Functional Analyses Of Magnaporthe Orvzae Zn₂Cys₆and BHLH Transcription Factors

Rice blast, caused by Magnaporthe oryzae, is one of the destructive rice diseases which threaten global cultured crop production. Its representative life cycle and infection process make it a suitable model of filamentous fungal pathogenesis and molecular biology research, as well as a model for host-fungal pathogen interactions. Further research on the mechanism of pathogenic process provides insights into the prevention of this pathogen. As the genomic sequences of both rice and M. oryzae provided substantial genome information, how to elucidate the gene function and their regulation network form the masses of DNA information becomes the trend of post-genome era. Transcription factors (TFs) are the major regulative pathway to control gene expression and research on the biological function and regulation network of TFs has significance in understanding the development and pathogenesis process of M. oryzae.Zn2Cys6transcription factor family is a fungal-specific and the largest TF family in the rice blast fungus. We identified163Zn2Cys6TF genes and developed a high-throughput gene knockout system to delete104genes of them in M. oryzae. Then we analyzed the phenotypes of these mutants with regard to mycelial growth, conidiogenesis, infection-related development, pathogenicity and9abiotic stresses. The resulting data showed25genes were involved in conidiation and three mutants of them (â–³gccl,â–³Mocodl andâ–³conx1) almost lost the ability to produce conidium. MoCODl null mutant lost pathogenicity on both barley and rice leaves.â–³ccal produced few multifarious and vacuolated conidia, which germinated and formed not fully melanized appressoria at a low ratio and the appressoria could not penetrate the host, but the mycelia could develop normal appressoria. GCC1, GPF1and GTA1were indispensable for the growth of mycelia, and GPF1, GTA1, CNF2, CNF1, CCA1, MoCOD1and CONx1were involved in pathogenicity.bHLH TFs are highly conserved proteins in eukaryotic organisms and9bHLH TF genes (named MoHLH1-MoHLH9respectively) were identified in M. oryzae genome. We had constructed a gene knockout mutant sets and characterized the function of the8null mutants (expect AMohlh4) and analyzed the regulatory pathway in pathogenicity of AMohlh6. Observation of the RFP-tagged PTS1and GFP-tagged PTS2inâ–³Mohlh6revealed the number of peroxisomes decreased in the conidia. The deletion of MoHLH6consequently impaired the lipid droplets mobilization and degradation, and could not supply enough appressorial turgor, which is the dominant factor for the fungus to penetrate the host cuticle. The Y2H assays showed that Mohlh6interacted with Osml, CpkA, Mocnb1and Mocmkk2, so Mohlh6may act as a downstream TF of many decisive signaling pathways to regulate the development of M. oryzae. Moreover, after combining the RNA-seq data in this study and the binding motif data of bHLH domain in previous reports, a number of Mohlh6-regulated genes were identified including several novel ones that were involved in pathogenicity or the related process.In a summary, we systematically identified and characterized the Zn2Cys6and bHLH TF genes in M. oryzae, and found lots of TF genes play key roles in mycelial growth, conidiation, appressorial development and pathogenicity. We explored the possible mechanism of MoHLH6functioned as a virulence determinant, and analyzed the upstream kinases and downstream regulated genes of Mohlh6to illustrate the regulatory network of transcription factors.