51 research outputs found
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N -acetyltransferase AAC(3)-I confers gentamicin resistance to Phytophthora palmivora and Phytophthora infestans
Abstract: Background: Oomycetes are pathogens of mammals, fish, insects and plants, and the potato late blight agent Phytophthora infestans and the oil palm and cocoa infecting pathogen Phytophthora palmivora cause economically impacting diseases on a wide range of crop plants. Increasing genomic and transcriptomic resources and recent advances in oomycete biology demand new strategies for genetic modification of oomycetes. Most oomycete transformation procedures rely on geneticin-based selection of transgenic strains. Results: We established N-acetyltransferase AAC(3)-I as a gentamicin-based selectable marker for oomycete transformation without interference with existing geneticin resistance. Strains carrying gentamicin resistance are fully infectious in plants. We further demonstrate the usefulness of this new antibiotic selection to super-transform well-characterized, already fluorescently-labelled P. palmivora strains and provide a comprehensive protocol for maintenance and zoospore electro-transformation of Phytophthora strains to aid in plant-pathogen research. Conclusions: N-acetyltransferase AAC(3)-I is functional in Phytophthora oomycetes. In addition, the substrate specificity of the AAC(3)-I enzyme allows for re-transformation of geneticin-resistant strains. Our findings and resources widen the possibilities to study oomycete cell biology and plant-oomycete interactions
The plant defense and pathogen counterdefense mediated by Hevea brasiliensis serine protease HbSPA and Phytophthora palmivora extracellular protease inhibitor PpEPI10.
Rubber tree (Hevea brasiliensis Muell. Arg) is an important economic crop in Thailand. Leaf fall and black stripe diseases caused by the aggressive oomycete pathogen Phytophthora palmivora, cause deleterious damage on rubber tree growth leading to decrease of latex production. To gain insights into the molecular function of H. brasiliensis subtilisin-like serine proteases, the HbSPA, HbSPB, and HbSPC genes were transiently expressed in Nicotiana benthamiana via agroinfiltration. A functional protease encoded by HbSPA was successfully expressed in the apoplast of N. benthamiana leaves. Transient expression of HbSPA in N. benthamiana leaves enhanced resistance to P. palmivora, suggesting that HbSPA plays an important role in plant defense. P. palmivora Kazal-like extracellular protease inhibitor 10 (PpEPI10), an apoplastic effector, has been implicated in pathogenicity through the suppression of H. brasiliensis protease. Semi-quantitative RT-PCR revealed that the PpEPI10 gene was significantly up-regulated during colonization of rubber tree by P. palmivora. Concurrently, the HbSPA gene was highly expressed during infection. To investigate a possible interaction between HbSPA and PpEPI10, the recombinant PpEPI10 protein (rPpEPI10) was expressed in Escherichia coli and purified using affinity chromatography. In-gel zymogram and co-immunoprecipitation (co-IP) assays demonstrated that rPpEPI10 specifically inhibited and interacted with HbSPA. The targeting of HbSPA by PpEPI10 revealed a defense-counterdefense mechanism, which is mediated by plant protease and pathogen protease inhibitor, in H. brasiliensis-P. palmivora interactions
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Deep learningâbased quantification of arbuscular mycorrhizal fungi in plant roots
Summary: Soil fungi establish mutualistic interactions with the roots of most vascular land plants. Arbuscular mycorrhizal (AM) fungi are among the most extensively characterised mycobionts to date. Current approaches to quantifying the extent of root colonisation and the abundance of hyphal structures in mutant roots rely on staining and human scoring involving simple yet repetitive tasks which are prone to variation between experimenters. We developed Automatic Mycorrhiza Finder (AMFinder) which allows for automatic computer visionâbased identification and quantification of AM fungal colonisation and intraradical hyphal structures on inkâstained root images using convolutional neural networks. AMFinder delivered highâconfidence predictions on image datasets of roots of multiple plant hosts (Nicotiana benthamiana, Medicago truncatula, Lotus japonicus, Oryza sativa) and captured the altered colonisation in ram1â1, str, and smax1 mutants. A streamlined protocol for sample preparation and imaging allowed us to quantify mycobionts from the genera Rhizophagus, Claroideoglomus, Rhizoglomus and Funneliformis via flatbed scanning or digital microscopy, including dynamic increases in colonisation in whole root systems over time. AMFinder adapts to a wide array of experimental conditions. It enables accurate, reproducible analyses of plant root systems and will support better documentation of AM fungal colonisation analyses. AMFinder can be accessed at https://github.com/SchornacklabSLCU/amfinder
Receptor-like cytoplasmic kinases of different subfamilies differentially regulate SOBIR1/BAK1-mediated immune responses in Nicotiana benthamiana
Cell-surface receptors form the front line of plant immunity. The leucine-rich repeat (LRR)-receptor-like kinases SOBIR1 and BAK1 are required for the functionality of the tomato LRR-receptor-like protein Cf-4, which detects the secreted effector Avr4 of the pathogenic fungus Fulvia fulva. Here, we show that the kinase domains of SOBIR1 and BAK1 directly phosphorylate each other and that residues Thr522 and Tyr469 of the kinase domain of Nicotiana benthamiana SOBIR1 are required for its kinase activity and for interacting with signalling partners, respectively. By knocking out multiple genes belonging to different receptor-like cytoplasmic kinase (RLCK)-VII subfamilies in N. benthamiana:Cf-4, we show that members of RLCK-VII-6, -7, and -8 differentially regulate the Avr4/Cf-4-triggered biphasic burst of reactive oxygen species. In addition, members of RLCK-VII-7 play an essential role in resistance against the oomycete pathogen Phytophthora palmivora. Our study provides molecular evidence for the specific roles of RLCKs downstream of SOBIR1/BAK1-containing immune complexes
Time-resolved dual transcriptomics reveal early induced Nicotiana benthamiana root genes and conserved infection-promoting Phytophthora palmivora effectors
BACKGROUND: Plant-pathogenic oomycetes are responsible for economically important losses in crops worldwide. Phytophthora palmivora, a tropical relative of the potato late blight pathogen, causes rotting diseases in many tropical crops including papaya, cocoa, oil palm, black pepper, rubber, coconut, durian, mango, cassava and citrus. Transcriptomics have helped to identify repertoires of host-translocated microbial effector proteins which counteract defenses and reprogram the host in support of infection. As such, these studies have helped in understanding how pathogens cause diseases. Despite the importance of P. palmivora diseases, genetic resources to allow for disease resistance breeding and identification of microbial effectors are scarce. RESULTS: We employed the model plant Nicotiana benthamiana to study the P. palmivora root infections at the cellular and molecular levels. Time-resolved dual transcriptomics revealed different pathogen and host transcriptome dynamics. De novo assembly of P. palmivora transcriptome and semi-automated prediction and annotation of the secretome enabled robust identification of conserved infection-promoting effectors. We show that one of them, REX3, suppresses plant secretion processes. In a survey for early transcriptionally activated plant genes we identified a N. benthamiana gene specifically induced at infected root tips that encodes a peptide with danger-associated molecular features. CONCLUSIONS: These results constitute a major advance in our understanding of P. palmivora diseases and establish extensive resources for P. palmivora pathogenomics, effector-aided resistance breeding and the generation of induced resistance to Phytophthora root infections. Furthermore, our approach to find infection-relevant secreted genes is transferable to other pathogen-host interactions and not restricted to plants.This work was supported by the Gatsby Charitable Foundation (RG62472),
by the Royal Society (RG69135) and by the European Research Council
(ERC-2014-STG, H2020, 637537)
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Sticking to it: phytopathogen effector molecules may converge on evolutionarily conserved host targets in green plants.
âą Phytopathogen effectors converge on similar sets of host proteins in angiosperms. âą Effectors may target host proteins and processes present across the green plant lineage. âą Bryophyte model plants are promising systems to investigate effectorâtarget relationships. Plant-associated microbes secrete effector proteins that subvert host cellular machinery to facilitate the colonization of plant tissues and cells. Accumulating data suggests that independently evolved effectors from bacterial, fungal, and oomycete pathogens may converge on a similar set of host proteins in certain angiosperm models, however, whether this concept is relevant throughout the green plant lineage is unknown. Here, we explore the idea that pathogen effector molecules target host proteins present across evolutionarily distant land plant lineages to promote disease. We discuss that host proteins targeted by phytopathogens or integrated into angiosperm immune receptors are likely found across green plant genomes, from early diverging non-vascular lineages (bryophytes) to flowering plants (angiosperms). This would suggest that independently evolved pathogens might manipulate their hosts by targeting `vulnerabilityâ hubs that are present across land plants. Future work focusing on accessible early divergent land plant model systems may therefore provide an insightful evolutionary backdrop for effectorâtarget research
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N -acetyltransferase AAC(3)-I confers gentamicin resistance to Phytophthora palmivora and Phytophthora infestans
Abstract: Background: Oomycetes are pathogens of mammals, fish, insects and plants, and the potato late blight agent Phytophthora infestans and the oil palm and cocoa infecting pathogen Phytophthora palmivora cause economically impacting diseases on a wide range of crop plants. Increasing genomic and transcriptomic resources and recent advances in oomycete biology demand new strategies for genetic modification of oomycetes. Most oomycete transformation procedures rely on geneticin-based selection of transgenic strains. Results: We established N-acetyltransferase AAC(3)-I as a gentamicin-based selectable marker for oomycete transformation without interference with existing geneticin resistance. Strains carrying gentamicin resistance are fully infectious in plants. We further demonstrate the usefulness of this new antibiotic selection to super-transform well-characterized, already fluorescently-labelled P. palmivora strains and provide a comprehensive protocol for maintenance and zoospore electro-transformation of Phytophthora strains to aid in plant-pathogen research. Conclusions: N-acetyltransferase AAC(3)-I is functional in Phytophthora oomycetes. In addition, the substrate specificity of the AAC(3)-I enzyme allows for re-transformation of geneticin-resistant strains. Our findings and resources widen the possibilities to study oomycete cell biology and plant-oomycete interactions
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FIRE mimics a 14-3-3-binding motif to promote Phytophthora palmivora infection
The oomycete Phytophthora palmivora infects a wide range of tropical crops worldwide. Like other filamentous plant pathogens, it secretes effectors to colonise plant tissues. Here we characterise FIRE, an RXLR effector that contains a canonical mode I 14-3-3 phospho-sensor binding motif that is conserved in effectors of several Phytophthora species. FIRE is phosphorylated in planta and interacts with multiple 14-3-3 proteins. Binding is sensitive to the R18 14-3-3 inhibitor. FIRE promotes plant susceptibility and co-localises with its target around haustoria. This work uncovers a new type of oomycete effector target mechanism. It demonstrates that substrate mimicry for 14-3-3 proteins is a cross-kingdom effector strategy used by both prokaryotic and eukaryotic plant pathogens to suppress host immunity.Gatsby Charitable Foundation (GAT3395/GLD)
European Research Council (ERC-2014-STG, H2020 and 637537)
Royal Society (UF110073 and UF160413)
a SLCU - SBS DTP PhD Studentship supported by Gatsby (GAT3395/GLD) and the University of Cambridge (G108721)
Candidate effector proteins from the oomycetes Plasmopara viticola and Phytophthora parasitica share similar predicted structures and induce cell death in Nicotiana species.
Acknowledgements: We thank Eric Galiana at Institut Sophia Agobiotech for P. parasitica strain 329, Olivier Lamotte at INRAE Dijon for B. cinerea strain BMM, Muriel Viaud at INRAE Versailles for B. cinerea strain B05-10-GFP and JĂ©rĂŽme Mutterer at IBMP Strasbourg for assistance with confocal microscopy. We are grateful to the UEAV at INRAE Colmar for technical support in the production of plants.Funder: INRA BAP DepartmentFunder: Region AlsaceFunder: Gatsby Charitable Foundation; funder-id: http://dx.doi.org/10.13039/501100000324Effector proteins secreted by plant pathogens are essential for infection. Cytoplasmic RXLR effectors from oomycetes are characterized by the presence of RXLR and EER motifs that are frequently linked to WY- and/or LWY-domains, folds that are exclusive to this effector family. A related family of secreted candidate effector proteins, carrying WY-domains and the EER motif but lacking the canonical RXLR motif, has recently been described in oomycetes and is mainly found in downy mildew pathogens. Plasmopara viticola is an obligate biotrophic oomycete causing grapevine downy mildew. Here we describe a conserved Pl. viticola secreted candidate non-RXLR effector protein with cell death-inducing activity in Nicotiana species. A similar RXLR effector candidate from the broad host range oomycete pathogen Phytophthora parasitica also induces cell death in Nicotiana. Through comparative tertiary structure modelling, we reveal that both proteins are predicted to carry WY- and LWY-domains. Our work supports the presence of LWY-domains in non-RXLR effectors and suggests that effector candidates with similar domain architecture may exert similar activities
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