Project description:Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. Due to particular physiological characteristics, no treatments against diseases caused by oomycetes are presently available. To develop such treatments, it appears essential to dissect the molecular mechanisms that determine the interaction between Phytophthora species and host plants. The present project is focused on the molecular mechanisms that underlie the compatible plant-oomycete interaction and plant disease. The laboratory developed a novel interaction system involving the model plant, Arabidopsis thaliana, and Phytophthora parasitica, a soil-borne pathogen infecting a wide host range, thus representing the majority of Phytophthora species. A characteristic feature of the compatible Arabidopsis/P. parasitica interaction is an extended biotrophic phase, before infection becomes necrotrophic. Because the initial biotrophic phase is extremely short on natural (e.g. solanaceous) hosts, the Arabidopsis system provides the opportunity to analyze, for both interaction partners, the molecular events that determine the initiation of infection and the switch to necrotrophy. The present project aims at analyzing the compatible interaction between A. thaliana roots and P. parasitica. The Affymetrix A. thaliana full genome chip will be used to characterize modulations of the transcriptome occurring over a period of 24h from the onset of plant root infection to the beginning of necrotrophy. Parallel to this study, a custom-designed P. parasitica biochip will enable analyzing of P. parasitica gene expression during the same stages. 10 samples were used in this experiment.
Project description:N6-methyladenosine (m6A) modification, as an important post-transcriptional regulator, is the most prevalent RNA modification in eukaryotes. However, the biological role of m6A in oomycetes remains unclear. Here, we characterized three m6A methyltransferases PsMETTL3A, PsMETTL3B, and PsMETTL16 that mainly affected the m6A levels in the CDS and 3’UTR in Phytophthora sojae. They play an important role in production of sporangia and oospores, mycelial growth, and virulence. PsMETTL3A/3B/16-mediated m6A positively regulated mRNA stability and translation efficiency of genes involved in chromatin remodeling and epigenetic regulation. Interestingly, PsMETTL3A, PsMETTL3B, or PsMETTL16 function in “independent” and “overlap” synergetic pattern. PsMETTL16 could interact with PsMETTL3A and PsMETTL3B to mediate m6A on some of the transcripts. Moreover, PsMETTL3A and PsMETTL3B positively regulated the expression of PsMETTL16 in an m6A-dependent manner. More importantly, m6A-modified PsBdf1 regulate chromatin accessibility to maintain DNA damage repair and tolerate host ROS stress in a PsMETTL3A/3B/16-dependent manner. Overall, these results indicate that m6A-mediated RNA metabolism are associated with development and pathogenicity of P. sojae, highlighting epigenetic variation as a mechanism of plant pathogenic oomycetes adaptive plasticity.
Project description:Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. Due to particular physiological characteristics, no treatments against diseases caused by oomycetes are presently available. To develop such treatments, it appears essential to dissect the molecular mechanisms that determine the interaction between Phytophthora species and host plants. The present project is focused on the molecular mechanisms that underlie the compatible plant-oomycete interaction and plant disease. The laboratory developed a novel interaction system involving the model plant, Arabidopsis thaliana, and Phytophthora parasitica, a soil-borne pathogen infecting a wide host range, thus representing the majority of Phytophthora species. A characteristic feature of the compatible Arabidopsis/P. parasitica interaction is an extended biotrophic phase, before infection becomes necrotrophic. Because the initial biotrophic phase is extremely short on natural (e.g. solanaceous) hosts, the Arabidopsis system provides the opportunity to analyze, for both interaction partners, the molecular events that determine the initiation of infection and the switch to necrotrophy. The present project aims at analyzing the compatible interaction between A. thaliana roots and P. parasitica. The Affymetrix A. thaliana full genome chip will be used to characterize modulations of the transcriptome occurring over a period of 24h from the onset of plant root infection to the beginning of necrotrophy. Parallel to this study, a custom-designed P. parasitica biochip will enable analyzing of P. parasitica gene expression during the same stages.
Project description:Background: The oomycete Phytophthora infestans possesses active RNA silencing pathways, which presumably enable this plant pathogen to control the large numbers of transposable elements present in its 240 Mb genome. Small RNAs (sRNAs), central molecules in RNA silencing, are known to also play key roles in this organism, notably in regulation of critical effector genes needed for infection of its potato host. Results: In order to identify additional classes of sRNAs in oomycetes, we mapped deep sequencing reads to transfer RNAs (tRNAs) thereby revealing the presence of 19-40 nt tRNA-derived RNA fragments (tRFs). Northern blot analysis identified abundant tRFs corresponding to half tRNA molecules. Some tRFs accumulated differentially during infection, as seen by examining sRNAs sequenced from P. infestans-potato interaction libraries. The putative connection between tRF biogenesis and the canonical RNA silencing pathways was investigated by employing hairpin RNA-mediated RNAi to silence the genes encoding P. infestans Argonaute (PiAgo) and Dicer (PiDcl) endoribonucleases. By sRNA sequencing we show that tRF accumulation is PiDcl1-independent, while Northern hybridizations detected reduced levels of specific tRNA-derived species in the PiAgo1 knockdown line. Conclusions: Our findings extend the sRNA diversity in oomycetes to include fragments derived from non-protein-coding RNA transcripts and identify tRFs whose levels are elevated during infection of P. infestans on potato. Small RNA sequence data from two Phytophthora infestans isolates. Four life-cycle stages from each isolate. No replicates. Total number of samples: 8.
Project description:Plant pathogens secreted effector proteins to overcome host immunity system and gain access to colonization. Although more than 300 hundred of RxLR effectors were encoded by the devastating soybean pathogen Phytophthora sojae, the patterns of conditionally expressed effector genes were well programmed. However, the effector gene regulation mechanisms were not fully dissected. In this study, we identified the H3K36me3 methyltransferase PsKMT3. Phenotypic assays showed PsKMT3 was involved in regulation of growth, sporulation, zoospore production and pathogenicity. ChIP-seq analysis, together with RNA-seq analysis, showed PsKMT3 controlled a set of RxLR gene expression. The transcriptome comparison showed large-scale mis-expression of RxLR waves during mycelium, 3 hpi and 6 hpi stages between WT and pskmt3. Our result supports a new RxLR gene regulation mechanisms in which methyltransferase PsKMT3 maintains well programmed RxLR gene expression in P. sojae.
Project description:Plant pathogens secreted effector proteins to overcome host immunity system and gain access to colonization. Although more than 300 hundred of RxLR effectors were encoded by the devastating soybean pathogen Phytophthora sojae, the patterns of conditionally expressed effector genes were well programmed. However, the effector gene regulation mechanisms were not fully dissected. In this study, we identified the H3K36me3 methyltransferase PsKMT3. Phenotypic assays showed PsKMT3 was involved in regulation of growth, sporulation, zoospore production and pathogenicity. ChIP-seq analysis, together with RNA-seq analysis, showed PsKMT3 controlled a set of RxLR gene expression. The transcriptome comparison showed large-scale mis-expression of RxLR waves during mycelium, 3 hpi and 6 hpi stages between WT and pskmt3. Our result supports a new RxLR gene regulation mechanisms in which methyltransferase PsKMT3 maintains well programmed RxLR gene expression in P. sojae.
Project description:Background: The oomycete Phytophthora infestans possesses active RNA silencing pathways, which presumably enable this plant pathogen to control the large numbers of transposable elements present in its 240 Mb genome. Small RNAs (sRNAs), central molecules in RNA silencing, are known to also play key roles in this organism, notably in regulation of critical effector genes needed for infection of its potato host. Results: To identify additional classes of sRNAs in oomycetes, we mapped deep sequencing reads to transfer RNAs (tRNAs) thereby revealing the presence of 19-40 nt tRNA-derived RNA fragments (tRFs). Northern blot analysis identified abundant tRFs corresponding to half tRNA molecules. Some tRFs accumulated differentially during infection, as seen by examining sRNAs sequenced from P. infestans-potato interaction libraries. The putative connection between tRF biogenesis and the canonical RNA silencing pathways was investigated by employing hairpin RNA-mediated RNAi to silence the genes encoding P. infestans Argonaute (PiAgo) and Dicer (PiDcl) endoribonucleases. By sRNA sequencing we show that tRF accumulation is PiDcl1-independent, while Northern hybridizations detected reduced levels of specific tRNA-derived species in the PiAgo1 knockdown line. Conclusions: Our findings extend the sRNA diversity in oomycetes to include fragments derived from non-protein-coding RNA transcripts and identify tRFs with elevated levels during infection of potato by P. infestans.
Project description:Background: The oomycete Phytophthora infestans possesses active RNA silencing pathways, which presumably enable this plant pathogen to control the large numbers of transposable elements present in its 240 Mb genome. Small RNAs (sRNAs), central molecules in RNA silencing, are known to also play key roles in this organism, notably in regulation of critical effector genes needed for infection of its potato host. Results: In order to identify additional classes of sRNAs in oomycetes, we mapped deep sequencing reads to transfer RNAs (tRNAs) thereby revealing the presence of 19-40 nt tRNA-derived RNA fragments (tRFs). Northern blot analysis identified abundant tRFs corresponding to half tRNA molecules. Some tRFs accumulated differentially during infection, as seen by examining sRNAs sequenced from P. infestans-potato interaction libraries. The putative connection between tRF biogenesis and the canonical RNA silencing pathways was investigated by employing hairpin RNA-mediated RNAi to silence the genes encoding P. infestans Argonaute (PiAgo) and Dicer (PiDcl) endoribonucleases. By sRNA sequencing we show that tRF accumulation is PiDcl1-independent, while Northern hybridizations detected reduced levels of specific tRNA-derived species in the PiAgo1 knockdown line. Conclusions: Our findings extend the sRNA diversity in oomycetes to include fragments derived from non-protein-coding RNA transcripts and identify tRFs whose levels are elevated during infection of P. infestans on potato.
Project description:Background: The oomycete Phytophthora infestans possesses active RNA silencing pathways, which presumably enable this plant pathogen to control the large numbers of transposable elements present in its 240 Mb genome. Small RNAs (sRNAs), central molecules in RNA silencing, are known to also play key roles in this organism, notably in regulation of critical effector genes needed for infection of its potato host. Results: To identify additional classes of sRNAs in oomycetes, we mapped deep sequencing reads to transfer RNAs (tRNAs) thereby revealing the presence of 19-40 nt tRNA-derived RNA fragments (tRFs). Northern blot analysis identified abundant tRFs corresponding to half tRNA molecules. Some tRFs accumulated differentially during infection, as seen by examining sRNAs sequenced from P. infestans-potato interaction libraries. The putative connection between tRF biogenesis and the canonical RNA silencing pathways was investigated by employing hairpin RNA-mediated RNAi to silence the genes encoding P. infestans Argonaute (PiAgo) and Dicer (PiDcl) endoribonucleases. By sRNA sequencing we show that tRF accumulation is PiDcl1-independent, while Northern hybridizations detected reduced levels of specific tRNA-derived species in the PiAgo1 knockdown line. Conclusions: Our findings extend the sRNA diversity in oomycetes to include fragments derived from non-protein-coding RNA transcripts and identify tRFs with elevated levels during infection of potato by P. infestans. Small RNA sequence data from Phytophthora infestans-infected potato leaf tissue and P. infestans mycelium tissue. Three infection stage time-points. Two P. infestans lines: 88089 (wild-type) and PiDcl1 (transformant PiDcl1 knock-down). No replicates. Total number of samples: 8.