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Differential Expression Analysis of a Histidine phosphotransferase RNAi mutant in Magnaporthe oryzae

ABSTRACT: Magnaporthe oryzae is notorious for its efficient evolution as a plant pathogen. Analysis of well organised fungal pathogenic mechanisms would enable the scientific understanding to fight against pathogens. Histidine phosphotransferase in fungal systems are good targets for antifungals since they are component of SAPK pathway, which is essential for survival of the fungus. The histidine Phosphotransferase YPD1 in M. oryzae is known to be light responsive and important for both asexual reproduction as well as pathogenicity. In this study, the knock-down (KD) mutant of YPD1 was highly sensitive to cell wall perturbing agents and were highly deficient in laccase expression and activity. A RNA-seq transcriptome screening of KD mutant compared to wild type revealed altered expression of about 146 genes important in biotrophic and necrotrophic phases of the pathogen. About 96 genes were found to be down regulated, of which several plant cell wall degrading genes vital for pathogenicity and fungal cell wall maintenance genes important for growth and fungal development were identified. Most of the genes identified are involved in biotrophic invasion and metabolism. Our results explain the reason for the deficiency of the KD mutant in asexual reproduction as well as pathogenicity. Analysis of differentially expressed genes in the KD YPD1 mutant suggests that YPD1 can be a good target for development of novel antifungal compounds, as YPD1 expression affects genes required for pathogenicity.

ORGANISM(S): Pyricularia oryzae

PROVIDER: GSE192934 | GEO | 2022/02/09

REPOSITORIES: GEO

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Project description:The hemibiotrophic fungus Magnaporthe oryzae produces specialized biotrophic invasive hyphae (IH) that alter membrane structure and defense responses in invaded rice cells. IH successively invade live neighbor cells, apparently through plasmodesmata. Understanding fungal and rice genes that contribute to biotrophic invasion has been a challenge because so few plant cells have encountered IH at the earliest infection stages. Using a rice sheath inoculation method, we successfully enriched for infected tissue RNA that contained ~20% fungal RNA at a point when most IH were still growing in first-invaded rice cells. The RNAs were analyzed using the whole-genome M. oryzae oligoarray and a rice oligoarray. Rice genes that were induced >50-fold during infection were enriched for genes involved in transferring information from sensors to cellular responses. Fungal genes that were induced >50-fold in IH included the PWL2 avirulence gene and genes encoding hypothetical secreted proteins. The IH-specific secreted proteins are candidate effectors, proteins that the fungus secretes into live host cells to control cellular processes. Gene knock-out analyses of three putative effector genes failed to show major effects on pathogenicity. Details of the blast interaction transcriptome will provide insights on the mechanisms of biotrophic plant disease. Experiment Overall Design: Our goal was to compare expression in infected rice sheath to expression in mock inoculated rice, and to compare expression in biotrophic IH to expression in mycelium grown in nutrient medium. Using the rice microarray (Agilent Technologies), we analyzed samples from three biological replicates of rice leaf sheath at 36 hours after inoculation with the rice blast fungus M. oryzae. The same samples were used with the Agilent M. oryzae microarray (see series GSE8517). To estimate the ratio of fungal to rice RNAs in the infected sheaths, we compared RT-PCR amplification of fungal genes in infected tissue to amplification in standards produced by mixing known ratios of pure mycelial RNA with mock-inoculated rice RNA. Using this assay, fungal RNA content in infected tissues were approximately 20% of the total RNAs. We prepared control mixtures by pooling 20% mycelial RNA and 80% RNA from mock-inoculated rice sheath. Complementary RNAs from infected tissues were labeled with Cy3 or Cy5 and hybridized together with the control mixture RNA labeled with the other dye. Three independent biological replications were performed, with separate hybridizations for 2 technical replications and corresponding dye swap experiments. Data were analyzed by Rosetta Resolver® and showed a correlation of over 80% between biological replications.

Project description:The hemibiotrophic fungus Magnaporthe oryzae produces specialized biotrophic invasive hyphae (IH) that alter membrane structure and defense responses in invaded rice cells. IH successively invade live neighbor cells, apparently through plasmodesmata. Understanding fungal and rice genes that contribute to biotrophic invasion has been a challenge because so few plant cells have encountered IH at the earliest infection stages. Using a rice sheath inoculation method, we successfully enriched for infected tissue RNA that contained ~20% fungal RNA at a point when most IH were still growing in first-invaded rice cells. The RNAs were analyzed using the whole-genome M. oryzae oligoarray and a rice oligoarray. Rice genes that were induced >50-fold during infection were enriched for genes involved in transferring information from sensors to cellular responses. Fungal genes that were induced >50-fold in IH included the PWL2 avirulence gene and genes encoding hypothetical secreted proteins. The IH-specific secreted proteins are candidate effectors, proteins that the fungus secretes into live host cells to control cellular processes. Gene knock-out analyses of three putative effector genes failed to show major effects on pathogenicity. Details of the blast interaction transcriptome will provide insights on the mechanisms of biotrophic plant disease. Keywords: Disease state analysis Our goal was to compare expression in biotrophic IH to expression in mycelium grown in nutrient medium, and to compare expression in infected rice sheath to expression in mock inoculated rice. Using version 2 of the fungal whole genome microarray (Agilent Technologies), we analyzed samples from three biological replicates of rice leaf sheath at 36 hours after inoculation with the rice blast fungus M. oryzae. The same samples were used with the Agilent rice microarray (see series GSE8518). To estimate the ratio of fungal to rice RNAs in the infected sheaths, we compared RT-PCR amplification of fungal genes in infected tissue to amplification in standards produced by mixing known ratios of pure mycelial RNA with mock-inoculated rice RNA. Using this assay, fungal RNA content in infected tissues were approximately 20% of the total RNAs. We prepared control mixtures by pooling 20% mycelial RNA and 80% RNA from mock-inoculated rice sheath. Complementary RNAs from infected tissues were labeled with Cy3 or Cy5 and hybridized together with the control mixture RNA labeled with the other dye. Three independent biological replications were performed, with separate hybridizations for 2 technical replications and corresponding dye swap experiments. Data were analyzed by Rosetta Resolver® and showed a correlation of over 80% between biological replications.

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Differential Expression Analysis of a Histidine phosphotransferase RNAi mutant in Magnaporthe oryzae

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