fc-fused therapeutic proteins in plants has shown limitations. Many Fc-fusion proteins expressed in plants show different degrees of instability resulting in high amounts of Fc-derived degradation products. To address this issue, we used erythropoietin (EPO) as a reporter protein and evaluated the efforts to enhance the expression of full-length EPO-Fc targeted to the apoplast of N.benthamiana. Our results show that the instability of the fusion protein is independent from the Fc origin or IgG subclass and from the peptide sequence used to link the two domains. We also show that a similar instability occurs upon the expression of individual heavy chains of monoclonal antibodies and ScFv-Fc that mimic the “Y”-shape of antibodies but lack the light chain. We propose that in this configuration, steric hindrance between the protein domains leads to physical instability. Indeed, mutations of critical residues located on the Fc dimerization interface allowed the expression of fully stable EPO monomeric Fc-fusion proteins. We discuss the limitations of Fc-fusion technology in N.benthamiana transient expression systems and suggest strategies to optimize the Fc-based scaffolds on their folding and aggregation resistance in order to improve the stability.
"Development and Validation of a Simple Bioaerosol Collection Filter System Using a Conventional Vacuum Cleaner for Sampling"
Abstract Although numerous bioaerosol samplers for counting and identifying airborne microorganisms are available, the considerably high purchase and maintenance costs for the sampler often prevent broad monitoring campaigns for occupational or environmental surveillance of bioaerosols. We present here a newly developed simple adapter and filter system (TOP filter system) designed to collect bioaerosol particles from a defined air volume using conventional vacuum cleaners as air pumps. We characterized the physical properties of the system using air flow measurements and validated the biological performance. The culture-based detection capacities for airborne fungal species were compared to a standard impaction sampler (MAS-100 NT) under controlled conditions in a bioaerosol chamber (using Trichoderma spores as the test organism) as well as in the field. In the chamber, an overall equivalent detection capacity between all tested filters was recorded, although a significant underrepresentation of the TOP filter system for Trichoderma spores were seen in comparison to the MAS-100 NT. In a comparative field study (n = 345), the system showed similar biological sampling efficiencies compared to the MAS-100 NT impactor, only the diversity of identified fungal communities was slightly lower on the filters. Thus, the system is suitable for large-scale environmental sampling operations where many samples have to be taken in parallel at a given time at distant locations. This system would allow endeavors such as antibiotics resistance monitoring or hygiene surveys in agricultural or occupational settings.
2. Preis für Krishna Mutanwad beim 20. CAS Semester-Touchdown
Am 18. Juni 2021 fand unser 20. CAS Semester-Touchdown der Agrarwissenschaften statt.
Auf dem Touchdown wurden 5 Abschlussarbeiten aus dem Sommersemester 2021 präsentiert, die aus den Bereichen der Pflanzenwissenschaften, Tierwissenschaften und den Wirtschafts- und Sozialwissenschaften kamen.
Preisträger*innen
1. Preis: Thomas Zankl 2. Preis: Dr. Krishna Vasant Mutanwad 3. Preis: Kerstin Kolkmann
» Lucyshyn Lab
Publication from David Canovas and Joseph Strauss
"Chromatin Regulation and Epigenetics" - a special issue for "Fungal Genetics and Biology"
"Proper protein folding in the endoplasmic reticulum is required for attachment of a glycosylphosphatidylinositol anchor in plants"
Abstract Endoplasmic reticulum (ER) quality control processes recognize and eliminate misfolded proteins to maintain cellular protein homeostasis and prevent the accumulation of defective proteins in the secretory pathway. Glycosylphosphatidylinositol (GPI)-anchored proteins carry a glycolipid modification, which provides an efficient ER export signal and potentially prevents the entry into ER-associated degradation (ERAD), which is one of the major pathways for clearance of terminally misfolded proteins from the ER. Here, we analyzed the degradation routes of different misfolded glycoproteins carrying a C-terminal GPI-attachment signal peptide in Arabidopsisthaliana. We found that a fusion protein consisting of the misfolded extracellular domain from Arabidopsis STRUBBELIG and the GPI-anchor attachment sequence of COBRA1 was efficiently targeted to hydroxymethylglutaryl reductase degradation protein 1 complex-mediated ERAD without the detectable attachment of a GPI anchor. Non-native variants of the GPI-anchored lipid transfer protein 1 (LTPG1) that lack a severely misfolded domain, on the other hand, are modified with a GPI anchor and targeted to the vacuole for degradation. Impaired processing of the GPI-anchoring signal peptide by mutation of the cleavage site or in a GPI-transamidase-compromised mutant caused ER retention and routed the non-native LTPG1 to ERAD. Collectively, these results indicate that for severely misfolded proteins, ER quality control processes are dominant over ER export. For less severely misfolded proteins, the GPI anchor provides an efficient ER export signal resulting in transport to the vacuole.
Long-distance transport is central to the mode of action of plant growth regulators. But in the case of brassinosteroids, spatiotemporal control of biosynthesis and local movements of the bioactive molecule or its precursors result in local hormone accumulation, functioning as a positional cue in root morphogenesis.
"Getting to the Root of Belowground High Temperature Responses in Plants"
Abstract The environment is continuously challenging plants. As a response, plants use various coping strategies, such as adaptation of their growth. Thermomorphogenesis is a specific growth adaptation that promotes organ growth in response to moderately high temperature. This would eventually enable plants to cool down by dissipating the heat. Although well understood for shoot organs, the thermomorphogenesis response in roots only recently obtained increasing research attention. Accordingly, in the last few years, the hormonal responses and underlying molecular players important for root thermomorphogenesis were revealed. Other responses triggered by high temperature in the root encompass modifications of overall root architecture and interactions with the soil environment, with consequences on the whole plant. Here, we review the scientific knowledge and highlight the current understanding on roots responding to moderately high and extreme temperature.
940098 Seminars in Molecular Biotechnology (SE) - Herta Steinkellner Content will be continuously adapted according to latest findings
Corona viruses: structure and life cycle
SARS CoV2 diagnostics (nucleic acid, antigen and antibody tests)
COVID 19 vaccination program (features of approved candidates)
Epidemiological and immunological data (infection and mortality rate, virus evolution and immunity)
940331 Cell factory plants (UE) - Herta Steinkellner, Richard Strasser, Jennifer Schoberer A key factor to contain COVID 19 pandemic is the rapid production of SARS CoV2 relevant proteins for both diagnostic (e.g. virus antigens) and therapeutic (protective antibodies) purposes.
The course refers to this need and focuses on the quick expression of:
a SARS CoV2 spike protein domain (which plays a key role in viral infection)
a specific antibody that binds to this domain
Determining the binding properties of the two components for diagnostic purposes (ELISA binding studies).
Examination of subcellular localization of recombinant proteins using confocal microscopy.
940003 Emerging Topics in RNA Biology (in Eng.) (SE) – Mariya Kalyna Topic: RNA vaccines – coming of age This year seminar topic is motivated by the coronavirus pandemic. What are RNA vaccines? How do they differ from conventional vaccine approaches? What are the disease targets? What are challenges and future directions in developing these new vaccine approaches?
FWF doc.funds Programme - with participation of Marie-Theres Hauser
DOC 111 „MENTOR – Molecular Mechanisms to Improve Plant Resilience“
Plant derived resources are an essential pillar for earth’s ecosystems and human survival. Food security in the future will depend on sustainable crop production to meet the projected demands. Due to climate change, the impact of “stress”, for instance temperature extremes, droughts, floods and pathogen infestations, on plant growth and development is a pressing issue. A better understanding of the molecular mechanisms of plant acclimation to stress will provide a significant key to future crop improvement. The MENTOR program will educate a cohort of future experts and equip them with the necessary expertise to evaluate and develop plant resilience strategies.
MENTOR will be the first PhD program in Austria dedicated to molecular plant sciences. Six of the nine groups (Bachmair, Ibl, Schlögelhofer, Teige, Weckwerth, Wienkoop) work at the University of Vienna (UNIVIE), one (Dagdas) at the Gregor Mendel Institute of Molecular Plant Biology (GMI), one (Hauser) at the University of Natural Resources and Life Sciences (BOKU), and one group (Jonak) is based at the AIT Austrian Institute of Technology in Tulln near Vienna.
"Auxin and Root Gravitropism: Addressing Basic Cellular Processes by Exploiting a Defined Growth Response"
Abstract: Root architecture and growth are decisive for crop performance and yield, and thus a highly topical research field in plant sciences. The root system of the model plant Arabidopsisthaliana is the ideal system to obtain insights into fundamental key parameters and molecular players involved in underlying regulatory circuits of root growth, particularly in responses to environmental stimuli. Root gravitropism, directional growth along the gravity, in particular represents a highly sensitive readout, suitable to study adjustments in polar auxin transport and to identify molecular determinants involved. This review strives to summarize and give an overview into the function of PIN-FORMED auxin transport proteins, emphasizing on their sorting and polarity control. As there already is an abundance of information, the focus lies in integrating this wealth of information on mechanisms and pathways. This overview of a highly dynamic and complex field highlights recent developments in understanding the role of auxin in higher plants. Specifically, it exemplifies, how analysis of a single, defined growth response contributes to our understanding of basic cellular processes in general.
Publication from Mach, Castilho and Steinkellner Lab
"Identification of two subtilisin‐like serine proteases engaged in the degradation of recombinant proteins in Nicotianabenthamiana"
Abstract The tobacco variant Nicotianabenthamiana has recently emerged as a versatile host for the manufacturing of protein therapeutics, but the fidelity of many recombinant proteins generated in this system is compromised by inadvertent proteolysis. Previous studies have revealed that the anti‐HIV‐1 antibodies 2F5 and PG9 as well as the protease inhibitor α1‐antitrypsin (A1AT) are particularly susceptible to N. benthamiana proteases. Here, we identify two subtilisin‐like serine proteases (NbSBT1 and NbSBT2) whose combined action is sufficient to account for all major cleavage events observed upon expression of 2F5, PG9 and A1AT in N. benthamiana. We propose that downregulation of NbSBT1 and NbSBT2 activities could constitute a powerful means to optimize the performance of this promising platform for the production of biopharmaceuticals.
"Generation of enzymatically competent SARS‐CoV‐2 decoy receptor ACE2‐Fc in glycoengineered Nicotianabenthamiana"
Abstract Human angiotensin‐converting enzyme 2 (ACE2) is the primary host cell receptor for Severe Acute Respiratory Syndrome Coronavirus‐2 (SARS‐CoV‐2) binding and cell entry. Administration of high concentrations of soluble ACE2 can be utilized as a decoy to block the interaction of the virus with cellular ACE2 receptors and potentially be used as a strategy for treatment or prevention of coronavirus disease 2019 (COVID‐19). Human ACE2 is heavily glycosylated and its glycans impact on binding to the SARS‐CoV‐2 spike protein and virus infectivity. Here, we describe the production of a recombinant soluble ACE2‐Fc variant in glycoengineered Nicotianabenthamiana. Our data reveal that the produced dimeric ACE2‐Fc variant is glycosylated with mainly complex human‐type N‐glycans and functional with regard to enzyme activity, affinity to the SARS‐CoV‐2 receptor‐binding domain (RBD) and wild‐type virus neutralization.
"The FLA4-FEI Pathway: A Unique and Mysterious Signaling Module Related to Cell Wall Structure and Stress Signaling"
Abstract Cell wall integrity control in plants involves multiple signaling modules that are mostly defined by genetic interactions. The putative co-receptors FEI1 and FEI2 and the extracellular glycoprotein FLA4 present the core components of a signaling pathway that acts in response to environmental conditions and insults to cell wall structure to modulate the balance of various growth regulators and, ultimately, to regulate the performance of the primary cell wall. Although the previously established genetic interactions are presently not matched by intermolecular binding studies, numerous receptor-like molecules that were identified in genome-wide interaction studies potentially contribute to the signaling machinery around the FLA4-FEI core. Apart from its function throughout the model plant Arabidopsisthaliana for the homeostasis of growth and stress responses, the FLA4-FEI pathway might support important agronomic traits in crop plants.
"Naphthylphthalamic acid associates with and inhibits PIN auxin transporters"
Significance The plant hormone auxin regulates many aspects of plant life and has the unique ability to flow throughout the plant in defined directions, as observed by Darwin over a century ago. The chemical NPA inhibits this directional flow, thereby severely affecting plant growth. In studying the specific effects of NPA, researchers have also uncovered general principles underlying plant development. Exactly how NPA inhibits directional auxin flow is unclear. NPA interacts with proteins that can transport auxin, such as ABCB transporters, and we show here that NPA also associates with and inhibits the major transporters that specialize in directional auxin flow—PINs. This explanation of NPA action will guide future research and may help reveal how PINs perform auxin transport.
Doris Lucyshyn auf Ö1 über die Folgen der UG-Novelle
Doris Lucyshyn und Christoph Reinprecht vom Institut für Soziologie (Universität Wien) haben in der Punkt eins Folge vom 20.01.2021 über die Folgen dier UG-Novelle für Studierende, Forschende und die Hochschulen selbst diskutiert.
Studierende müssen künftig jedes Jahr eine Mindestleistung erbringen, ansonsten erlischt ihre Zulassung; die Senate der Universitäten verlieren Mitbestimmungsrechte; die maximale Dauer befristeter Arbeitsverhältnisse wird auf acht Jahre gesetzt. Diese Neuerungen und einige andere Vorhaben, die sich in der Novelle des Universitätsgesetzes finden, sorgen bei vielen für Unmut und Kritik, bei anderen für Zustimmung.
Immer mehr Bakterien werden gegen die bekannten Antibiotika resistent und damit nicht mehr behandelbar. Das BIMM-Team in Tulln forscht mit Robotertechnik an Schimmelpilzen, die das ändern sollen. Sie könnten die Antibiotika der Zukunft werden – unter anderem.
BiMM cooperation paper with Italian Fusarium group on the mycotoxin repressing function of thymol
"Naturally Occurring Phenols Modulate Vegetative Growth and Deoxynivalenol Biosynthesis in Fusariumgraminearum"
This paper reports a couple of natural substances from plants that repress the production of the main Fusarium mycotoxin DON but does not inhibit growth of the fungus. Application perspectives are open and we also will try to understand the underlying mechanisms.
Abstract To assess the in vitro activity of five naturally occurring phenolic compounds (ferulic acid, apocynin, magnolol, honokiol, and thymol) on mycelial growth and type B trichothecene mycotoxin accumulation by Fusariumgraminearum, three complementary approaches were adopted. First, a high-throughput photometric continuous reading array allowed a parallel quantification of F.graminearum hyphal growth and reporter TRI5 gene expression directly on solid medium. Second, RT-qPCR confirmed the regulation of TRI5 expression by the tested compounds. Third, liquid chromatography–tandem mass spectrometry analysis allowed quantification of deoxynivalenol (DON) and its acetylated forms released upon treatment with the phenolic compounds. Altogether, the results confirmed the activity of thymol and an equimolar mixture of thymol–magnolol at 0.5 mM, respectively, in inhibiting DON production without affecting vegetative growth. The medium pH buffering capacity after 72–96 h of incubation is proposed as a further element to highlight compounds displaying trichothecene inhibitory capacity with no significant fungicidal effect.
Revealing a complex interplay between alternative splicing, RNAi, mRNA isoform compartmentalization and nonsense-mediated mRNA decay - Paper by Maria Kalyna
"Targeting alternative splicing by RNAi: from the differential impact on splice variants to triggering artificial pre-mRNA splicing"
Abstract Alternative splicing generates multiple transcript and protein isoforms from a single gene and controls transcript intracellular localization and stability by coupling to mRNA export and nonsense-mediated mRNA decay (NMD). RNA interference (RNAi) is a potent mechanism to modulate gene expression. However, its interactions with alternative splicing are poorly understood. We used artificial microRNAs (amiRNAs, also termed shRNAmiR) to knockdown all splice variants of selected target genes in Arabidopsisthaliana. We found that splice variants, which vary by their protein-coding capacity, subcellular localization and sensitivity to NMD, are affected differentially by an amiRNA, although all of them contain the target site. Particular transcript isoforms escape amiRNA-mediated degradation due to their nuclear localization. The nuclear and NMD-sensitive isoforms mask RNAi action in alternatively spliced genes. Interestingly, Arabidopsis SPL genes, which undergo alternative splicing and are targets of miR156, are regulated in the same manner. Moreover, similar results were obtained in mammalian cells using siRNAs, indicating cross-kingdom conservation of these interactions among RNAi and splicing isoforms. Furthermore, we report that amiRNA can trigger artificial alternative splicing, thus expanding the RNAi functional repertoire. Our findings unveil novel interactions between different post-transcriptional processes in defining transcript fates and regulating gene expression.
Hertha Firnberg fellowship for Lena Studt (Strauss Lab)
EpiVit: Role, function and crosstalk of Kmt6 in Fusarium spp.
Phytopathogenic fusaria produce a plethora of natural compounds, referred to as secondary metabolites (SMs), including harmful mycotoxins that frequently contaminate food and feed. A crucial step to combat mycotoxin contaminations is to understand the regulatory network that orchestrates their biosynthesis. Here, morphological changes in chromatin structure in general and Kmt6-mediated histone 3 lysine 27 methylation in particular play a key role in fungal SM gene regulation. Within a recently FWF-funded Hertha-Firnberg-Stipend to Lena Studt, we will explore Kmt6-mediated gene silencing and study in depth SM gene expression at the level of chromatin in the genus Fusarium, to unravel regulatory circuits that orchestrate SM biosynthesis. Results from this study will thus provide foundational knowledge to advance the understanding of epigenetic mechanisms ruling fungal life traits, but also for novel drug-discovery strategies.
Publication on the role of nucleosome positioning in regulation of alternative splicing in response to cold in Arabidopsis with contribution from Maria Kalyna
"Differential nucleosome occupancy modulates alternative splicing in Arabidopsisthaliana"
Summary
Alternative splicing (AS) is a major gene regulatory mechanism in plants. Recent evidence supports co‐transcriptional splicing in plants, hence the chromatin state can impact AS. However, how dynamic changes in the chromatin state such as nucleosome occupancy influence the cold‐induced AS remains poorly understood.
Here, we generated transcriptome (RNA‐Seq) and nucleosome positioning (MNase‐Seq) data for Arabidopsisthaliana to understand how nucleosome positioning modulates cold‐induced AS.
Our results show that characteristic nucleosome occupancy levels are strongly associated with the type and abundance of various AS events under normal and cold temperature conditions in Arabidopsis. Intriguingly, exitrons, alternatively spliced internal regions of protein‐coding exons, exhibit distinctive nucleosome positioning pattern compared to other alternatively spliced regions. Likewise, nucleosome patterns differ between exitrons and retained introns pointing to their distinct regulation.
Collectively, our data show that characteristic changes in nucleosome positioning modulate AS in plants in response to cold.
H2A.Z is essential there, but it was possible to select strains that survive despite having a H2A.Z deletion. WGS of several revertants revealed a number of possible compensatory mutations, all of them affected in components of the chromatin remodelling machinery. Hence, a new molecular link between H2A.Z and chromatin remodeller function is popping up.
Publication with Contribution from Gergely Molnár (Strauss Lab)
"Receptor kinase module targets PIN-dependent auxin transport during canalization"
Spontaneously arising channels that transport the phytohormone auxin provide positional cues for selforganizing aspects of plant development such as flexible vasculature regeneration or its patterning during leaf venation. The auxin canalization hypothesis proposes a feedback between auxin signaling and transport as the underlying mechanism, but molecular players await discovery. We identified part of the machinery that routes auxin transport. The auxin-regulated receptor CAMEL (Canalization-related Auxin-regulated Malectin-type RLK) together with CANAR (Canalization-related Receptor-like kinase) interact with and phosphorylate PIN auxin transporters. camel and canar mutants are impaired in PIN1 subcellular trafficking and auxin-mediated PIN polarization, which macroscopically manifests as defects in leaf venation and vasculature regeneration after wounding. The CAMEL-CANAR receptor complex is part of the auxin feedback that coordinates polarization of individual cells during auxin canalization.
"Beyond the visible and below the peel: how UV-B radiation influences the phenolic profile in the pulp of peach fruit. A biochemical and molecular study"
In the last decades, UV-B radiation has attracted attention due to its potential to increase nutraceutical values of fruit and vegetables, especially by inducing the accumulation of phenolics in a structure-dependent way. However, most current studies have investigated the UV-B-driven changes only in the peel or focusing on individual phenolic classes. Adopting an “-omics” approach, this work aimed to deepen the knowledge about the effects of UV-B radiation on the phenolic profile in the pulp of peach fruit. Based on these considerations, melting flesh yellow peaches (Prunuspersica L., cv. Fairtime) were subjected to either a 10- or 60-min UV-B treatment (1.39 and 8.33 kJ m–2, respectively), and sampled at different time points from the exposure. A UHPLC-ESI/QTOF-MS analysis coupled with a phenolics-specific database for the annotation of compounds and a multivariate discriminant analysis revealed a marked effect of UV-B radiation on the phenolic profiles of peach pulp. Particularly, a general, transient increase was observed after 24 h from the irradiation, especially for flavanols, flavonols, and flavones. Such behavior diverges from what was observed in the peel, where an overall increase of phenolics was observed after 36 h from the irradiation. Concerning the flavonols in the pulp, UV-B exposure stimulated a specific accumulation of isorhamnetin and kaempferol derivatives, with variations imposed by the different sugar moiety bound. Anthocyanins, which were the second most abundant flavonoid group after flavonols, displayed a general decrease after 36 h that was not attributable to specific molecules. The UV-B treatments also increased the glycoside/aglycone ratio of flavonols and anthocyanins after 24 h, by increasing the glycoside concentration of both, flavonols and anthocyanins, and decreasing the aglycone concentration of anthocyanins. In support of the biochemical results, targeted gene expression analysis by RT-qPCR revealed an UV-B-induced activation of many genes involved in the flavonoid pathway, e.g., CHS, F3H, F3′H, DFR, as well as some MYB transcription factors and few genes involved in the UV-B perception. Generally, all the flavonoid-related and MYB genes showed a transient UV-B dose-dependent activation after 6 h from the irradiation, similarly to what was observed in the peel.
We kindly acknowledge the technical support of Julia Richter and Nataliia Konstantinova for help with the sampling. Marco Santin was further supported by an Erasmus+ traineeship.
The Internet of Fungi - communication and memory within a fungal network
Our contribution to this year´s "Lange Nacht der Forschung": the simplified 3D animation movie of the inner life of a fungal cell. It was created in the course of the "Sparkling Science" project "The Internet of Fungi - communication and memory within a fungal network". The tour is of course most enjoyable with a real VR equipment, however, also on your 2D screen, it is a nice cell biology travel experience.
"A novel fungal gene regulation system based on inducible VPR-dCas9 and nucleosome map-guided sgRNA positioning"
Abstract Programmable transcriptional regulation is a powerful tool to study gene functions. Current methods to selectively regulate target genes are mainly based on promoter exchange or on overexpressing transcriptional activators. To expand the discovery toolbox, we designed a dCas9-based RNA-guided synthetic transcription activation system for Aspergillusnidulans that uses enzymatically disabled “dead” Cas9 fused to three consecutive activation domains (VPR-dCas9). The dCas9-encoding gene is under the control of an estrogen-responsive promoter to allow induction timing and to avoid possible negative effects by strong constitutive expression of the highly active VPR domains. Especially in silent genomic regions, facultative heterochromatin and strictly positioned nucleosomes can constitute a relevant obstacle to the transcriptional machinery. To avoid this negative impact and to facilitate optimal positioning of RNA-guided VPR-dCas9 to targeted promoters, we have created a genome-wide nucleosome map from actively growing cells and stationary cultures to identify the cognate nucleosome-free regions (NFRs). Based on these maps, different single-guide RNAs (sgRNAs) were designed and tested for their targeting and activation potential. Our results demonstrate that the system can be used to regulate several genes in parallel and, depending on the VPR-dCas9 positioning, expression can be pushed to very high levels. We have used the system to turn on individual genes within two different biosynthetic gene clusters (BGCs) which are silent under normal growth conditions. This method also opens opportunities to stepwise activate individual genes in a cluster to decipher the correlated biosynthetic pathway.
"A trimeric CrRLK1L-LLG1 complex genetically modulates SUMM2-mediated autoimmunity"
Abstract Cell death is intrinsically linked with immunity. Disruption of an immune-activated MAPK cascade, consisting of MEKK1, MKK1/2, and MPK4, triggers cell death and autoimmunity through the nucleotide-binding leucine-rich repeat (NLR) protein SUMM2 and the MAPK kinase kinase MEKK2. In this study, we identify a Catharanthusroseus receptor-like kinase 1-like (CrRLK1L), named LETUM2/MEDOS1 (LET2/MDS1), and the glycosylphosphatidylinositol (GPI)-anchored protein LLG1 as regulators of mekk1-mkk1/2-mpk4 cell death. LET2/MDS1 functions additively with LET1, another CrRLK1L, and acts genetically downstream of MEKK2 in regulating SUMM2 activation. LET2/MDS1 complexes with LET1 and promotes LET1 phosphorylation, revealing an intertwined regulation between different CrRLK1Ls. LLG1 interacts with the ectodomain of LET1/2 and mediates LET1/2 transport to the plasma membrane, corroborating its function as a co-receptor of LET1/2 in the mekk1-mkk1/2-mpk4 cell death pathway. Thus, our data suggest that a trimeric complex consisting of two CrRLK1Ls LET1, LET2/MDS1, and a GPI-anchored protein LLG1 that regulates the activation of NLR SUMM2 for initiating cell death and autoimmunity.
Abstract Root hairs are able to sense soil composition and play an important role for water and nutrient uptake. In Arabidopsisthaliana, root hairs are distributed in the epidermis in a specific pattern, regularly alternating with non-root hair cells in continuous cell files. This patterning is regulated by internal factors such as a number of hormones, as well as external factors like nutrient availability. Thus, root hair patterning is an excellent model for studying the plasticity of cell fate determination in response to environmental changes. Here, we report that loss-of-function mutants in the Protein O-fucosyltransferase, SPINDLY (SPY) show defects in root hair patterning. Using transcriptional reporters, we show that patterning in spy-22 is affected upstream of the GLABRA2 (GL2) and WEREWOLF (WER). O-fucosylation of nuclear and cytosolic proteins is an important post-translational modification that is still not very well understood. So far, SPY is best characterized for its role in gibberellin signaling via fucosylation of the growth-repressing DELLA protein REPRESSOR OF ga1-3 (RGA). Our data suggests that the epidermal patterning defects in spy-22 are independent of RGA and gibberellin signaling.
"Strigolactones inhibit auxin feedback on PIN-dependent auxin transport canalization"
Abstract Directional transport of the phytohormone auxin is a versatile, plant-specific mechanism regulating many aspects of plant development. The recently identified plant hormones, strigolactones (SLs), are implicated in many plant traits; among others, they modify the phenotypic output of PIN-FORMED (PIN) auxin transporters for fine-tuning of growth and developmental responses. Here, we show in pea and Arabidopsis that SLs target processes dependent on the canalization of auxin flow, which involves auxin feedback on PIN subcellular distribution. D14 receptor- and MAX2 F-box-mediated SL signaling inhibits the formation of auxin-conducting channels after wounding or from artificial auxin sources, during vasculature de novo formation and regeneration. At the cellular level, SLs interfere with auxin effects on PIN polar targeting, constitutive PIN trafficking as well as clathrin-mediated endocytosis. Our results identify a non-transcriptional mechanism of SL action, uncoupling auxin feedback on PIN polarity and trafficking, thereby regulating vascular tissue formation and regeneration.
"Establishment of Proximity-dependent Biotinylation Approaches in Different Plant Model Systems"
Abstract Proximity labeling is a powerful approach for detecting protein-protein interactions. Most proximity labeling techniques use a promiscuous biotin ligase (PBL) or a peroxidase fused to a protein of interest, enabling the covalent biotin labelling of proteins and subsequent capture and identification of interacting and neighbouring proteins without the need for the protein complex to remain intact. To date, only few papers report on the use of proximity labeling in plants. Here, we present the results of a systematic study applying a variety of biotin-based proximity labeling approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols which combine Mass Spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as test-case. We further benchmark the efficiency of various PBLs in comparison with one-step affinity purification approaches. We identified both known as well as novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both non-biotinylated as well as biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to infer structural information of protein complexes.
"On the Potential Function of Type II Arabinogalactan O-Glycosylation in Regulating the Fate of Plant Secretory Proteins"
In a plant-specific mode of protein glycosylation, various sugars and glycans are attached to hydroxyproline giving rise to a variety of diverse O-glycoproteins. The sub-family of arabinogalactan proteins is implicated in a multitude of biological functions, however, the mechanistic role of O-glycosylation on AGPs by type II arabinogalactans is largely elusive. Some models suggest roles of the O-glycans such as in ligand-receptor interactions and as localized calcium ion store. Structurally different but possibly analogous types of protein O-glycosylation exist in animal and yeast models and roles for O-glycans were suggested in determining the fate of O-glycoproteins by affecting intracellular sorting or proteolytic activation and degradation. At present, only few examples exist that describe how the fate of artificial and endogenous arabinogalactan proteins is affected by O-glycosylation with type II arabinogalactans. In addition to other roles, these glycans might act as a molecular determinant for cellular localization and protein lifetime of many endogenous proteins.
Topic Editors: Ezequiel Petrillo, CONICET Institute of Physiology, Molecular Biology and Neurosciences (IFIBYNE), Argentina Maria Kalyna, University of Natural Resources and Life Sciences Vienna, Austria Craig G. Simpson, The James Hutton Institute, United Kingdom Shih-Long Tu, Institute of Plant and Microbial Biology, Academia Sinica, Taiwan Kranthi Kiran Mandadi, Texas A&M University, United States
"The Beginning of the End: Initial Steps in the Degradation of Plasma Membrane Proteins"
The plasma membrane (PM), as border between the inside and the outside of a cell, is densely packed with proteins involved in the sensing and transmission of internal and external stimuli, as well as transport processes and is therefore vital for plant development as well as quick and accurate responses to the environment. It is consequently not surprising that several regulatory pathways participate in the tight regulation of the spatiotemporal control of PM proteins. Ubiquitination of PM proteins plays a key role in directing their entry into the endo-lysosomal system, serving as a signal for triggering endocytosis and further sorting for degradation. Nevertheless, a uniting picture of the different roles of the respective types of ubiquitination in the consecutive steps of down-regulation of membrane proteins is still missing. The trans-Golgi network (TGN), which acts as an early endosome (EE) in plants receives the endocytosed cargo, and here the decision is made to either recycled back to the PM or further delivered to the vacuole for degradation. A multi-complex machinery, the endosomal sorting complex required for transport (ESCRT), concentrates ubiquitinated proteins and ushers them into the intraluminal vesicles of multi-vesicular bodies (MVBs). Several ESCRTs have ubiquitin binding subunits, which anchor and guide the cargos through the endocytic degradation route. Basic enzymes and the mode of action in the early degradation steps of PM proteins are conserved in eukaryotes, yet many plant unique components exist, which are often essential in this pathway. Thus, deciphering the initial steps in the degradation of ubiquitinated PM proteins, which is the major focus of this review, will greatly contribute to the larger question of how plants mange to fine-tune their responses to their environment.
"The outer influences the inner: postharvest UV-B irradiation modulates peach flesh metabolome although shielded by the skin"
Highlights • Peach flesh metabolome is strongly influenced by UV-B exposure • Phenolics, terpenoids and phytoalexins highly responded to UV-B • Decreased levels of most metabolic classes were detected after 24 h • Accumulation of most metabolic classes was observed after 36 h • UV-B radiation does not penetrate peach skin.
Abstract UV-B-driven modulation of secondary metabolism in peach fruit by enhancing the biosynthesis of specific phenolic subclasses, is attracting interest among consumers. However, current literature explored the UV-B-induced metabolic changes only in peach skin subjected to direct UV-B irradiation. Accordingly, this study aimed to understand whether UV-B radiation penetrates the fruit skin and is able to induce metabolic changes also within the inner flesh. Peaches were UV-B-irradiated either 10 or 60 min, and the flesh was sampled after 24 and 36 h. Non-targeted metabolomics revealed that UV-B has a strong impact on peach flesh metabolome, determining an initial decrease after 24 h, followed by an overall increase after 36 h, particularly for terpenoids, phenylpropanoids, phytoalexins and fatty acids in the 60 min UV-B-treated samples (+150.02, +99.14, +43.79 and +25.44 log2FC, respectively). Transmittance analysis indicated that UV-B radiation does not penetrate below the skin, suggesting a possible signalling pathway between tissues.
"Suppression of Trichothecene-Mediated Immune Response by the Fusarium Secondary Metabolite Butenolide in Human Colon Epithelial Cells"
Abstract Butenolide (BUT, 4-acetamido-4-hydroxy-2-butenoic acid gamma-lactone) is a secondary metabolite produced by several Fusarium species and is co-produced with the major trichothecene mycotoxin deoxynivalenol (DON) on cereal grains throughout the world. BUT has low acute toxicity and only very limited occurrence and exposure data are available. The intestinal epithelium represents the first physiological barrier against food contaminants. We aimed to elucidate the intestinal inflammatory response of the human, non-cancer epithelial HCEC-1CT cells to BUT and to characterize potential combinatory interactions with co-occurring trichothecenes, such as DON and NX-3. Using a reporter gene approach, BUT (≥5 μM, 20 h) was found to decrease lipopolysaccharide (LPS; 10 ng/mL) induced nuclear factor kappa B (NF-κB) activation in a dose-dependent manner, and in combinatory treatments BUT represses trichothecene-induced enhancement of this important inflammatory pathway. Analysis of transcription and secretion levels of NF-κB-dependent, pro-inflammatory cytokines, revealed a significant down-regulation of IL-1β, IL-6, and TNF-α in IL-1β-stimulated (25 ng/mL) HCEC-1CT cells after BUT exposure (10 μM). Trichothecene-induced expression of pro-inflammatory cytokines by the presence of 1 μM DON or NX-3 was substantially suppressed in the presence of 10 μM BUT. The emerging mycotoxin BUT has the ability to suppress NF-κB-induced intestinal inflammatory response mechanisms and to modulate substantially the immune responsiveness of HCEC-1CT cells after trichothecene treatment. Our results suggest that BUT, present in naturally occurring mixtures of Fusarium fungal metabolites, should be increasingly monitored, and the mechanism of inhibition of NF-κB that might affect the pathogenesis or progression of intestinal inflammatory disorders, should be further investigated.
Editorial on the Research Topic: Alternative Splicing Regulation in Plants
Research on alternative splicing (AS) in plants has bloomed during the past decade, largely fueled by the advance of high-throughput sequencing (HTS) technologies and pioneering papers demonstrating an unexpectedly high frequency of AS in plants (Filichkin et al., 2010; Lu et al., 2010; Marquez et al., 2012; Syed et al., 2012). The formation and regulation of alternative transcripts from individual transcribed genes by alternative splice site selection pervades all aspects of a eukaryote's development and adaptive response to its changing environment. This is particularly relevant to sessile plant species that must be able to rapidly respond to abiotic, biotic, diurnal, and seasonal fluctuations. The mechanism of pre-mRNA splicing and the process of splice site selection has existed since its divergence from metazoans (Fedorov et al., 2002) and is regulated by splicing factors that are components of the assembling spliceosome. Many knowledge gaps remain to be addressed, not only to define the AS prevalence in different plant species and its impact on various biological processes, but also to understand its mechanistic basis with the aim of manipulating crops for important traits required for food security. Here, we share with the plant biology community a Research Topic that aims to showcase current findings, emerging questions, and technical advances, in the field of AS in plants.
On the discovery of an endomembrane compartment in plants
To maintain homeostasis and to react to external stimuli, eukaryotic cells have evolved a complex internal membrane system. Among them, lytic compartments are hallmarks of eukaryotic cells: Animals possess lysosomes, whereas fungi and plants build up vacuoles. Although largely molecularly conserved, the plant endomembrane system displays unique features, among them the presence of large vacuoles. The central vacuole can occupy up to 90% of the cellular volume in mature vegetative tissues and is involved in numerous essential functions. These include degradation of cellular waste, storage of ions and proteins, plant growth, defense against pathogens, and intracellular pH homeostasis. Given the importance of vacuoles for plants, it is not surprising that trafficking to this compartment has been intensively studied. Despite significant progress toward the identification of different trafficking routes, the underlying molecular machinery is far from being understood and is, in part, controversially discussed in the field. In PNAS, Delgadillo et al. (1) elegantly identify and characterize a set of mutants impaired in vacuolar transport. Most identified mutant alleles were known trafficking factors, but subsequent localization analysis proposes the identification of a previously unknown plant compartment.
The lipid code-dependent phosphoswitch PDK1–D6PK activates PIN-mediated auxin efflux in Arabidopsis
Abstract Directional intercellular transport of the phytohormone auxin mediated by PIN-FORMED (PIN) efflux carriers has essential roles in both coordinating patterning processes and integrating multiple external cues by rapidly redirecting auxin fluxes. PIN activity is therefore regulated by multiple internal and external cues, for which the underlying molecular mechanisms are not fully elucidated. Here, we demonstrate that 3′-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1 (PDK1), which is conserved in plants and mammals, functions as a molecular hub that perceives upstream lipid signalling and modulates downstream substrate activity through phosphorylation. Using genetic analysis, we show that the loss-of-function Arabidopsis pdk1.1 pdk1.2 mutant exhibits a plethora of abnormalities in organogenesis and growth due to defective polar auxin transport. Further cellular and biochemical analyses reveal that PDK1 phosphorylates D6 protein kinase, a well-known upstream activator of PIN proteins. We uncover a lipid-dependent phosphorylation cascade that connects membrane-composition-based cellular signalling with plant growth and patterning by regulating morphogenetic auxin fluxes.
Mag. Dr. Barbara Korbei BOKU-Institut für Molekulare Pflanzenbiologie für das wissenschaftliche Fach Molekulare Zellbiologie
mit dem Lehrvortrag: Mechanismus der Endozytose in Pflanzen
und dem Fachvortrag: Ubiquitin receptors in the early steps of plasma membrane protein degradation in plants
Donnerstag, 14. Mai 2020, 13:15 Uhr Per Zoom-Meeting: bokuvienna.zoom.us/j/95315229552 ************************************************************* Die Vorsitzende der Habilitationskommission Univ.-Prof. Mag. Dr. Eva Stöger Department für Angewandte Genetik und Zellbiologie
Publication from Sascha Waidmann (Kleine-Vehn Lab)
"Asymmetric cytokinin signaling opposes gravitropism in roots"
Abstract Plants depend on gravity to provide the constant landmark for downward root growth and upward shoot growth. The phytohormone auxin and its cell‐to‐cell transport machinery are central determinants ensuring gravitropic growth. Statolith sedimentation toward gravity is sensed in specialized cells. This positional cue is translated into the polar distribution of PIN auxin efflux carriers at the plasma membrane, leading to asymmetric auxin distribution and consequently, differential growth and organ bending. While we have started to understand the general principles of how primary organs execute gravitropism, we currently lack basic understanding of how lateral plant organs can defy gravitropic responses. Here we briefly review the establishment of the oblique gravitropic set point angle in lateral roots and particularly discuss the emerging role of asymmetric cytokinin signaling as a central anti‐gravitropic signal. Differential cytokinin signaling is co‐opted in gravitropic lateral and hydrotropic primary roots to counterbalance gravitropic root growth.
"PIN-LIKES Coordinate Brassinosteroid Signaling with Nuclear Auxin Input in Arabidopsisthaliana"
Highlights • Impaired BR perception enhances PILS5 overexpression phenotypes • BR signaling increases PILS protein turnover • BR signaling defines PILS-dependent nuclear abundance and signaling of auxin • PILS-dependent BR-auxin crosstalk affects organ growth
Summary Auxin and brassinosteroids (BR) are crucial growth regulators and display overlapping functions during plant development. Here, we reveal an alternative phytohormone crosstalk mechanism, revealing that BR signaling controls PIN-LIKES (PILS)-dependent nuclear abundance of auxin. We performed a forward genetic screen for imperial pils (imp) mutants that enhance the overexpression phenotypes of PILS5 putative intracellular auxin transport facilitator. Here, we report that the imp1 mutant is defective in the BR-receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1). Our set of data reveals that BR signaling transcriptionally and post-translationally represses the accumulation of PILS proteins at the endoplasmic reticulum, thereby increasing nuclear abundance and signaling of auxin. We demonstrate that this alternative phytohormonal crosstalk mechanism integrates BR signaling into auxin-dependent organ growth rates and likely has widespread importance for plant development.
"High Throughput Screening for New Fungal Polyester Hydrolyzing Enzymes"
here is a strong need for novel and more efficient polyester hydrolyzing enzymes in order to enable the development of more environmentally friendly plastics recycling processes allowing the closure of the carbon cycle. In this work, a high throughput system on microplate scale was used to screen a high number of fungi for their ability to produce polyester-hydrolyzing enzymes. For induction of responsible enzymes, the fungi were cultivated in presence of aliphatic and aromatic polyesters [poly(1,4-butylene adipate co terephthalate) (PBAT), poly(lactic acid) (PLA) and poly(1,4-butylene succinate) (PBS)], and the esterase activity in the culture supernatants was compared to the culture supernatants of fungi grown without polymers. The results indicate that the esterase activity of the culture supernatants was induced in about 10% of the tested fungi when grown with polyesters in the medium, as indicated by increased activity (to >50 mU/mL) toward the small model substrate para-nitrophenylbutyrate (pNPB). Incubation of these 50 active culture supernatants with different polyesters (PBAT, PLA, PBS) led to hydrolysis of at least one of the polymers according to liquid chromatography-based quantification of the hydrolysis products terephthalic acid, lactic acid and succinic acid, respectively. Interestingly, the specificities for the investigated polyesters varied among the supernatants of the different fungi.
"Efficient N-Glycosylation of the Heavy Chain Tailpiece Promotes the Formation of Plant-Produced Dimeric IgA"
Production of monomeric IgA in mammalian cells and plant expression systems such as Nicotianabenthamiana is well-established and can be achieved by co-expression of the corresponding light and heavy chains. In contrast, the assembly of dimeric IgA requires the additional expression of the joining chain and remains challenging especially in plant-based systems. Here, we examined factors affecting the assembly and expression of HER2 binding dimeric IgA1 and IgA2m(2) variants transiently produced in N.benthamiana. While co-expression of the joining chain resulted in efficient formation of dimeric IgAs in HEK293F cells, a mixture of monomeric, dimeric and tetrameric variants was detected in plants. Mass-spectrometric analysis of site-specific glycosylation revealed that the N-glycan profile differed between monomeric and dimeric IgAs in the plant expression system. Co-expression of a single-subunit oligosaccharyltransferase from the protozoan Leishmaniamajor in N.benthamiana increased the N-glycosylation occupancy at the C-terminal heavy chain tailpiece and changed the ratio of monomeric to dimeric IgAs. Our data demonstrate that N-glycosylation engineering is a suitable strategy to promote the formation of dimeric IgA variants in plants.
"Plant-based production of highly potent anti-HIV antibodies with engineered posttranslational modifications"
Abstract Broadly neutralising antibodies (bNAbs) against human immunodeficiency virus type 1 (HIV-1), such as CAP256-VRC26 are being developed for HIV prevention and treatment. These Abs carry a unique but crucial post-translational modification (PTM), namely O-sulfated tyrosine in the heavy chain complementarity determining region (CDR) H3 loop. Several studies have demonstrated that plants are suitable hosts for the generation of highly active anti-HIV-1 antibodies with the potential to engineer PTMs. Here we report the expression and characterisation of CAP256-VRC26 bNAbs with posttranslational modifications (PTM). Two variants, CAP256-VRC26 (08 and 09) were expressed in glycoengineered Nicotianabenthamiana plants. By in planta co-expression of tyrosyl protein sulfotransferase 1, we installed O-sulfated tyrosine in CDR H3 of both bNAbs. These exhibited similar structural folding to the mammalian cell produced bNAbs, but non-sulfated versions showed loss of neutralisation breadth and potency. In contrast, tyrosine sulfated versions displayed equivalent neutralising activity to mammalian produced antibodies retaining exceptional potency against some subtype C viruses. Together, the data demonstrate the enormous potential of plant-based systems for multiple posttranslational engineering and production of fully active bNAbs for application in passive immunisation or as an alternative for current HIV/AIDS antiretroviral therapy regimens.
"Steric Accessibility of the Cleavage Sites Dictates the Proteolytic Vulnerability of the Anti‐HIV‐1 Antibodies 2F5, 2G12, and PG9 in Plants"
Abstract Broadly neutralizing antibodies (bNAbs) to human immunodeficiency virus type 1 (HIV‐1) hold great promise for immunoprophylaxis and the suppression of viremia in HIV‐positive individuals. Several studies have demonstrated that plants as Nicotianabenthamiana are suitable hosts for the generation of protective anti‐HIV‐1 antibodies. However, the production of the anti‐HIV‐1 bNAbs 2F5 and PG9 in N. benthamiana is associated with their processing by apoplastic proteases in the complementarity‐determining‐region (CDR) H3 loops of the heavy chains. Here, it is shown that apoplastic proteases can also cleave the CDR H3 loop of the bNAb 2G12 when the unusual domain exchange between its heavy chains is prevented by the replacement of Ile19 with Arg. It is demonstrated that CDR H3 proteolysis leads to a strong reduction of the antigen‐binding potencies of 2F5, PG9, and 2G12‐I19R. Inhibitor profiling experiments indicate that different subtilisin‐like serine proteases account for bNAb fragmentation in the apoplast. Differential scanning calorimetry experiments corroborate that the antigen‐binding domains of wild‐type 2G12 and 4E10 are more compact than those of proteolysis‐sensitive antibodies, thus shielding their CDR H3 regions from proteolytic attack. This suggests that the extent of proteolytic inactivation of bNAbs in plants is primarily dictated by the steric accessibility of their CDR H3 loops.
"Pro-Inflammatory Effects of NX-3 Toxin Are Comparable to Deoxynivalenol and not Modulated by the Co-Occurring Pro-Oxidant Aurofusarin"
Abstract The type A trichothecene NX-3, produced by certain Fusariumgraminearum strains, is similar to the mycotoxin deoxynivalenol (DON), with the exception that it lacks the carbonyl moiety at the C-8 position. NX-3 inhibits protein biosynthesis and induces cytotoxicity to a similar extent as DON, but so far, immunomodulatory effects have not been assessed. In the present study, we investigated the impact of NX-3 on the activity of the nuclear factor kappa B (NF-κB) signaling pathway in direct comparison to DON. Under pro-inflammatory conditions (IL-1β treatment), the impact on cytokine mRNA levels of NF-κB downstream genes was studied in human colon cell lines, comparing noncancer (HCEC-1CT) and cancer cells (HT-29). In addition, potential combinatory effects with the co-occurring Fusarium secondary metabolite aurofusarin (AURO), a dimeric naphthoquinone known to induce oxidative stress, were investigated. NX-3 and DON (1 μM, 20 h) significantly activated a NF-κB regulated reporter gene to a similar extent. Both trichothecenes also enhanced transcript levels of the known NF-κB-dependent pro-inflammatory cytokines IL-8, IL-6, TNF-α and IL-1β. Comparing the colon cancer HT-29 and noncancer HCEC-1CT cells, significant differences in cytokine signaling were identified. In contrast, AURO did not affect NF-κB pathway activity and respective cytokine expression levels at the tested concentration. Despite its pro-oxidant potency, the combination with AURO did not significantly affect the immunomodulatory effects of the tested trichothecenes. Taken together, the present study reveals comparable potency of DON and NX-3 with respect to immunomodulatory and pro-inflammatory potential. Consequently, not only DON but also NX-3 should be considered as factors contributing to intestinal inflammatory processes.
"Current Challenges in Studying Alternative Splicing in Plants: The Case of Physcomitrellapatens SR Proteins"
To colonize different terrestrial habitats, early land plants had to overcome the challenge of coping with harsh new environments. Alternative splicing – an RNA processing mechanism through which splice sites are differentially recognized, originating multiple transcripts and potentially different proteins from the same gene – can be key for plant stress tolerance. Serine/arginine-rich (SR) proteins constitute an evolutionarily conserved family of major alternative splicing regulators that in plants subdivides into six subfamilies. Despite being well studied in animals and a few plant species, such as the model angiosperm Arabidopsisthaliana and the crop Oryzasativa, little is known of these splicing factors in early land plants. Establishing the whole complement of SR proteins in different species is essential to understand the functional and evolutionary significance of alternative splicing. An insilico search for SR proteins in the extant moss Physcomitrella patens revealed inconsistencies both in the published data and available databases, likely arising from automatic annotation lacking adequate manual curation. These misannotations interfere with the description not only of the number and subfamily classification of Physcomitrella SR proteins but also of their domain architecture, potentially hindering the elucidation of their molecular functions. We therefore advise caution when looking into P.patens genomic resources. Our systematic survey nonetheless confidently identified 16 P. patens SR proteins that fall into the six described subfamilies and represent counterparts of well-established members in Arabidopsis and rice. Intensified research efforts should disclose whether SR proteins were already determining alternative splicing modulation and stress tolerance in early land plants.
"A cell surface arabinogalactan‐peptide influences root hair cell fate"
Summary
Root hairs (RHs) develop from specialized epidermal trichoblast cells, whereas epidermal cells that lack RHs are known as atrichoblasts. The mechanism controlling RH cell fate is only partially understood.
RH cell fate is regulated by a transcription factor complex that promotes the expression of the homeodomain protein GLABRA 2 (GL2), which blocks RH development by inhibiting ROOT HAIR DEFECTIVE 6 (RHD6). Suppression of GL2 expression activates RHD6, a series of downstream TFs including ROOT HAIR DEFECTIVE 6 LIKE‐4 (RSL4) and their target genes, and causes epidermal cells to develop into RHs. Brassinosteroids (BRs) influence RH cell fate. In the absence of BRs, phosphorylated BIN2 (a Type‐II GSK3‐like kinase) inhibits a protein complex that regulates GL2 expression.
Perturbation of the arabinogalactan peptide (AGP21) in Arabidopsis thaliana triggers aberrant RH development, similar to that observed in plants with defective BR signaling. We reveal that an O‐glycosylated AGP21 peptide, which is positively regulated by BZR1, a transcription factor activated by BR signaling, affects RH cell fate by altering GL2 expression in a BIN2‐dependent manner.
Changes in cell surface AGP disrupts BR responses and inhibits the downstream effect of BIN2 on the RH repressor GL2 in root epidermis.
"Stable isotope-assisted metabolomics for deciphering xenobiotic metabolism in mammalian cell culture"
Abstract Xenobiotics are ubiquitous in the environment and modified in the human body by phase I and II metabolism. Liquid chromatography coupled to high resolution mass spectrometry is a powerful tool to investigate these biotransformation products. We present a workflow based on stable isotope-assisted metabolomics and the bioinformatics tool MetExtract II for deciphering xenobiotic metabolites produced by human cells. Its potential was demonstrated by the investigation of the metabolism of deoxynivalenol (DON), an abundant food contaminant, in a liver carcinoma cell line (HepG2) and a model for colon carcinoma (HT29). Detected known metabolites included DON-3-sulfate, DON-10-sulfonate, and DON-10-glutathione as well as DON-cysteine. Conjugation with amino acids and antibiotics was confirmed for the first time. The approach allows the untargeted elucidation of human xenobiotic products in tissue culture. It may be applied to other fields of research including drug metabolism, personalized medicine and systems biology to better understand the relevance of in vitro experiments.
"PIN-LIKES Coordinate Brassinosteroid Signaling with Nuclear Auxin Input in Arabidopsisthaliana"
SUMMARY Auxin and brassinosteroids (BR) are crucial growth regulators and display overlapping functions during plant development. Here, we reveal an alternative phytohormone crosstalk mechanism, revealing that BR signaling controls PIN-LIKES (PILS)-dependent nuclear abundance of auxin. We performed a forward genetic screen for imperial pils (imp) mutants that enhance the overexpression phenotypes of PILS5 putative intracellular auxin transport facilitator. Here, we report that the imp1 mutant is defective in the BR-receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1). Our set of data reveals that BR signaling transcriptionally and post-translationally represses the accumulation of PILS proteins at the endoplasmic reticulum, thereby increasing nuclear abundance and signaling of auxin. We demonstrate that this alternative phytohormonal crosstalkmechanism integrates BR signaling into auxin-dependent organ growth rates and likely has widespread importance for plant development.
"Alternative Splicing and DNA Damage Response in Plants"
Plants are exposed to a variety of abiotic and biotic stresses that may result in DNA damage. Endogenous processes - such as DNA replication, DNA recombination, respiration, or photosynthesis - are also a threat to DNA integrity. It is therefore essential to understand the strategies plants have developed for DNA damage detection, signaling, and repair. Alternative splicing (AS) is a key post-transcriptional process with a role in regulation of gene expression. Recent studies demonstrate that the majority of intron-containing genes in plants are alternatively spliced, highlighting the importance of AS in plant development and stress response. Not only does AS ensure a versatile proteome and influence the abundance and availability of proteins greatly, it has also emerged as an important player in the DNA damage response (DDR) in animals. Despite extensive studies of DDR carried out in plants, its regulation at the level of AS has not been comprehensively addressed. Here, we provide some insights into the interplay between AS and DDR in plants.
"TOLs function as ubiquitin receptors in the early steps of the ESCRT pathway in higher plants"
Abstract Protein abundance and localization at the plasma membrane (PM) shapes plant development and mediates adaptation to changing environmental conditions. It is regulated by ubiquitination, a post-translational modification crucial for the proper sorting of endocytosed PM proteins to the vacuole for subsequent degradation. To understand the significance and the variety of roles played by this reversible modification, the function of ubiquitin receptors, which translate the ubiquitin signature into a cellular response, needs to be elucidated. In this study, we show that TOL (TOM1-like) proteins function in plants as multivalent ubiquitin receptors, governing ubiquitinated cargo delivery to the vacuole via the conserved Endosomal Sorting Complex Required for Transport (ESCRT) pathway. TOL2 and TOL6 interact with components of the ESCRT machinery and bind to K63-linked ubiquitin, via two tandemly arranged conserved ubiquitin binding domains. Mutation of these domains results not only in a loss of ubiquitin binding, but also altered localization, abolishing TOL6 ubiquitin receptor activity. Function and localization of TOL6 is itself regulated by ubiquitination, whereby TOL6 ubiquitination potentially modulates degradation of PM-localized cargos, assisting in the fine-tuning of the delicate interplay between protein recycling and downregulation. Our findings demonstrate the function and regulation of a ubiquitin receptor, mediating vacuolar degradation of PM proteins in higher plants.
"Nitric oxide homeostasis is required for light-dependent regulation of conidiation in Aspergillus"
Abstract Nitric oxide (NO) can be biologically synthesized from nitrite or from arginine. Although NO is involved as a signal in many biological processes in bacteria, plants, and mammals, still little is known about the role of NO in fungi. Here we show that NO levels are regulated by light as an environmental signal in Aspergillusnidulans. The flavohaemoglobin-encoding fhbB gene involved in NO oxidation to nitrate, and the arginine-regulated arginase encoded by agaA, which controls the intracellular concentration of arginine, are both up-regulated by light. The phytochrome fphA is required for the light-dependent induction of fhbB and agaA, while the white-collar gene lreA acts as a repressor when arginine is present in the media. The intracellular arginine pools increase upon induction of both developmental programs (conidiation and sexual development), and the increase is higher under conditions promoting sexual development. The presence of low concentrations of arginine does not affect the light-dependent regulation of conidiation, but high concentrations of arginine overrun the light signal. Deletion of fhbB results in the partial loss of the light regulation of conidiation on arginine and on nitrate media, while deletion of fhbA only affects the light regulation of conidiation on nitrate media. Our working model considers a cross-talk between environmental cues and intracellular signals to regulate fungal reproduction.
Publication from Gergely Molnar (Strauss Lab) and Lindy Abas (Mach Lab)
"Salicylic Acid Targets Protein Phosphatase 2A to Attenuate Growth in Plants"
Summary Plants, like other multicellular organisms, survive through a delicate balance between growth and defense against pathogens. Salicylic acid (SA) is a major defense signal in plants, and the perception mechanism as well as downstream signaling activating the immune response are known. Here, we identify a parallel SA signaling that mediates growth attenuation. SA directly binds to A subunits of protein phosphatase 2A (PP2A), inhibiting activity of this complex. Among PP2A targets, the PIN2 auxin transporter is hyperphosphorylated in response to SA, leading to changed activity of this important growth regulator. Accordingly, auxin transport and auxin-mediated root development, including growth, gravitropic response, and lateral root organogenesis, are inhibited. This study reveals how SA, besides activating immunity, concomitantly attenuates growth through crosstalk with the auxin distribution network. Further analysis of this dual role of SA and characterization of additional SA-regulated PP2A targets will provide further insights into mechanisms maintaining a balance between growth and defense.
Publication from Sascha Waidmann (Kleine-Vehn Lab)
"Same same, but different: growth responses of primary and lateral roots"
Abstract The root system architecture describes the shape and spatial arrangement of roots within the soil. Its spatial distribution depends on growth and branching rates as well as directional organ growth. The embryonic primary root gives rise to lateral (secondary) roots, and the ratio of both root types changes over the life span of a plant. Most studies have focused on the growth of primary roots and lateral root primordia development. Comparably less is known about the growth regulation of secondary root organs. Here, we review similarities and differences between primary and lateral root organ growth and particularly emphasize on how external stimuli and internal signals differentially integrate root system growth.
Wenn ein Pilzgeflecht eine Nahrungsquelle gegen Bakterien verteidigt oder auf sonstige Art mit seiner Umwelt interagiert, dann sehen Forscherinnen und Forscher der Plattform BiMM (Bioactive Microbial Metabolites) in Tulln ganz genau hin. Das Wissen um die molekularen Prozesse und die "Links" im "Internet der Pilze" könnte sich sogar als Waffe gegen Krebs oder multiresistente Keime erweisen...
"BGAL1 depletion boosts the level of β‐galactosylation of N‐ and O‐glycans in N.benthamiana"
Abstract Glyco‐design of proteins is a powerful tool in fundamental studies of structure‐function relationship and in obtaining profiles optimized for efficacy of therapeutic glycoproteins. Plants and particularly Nicotianabenthamiana, are attractive hosts to produce recombinant glycoproteins and recent advances in glyco‐engineering facilitate customized N‐glycosylation of plant‐derived glycoproteins. However, with exception of monoclonal antibodies, homogenous human‐like β1,4‐galactosylation is very hard to achieve in recombinant glycoproteins. Despite significant efforts to optimize the expression of β1,4‐galactosyltransferase, many plant‐derived glycoproteins still exhibit incomplete processed N‐glycans with heterogeneous terminal galactosylation. The most obvious suspects to be involved in trimming terminal galactose residues are β‐galactosidases (BGALs) from the glycosyl‐hydrolase family GH35.
To elucidate the so far uncharacterized mechanisms leading to the trimming of terminal galactose residues from glycans of secreted proteins, we studied a N.benthamiana BGAL known to be active in the apoplast (NbBGAL1). Here we determined the NbBGAL1 subcellular localization, substrate specificity and inplanta biological activity. We show that NbBGAL1 can remove β1,4‐ and β1,3‐galactose residues on both N‐ and O‐glycans. Transient BGAL1 downregulation by RNA interference (RNAi) and BGAL1 depletion by genome editing drastically reduce β‐galactosidase activity in N.benthamiana and increase the amounts of fully galactosylated complex N‐glycans on several plant‐produced glycoproteins. Altogether, our data demonstrate that NbBGAL1 acts on galactosylated complex N‐glycans of plant‐produced glycoproteins.
"Brassinosteroid signaling delimits root gravitropism via sorting of the Arabidopsis PIN2 auxin transporter"
Abstract Arabidopsis PIN2 protein directs transport of the phytohormone auxin from the root tip into the root elongation zone. Variation in hormone transport, which depends on a delicate interplay between PIN2 sorting to and from polar plasma membrane domains, determines root growth. By employing a constitutively degraded version of PIN2, we identify brassinolides as antagonists of PIN2 endocytosis. This response does not require de novo protein synthesis, but involves early events in canonical brassinolide signaling. Brassinolide-controlled adjustments in PIN2 sorting and intracellular distribution governs formation of a lateral PIN2 gradient in gravistimulated roots, coinciding with adjustments in auxin signaling and directional root growth. Strikingly, simulations indicate that PIN2 gradient formation is no prerequisite for root bending but rather dampens asymmetric auxin flow and signaling. Crosstalk between brassinolide signaling and endocytic PIN2 sorting, thus, appears essential for determining the rate of gravity-induced root curvature via attenuation of differential cell elongation.
"Involvement of the eIF2α Kinase GCN2 in UV-B Responses"
GCN2 (general control nonrepressed 2) is a serine/threonine-protein kinase that regulates translation in response to stressors such as amino acid and purin deprivation, cold shock, wounding, cadmium, and UV-C exposure. Activated GCN2 phosphorylates the α-subunit of the eukaryotic initiation factor 2 (eIF2) leading to a drastic inhibition of protein synthesis and shifting translation to specific mRNAs. To investigate the role of GCN2 in responses to UV-B radiation its activity was analyzed through eIF2α phosphorylation assays in mutants of the specific UV-B and stress signaling pathways of Arabidopsisthaliana. EIF2α phosphorylation was detectable 30 min after UV-B exposure, independent of the UV-B photoreceptor UVRESISTANCELOCUS8 and its downstream signaling components. GCN2 dependent phosphorylation of eIF2α was also detectable in mutants of the stress related MAP kinases, MPK3 and MPK6 and their negative regulator map kinase phosphatase1 (MKP1). Transcription of downstream components of the UV-B signaling pathway, the Chalcone synthase (CHS) was constitutively higher in gcn2-1 compared to wildtype and further increased upon UV-B while GLUTATHIONEPEROXIDASE7 (GPX7) behaved similarly to wildtype. The UVR8 independent FAD-LINKEDOXIDOREDUCTASE (FADox) had a lower basal expression in gcn2-1 which was increased upon UV-B. Since high fluence rates of UV-B induce DNA damage the expression of the RASASSOCIATEDWITHDIABETESPROTEIN51 (RAD51) was quantified before and after UV-B. While the basal expression was similar to wildtype it was significantly less induced upon UV-B in the gcn2-1 mutant. This expression pattern correlates with the finding that gcn2 mutants develop less cyclobutane pyrimidine dimers after UV-B exposure. Quantification of translation with the puromycination assay revealed that gcn2 mutants have an increased rate of translation which was also higher upon UV-B. Growth of gcn2 mutants to chronic UV-B exposure supports GCN2’s role as a negative regulator of UV-B responses. The elevated resistance of gcn2 mutants towards repeated UV-B exposure points to a critical role of GCN2 in the regulation of translation upon UV-B.
"Stable Isotope–Assisted Plant Metabolomics: Combination of Global and Tracer-Based Labeling for Enhanced Untargeted Profiling and Compound Annotation"
Untargeted approaches and thus biological interpretation of metabolomics results are still hampered by the reliable assignment of the global metabolome as well as classification and (putative) identification of metabolites. In this work we present an liquid chromatography-mass spectrometry (LC-MS)–based stable isotope assisted approach that combines global metabolome and tracer based isotope labeling for improved characterization of (unknown) metabolites and their classification into tracer derived submetabolomes. To this end, wheat plants were cultivated in a customized growth chamber, which was kept at 400 ± 50 ppm 13CO2 to produce highly enriched uniformly 13C-labeled sample material. Additionally, native plants were grown in the greenhouse and treated with either 13C9-labeled phenylalanine (Phe) or 13C11-labeled tryptophan (Trp) to study their metabolism and biochemical pathways. After sample preparation, liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis and automated data evaluation, the results of the global metabolome- and tracer-labeling approaches were combined. A total of 1,729 plant metabolites were detected out of which 122 respective 58 metabolites account for the Phe- and Trp-derived submetabolomes. Besides m/z and retention time, also the total number of carbon atoms as well as those of the incorporated tracer moieties were obtained for the detected metabolite ions. With this information at hand characterization of unknown compounds was improved as the additional knowledge from the tracer approaches considerably reduced the number of plausible sum formulas and structures of the detected metabolites. Finally, the number of putative structure formulas was further reduced by isotope-assisted annotation tandem mass spectrometry (MS/MS) derived product ion spectra of the detected metabolites. A major innovation of this paper is the classification of the metabolites into submetabolomes which turned out to be valuable information for effective filtering of database hits based on characteristic structural subparts. This allows the generation of a final list of true plant metabolites, which can be characterized at different levels of specificity.
"Stable Isotope-Assisted Plant Metabolomics: Investigation of Phenylalanine-Related Metabolic Response in Wheat Upon Treatment With the Fusarium Virulence Factor Deoxynivalenol"
The major Fusarium mycotoxin deoxynivalenol (DON) is a virulence factor in wheat and has also been shown to induce defense responses in host plant tissue. In this study, global and tracer labeling with 13C were combined to annotate the overall metabolome of wheat spikes and to evaluate the response of phenylalanine-related pathways upon treatment with DON. At anthesis, spikes of resistant and susceptible cultivars as well as two related near isogenic wheat lines (NILs) differing in the presence/absence of the major resistance QTL Fhb1 were treated with 1 mg DON or water (control), and samples were collected at 0, 12, 24, 48, and 96 h after treatment (hat). A total of 172 Phe-derived wheat constituents were detected with our untargeted approach employing 13C-labeled phenylalanine and subsequently annotated as flavonoids, lignans, coumarins, benzoic acid derivatives, hydroxycinnamic acid amides (HCAAs), as well as peptides. Ninety-six hours after the DON treatment, up to 30% of the metabolites biosynthesized from Phe showed significantly increased levels compared to the control samples. Major metabolic changes included the formation of precursors of compounds implicated in cell wall reinforcement and presumed antifungal compounds. In addition, also dipeptides, which presumably are products of proteolytic degradation of truncated proteins generated in the presence of the toxin, were significantly more abundant upon DON treatment. An in-depth comparison of the two NILs with correlation clustering of time course profiles revealed some 70 DON-responsive Phe derivatives. While several flavonoids had constitutively different abundance levels between the two NILs differing in resistance, other Phe-derived metabolites such as HCAAs and hydroxycinnamoyl quinates were affected differently in the two NILs after treatment with DON. Our results suggest a strong activation of the general phenylpropanoid pathway and that coumaroyl-CoA is mainly diverted towards HCAAs in the presence of Fhb1, whereas the metabolic route to monolignol(-conjugates), lignans, and lignin seems to be favored in the absence of the Fhb1 resistance quantitative trait loci.
Die Auszeichnung BOKU Erfinderin 2019 ging dieses Jahr ex aequo an Prof.in Hertha Steinkellner, Prof.in Grabherr und Prof.in Gasser. Ihr Einsatz und ihre Vorbildwirkung wird neben einem Geldpreis mit einem persönlichen Video, das beim Tullner Herbstfest vorgestellt wurde, belohnt ⇒ zur BOKU Top Story
"Zearalenone and ß-Zearalenol But Not Their Glucosides Inhibit Heat Shock Protein 90 ATPase Activity"
Abstract The mycotoxin zearalenone (ZEN) is produced by many plant pathogenic Fusarium species. It is well known for its estrogenic activity in humans and animals, but whether ZEN has a role in plant–pathogen interaction and which process it is targeting in planta was so far unclear. We found that treatment of Arabidopsisthaliana seedlings with ZEN induced transcription of the AtHSP90.1 gene. This heat shock protein (HSP) plays an important role in plant–pathogen interaction, assisting in stability and functionality of various disease resistance gene products. Inhibition of HSP90 ATPase activity impairs functionality. Because HSP90 inhibitors are known to induce HSP90 gene expression and due to the structural similarity with the known HSP90 inhibitor radicicol (RAD), we tested whether ZEN and its phase I metabolites α- and ß-zearalenol are also HSP90 ATPase inhibitors. Indeed, AtHSP90.1 and wheat TaHSP90-2 were inhibited by ZEN and ß-zearalenol, while α-zearalenol was almost inactive. Plants can efficiently glycosylate ZEN and α/ß-zearalenol. We therefore tested whether glucosylation has an effect on the inhibitory activity of these metabolites. Expression of the A.thaliana glucosyltransferase UGT73C6 conferred RAD resistance to a sensitive yeast strain. Glucosylation of RAD, ZEN, and α/ß-zearalenol abolished the in vitro inhibitory activity with recombinant HSP90 purified from Escherichiacoli. In conclusion, the mycotoxin ZEN has a very prominent target in plants, HSP90, but it can be inactivated by glycosylation. This may explain why there is little evidence for a virulence function of ZEN in host plants.
"Auxin-dependent xyloglucan remodelling defines differential tissue expansion in Arabidopsisthaliana"
Abstract Size control is a fundamental question in biology, showing incremental complexity in case of cell wall surrounded plant cells. Here we show that auxin signalling restricts the complexity of extracellular xyloglucans, which defines cell wall properties and tissue expansion. Our work uncovers an alternative mechanism of how the phytohormone auxin modulates the cell wall for steering differential growth control in gravitropic hypocotyls.
"In Planta Glycan Engineering and Functional Activities of IgE Antibodies"
Abstract Human immunoglobulin E (IgE) is the most extensively glycosylated antibody isotype so glycans attached to the seven N-glycosites (NGS) in its Fab and Fc domains may modulate its functions. However, targeted modification of glycans in multiply glycosylated proteins remains a challenge. Here, we applied an in vivo approach that allows the manipulation of IgE N-glycans, using a trastuzumab equivalent IgE (HER2-IgE) as a model. Taking advantage of plant inherent features, i.e., synthesis of largely homogeneous complex N-glycans and susceptibility to glycan engineering, we generated targeted glycoforms of HER2-IgE largely resembling those found in serum IgE. Plant-derived HER2-IgE exhibited N-glycans terminating with GlcNAc, galactose or sialic acid, lacking, or carrying core fucose and xylose. We were able to not only modulate the five NGSs naturally decorated with complex N-glycans, but to also induce targeted glycosylation at the usually unoccupied NGS6, thus increasing the overall glycosylation content of HER2-IgE. Recombinant human cell-derived HER2-IgE exhibited large N-glycan heterogeneity. All HER2-IgE variants demonstrated glycosylation-independent binding to the target antigen and the high affinity receptor FcεRI, and subsequent similar capacity to trigger mast cell degranulation. In contrast, binding to the low affinity receptor CD23 (FcεRII) was modulated by the glycan profile, with increased binding to IgE variants with glycans terminating with GlcNAc residues. Here we offer an efficient in planta approach to generate defined glycoforms on multiply glycosylated IgE, allowing the precise exploration of glycosylation-dependent activities.
"NET4 Modulates the Compactness of Vacuoles in Arabidopsisthaliana"
Abstract The dimension of the plants largest organelle—the vacuole—plays a major role in defining cellular elongation rates. The morphology of the vacuole is controlled by the actin cytoskeleton, but molecular players remain largely unknown. Recently, the Networked (NET) family of membrane-associated, actin-binding proteins has been identified. Here, we show that NET4A localizes to highly constricted regions of the vacuolar membrane and contributes to vacuolar morphology. Using genetic interference, we found that deregulation of NET4 abundance increases vacuolar occupancy, and that overexpression of NET4 abundance decreases vacuolar occupancy. Our data reveal that NET4A induces more compact vacuoles, correlating with reduced cellular and organ growth in Arabidopsisthaliana.
Aaron Rashotte (Auburn University, USA) will talk about:
"Cytokinin Response Factors and their expanding roles in stress response."
Tuesday, 8th October 2019, 09:00 am Muthgasse 18, 1190 Vienna DAGZ Seminar room 4th floor Host: Sascha Waidmann
» Kleine-Vehn Lab
Publication from Kai Dünser (Kleine-Vehn Lab)
"Leucine-Rich Repeat Extensin Proteins and Their Role in Cell Wall Sensing"
Abstract Plant cells are surrounded by a cell wall that provides shape and physically limits cell expansion. To sense the environment and status of cell wall structures, plants have evolved cell wall integrity-sensing mechanisms that involve a number of receptors at the plasma membrane. These receptors can bind cell wall components and/or hormones to coordinate processes in the cell wall and the cytoplasm. This review focuses on the role of leucine-rich repeat extensins (LRXs) during cell wall development. LRXs are chimeric proteins that insolubilize in the cell wall and form protein–protein interaction platforms. LRXs bind RALF peptide hormones that modify cell wall expansion and also directly interact with the transmembrane receptor FERONIA, which is involved in cell growth regulation. LRX proteins, therefore, also represent a link between the cell wall and plasma membrane, perceiving extracellular signals and indirectly relaying this information to the cytoplasm.
"Biochemical Characterization of the Fusariumgraminearum Candidate ACC-Deaminases and Virulence Testing of Knockout Mutant Strains"
Abstract Fusariumgraminearum is a plant pathogenic fungus which is able to infect wheat and other economically important cereal crop species. The role of ethylene in the interaction with host plants is unclear and controversial. We have analyzed the inventory of genes with a putative function in ethylene production or degradation of the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC). F. graminearum, in contrast to other species, does not contain a candidate gene encoding ethylene-forming enzyme. Three genes with similarity to ACC synthases exist; heterologous expression of these did not reveal enzymatic activity. The F.graminearum genome contains in addition two ACC deaminase candidate genes. We have expressed both genes in E.coli and characterized the enzymatic properties of the affinity-purified products. One of the proteins had indeed ACC deaminase activity, with kinetic properties similar to ethylene-stress reducing enzymes of plant growth promoting bacteria. The other candidate was inactive with ACC but turned out to be a D-cysteine desulfhydrase. Since it had been reported that ethylene insensitivity in transgenic wheat increased Fusarium resistance and reduced the content of the mycotoxin deoxynivalenol (DON) in infected wheat, we generated single and double knockout mutants of both genes in the F.graminearum strain PH-1. No statistically significant effect of the gene disruptions on fungal spread or mycotoxin content was detected, indicating that the ability of the fungus to manipulate the production of the gaseous plant hormones ethylene and H2S is dispensable for full virulence.
"Biotransformation of the Mycotoxin Zearalenone to its Metabolites Hydrolyzed Zearalenone (HZEN) and Decarboxylated Hydrolyzed Zearalenone (DHZEN) Diminishes its Estrogenicity In Vitro and In Vivo"
Abstract Zearalenone (ZEN)-degrading enzymes are a promising strategy to counteract the negative effects of this mycotoxin in livestock. The reaction products of such enzymes need to be thoroughly characterized before technological application as a feed additive can be envisaged. Here, we evaluated the estrogenic activity of the metabolites hydrolyzed zearalenone (HZEN) and decarboxylated hydrolyzed zearalenone (DHZEN) formed by hydrolysis of ZEN by the zearalenone-lactonase Zhd101p. ZEN, HZEN, and DHZEN were tested in two in vitro models, the MCF-7 cell proliferation assay (0.01–500 nM) and an estrogen-sensitive yeast bioassay (1–10,000 nM). In addition, we compared the impact of dietary ZEN (4.58 mg/kg) and equimolar dietary concentrations of HZEN and DHZEN on reproductive tract morphology as well as uterine mRNA and microRNA expression in female piglets (n = 6, four weeks exposure). While ZEN increased cell proliferation and reporter gene transcription, neither HZEN nor DHZEN elicited an estrogenic response, suggesting that these metabolites are at least 50–10,000 times less estrogenic than ZEN in vitro. In piglets, HZEN and DHZEN did not increase vulva size or uterus weight. Moreover, RNA transcripts altered upon ZEN treatment (EBAG9, miR-135a-5p, miR-187-3p and miR-204-5p) were unaffected by HZEN and DHZEN. Our study shows that both metabolites exhibit markedly reduced estrogenicity in vitro and in vivo, and thus provides an important basis for further evaluation of ZEN-degrading enzymes.
Publication from Jennifer Schoberer & Strasser Lab
"A signal motif retains Arabidopsis ER-α-mannosidase I in the cis-Golgi and prevents enhanced glycoprotein ERAD"
Abstract The Arabidopsis ER-α-mannosidase I (MNS3) generates an oligomannosidic N-glycan structure that is characteristically found on ER-resident glycoproteins. The enzyme itself has so far not been detected in the ER. Here, we provide evidence that in plants MNS3 exclusively resides in the Golgi apparatus at steady-state. Notably, MNS3 remains on dispersed punctate structures when subjected to different approaches that commonly result in the relocation of Golgi enzymes to the ER. Responsible for this rare behavior is an amino acid signal motif (LPYS) within the cytoplasmic tail of MNS3 that acts as a specific Golgi retention signal. This retention is a means to spatially separate MNS3 from ER-localized mannose trimming steps that generate the glycan signal required for flagging terminally misfolded glycoproteins for ERAD. The physiological importance of the very specific MNS3 localization is demonstrated here by means of a structurally impaired variant of the brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1.
"Evidence of a Demethylase-Independent Role for the H3K4-Specific Histone Demethylases in Aspergillusnidulans and Fusariumgraminearum Secondary Metabolism"
Abstract Fungi produce a plethora of secondary metabolites (SMs) involved in cellular protection, defense, and signaling. Like other metabolic processes, transcription of SM biosynthesis genes is tightly regulated to prevent an unnecessary use of resources. Genes involved in SM biosynthesis are usually physically linked, arranged in secondary metabolite gene clusters (SMGCs). Research over the last decades has shown that chromatin structure and posttranslational modifications (PTMs) of histones represent important layers of SMGC regulation. For instance, trimethylation of histone H3 lysine 4 (H3K4me3) is a PTM typically associated with promoter regions of actively transcribed genes. Previously, we have shown that the H3K4me3-specific, JmjC domain-containing histone demethylase KdmB functions not only in repression but also in activation of secondary metabolism in Aspergillusnidulans, suggesting that KdmB has additional functions apart from histone demethylation. In this study, we identified demethylase-independent functions of KdmB in transcriptional regulation of SM gene clusters. Furthermore, we show that this activating and demethylase-independent role of the H3K4 demethylase is also conserved in the phytopathogenic fungus Fusariumgraminearum. Lack of FgKdm5 resulted in significant downregulation of five of seven analyzed SMs, whereby only one SMGC depends on a functional JmjC-domain. In A. nidulans strains deficient in H3K4 methylation, i.e., cclA∆, largely phenocopied kdmB∆, while this is not the case for most of the SMs analyzed in Fusarium spp. Notably, KdmB could not rescue the demethylase function in ∆fgkdm5 but restored all demethylase-independent phenotypes.
Publication from Sascha Waidmann (Kleine-Vehn Lab)
"Cytokinin functions as an asymmetric and anti-gravitropic signal in lateral roots"
Abstract Directional organ growth allows the plant root system to strategically cover its surroundings. Intercellular auxin transport is aligned with the gravity vector in the primary root tips, facilitating downward organ bending at the lower root flank. Here we show that cytokinin signaling functions as a lateral root specific anti-gravitropic component, promoting the radial distribution of the root system. We performed a genome-wide association study and reveal that signal peptide processing of Cytokinin Oxidase 2 (CKX2) affects its enzymatic activity and, thereby, determines the degradation of cytokinins in natural Arabidopsisthaliana accessions. Cytokinin signaling interferes with growth at the upper lateral root flank and thereby prevents downward bending. Our interdisciplinary approach proposes that two phytohormonal cues at opposite organ flanks counterbalance each other’s negative impact on growth, suppressing organ growth towards gravity and allow for radial expansion of the root system.
"Distinct Fc alpha receptor N-glycans modulate the binding affinity to immunoglobulin A (IgA) antibodies"
Abstract Human immunoglobulin A (IgA) is the most prevalent antibody class at mucosal sites with an important role in mucosal defense. Little is known about the impact of N-glycan modifications of IgA1 and IgA2 on binding to the Fc alpha receptor (FcαRI) which is also heavily glycosylated at its extracellular domain. Here, we transiently expressed human epidermal growth factor receptor 2 (HER2)-binding monomeric IgA1, IgA2m(1) and IgA2m(2) variants in Nicotianabenthamiana ΔXT/FT plants lacking the enzymes responsible for generating non-human N-glycan structures. By co-infiltrating IgA with the respective glycan modifying enzymes, we generated IgA carrying distinct homogenous N-glycans. We demonstrate that distinctly different N-glycan profiles do not influence antigen binding or the overall structure and integrity of the IgA antibodies, but did affect their thermal stability. Using size-exclusion chromatography, differential scanning and isothermal titration calorimetry, surface plasmon resonance spectroscopy, and molecular modeling, we probed distinct IgA1 and IgA2 glycoforms for binding to four different FcαRI glycoforms and investigated the thermodynamics and kinetics of complex formation. Our results suggest that different N-glycans on the receptor significantly contribute to binding affinities for its cognate ligand. We also noted that full-length IgA and FcαRI form a mix of 1:1 and 1:2 complexes tending toward a 1:1 stoichiometry due to different IgA tailpiece conformations that make it less likely that both binding sites are simultaneously occupied. In conclusion, N-glycans of human IgA do not affect its structure and integrity, but its thermal stability, and FcαRI N-glycans significantly modulate binding affinity to IgA.
"PIN-FORMED and PIN-LIKES auxin transport facilitators"
Abstract The phytohormone auxin influences virtually all aspects of plant growth and development. Auxin transport across membranes is facilitated by, among other proteins, members of the PIN-FORMED (PIN) and the structurally similar PIN-LIKES (PILS) families, which together govern directional cell-to-cell transport and intracellular accumulation of auxin. Canonical PIN proteins, which exhibit a polar localization in the plasma membrane, determine many patterning and directional growth responses. Conversely, the less-studied non-canonical PINs and PILS proteins, which mostly localize to the endoplasmic reticulum, attenuate cellular auxin responses. Here, and in the accompanying poster, we provide a brief summary of current knowledge of the structure, evolution, function and regulation of these auxin transport facilitators.
"Genome and transcriptome characterization of the glycoengineered Nicotianabenthamiana line ΔXT/FT"
Results: Based on an existing draft assembly of the N.benthamiana genome we predict 50,516 protein –encoding genes (62,216 transcripts) supported by expression data derived from 2.35 billion mRNA-seq reads. Using singlecopy core genes we show high completeness of the predicted gene set. We functionally annotate more than two thirds of the gene set through sequence homology to genes from other Nicotiana species. We demonstrate that the expression profiles from leaf tissue of ΔXT/FT and its wild type progenitor only show minimal differences. We identify the transgene insertion sites in ΔXT/FT and show that one of the transgenes was inserted inside another predicted gene that most likely lost its function upon insertion. Based on publicly available mRNA-seq data, we confirm that the N.benthamiana accessions used by different research institutions most likely derive from a single source.
"NET4 modulates the compactness of vacuoles in Arabidopsis thaliana"
Abstract The dimension of the plants largest organelle – the vacuole, plays a major role in defining cellular elongation rates. The morphology of the vacuole is controlled by the actin cytoskeleton but the mechanistic connection between them remains largely elusive. Recently, the NETWORKED (NET) family of membrane-associated, actin-binding proteins has been identified and represent potential candidates to impact on vacuolar morphology. Here, we show that NET4A localizes to highly constricted regions in the vacuolar membrane and contributes to the compactness of the vacuole. Using genetic interference, we found that deregulation of NET4 abundance impacts on vacuole morphogenesis and overexpression leads to more compact vacuoles. We moreover show that the NET4A-induced changes in vacuolar shape correlates with reduced cellular and organ growth in Arabidopsis thaliana. Our results demonstrate that NET4 modulates the compactness of vacuoles and reveal higher complexity in the regulation of actin-reliant vacuolar morphology.
Am Montag und Dienstag (17./18.06.2019) waren je eine Klasse der Volksschule Familienschule Leopoldstadt bei uns zu Gast, jeweils für den ganzen Vormittag. Die Kinder waren zwischen 8 und 10 Jahre alt (3. und 4. Klasse), es waren einmal 20 und einmal 21 Kinder, und wir hatten ein Programm mit Experimenten zum Thema Pflanzen und Pilze, und generell zur Arbeit in einem Biologie-Labor.
Die Mitwirkenden waren: Jeanette Moulinier-Anzola, Barbara Korbei, Elena Feraru, Sabine Strauss-Goller, Max Schwihla, Kai Dünser und Doris Lucyshyn.
Wissenschaftskommunikation "extended" - Wie beeinflussen Bakterien die Genexpression in assoziierten Pilzen?
Die eLIFE Publikation über Bakterien-Pilz Interaktion der Arbeitsgruppe Strauss, wurde als Highlight in der Märzausgabe der Zeitschrift Biospektrum vorgestellt.
"Brassinosteroid signaling controls PILS-dependent nuclear auxin input in Arabidopsisthaliana"
Abstract Auxin and brassinosteroids (BR) are crucial growth regulators and display overlapping functions during plant development. Here, we reveal an alternative phytohormone crosstalk mechanism, revealing that brassinosteroid signaling controls nuclear abundance of auxin. We performed a forward genetic screen for imperialpils (imp) mutants that enhance the overexpression phenotypes of PIN-LIKES (PILS) putative intracellular auxin transport facilitator. Here we report that the imp1 mutant is defective in the brassinosteroid-receptor BRI1. BR signaling transcriptionally and posttranslationally represses accumulation of PILS proteins at the endoplasmic reticulum, thereby increasing nuclear abundance and signaling of auxin. We demonstrate that this alternative phytohormonal crosstalk mechanism integrates BR signaling into auxin-dependent organ growth rates and likely has widespread importance for plant development.
Qiang Chen The Biodesign Institute and School of Life Sciences
"Improving effector functions of antibodies against flavi-and alphaviruses"
Host: Herta Steinkellner
Muthgasse18, 1190 Vienna Seminar-room DAGZ (4thfloor) June 6th2019, 11:30 h
Publication from Strauss Lab
"Fusaoctaxin A, an Example of a Two-Step Mechanism for Non-Ribosomal Peptide Assembly and Maturation in Fungi"
Abstract: Fungal non-ribosomal peptide synthetase (NRPS) clusters are spread across the chromosomes, where several modifying enzyme-encoding genes typically flank one NRPS. However, a recent study showed that the octapeptide fusaoctaxin A is tandemly synthesized by two NRPSs in Fusariumgraminearum. Here, we illuminate parts of the biosynthetic route of fusaoctaxin A, which is cleaved into the tripeptide fusatrixin A and the pentapeptide fusapentaxin A during transport by a cluster-specific ABC transporter with peptidase activity. Further, we deleted the histone H3K27 methyltransferase kmt6, which induced the production of fusaoctaxin A.
"Simple validated method for simultaneous determination of deoxynivalenol, nivalenol, and their 3-β-D-glucosides in baby formula and Korean rice wine via HPLC-UV with immunoaffinity cleanup"
Abstract A simple and reliable method for the simultaneous determination of major type B trichothecene mycotoxins, deoxynivalenol (DON) and nivalenol (NIV), along with their 3-β-d-glucosides (DON-3-glucoside (DON3G) and NIV-3-glucoside (NIV3G)) in baby formula and Korean rice wine was validated in the present study. The method was based on immunoaffinity cleanup followed by analysis using an HPLC-UV technique. The method was validated in-house for two matrices as follows: linearity (R2 > 0.99) was established in the range of 20-1000 μg kg-1; accuracy (expressed as recovery) ranged from 78.7 to 106.5% for all the analytes; good intermediate precision (relative standard deviation < 12%), and adequate detection and quantitation limits (< 4.4 and < 13.3 μg kg-1, respectively) were achieved. Furthermore, the estimated measurement expanded uncertainty was determined to be 4-24%. The validated method was successfully applied to the analysis of 31 baby formulas and Korean rice wines marketed in Korea.
"The Fusarium metabolite culmorin suppresses the in vitro glucuronidation of deoxynivalenol"
Abstract Glucuronidation is a major phase II conjugation pathway in mammals, playing an important role in the detoxification and biotransformation of xenobiotics including mycotoxins such as deoxynivalenol (DON). Culmorin (CUL), a potentially co-occurring Fusarium metabolite, was recently found to inhibit the corresponding detoxification reaction in plants, namely DON-glucoside formation, raising the question whether CUL might affect also the mammalian counterpart. Using cell-free conditions, CUL when present equimolar (67 µM) or in fivefold excess, suppressed DON glucuronidation by human liver microsomes, reducing the formation of DON-15-glucuronide by 15 and 50%, and DON-3-glucuronide by 30 and 50%, respectively. Substantial inhibitory effects on DON glucuronidation up to 100% were found using the human recombinant uridine 5'-diphospho-glucuronosyltransferases (UGT) 2B4 and 2B7, applying a tenfold excess of CUL (100 µM). In addition, we observed the formation of a novel metabolite of CUL, CUL-11-glucuronide, identified for the first time in vitro as well as in vivo in piglet and human urine samples. Despite the observed potency of CUL to inhibit glucuronidation, no significant synergistic toxicity on cell viability was observed in combinations of CUL (0.1-100 µM) and DON (0.01-10 µM) in HT-29 and HepG2 cells, presumably reflecting the limited capacity of the tested cell lines for DON glucuronidation. However, in humans, glucuronidation is known to represent the main detoxification pathway for DON. The present results, including the identification of CUL-11-glucuronide in urine samples of piglets and humans, underline the necessity of further studies on the relevance of CUL as a potentially co-occurring modulator of DON toxicokinetics in vivo.itro glucuronidation of deoxynivalenol.
"Comparative Antigenicity of Thiourea and Adipic Amide Linked Neoglycoconjugates Containing Modified Oligomannose Epitopes for the Carbohydrate-Specific anti-HIV Antibody 2G12"
Abstract: Novel neoglycoproteins containing oligomannosidic penta- and heptasaccharides as structural variants of oligomannose-type N-glycans found on human immunodeficiency virus type 1 gp120 have been prepared using different conjugation methods. Two series of synthetic ligands equipped with 3-aminopropyl spacer moieties and differing in the anomeric configuration of the reducing mannose residue were activated either as isothiocyanates or as adipic acid succinimidoyl esters and coupled to bovine serum albumin. Coupling efficiency for adipic acid connected neoglycoconjugates was better than for the thiourea-linked derivatives; the latter constructs, however, exhibited higher reactivity toward antibody 2G12, an HIV-neutralizing antibody with exquisite specificity for oligomannose-type glycans. 2G12 binding avidities for the conjugates, as determined by Bio-Layer Interferometry, were mostly higher for the β-linked ligands and, as expected, increased with the numbers of covalently linked glycans, leading to approximate KD values of 10 to 34 nM for optimized ligand-to-BSA ratios. A similar correlation was observed by enzyme-linked immunosorbent assays. In addition, dendrimer-type ligands presenting trimeric oligomannose epitopes were generated by conversion of the amino-spacer group into a terminal azide, followed by triazole formation using “click chemistry”. The severe steric bulk of the ligands, however, led to poor efficiency in the coupling step and no increased antibody binding by the resulting neoglycoconjugates, indicating that the low degree of substitution and the spatial orientation of the oligomannose epitopes within these trimeric ligands are not conducive to multivalent 2G12 binding
Publication from Jennifer Schoberer & Strasser Lab
"Golgi localization of GnTI requires a polar amino acid residue within its transmembrane domain"
Abstract The Golgi apparatus consists of stacked cisternae filled with enzymes to facilitate the sequential and highly controlled modification of glycans from proteins that transit through the organelle. Although the glycan processing pathways have been extensively studied, the underlying mechanisms that concentrate Golgi resident glycosyltransferases and glycosidases in distinct Golgi compartments are unclear. The single-pass transmembrane domain of N-acetylglucosaminyltransferase I (GnTI) accounts for its steady-state distribution in the cis/medial-Golgi. Here, we investigated the contribution of individual amino acid residues from the GnTI transmembrane domain for Golgi localization and N-glycan processing. Conserved sequence motifs within the transmembrane domain were replaced with those from the well-known trans-Golgi enzyme alpha2,6-sialyltransferase (ST) and site-directed mutagenesis was used to exchange individual amino acid residues. Subcellular localization of fluorescent fusion proteins and N-glycan profiling revealed that a conserved glutamine residue in the GnTI transmembrane domain is essential for its cis/medial-Golgi localization. Replacing the crucial glutamine residue with other amino acids results in mislocalization to the vacuole and impaired N-glycan processing in vivo. Our results suggest that sequence-specific features in the transmembrane domain of GnTI are required for interaction with a Golgi-resident adaptor protein or specific lipid environment that likely promotes COPI-mediated retrograde transport to maintain the steady-state distribution of GnTI in the cis/medial-Golgi.
“I see the light! Cell wall engineering, pH and fluorescence”
Wednesday 24.04.2019, 09:00 am DAGZ Seminar room, 4th floor (MUG2‐04/54) Muthgasse 18, 1190 Wien Hosts: Elke Barbez, Sascha Waidmann
» Kleine-Vehn Lab
BiRT Seminar Series
Prof. Dr. Gerhard H. Braus
Georg-August-University Göttingen, Institute of Microbiology and Genetics "Aspergillus as model for coordinated development and secondary metabolism" Donnerstag: 04. April 2019, 09:30 Campus Tulln / UFT – Seminarraum 14
"UV-B exposure reduces the activity of several cell wall-dismantling enzymes and affects the expression of their biosynthetic genes in peach fruit (Prunuspersica L., cv. Fairtime, melting phenotype)"
Abstract Softening processes after ripening are a major factor contributing to the perishability of fleshy fruits and, together with mechanical damage, represents the onset of physiological decay. Softening involves multiple co-ordinated events leading to modifications of the cell wall architecture. Several studies described that UV-B radiation positively affects both the nutraceutical and aesthetical qualities of fruits. However, very few studies investigated the effect of UV-B irradiation on the activity of cell wall-related enzymes. This research aimed to study how different UV-B treatments (10 min and 60 min) affect the activity of cell wall-modifying enzymes (pectin methylesterase, polygalacturonase and β-galactosidase) together with the expression of some of their isoforms up to 36 h after UV-B treatment of peach (cv. Fairtime, melting phenotype) fruits. Results revealed that UV-B radiation did not affect the soluble solid content and the titratable acidity, two important parameters influencing consumers choice and taste. On the contrary, UV-B was effective in reducing the loss of firmness 24 h after the 60 min irradiation. Generally, a lower activity of the hydrolytic enzymes compared to untreated fruits was observed, regardless of the UV-B dose. However, gene expression did not reflect the corresponding enzymatic activity. Based on these results, UV-B irradiation might be a successful tool in reducing the loss of firmness of peach fruit in post-harvest, thus improving their quality and shelf-life.
"Cytokinin functions as an asymmetric and anti-gravitropic signal in lateral roots"Abstract Directional organ growth allows the plant root system to strategically cover its surroundings. Intercellular auxin transport is aligned with the gravity vector in the primary root tips, facilitating downward organ bending at the lower root flank. Here we show that cytokinin signaling functions as a lateral root specific anti-gravitropic component, promoting the radial distribution of the root system. We performed a genome-wide association study and revealed that signal peptide processing of Cytokinin Oxidase 2 (CKX2) affects its enzymatic activity and, thereby, determines the degradation of cytokinins in natural Arabidopsisthaliana accessions. Cytokinin signaling interferes with growth at the upper lateral root flank and thereby prevents downward bending. Our interdisciplinary approach revealed that two phytohormonal cues at opposite organ flanks counterbalance each others negative impact on growth, suppressing organ growth towards gravity and allow for radial expansion of the root system. » https://doi.org/10.1101/572941 » Kleine-Vehn Lab
Publication from Kleine-Vehn Lab
"Extracellular matrix sensing by FERONIA and Leucine‐Rich Repeat Extensins controls vacuolar expansion during cellular elongation in Arabidopsisthaliana"Abstract Cellular elongation requires the defined coordination of intra‐ and extracellular processes, but the underlying mechanisms are largely unknown. The vacuole is the biggest plant organelle, and its dimensions play a role in defining plant cell expansion rates. Here, we show that the increase in vacuolar occupancy enables cellular elongation with relatively little enlargement of the cytosol in Arabidopsisthaliana. We demonstrate that cell wall properties are sensed and impact on the intracellular expansion of the vacuole. Using vacuolar morphology as a quantitative read‐out for intracellular growth processes, we reveal that the underlying cell wall sensing mechanism requires interaction of extracellular leucine‐rich repeat extensins (LRXs) with the receptor‐like kinase FERONIA (FER). Our data suggest that LRXs link plasma membrane‐localised FER with the cell wall, allowing this module to jointly sense and convey extracellular signals to the cell. This mechanism coordinates the onset of cell wall acidification and loosening with the increase in vacuolar size. » https://doi.org/10.15252/embj.2018100353 » Kleine-Vehn Lab
Die Umwelt.Wissen Tage für Kids geben Kindern und Jugendlichen die Möglichkeit, auf spannende Art Umwelt-, Klima- und Energie-Themen zu erforschen und Organisationen kennen zu lernen, die in diesen Themenfeldern aktiv sind. „Liquid handling“ oder Was tun die im Labor den ganzen Tag? Kannst Du mit kleinen Mengen Substanz umgehen? In unserer Forschung analysieren wir Pilze, Bakterien sowie Proben aller Art. Wir verwenden oft kleine Mengen Flüssigkeit und beobachten Reaktionen. An der Station wird anhand eines Pipettierschemas der Umgang mit Pipetten geübt. Wir verwenden Farblösungen und kreieren eine bunte Platte mit bis zu 96 Positionen. So kann ein Eindruck der wesentlichsten Arbeitsschritte in der molekularen und mikrobiologischen Forschung gewonnen werden. Die BiMM hat mit 120 Kindern pipettieren geübt. Jetzt wissen die was wir im Labor so machen. » http://www.umweltwissenkids.at/ » Videoclip der Umwelt.Wissen Tage » https://www.bimm-research.at/
Publication from Kalyna Lab
"Does co-transcriptional regulation of alternative splicing mediate plant stress responses?"Abstract Plants display exquisite control over gene expression to elicit appropriate responses under normal and stress conditions. Alternative splicing (AS) of pre-mRNAs, a process that generates two or more transcripts from multi-exon genes, adds another layer of regulation to fine-tune condition-specific gene expression in animals and plants. However, exactly how plants control splice isoform ratios and the timing of this regulation in response to environmental signals remains elusive. In mammals, recent evidence indicate that epigenetic and epitranscriptome changes, such as DNA methylation, chromatin modifications and RNA methylation, regulate RNA polymerase II processivity, co-transcriptional splicing, and stability and translation efficiency of splice isoforms. In plants, the role of epigenetic modifications in regulating transcription rate and mRNA abundance under stress is beginning to emerge. However, the mechanisms by which epigenetic and epitranscriptomic modifications regulate AS and translation efficiency require further research. Dynamic changes in the chromatin landscape in response to stress may provide a scaffold around which gene expression, AS and translation are orchestrated. Finally, we discuss CRISPR/Cas-based strategies for engineering chromatin architecture to manipulate AS patterns (or splice isoforms levels) to obtain insight into the epigenetic regulation of AS. » https://doi.org/10.1093/nar/gkz121 » Kalyna Lab
DAGZ Tulln Seminar
"Key findings on how different nitrogen sources affect root development in Arabidopsis thaliana" Great insights how different forms of nitrogen nutrition influence plant root development in the yeasterday DAGZ Tulln seminar given by IST researcher Krisztina Ötvös. » https://www.researchgate.net/profile/Krisztina_Oetvoes » Strauss Lab
Publication with contribution of Strauss Lab (Simone Bachleitner & Lena Studt)
"Regulation of a novel Fusarium cytokinin in Fusarium pseudograminearum"Abstract Fusariumpseudograminearum is an agronomically important fungus, which infects many crop plants, including wheat, where it causes Fusarium crown rot. Like many other fungi, the Fusarium genus produces a wide range of secondary metabolites of which only few have been characterized. Recently a novel gene cluster was discovered in F. pseudograminearum, which encodes production of cytokinin-like metabolites collectively named Fusarium cytokinins. They are structurally similar to plant cytokinins and can activate cytokinin signalling in vitro and in planta. Here, the regulation of Fusarium cytokinin production was analysed in vitro. This revealed that, similar to deoxynivalenol (DON) production in Fusariumgraminearum, cytokinin production can be induced in vitro by specific nitrogen sources in a pH-dependent manner. DON production was also induced in both F. graminearum and F.pseudograminearum in cytokinin-inducing conditions. In addition, microscopic analyses of wheat seedlings infected with a F.pseudograminearum cytokinin reporter strain showed that the fungus specifically induces its cytokinin production in hyphae, which are in close association with the plant, suggestive of a function of Fusarium cytokinins during infection. » https://doi.org/10.1016/j.funbio.2018.12.009 » Strauss Lab
Publication with contribution of Adam Lab
"Metabolism of nivalenol and nivalenol-3-glucoside in rats"Highlights
Characterization of 7 novel nivalenol (NIV)- and NIV-3-glucoside (NIV3 G) metabolites in rats.
Development of LC-MS/MS based methods for analysis of NIV(3 G) metabolites in rat excrements.
Similar metabolization of NIV and deoxynivalenol in rats, but with different metabolite patterns.
NIV3 G is largely hydrolyzed in the GI tract of rats, but also extensively further metabolized.
The systemic exposure to NIV is roughly 30 times lower after NIV3 G consumption than after NIV dosage.
"PILS6 is a temperature-sensitive regulator of nuclear auxin input and organ growth in Arabidopsisthaliana"Significance High temperature (HT) strongly modulates plant growth and eventually threatens yield stability. HT induces biosynthesis of the phytohormone auxin, which in turn increases cellular auxin levels and growth rates in shoots. This mechanism does not control HT-induced root growth and, hence, the role of auxin in this process is currently controversial. Here we show that the putative auxin carrier PIN-LIKES 6 (PILS6) localizes to the endoplasmic reticulum, where it gates nuclear auxin accumulation and perception. HT decreases the abundance of PILS6 proteins, consequently increasing nuclear auxin signaling and root growth. Our data dismantle current controversy, revealing an alternative subcellular mechanism in roots, which links PILS6-dependent control of cellular auxin sensitivity with HT-induced organ growth. » https://doi.org/10.1073/pnas.1814015116 » Presseaussendung » Kleine-Vehn Lab
Publication from Kleine-Vehn Lab
"Identification of novel inhibitors of auxin-induced Ca2+ signaling via a plant-based chemical screen"Abstract Many signal perception mechanisms are connected to Ca2+-based second messenger signaling to modulate specific cellular responses. The well-characterized plant hormone auxin elicits a very rapid Ca2+ signal. However, the cellular targets of auxin-induced Ca2+ are largely unknown. Here, we screened a biologically annotated chemical library for inhibitors of auxin-induced Ca2+ entry in plant cell suspensions to better understand the molecular mechanism of auxin-induced Ca2+ and to explore the physiological relevance of Ca2+ in auxin signal transduction. Using this approach, we defined a set of diverse, small molecules that interfere with auxin-induced Ca2+ entry. Based on annotated biological activities of the hit molecules, we found that auxin-induced Ca2+ signaling is, among others, highly sensitive to disruption of membrane proton gradients and the mammalian Ca2+ channel inhibitor bepridil. Whereas protonophores nonselectively inhibited auxin-induced and osmotic-stress-induced Ca2+ signals, bepridil specifically inhibited auxin-induced Ca2+. We found evidence that bepridil severely alters vacuolar morphology and antagonized auxin-induced vacuolar remodeling. Further exploration of this plant-tailored collection of inhibitors will lead to a better understanding of auxin-induced Ca2+ entry and its relevance for auxin responses. » https://doi.org/10.1104/pp.18.01393 » Kleine-Vehn Lab
Publication from Kalyna Lab
"VARIFI-Web-Based Automatic Variant Identification, Filtering and Annotation of Amplicon Sequencing Data"Abstract Fast and affordable benchtop sequencers are becoming more important in improving personalized medical treatment. Still, distinguishing genetic variants between healthy and diseased individuals from sequencing errors remains a challenge. Here we present VARIFI, a pipeline for finding reliable genetic variants (single nucleotide polymorphisms (SNPs) and insertions and deletions (indels)). We optimized parameters in VARIFI by analyzing more than 170 amplicon-sequenced cancer samples produced on the Personal Genome Machine (PGM). In contrast to existing pipelines, VARIFI combines different analysis methods and, based on their concordance, assigns a confidence score to each identified variant. Furthermore, VARIFI applies variant filters for biases associated with the sequencing technologies (e.g., incorrectly identified homopolymer-associated indels with Ion Torrent). VARIFI automatically extracts variant information from publicly available databases and incorporates methods for variant effect prediction. VARIFI requires little computational experience and no in-house compute power since the analyses are conducted on our server. VARIFI is a web-based tool available at varifi.cibiv.univie.ac.at. » https://doi.org/10.3390/jpm9010010 » Kalyna Lab
Welcome Felipe dos Santos Maraschin!
The Kleine-Vehn lab is currently hosting Prof. Felipe dos Santos Maraschin from the Federal University of Rio Grande do Sul (Porto Alegre, Brazil). In the frame of an ERC project, the labs are collaborating on light signalling in roots. » https://www.researchgate.net/profile/Felipe_Maraschin » Kleine-Veh Lab
Impromptu seminar by Toshiki Kameyama
"Cancer-specific aberrant mRNA re-splicing and its repressors: What are the splicing termination mechanisms destroyed in cancer cells?" (Fujita Health University, Japan) Talk synopsis: "We have recently discovered the cancer-specific aberrant splicing that occurs through re-splicing in the mature spliced mRNAs. In normal cells, spliced mRNA must export to the cytoplasm to serve as a template for the protein biosynthesis. However, such mature mRNA is spliced again and generates aberrant truncated mRNA in cancer cells. This finding implicates the control mechanism to prevent deleterious extra mRNA splicing that must operate in normal cells. We are currently identifying repressor candidates of mRNA re-splicing." About speaker: Toshiki Kameyama is an Assistant Professor at the Division of Gene Expression Mechanism, Institute for Comprehensive Medical Science, Fujita Health University, Japan. His research focuses on aberrant splicing in cancer and transcriptional and post-transcriptional regulation of gene expression in the nervous system development and in neurological diseases. 27. February 2019, 11:00 am Seminarraum Angewandte Genetik (MUG2-04/54) Muthgasse 18, 1190 Wien
Publication from Strauss Lab
"A novel laminar-flow-based bioaerosol test system to determine biological sampling efficiencies of bioaerosol samplers"Abstract In this work, we describe a novel type of bioaerosol test system based on a laminar airflow chamber that provides a homogenous aerosol of microbial cells with known concentrations and defined culturability to bioaerosol samplers positioned in the chamber. In the system, three control and monitoring points (CMPs) are implemented in which the number and culturability of microbes can be determined by combining optical particle counting with microscopic and culture-based microbiological analyses... » https://doi.org/10.1080/02786826.2018.1562151 » Strauss Lab
Paper from BiMM and Schüller Lab with AKH
"Antifungal susceptibility of yeast blood stream isolates collected during a 10 year period in Austria"Background Candida‐associated infections put a significant burden on western health care systems. Development of (multi‐) resistant fungi can become untreatable and threaten especially vulnerable target groups, such as the immunocompromised. Objectives We assessed antifungal susceptibility and explored possible influence factors of clinical Candida isolates collected from Austrian hospitals between 2007 and 2016 » https://doi.org/10.1111/myc.12892 » BiMM Bioactive Microbial Metabolites » Schüller Lab
Publication with contribution from Hauser Lab
"Comparative “phenol-omics” and gene expression analyses in peach (Prunus persica) skin in response to different postharvest UV-B treatments"Highlights
UV-B exposure increased expression of flavonoid and UVR8 genes
Accumulation of most phenolics occurred 36 h after UV-B irradiation
60 min-UVB-exposure determined a greater phenolic accumulation than 10 min-UVB
Flavonoids, particularly anthocyanins, were the most UV-B-affected phenolics
UV-B treatment determined a higher complexity level of anthocyanins after 36 h
"The role of roughage provision on the absorption and disposition of the mycotoxin deoxynivalenol and its acetylated derivatives in calves: from field observations to toxicokinetics."Abstract A clinical case in Belgium demonstrated that feeding a feed concentrate containing considerable levels of deoxynivalenol (DON, 1.13 mg/kg feed) induced severe liver failure in 2- to 3-month-old beef calves. Symptoms disappeared by replacing the highly contaminated corn and by stimulating ruminal development via roughage administration. A multi-mycotoxin contamination was demonstrated in feed samples collected at 15 different veal farms in Belgium... » https://doi.org/10.1007/s00204-018-2368-8 » Adam Lab
Publication with contribution from Adam Lab
"Untargeted LC–MS based 13C labelling provides a full mass balance of deoxynivalenol and its degradation products formed during baking of crackers, biscuits and bread"Highlights
Deoxynivalenol (DON) is mostly stable during the baking process.
isoDON, norDON B and norDON C were identified as DON degradation products.
The major thermal degradation product of DON is isoDON.
"Cross-reactivity of commercial and non-commercial deoxynivalenol-antibodies to emerging trichothecenes and common deoxynivalenol-derivatives"Abstract Immunoassay based techniques are an important and fast option for the detection and quantification of mycotoxins. They are frequently used as on-site screening tools in grain elevators, storage and production facilities. However, accurate quantification may be hampered by the co-recognition of structurally related metabolites by the used antibodies. Therefore, it is crucial to assess their cross-reactivity to avoid misinterpretation of the results... » https://www.wageningenacademic.com/doi/pdf/10.3920/WMJ2018.2363 » Adam Lab
International PhD Programme “Biomolecular Technology of Proteins (BioToP)”
at the Vienna Institute of BioTechnology of BOKU University of Natural Resources and Life Sciences, Vienna, Austria » fully-funded PhD student positions for three years BioToP offers an inter- and multi-disciplinary research-based doctoral education at the interface of basic and applied science in the field of protein biotechnology. BioToP offers a challenging scientific environment with state-of-the-art facilities and provides comprehensive and thorough up-to-date research training in the fields of:
structure-function analysis, engineering and design of proteins
protein synthesis, targeting and post-translational modifications
expression systems and cell factories
bioinformatics and molecular modelling
The BioToP-specific educational programme comprises lectures, seminars and instructional courses that complement the research work in the participating groups. Highly qualified and motivated students of any nationality are invited to apply for the 3-year studentships. Funding will be according to the salary scheme of the Austrian Science Fund. Additionally students will receive funding for research stays abroad and for the participation at international conferences. Further information on research projects as well as application guidelines and forms are available at: http://biotop.boku.ac.at Application deadline: February 13th, 2019
Publication from Lena Studt (Strauss Lab)
"The putative H3K36 demethylase BcKDM1 affects virulence, stress responses and photomorphogenesis in Botrytiscinerea"Highlights:
T-DNA insertion in the virulence-attenuated mutant PA2810 disrupts bckdm1.
Bckdm1 encodes a putative histone 3 lysine 36-specific demethylase.
Bckdm1 is required for virulence, stress responses and photomorphogenesis.
Orthologs from other Ascomycetes cannot replace BcKDM1.
"Maize and Sorghum Flavonoid Pigments: From Genetic and Epigenetic Markers to Plant Health Applications" (Penn State University, Department of Plant Sciences) The goal of Dr. Surinder Chopra’s research is to develop a better understanding of metabolic coordination and role of secondary metabolites in plant developmental process as well as plant resistance to biotic and abiotic stresses. His group uses a flavonoid biosynthetic pathway in maize and sorghum as a model system. The research has led to the identification of a genetic factor that is involved in the biosynthesis of a class of plant flavonoid compounds, which act as anti-fungal agents in sorghum. These antifungal metabolites are also known as phytoalexins and in sorghum these compounds belong to the 3-deoxyanthocyanidin category. The group is now generating sorghum and maize mutants and introgression lines/over expression breeding lines to understand the regulation of defense compounds. In addition to the biotic stress, they are screening and developing new sorghum accessions that are tolerant to colder climatic regions. 19. December 2018, 11:00-11:45 Seminarraum Angewandte Genetik (MUG2-04/54) Muthgasse 18, 1190 Wien
Guest Professor at the DAGZ
David Cánovas research interests are focused on the signals that control fungal development and reproduction using Aspergillusnidulans and Neurosporacrassa as model organisms. 941001VUGenetics of fungal development (in Eng.)›BOKU online 06.12.2018 - 14.12.2018, UFT Tulln, 3 ECTS
06.12.2018: 09:00 14:00 (Lab E2.4.170)
07.12.2018: 09:00 14:00 (Lab E2.4.170)
10.12.2018: 09:00 15:00 (Lab E2.4.170)
11.12.2018: 09:00 15:00 (Lab E2.4.170)
12.12.2018: 09:00 14:00 (Lab E2.4.170)
13.12.2018: 09:00 14:00 (Lab E2.4.170)
14.12.2018: 09:00 14:00 (Lab E2.4.170)
David Cánovas studied Pharmacology at the University of Sevilla and received his Ph.D. degree in Pharmacology with a focus on Molecular and Cellular Biology in 1999. He received further training as a postdoc, first in environmental microbiology and then in fungal genetics, in the National Center for Biotechnology – CSIC (Madrid, Spain) and the University of Melbourne (Australia). He was appointed as a tenure track (within the competitive national “Ramon y Cajal” program) in the Department of Genetics – University of Sevilla in 2008. He was appointed Associate Professor in 2012. Currently he is associate editor of several international journals. He was recently the recipient of a Lise Meitner project from the FWF to study the role of nitric oxide in fungal reproduction at the DAGZ, which finished in 2017 and has already resulted in several publications.
Preprint from Kalyna Lab
"Remote control of alternative splicing in roots through TOR kinase"Abstract For plants, light is the source of energy and the most relevant regulator of growth and adaptations to the environment by inducing changes in gene expression at various levels, including alternative splicing. Chloroplasts trigger retrograde signals that control alternative splicing in leaves and roots in response to light. https://doi.org/10.1101/472126 Kalyna Lab
Publication From Strauss Lab
"Chromatin mapping identifies BasR, a key regulator of bacteria-triggered production of fungal secondary metabolites" The eukaryotic epigenetic machinery can be modified by bacteria to reprogram the response of eukaryotes during their interaction with microorganisms. We discovered that the bacterium Streptomycesrapamycinicus triggered increased chromatin acetylation and thus activation of the silent secondary metabolism ors gene cluster in the fungus Aspergillus nidulans. https://www.ncbi.nlm.nih.gov/pubmed/30311911Strauss Lab
DAGZ Seminar 11.12.2018
Alexander Jones Sainsbury Laboratory "The makings of a gradient: visualising plant hormones in vivo"" Tuesday 11.12.2018 09:00 am DAGZ seminar room (4th floor, Muthgasse 18, 1190 Wien) Host: Sascha Waidmann Kleine-Vehn Lab
BiRT Lecture Series
Dr. Jan-Peter George BFW Bundesforschungszentrum für Wald Institut für Waldgenetik "Molecular genetics reveal putative adaptive variation in resistance against Chrysomyxarhododendri in Norway spruce" Thursday November 22nd, 2018 Time: 10:00 Campus Tulln / UFT – Seminar Room 14 Host: Joseph Strauss & Markus Gorfer Strauss Lab
VIBT Seminar
Kris Vissenberg IMPRES (Integrated Molecular Plant physiology Research) Biology Department, University of Antwerp, Belgium "Control of root hair development in Arabidopsisthaliana" Friday 09.11.2018, 13:00 DAGZ seminar room (MUG2-04/54) Host: Marie-Theres Hauser Hauser Lab
BiRT Seminar Series „die Welt der biologischen Interaktionen“
Friedrich Kragler Univ. Doz. Dr. Max Planck Institute of Molecular Plant Physiology, Wissenschaftspark Golm Germany "Lost in translation: messenger RNA moving to distant tissues" Wann: 14. November 2018, 10:00 Wo: Campus Tulln / UFT – Seminarraum 17' Host: Christoph Schüller Schüller Lab
Die BiMM und das Internet der Pilze
Auf der Suche nach Links im Internet der Pilze Können Schimmelpilze Informationen über die Abwehr von Bakterien an das gesamte Pilznetzwerk weitergeben und speichern? Die Antwort auf diese Frage könnte bei der Entwicklung neuer Antibiotika und Krebstherapien helfen » zum ArtikelStrauss LabBiMM Bioactive Microbial Metabolites
Publication from Kalyna Lab
"PRP4KA, a Putative Spliceosomal Protein Kinase, Is Important for Alternative Splicing and Development in Arabidopsisthaliana" Splicing of pre-mRNAs is an essential step in the expression of most eukaryotic genes. Both constitutive splicing and alternative splicing, which produces multiple mRNA isoforms from a single primary transcript, are modulated by reversible protein phosphorylation. Although the plant splicing machinery is known to be a target for phosphorylation, the protein kinases involved remain to be fully defined... The article has been chosen as a highlight for the December issue of GENETICS. http://www.genetics.org/content/210/4/NPhttps://doi.org/10.1534/genetics.118.301515 Kalyna Lab
Publication from Schüller Lab
"A constitutive active allele of the transcription factor Msn2 mimicking low PKA activity dictates metabolic remodeling in yeast" In yeast, protein kinase A (PKA) adjusts transcriptional profiles, metabolic rates and cell growth in concordance with carbon source availability. PKA affects gene expression mostly via the transcription factors Msn2 and Msn4, two key regulators of the environmental stress response. Here we analyzed the role of the PKA-Msn2 signaling module by using an Msn2 allele that harbors serine to alanine substitutions at six functionally important PKA motifs (Msn2A6)... https://doi.org/10.1091/mbc.E18-06-0389Schüller Lab
FWF Einzelprojekt für Richard Strasser
"Der Oligosaccharyltransferase-Komplex von Pflanzen"
“Endoplasmic reticulum-associated degradation of glycoproteins”
Richard Strasser talks about “Endoplasmic reticulum-associated degradation of glycoproteins” at the GMI seminar. Friday, 19 October 2018, 11:30am @ GMI, Orange Seminar Room. Gregor Mendel Institute of Molecular Plant Biology GmbH Dr. Bohr-Gasse 3, 1030 Vienna Strasser Lab
BiRT Seminar Series „die Welt der biologischen Interaktionen“
Prof. Ivo Frébort Palacký University Olomouc, Czech Republic "Plants as a tool for sustainable global development - Genetic improvement of barley and molecular farming" Wann: 18. Oktober 2018, 9:30 Wo: Campus Tulln / UFT – Seminarraum 14
Publication from Strauss Lab
Fungus found floating in the Danube river - now identified and described by BiMM team as new fungal species: "Metapochonialutea, a new species isolated from the Danube river in Austria" A new species Metapochonialutea (Ascomycota, Hypocreales) is described and illustrated. This fungus differs from the other taxa in the genus Metapochonia by its production of intensive yellow pigment in culture, conidiophores with relatively complex verticillate branching, bean-shaped conidia, and by delayed development of one-celled and prominent thick-walled submerged chlamydospores usually in chains or irregular clusters of 3–5 cells... https://www.schweizerbart.de/journals/nova_hedwigiaStrauss Lab
Publication from Adam Lab
"Impact of glutathione modulation on the toxicity of the Fusarium mycotoxins deoxynivalenol (DON), NX-3 and butenolide in human liver cells." DON, NX-3 and butenolide (BUT) are secondary metabolites formed by Fusariumgraminearum. Evidence for formation of DON-glutathione adducts exists in plants, and also in human liver (HepG2) cells mass spectrometric evidence for GSH-adduct formation was reported. NX-3 is a DON derivative lacking structural features for Thiol-Michael addition, while BUT has the structural requirements (conjugated double bond and keto group)... https://www.ncbi.nlm.nih.gov/pubmed/30244016Adam Lab
Publication from Adam Lab
"New Plasmids for Fusarium Transformation Allowing Positive-Negative Selection and Efficient Cre-loxP Mediated Marker Recycling" In filamentous fungi such as Fusariumgraminearum, disruption of multiple genes of interest in the same strain (e.g., to test for redundant gene function) is a difficult task due to the limited availability of reliable selection markers. We have created a series of transformation vectors that allow antibiotic-based selection of transformants and subsequent negative selection for marker removal using thymidine kinase fusions combined with the Cre-loxP system... https://doi.org/10.3389/fmicb.2018.01954Adam Lab
Hands-on Exhibition for children "Earth & Soil"
Our department is a cooperation partner for an upcoming exhibition of the Zoom museum. This hands on exhibition on "Earth and Soil" is for children ages 6-12. Running time: 27. September 2018 - 24. February 2019 Zoom Kindermuseum Kleine-Vehn Lab
Congratulation to Verena Unterwurzacher to finalzing her PhD Thesis with an excellent defense! Thesis was done over the last years in collaboration with the AIT group of Markus Gorfer. Topic: "Development of a multiplex qPCR detection systems for fungal indoor contamination" Strauss Lab
At the 10th ÖGMBT meeting Somanath Kallolimath was awarded with the ÖGMBT Life Science PhD Award 2018 (sponsored by THP medical products). Steinkellner Lab
WKO Preis 2018 für Andreas Schüller (Strauss Lab)
Andreas Schüller stellt sein Projekt vor, welches mit dem Wirtschaftskammerpreis 2018 an der BOKU ausgezeichnet wurde. Es geht um die Etablierung eines neuen Systems zur gezielten Aktivierung und Entdeckung von bioaktiven Stoffen in filamentösen Pilzen. https://www.youtube.com/watch?v=3BCayifmbxoStrauss Lab
"Development of the Next Generation Influenza Vaccine"
Seminar of Marc-André D’Aoust Vice-President, Research and Innovation Medicago, inc, CAN
Thurs, 4th Oct 2018, 2 pm Seminar room Department of Applied Genetics and Cell Biology (Muthgasse 18, 4th floor)
Host: Herta Steinkellner, Richard Strasser, Alexandra Castilho
The BOKU (Jürgen Kleine-Vehn), GMI (Yasin Dagdas) and the University of Vienna (Verena Ibl) have organized (4th-7th September 2018) together the 21st Meeting of the European Network of Plant Endomembrane Research (ENPER) #ENPER 2018 150 plant cell biologists attended the meeting. » Kleine-Vehn Lab » Dagdas Lab » Ibl Lab
"A speculation on the tandem fasciclin 1 repeat of FLA4 proteins in angiosperms"Abstract The Arabidopsisthaliana Fasciclin like arabinogalactan protein 4 (FLA4) locus is required for normal root growth in a linear genetic pathway with the FEI1 and FEI2 loci coding for receptor-like kinases. The two Fas1 domains of FLA4 are onserved among angiosperms but only the C-terminal Fas1 domain is required for genetic function... » https://doi.org/10.1080/15592324.2018.1507403 » Seifert Lab
»Just cut it - an easy way to turn a negative regulator to positive mode-of-action« Publication from Strauss Lab
"Truncation of the transcriptional repressor protein Cre1 in Trichodermareesei Rut-C30 turns it into an activator"Background The filamentous fungus Trichodermareesei is a natural producer of cellulolytic and xylanolytic enzymes and is therefore industrially used. Many industries require high amounts of enzymes, in particular cellulases. Strain improvement strategies by random mutagenesis yielded the industrial ancestor strain Rut-C30... » https://doi.org/10.1186/s40694-018-0059-0 » Strauss Lab
Publication from Schüller Lab
"Competition of Candidaglabrata against Lactobacillus is Hog1 dependent"Abstract Candidaglabrata is a common human fungal commensal and opportunistic pathogen. This fungus shows remarkable resilience as it can form recalcitrant biofilms on indwelling catheters, has intrinsic resistance against azole antifungals and is causing vulvo‐vaginal candidiasis. As a nosocomial pathogen, it can cause life‐threatening bloodstream infections in immune‐compromised patients... » https://doi.org/10.1111/cmi.12943 » Schüller Lab
Publication from Hauser Lab
"Multiplex mutagenesis of four clustered CrRLK1L with CRISPR/Cas9 exposes their growth regulatory roles in response to metal ions"Abstract Resolving functions of closely linked genes is challenging or nearly impossible with classical genetic tools. Four members of the Catharanthusroseus receptor-like kinase 1-like (CrRLK1L) family are clustered on Arabidopsis chromosome five. To resolve the potentially redundant functions of this subclass of CrRLK1Ls named MEDOS1 to 4 (MDS1 to 4), we generated a single CRISPR/Cas9 transformation vector using a Golden Gate based cloning system to target all four genes simultaneously... » https://doi.org/10.1038/s41598-018-30711-3 » Hauser Lab
Publication from Lena Studt (Strauss Lab)
"Set1 and Kdm5 are antagonists for H3K4 methylation and regulators of the major conidiation‐specific transcription factor gene ABA1 in Fusariumfujikuroi"Summary Here we present the identification and characterisation of the H3K4‐specific histone methyltransferase Set1 and its counterpart, the Jumonji C demethylase Kdm5, in the rice pathogen Fusariumfujikuroi. While Set1 is responsible for all detectable H3K4me2/me3 in this fungus, Kdm5 antagonises the H3K4me3 mark. Notably, deletion of both SET1 and KDM5 mainly resulted in the upregulation of genome‐wide transcription, also affecting a large set of secondary metabolite (SM) key genes... » https://doi.org/10.1111/1462-2920.14339 » Strauss Lab
Preprint from Korbei Lab
"TOL proteins mediate vacuolar sorting of the borate transporter BOR1 in Arabidopsisthaliana"Abstract Boron (B) is an essential micronutrient for plants, however, it shows cytotoxicity at high concentrations. A borate transporter BOR1 is required for efficient transport of boron (B) toward the root stele in Arabidopsisthaliana. BOR1 shows polar localization in the plasma membrane of various root cells toward the stele-side under B limitation. » https://doi.org/10.1101/342345 » Korbei Lab
Josef Glössl zum Urteil des Europäischen Gerichtshofs über den Einsatz neuer Mutagenese Methoden:
"Ich bin von diesem Urteil sehr enttäuscht, weil ich es in den wesentlichen Punkten nicht für sachlich begründet und nachvollziehbar halte" ⇒ zum Artikel ⇒ zum Urteil im Wortlaut
"Response of intestinal HT-29 cells to the trichothecene mycotoxin deoxynivalenol and its sulfated conjugates" The sulfated forms of the Fusarium toxin deoxynivalenol (DON), deoxynivalenol-3-sulfate (DON-3-Sulf) and deoxynivalenol-15-sulfate (DON-15-Sulf) were recently described, however little is known about their mechanism of action in mammalian cells... » https://doi.org/10.1016/j.toxlet.2018.07.007 » Adam Lab
PhD Defense of Julia Richter - passed with distinction!
"The role of cell wall integrity receptors in responses to heavy metals" Zeit: Mittwoch, 11.07.2018, 10:00 Ort: Seminarraum Angewandte Genetik (MUG2-04/54) Muthgasse 18, 4. Stock Supervisor: Marie-Theres Hauser
Publication from Mach Lab
"The two cathepsin B-like proteases of Arabidopsis thaliana are closely related enzymes with discrete endopeptidase and carboxydipeptidase activities" The genome of the model plant Arabidopsisthaliana encodes three paralogues of the papain-like cysteine proteinase cathepsin B (AtCathB1, AtCathB2 and AtCathB3), whose individual functions are still largely unknown. » https://doi.org/10.1515/hsz-2018-0186 » Mach Lab
FWF Elise-Richter Programm for Elena Feraru
Auxin homeostasis in plant organ responses to high temperature project number: V 690 Richter-Programm decision board: 25.06.2018 » Elena Feraru
FWF Elise-Richter Programm for Doris Lucyshyn
O-GlcNAc and O-fucose binding plant lectins project number: V 658 Richter-Programm decision board: 25.06.2018 » Lucyshyn Lab
Das war auch heuer wieder ein spannender Exkursionstag im Zoo Schönbrunn mit Vorträgen und Backstage-Führungen für unsere Studierenden des Bachelorstudiums Agrarwissenschaften. Die Themen: Warum ist Biodiversität nur mit genetischer Diversität denkbar, warum ist Artenschutz essentiell und was sind die Gründe für die „Kernschmelze der Agrar-Biodiversität“ in den letzten Jahrzehnten?
Diese und viele weitere Fragen wurden von den Studierenden mit den Veranstaltern diskutiert. Ein Angebot des DAGZ im Zuge der Lehrveranstaltung "941091 Agrargenetik und Biodiversität - Exkursion".
Publication from Steinkellner Lab
"Advanced Plant-Based Glycan Engineering" With respect to biomanufacturing, glycosylation is one of the most addressed post-translational modifications, since it is well-known that the attachment of sugar residues efficiently affects protein homogeneity and functionality. Much effort has been taken into engineering various expression systems to control glycosylation and to generate molecules with targeted sugar profiles. » https://doi.org/10.3389/fbioe.2018.00081 » Steinkellner Lab
Vortrag zum Thema "Der EU-Sortenschutz, gewerblicher Rechtsschutz für Pflanzensorten"
Der dazu eingeladene Experte, Dirk Theobal kommt vom Gemeinschaftlichen Sortenamt (CPVO) und wird über die Funktionsweise des gemeinschaftlichen Systems zum Schutz von Pflanzensorten, die technischen Schutzanforderungen an Sorten und die rechtlichen Verfahren berichten (die Agenda finden Sie im Anhang) Donnerstag, 14.06.2018, von 09:00 bis 12:00 Uhr Department für Angewandte Genetik und Zellbiologie (DAGZ): Muthgasse 18, (MUG2-04/54)
Publication from Seifert Lab
"Fascinating Fasciclins: A Surprisingly Widespread Family of Proteins that Mediate Interactions between the Cell Exterior and the Cell Surface" The Fas1 proteins act as mediators between the cell and its environment and function in similar fashion in processes as diverse as human brain cancer and pollen formation in plants.This review for the first time compares the biology of Fas1 proteins from all kingdoms of life. » https://doi.org/10.3390/ijms19061628 » Seifert Lab
Josef Glössl zum stellvertretenden Vorsitzenden des Universitätsrats der Medizinischen Universität Innsbruck gewählt Professor JosefGlössl, gerade eben aus dem Rektorat der BOKU (Vizerektor für Forschung und Internationale Beziehungen) an das DAGZ zurückgekehrt, wurde vor Kurzem zum stellvertretenden Vorsitzenden des Universitätsrats der Medizinischen Universität Innsbruck gewählt. Herzliche Gratulation! » Universitätsrat der Medizinischen Universität Innsbruck
Visit from the Wake Forest University
ElizabethSarkel, a junior biochemistry and molecular biology major from Columbus, Ohio is joining the Kleine-Vehn Lab again this summer. Elizabeth received a Fulbright Program fellowship and will work on gravitropic set point angle of lateral roots. ______________ Twitter: • The Fulbright Program @FulbrightPrgrm • Wake Forest University @WakeForest • Kleine-Vehn Lab @KleineVehnLab
"Mad moves of the building blocks – nucleotide sugars find unexpected paths into cell walls" This short review tracks the unexpectedly yet purposefully complex, intracellular journey of an activated sugar before it safely reaches its destination in plant fibre polysaccharides. » https://doi.org/10.1093/jxb/ery026 » Seifert Lab
Publication from Kleine-Vehn Lab
"The Road to Auxin-Dependent Growth Repression and Promotion in Apical Hooks" The phytohormone auxin controls growth rates within plant tissues, but the underlying mechanisms are still largely enigmatic. The apical hook is a superb model to understand differential growth, because it displays both auxin-dependent growth repression and promotion. In this special issue on membranes, we illustrate how the distinct utilization of vesicle trafficking contributes to the spatial control of polar auxin transport, thereby pinpointing the site of growth repression in apical hooks. » https://doi.org/10.1016/j.cub.2018.01.069 » Kleine-Vehn Lab
Publication from Kleine-Vehn Lab
"PIN7 Auxin Carrier Has a Preferential Role in Terminating Radial Root Expansion in Arabidopsis thaliana" Directional growth of lateral roots is critical for radial expansion and soil coverage. Despite its importance, almost nothing is known about its molecular determinants. Initially, young lateral roots (LRs) grow away from the parental root, maintaining the angle acquired shortly after emergence. A second downwards bending response to gravity terminates the so-called plateau phase and thereby limits radial root expansion » doi:10.3390/ijms19041238 » Kleine-Vehn Lab
Publication from Hauser Lab
"Arabidopsis ILITHYIA protein is necessary for proper chloroplast biogenesis and root development independent of eIF2α phosphorylation" One of the main mechanisms blocking translation after stress situations is mediated by phosphorylation of the α-subunit of the eukaryotic initiation factor 2 (eIF2), performed in Arabidopsis by the protein kinase GCN2 which interacts and is activated by ILITHYIA(ILA). ILA is involved in plant immunity and its mutant lines present phenotypes not shared by the gcn2 mutants » doi.org/10.1016/j.jplph.2018.04.003 » Hauser Lab
Faszination Pflanze: Warum die Karotte orange ist
Jürgen Kleine-Vehn im Blog der Junge Akademie der ÖAW über die Faszination Pflanze. Hier geht es zum »Blog ______________ Twitter: • Jürgen Kleine-Vehn @KleineVehnLab • Junge Akademie der ÖAW @ya_OeAW
Book on Root Development edited by Elke Barbez (Kleine-Vehn Lab)
Introduction This detailed volume provides diverse elegant methods, complemented with existing protocols, which are optimized for the current needs in plant root biology as well as for use in plant species other than Arabidopsis thaliana. The collection covers methods ranging from genetic screens, phenotypic analysis, and cell biology methods to systems biology tools and genome-wide approaches. The collection contains a range of complexity from fundamental methods for quantification of different root developmental processes to complex methods that require sophisticated equipment » Methods in Molecular Biology, vol 1761
Publication from Korbei Lab and Kleine-Vehn Lab
Immunoprecipitation of Membrane Proteins from Arabidopsisthaliana Root TissueAbstract Here, we present different methods for immunoprecipitating membrane proteins of Arabidopsisthaliana root material. We describe two extraction methods for the precipitation either for an integral membrane protein of the endoplasmic reticulum (ER) or a peripheral membrane protein partially localized at the plasma membrane, where we precipitate the protein out of the total membrane as well as total cytosolic fractions » doi.org/10.1007/978-1-4939-7747-5_16
Publication from Kleine-Vehn Lab
Growth Rate Normalization Method to Assess Gravitropic Root GrowthAbstract Time-lapse imaging of roots is highly suitable for depicting gravitropic growth behaviors. However, roots may show faster or slower bending kinetics when compared to control as a result of differences in overall root growth. Accordingly, conditions that cause differential organ growth require growth rate normalization to compare gravitropic curvature » doi.org/10.1007/978-1-4939-7747-5_15
Publication from Kleine-Vehn Lab
Cortical Cell Length Analysis During Gravitropic Root Growth
Abstract The typical parameter used to evaluate the root growth response to gravity is the degree of root bending in time. This employs the quantification of the root tip angle toward gravity and, hence, does not directly assess the actual differential growth process. Here, we describe the cortical cell length as a parameter to quantify cell elongation during the gravitropic response, using median longitudinal confocal sections » doi.org/10.1007/978-1-4939-7747-5_14
Congratulations Alexandra!
New FWF Stand-Alone Project:
N. benthamiana ß-galactosidases acting on glycoproteins
UDP-Glucosyltransferases from Rice, Brachypodium, and Barley: Substrate Specificities and Synthesis of Type A and B Trichothecene-3-O-β-D-glucosidesAbstract Trichothecene toxins are confirmed or suspected virulence factors of various plant-pathogenic Fusarium species. Plants can detoxify these to a variable extent by glucosylation, a reaction catalyzed by UDP-glucosyltransferases (UGTs). Due to the unavailability of analytical standards for many trichothecene-glucoconjugates, information on such compounds is limited » full text
PhD Defense of Somanath Kallolimath
Production of recombinant proteins with polysialylated N-glycans in Nicotianabenthamiana 23.02.2018 - 14:00 DAGZ-Seminarroom (MUG2-04/54) Muthgasse 18, 4th floor Supervisor: Herta Steinkellner
PhD Defense of Chloé Béziat
Characterization of PILS putative intracellular auxin carrier family during developmental processes in Arabidopsis 22.02.2018 - 14:30 DAGZ-Seminarroom (MUG2-04/54) Muthgasse 18, 4th floor Supervisor: Jürgen Kleine-Vehn
Publication from Steinkellner/Strasser Lab
An oligosaccharyltransferase from Leishmania major increases the N-glycan occupancy on recombinantglycoproteins produced in Nicotianabenthamiana.Summary N-glycosylation is critical for recombinant glycoprotein production as it influences the heterogeneity of products and affects their biological function. In most eukaryotes, the oligosaccharyltransferase is the central protein-complex facilitating the N-glycosylation of proteins in the lumen of the endoplasmic reticulum (ER) » doi.org/10.1111/pbi.12906
Publication from Adam Lab:
Chemical synthesis of culmorin metabolites and their biologic role in culmorin and acetyl-culmorin treated wheat cells The Fusarium metabolite culmorin (1) is receiving increased attention as an “emerging mycotoxin”. It cooccurs with trichothecene mycotoxins and potentially influences their toxicity. Its ecological role and fate in plants is unknown. We synthesized sulfated and glucosylated culmorin conjugates as potential metabolites, which are expected to be formed in planta, and used them as reference compounds. An efficient procedure for the synthesis of culmorin sulfates was developed » DOI: 10.1039/c7ob02460f
Publication from Strasser Lab
The glycan-dependent ERAD machinery degrades topologically diverse misfolded proteins.Summary A great number of soluble and integral membrane proteins fold in the endoplasmic reticulum (ER) with the help of chaperones and folding factors. Despite these efforts, protein folding is intrinsically error prone and amino acid changes, alterations in posttranslational modifications or cellular stress can cause protein misfolding. Folding-defective non-native proteins are cleared from the ER and typically undergo ER-associated degradation (ERAD) » doi.org/10.1111/tpj.13851
Publication from Hauser Lab:
Root hair abundance impacts cadmium accumulation in Arabidopsis thaliana shootsBackground and Aims Root hairs increase the contact area of roots with soil and thereby enhance the capacity for solute uptake. The strict hair/non-hair pattern of Arabidopsis thaliana can change with nutrient deficiency or exposure to toxic elements, which modify root hair density. The effects of root hair density on cadmium (Cd) accumulation in shoots of arabidopsis genotypes with altered root hair development and patterning were studied » doi.org/10.1093/aob/mcx220
Pre-print from Elena Feraru (Kleine-Vehn Lab):
PILS6 is a temperature-sensitive regulator of nuclear auxin input and organ growth in Arabidopsisthaliana Abstract Global warming is threatening plant productivity, because plant growth is highly sensitive to elevated temperatures. High temperature (HT) triggers the auxin biosynthesis-dependent growth in aerial tissues. On the other hand, the contribution of auxin to HT-induced root growth is currently under debate. Here we show that the putative intracellular auxin carrier PIN-LIKES 6 (PILS6) is a negative regulator of organ growth and that its abundance is highly sensitive to HT » get the pre-print
