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

Alternative oxidase involvement in Daucus carota somatic embryogenesis

Plant alternative oxidase (AOX) is a mitochondrial inner membrane enzyme involved in alternative respiration.The critical importance of the enzyme during acclimation upon stress of plant cells is not fully understood and is still an issue of intensive research and discussion. Recently, a role of AOX was suggested for the ability of plant cells to change easily its fate upon stress. In order to get new insights about AOX involvement in cell reprogramming, quantitative real-time polymerase chain reaction (PCR) and inhibitor studies were performed during cell redifferentiation and developmental stages of Daucus carota L. somatic embryogenesis. Transcript level analysis shows that D. carota AOX genes (DcAOX1a and DcAOX2a) are differentially expressed during somatic embryogenesis. DcAOX1a shows lower expression levels, being mainly down- regulated, whereas DcAOX2a presented a large up-regulation during initiation of the realization phase of somatic embryogenesis. However, when globular embryos start to develop, both genes are down-regulated, being this state transient for DcAOX2a. In addition, parallel studies were performed using salicylhydroxamic acid (SHAM) in order to inhibit AOX activity during the realization phase of somatic embryogenesis. Embryogenic cells growing in the presence of the inhibitor were unable to develop embryogenic structures and its growth rate was diminished. This effect was reversible and concentration dependent. The results obtained contribute to the hypothesis that AOX activity supports metabolic reorganization as an essential part of cell reprogramming and, thus, enables restructuring and de novo cell differentiation

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

Hordeum vulgare differentiates its response to beneficial bacteria

Background In nature, beneficial bacteria triggering induced systemic resistance (ISR) may protect plants from potential diseases, reducing yield losses caused by diverse pathogens. However, little is known about how the host plant initially responds to different beneficial bacteria. To reveal the impact of different bacteria on barley (Hordeum vulgare), bacterial colonization patterns, gene expression, and composition of seed endophytes were explored. Results This study used the soil-borne Ensifer meliloti, as well as Pantoea sp. and Pseudomonas sp. isolated from barley seeds, individually. The results demonstrated that those bacteria persisted in the rhizosphere but with different colonization patterns. Although root-leaf translocation was not observed, all three bacteria induced systemic resistance (ISR) against foliar fungal pathogens. Transcriptome analysis revealed that ion- and stress-related genes were regulated in plants that first encountered bacteria. Iron homeostasis and heat stress responses were involved in the response to E. meliloti and Pantoea sp., even if the iron content was not altered. Heat shock protein-encoding genes responded to inoculation with Pantoea sp. and Pseudomonas sp. Furthermore, bacterial inoculation affected the composition of seed endophytes. Investigation of the following generation indicated that the enhanced resistance was not heritable. Conclusions Here, using barley as a model, we highlighted different responses to three different beneficial bacteria as well as the influence of soil-borne Ensifer meliloti on the seed microbiome. In total, these results can help to understand the interaction between ISR-triggering bacteria and a crop plant, which is essential for the application of biological agents in sustainable agriculture

Insect peptide metchnikowin confers on barley a selective capacity for resistance to fungal ascomycetes pathogens

The potential of metchnikowin, a 26-amino acid residue proline-rich antimicrobial peptide synthesized in the fat body of Drosophila melanogaster was explored to engineer disease resistance in barley against devastating fungal plant pathogens. The synthetic peptide caused strong in vitro growth inhibition (IC50 value ∼1 μM) of the pathogenic fungus Fusarium graminearum. Transgenic barley expressing the metchnikowin gene in its 52-amino acid pre-pro-peptide form under the control of the inducible mannopine synthase (mas) gene promoter from the Ti plasmid of Agrobacterium tumefaciens displayed enhanced resistance to powdery mildew as well as Fusarium head blight and root rot. In response to these pathogens, metchnikowin accumulated in plant apoplastic space, specifying that the insect signal peptide is functional in monocotyledons. In vitro and in vivo tests revealed that the peptide is markedly effective against fungal pathogens of the phylum Ascomycota but, clearly, less active against Basidiomycota fungi. Importantly, germination of the mutualistic basidiomycete mycorrhizal fungus Piriformospora indica was affected only at concentrations beyond 50 μM. These results suggest that antifungal peptides from insects are a valuable source for crop plant improvements and their differential activities toward different phyla of fungi denote a capacity for insect peptides to be used as selective measures on specific plant diseases

New insights into the subcellular localization of Tubby-like proteins and their participation in the Arabidopsis

Tubby-like proteins (TLPs) have been associated with hormone signaling and responses to abiotic and biotic stress in plants. Recently, Arabidopsis thaliana TLP3 was found to translocate from the plasma membrane of cells in response to distinct abiotic stresses, thereby activating cellular signaling. In addition, several AtTLPs were demonstrated to be necessary for normal colonization of roots by the mutualistic fungus Piriformospora indica. Here, we present evidence for the involvement of another two AtTLPs in this interaction. Furthermore, we show that plasma membrane targeting of TLPs might be conserved in other plant species, although we did not find it for all members of the protein family. Finally, the position of a GFP-tag influences the localization of AtTLP3, which needs to be considered when working with TLPs

New insights into the subcellular localization of Tubby-like proteins and their participation in the Arabidopsis-Piriformospora indica interaction

Tubby-like proteins (TLPs) have been associated with hormone signaling and responses to abiotic and biotic stress in plants. Recently, Arabidopsis thaliana TLP3 was found to translocate from the plasma membrane of cells in response to distinct abiotic stresses, thereby activating cellular signaling. In addition, several AtTLPs were demonstrated to be necessary for normal colonization of roots by the mutualistic fungus Piriformospora indica. Here, we present evidence for the involvement of another two AtTLPs in this interaction. Furthermore, we show that plasma membrane targeting of TLPs might be conserved in other plant species, although we did not find it for all members of the protein family. Finally, the position of a GFP-tag influences the localization of AtTLP3, which needs to be considered when working with TLPs

Evaluation of dsRNA delivery methods for targeting macrophage migration inhibitory factor MIF in RNAi-based aphid control

Macrophage migration inhibitory factors (MIFs) are multifunctional proteins regulating major processes in mammals, including activation of innate immune responses. In invertebrates, MIF proteins participate in the modulation of host immune responses when secreted by parasitic organisms, such as aphids. In this study, we assessed the possibility to use MIF genes as targets for RNA interference (RNAi)-based control of the grain aphid Sitobion avenae (Sa) on barley (Hordeum vulgare). When nymphs were fed on artificial diet containing double-stranded (ds)RNAs (SaMIF-dsRNAs) that target sequences of the three MIF genes SaMIF1, SaMIF2 and SaMIF3, they showed higher mortality rates and these rates correlated with reduced MIF transcript levels as compared to the aphids feeding on artificial diet containing a control dsRNA (GFP-dsRNA). Comparison of different feeding strategies showed that nymphs' survival was not altered when they fed from barley seedlings sprayed with naked SaMIF-dsRNAs, suggesting they did not effectively take up dsRNA from the sieve tubes of these plants. Furthermore, aphids' survival was also not affected when the nymphs fed on leaves supplied with dsRNA via basal cut ends of barley leaves. Consistent with this finding, the use of sieve tube-specific YFP-labeled Arabidopsis reporter lines confirmed that fluorescent 21 nt dsRNA(Cy3), when supplied via petioles or spraying, co-localized with xylem structures, but not with phloem tissue. Our results suggest that MIF genes are a potential target for insect control and also imply that application of naked dsRNA to plants for aphid control is inefficient. More efforts should be put into the development of effective dsRNA formulations.Peer reviewe

The Piriformospora indica effector PIIN_08944 promotes the mutualistic Sebacinalean symbiosis

Pathogenic and mutualistic microbes actively suppress plant defense by secreting effector proteins to manipulate the host responses for their own benefit. Current knowledge about fungal effectors has been mainly derived from biotrophic and hemibiotrophic plant pathogenic fungi and oomycetes with restricted host range. We studied colonization strategies of the root endophytic basidiomycete Piriformospora indica that colonizes a wide range of plant species thereby establishing long-term mutualistic relationships. The release of P. indica’s genome helped to identify hundreds of genes coding for candidate effectors and provides an opportunity to investigate the role of those proteins in a mutualistic symbiosis. We demonstrate that the candidate effector PIIN_08944 plays a crucial role during fungal colonization of Arabidopsis thaliana roots. PIIN_08944 expression was detected during chlamydospore germination, and fungal deletion mutants (Pi∆08944) showed delayed root colonization. Constitutive over-expression of PIIN_08944 in Arabidopsis rescued the delayed colonization phenotype of the deletion mutant. PIIN_08944-expressing Arabidopsis showed a reduced expression of flg22-induced marker genes of pattern-triggered immunity (PTI) and the salicylic acid (SA) defense pathway, and expression of PIIN_08944 in barley reduced the burst of reactive oxygen species (ROS) triggered by flg22 and chitin. These data suggest that PIIN_08944 contributes to root colonization by P. indica by interfering with SA-mediated basal immune responses of the host plant. Consistent with this, PIIN_08944-expressing Arabidopsis also supported the growth of the biotrophic oomycete Hyaloperonospora arabidopsidis while growth of the necrotrophic fungi Botrytis cinerea on Arabidopsis and Fusarium graminearum on barley was not affected