Charakterisierung und Nutzung von bakteriellen Quorum Sensing Molekülen für die Weiterentwicklung eines umweltgerechten Pflanzenbaus

Die Aktivierung und Stärkung des pflanzlichen Immunsystems ist eine vielversprechende Alternative im Pflanzenschutz. Das Konzept umfasst eine Sensibilisierung (priming) der Kulturpflanzen gegenüber Pathogenen, die einen hohen Ernteertrag auch unter Pathogendruck gewährleistet. Bakterielle quorum sensing Moleküle haben die Eigenschaft, Pflanzen zu sensibilisieren und sind deshalb eine mögliche Ergänzung zum Einsatz von Pflanzenschutzmaßnahmen. Das Ziel dieses Vorhabens war es, Immunantwort und physiologische Veränderungen in wichtigen Kulturpflanzen, wie Gerste, Weizen, Tomaten und Luzerne nach einer Sensibilisierung mit QS Molekülen zu analysieren. Eingesetzt wurden einerseits langkettige Acyl-Homoserinlaktone, wie das oxo-C14-HSL, und andererseits nützliche, nicht-pathogene oxo-C14-HSL-produzierende Rhizobakterium, wie Sinorhizobium meliloti und Rhizobium radiobacter F4 (RrF4), das aus dem nützlichen Pilz Piriformospora indica isoliert wurde. Wir konnten eine oxo-C14-HSL-induzierte Resistenz von Arabidopsis Pflanzen demonstrieren und zeigen, dass diese Resistenzinduktion auf der Basis eines Salicylsäure/Oxylipin-abhängigen systemischen Signal generiert wird. Darüber hinaus bewirkt eine oxo-C14-HSL Behandlung bei Gerste und Weizen eine verstärkte Produktion von reaktiven Sauerstoffspezies und eine transkriptionelle Regulation verteidigungsrelevanter Genen. Die oxo-C14-HSL-induzierte Sensibilisierung bewirkt eine Verstärkung der Pflanzenzellwand und eine Abwehrreaktion in den Stomata, wodurch ein Eindringen von bakteriellen und pilzlichen Krankheitserregern und deren Proliferation in der Pflanze stark gehemmt werden. Zudem wurde überraschenderweise gefunden, dass oxo-C14-HSL auch die Vermehrung des humanpathogenen Erregers Salmonella enterica serovar Thyphimurium in Arabidopsis verhindert. Dieser Effekt war starker ausgeprägt bei direkter Behandlung mit oxo-C14-HSL, während eine Behandlung mit oxo-C14-HSL-produzierendem S. meliloti keine gute Wirkung gegen Salmonellen zeigte. Unsere Ergebnisse verdeutlichen, das bakterielle quorum sensing Moleküle eine positive Wirkung auf Pflanzen haben. Die hier beschriebene Acyl-Homoserinlakton-induzierte Resistenz (AIR) ist ein neues Modell zur pflanzlichen „Sensibilisierung“ für schnellere und stärkere Abwehrreaktionen auf künftige Stresssituationen und bahnt somit einen vielversprechenden Weg im modernen Pflanzenschutz

The mutualistic fungus Piriformospora indica colonizes Arabidopsis roots by inducing an endoplasmic reticulum stress-triggered caspase-dependent cell death

In Arabidopsis thaliana roots, the mutualistic fungus Piriformospora indica initially colonizes living cells, which die as the colonization proceeds. We aimed to clarify the molecular basis of this colonization-associated cell death. Our cytological analyses revealed endoplasmic reticulum (ER) swelling and vacuolar collapse in invaded cells, indicative of ER stress and cell death during root colonization. Consistent with this, P. indica–colonized plants were hypersensitive to the ER stress inducer tunicamycin. By clear contrast, ER stress sensors bZIP60 and bZIP28 as well as canonical markers for the ER stress response pathway, termed the unfolded protein response (UPR), were suppressed at the same time. Arabidopsis mutants compromised in caspase 1–like activity, mediated by cell death–regulating vacuolar processing enzymes (VPEs), showed reduced colonization and decreased cell death incidence. We propose a previously unreported microbial invasion strategy during which P. indica induces ER stress but inhibits the adaptive UPR. This disturbance results in a VPE/caspase 1–like-mediated cell death, which is required for the establishment of the symbiosis. Our results suggest the presence of an at least partially conserved ER stress–induced caspase-dependent cell death pathway in plants as has been reported for metazoans

Using Copper in Organic Viticulture: Doing it best with less?

For many years, applications of copper fungicides have been used to control downy mildew on grapes. Nowadays, its intensive use is under consideration due to ecotoxicological aspects, especially in organic viticulture. As a result, this project made allowance for consumer perception about organic viticulture and is seeking suitable alternatives. The project includes an association of six research facilities, four organic wineries, several consultant agencies for organic viticulture as well as SME (small and medium-sized enterprise) partners, which are working together in four work packages. The aim of this work is to obtain an array of products that provide sufficient control of Plasmopara viticola with the lowest possible input of copper. As a longterm intention, copper-containing products should be totally replaced by other effective agents. Progress to-date suggests that some non-copper products and several copper-based products using less than 3 kg/(ha*a) have potential to effectively control the disease with less ecological residue

Further analysis of barley MORC1 using a highly efficient RNA-guided Cas9 gene-editing system

Summary Microrchidia (MORC) proteins comprise a family of proteins that have been identified in prokaryotes and eukaryotes. They are defined by two hallmark domains: a GHKL-type ATPase and an S5-fold. In plants, MORC proteins were first discovered in a genetic screen for Arabidopsis thaliana mutants compromised for resistance to a viral pathogen. Subsequent studies expanded their role in plant immunity and revealed their involvement in gene silencing and genome stabilization. Little is known about the role of MORC proteins of cereals, especially because knockout (KO) mutants were not available and assessment of loss of function relied only on RNAi strategies, which were arguable, given that MORC proteins in itself are influencing gene silencing. Here, we used a Streptococcus pyogenes Cas9 (SpCas9)-mediated KO strategy to functionally study HvMORC1, one of the current seven MORC members of barley. Using a novel barley RNA Pol III-dependent U3 small nuclear RNA (snRNA) promoter to drive expression of the synthetic single guide RNA (sgRNA), we achieved a very high mutation frequency in HvMORC1. High frequencies of mutations were detectable by target sequencing in the callus, the T0 generation (77%) and T1 generation (70% - 100%), which constitutes an important improvement of the gene-editing technology in cereals. Corroborating and extending earlier findings, SpCas9-edited hvmorc1-KO barley, in clear contrast to Arabidopsis atmorc1 mutants, had a distinct phenotype of increased disease resistance to fungal pathogens, while morc1 mutants of either plant showed de-repressed expression of transposable elements (TEs), substantiating that plant MORC proteins contribute to genome stabilization in monocotyledonous and dicotyledonous plants

The mutualistic fungus Piriformospora indica protects barley roots from a loss of antioxidant capacity caused by the necrotrophic pathogen Fusarium culmorum

Fusarium culmorum causes root rot in barley (Hordeum vulgare), resulting in severely reduced plant growth and yield. Pretreatment of roots with chlamydospores of the mutualistic root-colonizing basidiomycete Piriformospora indica (Agaricomycotina) prevented necrotization of root tissues and plant growth retardation commonly associated with Fusarium root rot. Quantification of Fusarium infections with a real-time PCR assay revealed a correlation between root rot symptoms and the relative amount of fungal DNA. Fusarium-infected roots showed reduced levels of ascorbate and glutathione (GSH), along with reduced activities of antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR). Consistent with this, Fusarium-infected roots showed elevated levels of lipid hydroperoxides and decreased ratios of reduced to oxidized forms of ascorbate and glutathione. In clear contrast, roots treated with P. indica prior to inoculation with F. culmorum showed levels of ascorbate and GSH that were similar to controls. Likewise, lipid peroxidation and the overall reduction in antioxidant enzyme activities were largely attenuated by P. indica in roots challenged by F. culmorum. These results suggest that P. indica protects roots from necrotrophic pathogens at least partly, through activating the plant’s antioxidant capacity

Analysis and purification of ssRNA and dsRNA molecules using asymmetrical flow field flow fractionation

Robust RNA purification and analysis methods are required to support the development of RNA vaccines and therapeutics as well as RNA interference-based crop protection solutions. Asymmetrical flow field -flow fractionation (AF4) is a gentle native purification method that applies liquid flows to separate sample components based on their hydrodynamic sizes. We recently showed that AF4 can be utilized to separate RNA molecules that are shorter than 110 nucleotides (nt), but the performance of AF4 in the analysis and purification of longer RNA molecules has not been previously evaluated. Here, we studied the perfor-mance of AF4 in separation of single-stranded (ss) and double-stranded (ds) RNA molecules in the size range of 75-6400 nt. In addition, we evaluated the power of AF4 coupling to different detectors, allow-ing separation to be combined with data collection on yield as well as molecular weight ( MW ) and size distribution. We show that AF4 method is applicable in RNA purification, quality control, and analytics, and results in good recoveries of ssRNA and dsRNA molecules. In addition, our results demonstrate the utility of AF4 multidetection platforms to study biophysical properties of long RNA molecules.(c) 2022 The Author(s). Published by Elsevier B.V.This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )Peer reviewe

Matrix metalloproteinases operate redundantly in Arabidopsis immunity against necrotrophic and biotrophic fungal pathogens

Matrix metalloproteinases (MMPs) are evolutionarily conserved and multifunctional effector molecules playing pivotal roles in development and homeostasis. In this study we explored the involvement of the five Arabidopsis thaliana At-MMPs in plant defence against microbial pathogens. Expression of At2-MMP was most responsive to inoculation with fungi and a bacterial pathogen followed by At3-MMP and At5-MMP, while At1-MMP and At4-MMP were non-responsive to these biotic stresses. Loss-of-function mutants for all tested At-MMPs displayed increased susceptibility to the necrotrophic fungus Botrytis cinerea and double mutant at2,3-mmp and triple mutant at2,3,5-mmp plants developed even stronger symptoms. Consistent with this, transgenic Arabidopsis plants that expressed At2-MMP constitutively under the Cauliflower mosaic virus 35S promoter showed enhanced resistance to the necrotrophic pathogen. Similarly, resistance to the biotrophic Arabidopsis powdery mildew fungus Golovinomyces orontii was also compromised particularly in the at2,3-mmp / at2,3,5-mmp multiplex mutants, and increased in At2-MMP overexpressor plants. The degree of disease resistance of at-mmp mutants and At2-MMP overexpressor plants also correlated positively with the degree of MAMP-triggered callose deposition in response to the bacterial flagellin peptide flg22, suggesting that matrix metalloproteinases contribute to pattern-triggered immunity (PTI) in interactions of Arabidopsis with necrotrophic and biotrophic pathogens

Silencing β1,2-xylosyltransferase in Transgenic Tomato Fruits Reveals xylose as Constitutive Component of Ige-Binding Epitopes

Complex plant N-glycans containing β1,2-xylose and core α1,3-fucose are regarded as the major class of the so-called “carbohydrate cross-reactive determinants” reactive with IgE antibodies in sera of many allergic patients, but their clinical relevance is still under debate. Plant glycosyltransferases, β1,2-xylosyltransferase (XylT), and core α1,3-fucosyltransferase (FucT) are responsible for the transfer of β1,2-linked xylose and core α1,3-linked fucose residues to N-glycans of glycoproteins, respectively. To test the clinical relevance of β1,2-xylose-containing epitopes, expression of the tomato β1,2-xylosyltransferase was down-regulated by RNA interference (RNAi) in transgenic plants. Fruits harvested from these transgenic plants were analyzed for accumulation of XylT mRNA, abundance of β1,2-xylose epitopes and their allergenic potential. Based on quantitative real-time PCR analysis XylT mRNA levels were reduced up to 10-fold in independent transgenic lines as compared to untransformed control, whereas no xylosylated N-glycans could be revealed by MS analysis. Immunoblotting using anti-xylose-specific IgG antibodies revealed a strong reduction of β1,2-xylose-containing epitopes. Incubating protein extracts from untransformed controls and XylT_RNAi plants with sera from tomato allergic patients showed a patient-specific reduction in IgE-binding, indicating a reduced allergenic potential of XylT_RNAi tomato fruits, in vitro. To elucidate the clinical relevance of β1,2-xylose-containing complex N-glycans skin prick tests were performed demonstrating a reduced responsiveness of tomato allergic patients, in vivo. This study provides strong evidence for the clinical relevance of β1,2-xylose-containing epitopes in vivo