| The filamentous ascomycete fungus Magnaporthe oryzae is the causal agent of rice blast disease, one of the most devastating diseases of rice cultivation. Owing to its economic and social significance, M. oryzae, which is easily grown in culture and is genetically tractable, has emerged as a model organism for studying fungal biology and pathogenicity. Here, we report the identification of key enzymes in the ergosterol biosynthesis pathway and the transcriptional regulators downstream of the cAMP-PKA signaling pathway and their important roles on plant infection-related morphogenesis and pathogenicity in the rice blast fungus.1. MoCYP51A is required for conidiation, virulence and for mediating sensitivity to sterol demethylation inhibitors in M. oryzaeIn fungi, ergosterol regulates cell membrane fluidity and permeability and is essential for cell survival. Sterol14-α demethylase is one of the key enzymes in the ergosterol biosynthesis pathway. The M. oryzae genome contains two homologous Saccharomyces cerevisiae CYP51(ERG11) genes, MoCYP51A and MoCYP51B that putatively encoding14-a sterol demethylase. Targeted gene deletion mutants of MoCYP51A were morphologically indistinguishable from the isogenic wild type M. oryzae strain Guy11on CM culture, but were impaired in both conidiation and virulence. Deletion of MoCYP51B did not result in any obvious phenotypic changes compared with Guyll. The Amocyp51A mutants were highly sensitive to sterol demethylation inhibitor (DMI) fungicides, while Amocyp51B mutants were unchanged in their sensitivity to these fungicides. Analysis of intracellular localization of MoCyp51A showed that MoCyp51A was mainly localized to the cytoplasm of hyphae and conidia. The fusion protein MoCyp51A-GFP mainly localizes in vacuoles under the inducement of fungicide, which is consistent with the result of over-expression of MoCYP51A-GFP by an enhanced promoter. Taken together, the results indicate that MoCYP51A is required for efficient conidiogenesis, full virulence and for mediating DMI sensibility by the rice blast fungus.In addition, the M. oryzae genome contains three homologous genes of S. cerevisiae ERG6(encoding24-C-methytransferase),24C-SMTA,24C-SMTB and24C-SMTC. By deleting each single gene of the ERG6homologs, however, no obvious phenotypes in fungal morphogenesis and pathogenicity were observed from the mutants. GFP tagging experiments showed that the fusion protein24C-SmtA-GFP expressed at a high level during fungal development, and the fluorescence was mainly observed in vacuole and membranes.2. MoSOMland MoCDTFl are two novel pathogenicity-related genes identified by T-DNA insertional mutagenesis in M. oryzae.To explore the unknown pathogenciy related genes and understand the interaction mechanism between M. oryzae and the host plant, a large T-DNA insertional mutagenesis library was constructed, and among them, Five transformants mutants were identified as mutants, which were incapable of causing disease. By amplifying the genomic DNAs flanking the borders of the integrated T-DNA, the patterns of T-DNA integrated into these mutants were determined successfully. Among the T-DNA tagged genes, MoSOMl and MoCDTFl were identified as two novel pathogenicity-related genes in M. oryzae. The results of gene replacement and gene complement assays showed that both MoSOMl and MoCDTF1are necessary for the development of spores and appressoria by M. oryzae and play roles in cell wall differentiation, regulating melanin pigmentation and cell surface hydrophobicity during spore formation. The Amosoml and Amocdtfl mutants were unable to form conidia, perithecia and were also unable to form appressoria from mycelium. They also lost the ability to infect the leaves of barley and rice. Furthermore, the Amosoml mutants performed more significant reduction in vegetative growth, melanin pigmentation and hydrophobicity compared with the Amocdtfl mutants.3. MoSoml strongly interacts with MoStul, an APSES transcription factor, and with MoCdtfl, and functions downstream of the cAMP-PKA signaling pathway to regulate cellular differentiation and pathogenicity.MoSom1strongly interacts with MoStu1(Mstu1) and with MoCdtf1in yeast two hybrid experiments. In S. cerevisiae, Flo8is a transcription factor directly interacts with the catalytic subunit Tpk2(CpkA), and functions downstream of the cAMP-PKA signaling pathway. Interestingly, a weak interaction between MoSoml and CpkA was also detected by the addition of exogenous cAMP. MoSoml showed low amino acid identity with S. cerevisiae Flo8,but the MoSOMl could overcome the phenotypic defects in haploid invasive growth and diploid pseudohyphal development of S. cerevisiae flo8. Moreover, qRT-PCR analysis showed that the expression of MoSOMl and MoCDTFl were significantly reduced in Amac1and AcpkA mutants. These results indicate that MoSoml interacts with the transcription factors, MoStul and MoCdtfl, and acts downstream of the cAMP-PKA pathway in M. oryzae.4. The domains of MoSoml and MoCdtf1are essential for protein function and FgSOM1of Fusarium graminearum can complement the defects of Δmosoml mutants.MoSoml-GFP and MoCdtfl-GFP fusion proteins localized to the nucleus of fungal cells. The experiments of site-directed mutagenesis showed that nuclear localization signals of these two proteins are essential for their subcellular localization and biological function. Like S. cerevisiae Flo8, MoSoml has a LisH motif, while MoCdtfl has a ZnF_C2H2domain. The results of site-directed mutagenesis suggested that these domains are required for the function and localization of the two proteins. Furthermore, transcriptional profiling revealed major changes in gene expression associated with loss of MoSOM1during infection-related development, such as MPG1, COS1, MoRIC8, MAC1and CPKA. We also found that the phenotypes of Amosoml mutants could be restored when introduced with FgSOM1of Fusarium graminearum, indicating that MoSoml homologs might be functionally conservative in plant pathogenic fungi. |
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