Identification, Molecular Cloning And Characterization Of Pathogenicity-Related Genes In Magnaporthe Oryzae Genome

Magnaporthe oryzae, the causal agent of the devastating rice blast disease, is a grave threat to global rice production, reported from about85countries worldwide causing70-80%of rice yield losses during an epidemic season. M. oryzae, an ascomycete phytopathogenic fungus with both scientific and economical value, is also an important model fungus for molecular-based studies of fungal-caused diseases and plant-microbe interaction. Rice is one of the most important food security crops providing23%calories to mankind. Both M. oryzae and rice are genetically tractable and their genome sequence information is available.M. oryzae causes infection through a specialized dome-shaped appressorium penetrating the host tissue through a penetration peg by using turgor pressure derived from lipid and glycerol degradation. Studies have shown that appressorium-mediated penetration events are controlled by highly conserved several signaling networks （G protein coupled receptors （GPCRs）, autophagy, mitogen-activated protein kinase （MAPK）, cyclic adenosyl monophosphate （cAMP） and Ca+-mediated signaling） and these signalings are regulated by genes. The disruption of such signaling genes results in the loss and/or reduction of pathogenicity as well as pleiotrophic effects on cellular processes including mating, conidiation and growth rate. The components of these signalings may represent targets for the development of antifungal drugs. Although several genes regulating these signalings have been characterized, further research is needed to get a better understanding of the biology and signaling-regulated genes to effectively control this fatal disease.Pathogenicity-related genes are of great interest not only to increase our understanding of infection process, but also because any such gene could become a target for disease control. Keeping in view the worldwide importance of this blast disease, we have conducted our research on the molecular characterization of several pathogenicity-related genes including FIS1（MGG₀6075）, YPL1（MGG₁4620）, NUC1（MGG₀5324）, AIF1（MGG₀8262）, ZAP I （MGG₀4456）, STM1（MGG₀5307）, CAPN1（MGG₁4872）, CAPN3（MGG₀8067） and CAPN4（MGG₀4818） based on their homology proteins functions and significance in other organisms. To find the role of these genes in blast disease, we firstly identified these genes using bioinformatic analysis and then disrupted through gene disruption method by cloning the targeted gene into the pBS vector containing reporter gene （HPH//SUR/BAR/NEO） harboring resistance to the growth medium-specific antibiotic. The recombinant fragment was released from the pBS vector by restriction digestion using appropriate enzymes and subcloned into a donor binary pCAMBIA-1300vector. The pCAMBIA-1300vector after confirmed （PCR and restriction digestion） for the existence of a single fragment （up fragment-reporter gene-dn fragment） was then transformed into the wild-type strain Guy-11conidia through ATMT （AGL-1） transformation and finally mutants were obtained through homologous recombination. The mutants were first screened on selective medium （CM/DCM） containing reporter gene specific antibiotic and then confirmed by PCR for deletion event. The mutants after mono-conidial isolation were finally confirmed by southern blotting.The mutants for all the deleted genes were functionally analyzed for the rice blast-related phenotypes such as mycelium radial growth on CM medium （9dpi）, conidiation （9dpi）, conidial germination （2and4hpi）, appressorium formation （6hpi）, appressorium turgor pressure （glycerol）, nucleus condensation （DAPI）, apoptotic cell death （farnesol）, cell wall integrity （congo red） test, stress response under chemical pressure （ZnSO4, CuSO4and CdCl2）, wettable phenotype/surface hydrophobicity （distilled water and0.2%gelatin）, osmotic and oxidative （NaCl and H2O2） stress response, pathogenicity test （rice and barley） and mating test （2539strain） and all these phenotypes were compared with those of the wild-type pathogenic Guy-11strain.Among all, only the ΔMoFISl mutants displayed different phenotypes in contrast to those of the Guy-11strain. To confirm that these phenotypic differences were due to the MoFIS1deletion, we reintroduced the parent MoFISl gene into the ΔMoFIS1mutant allele through ATMT transformation. The resulting rescued transformants were screened by phenotype characterization and PCR amplification. For subcellular localization of the MoFIS1inside the hyphal and conidial cell, the full coding sequence of the targeted gene tagged with GFP was transformed into the mutant AMoFIS1conidia using ATMT （AGL-1） transformation. Conidia and hyphae of the strains expressing GFP were observed under a laser scanning confocal microscope at40X magnification. The AMoFISl deleted mutants showed significantly reduced conidiation compared to that of the Guy-11and rescued strain, showing its involvement in conidiation. The ΔMoFISl mutants also showed reduced mycelium radial growth on CM medium at9dpi compared to that in the Guy-11and rescued strain. Disease density and severity in the ΔMoFISl mutants on blast susceptible rice and barley leaves were significantly reduced showing small, reduced and non-proliferating blast lesions compared to necrotic, coalesced and more proliferating lesions in the Guy-11and rescued strain which obviously suggested that the gene was required for full pathogenicity. Conidial germination （2and4hpi） and appressorium formation （6hpi） of the ΔMoFIS1mutants were not significantly different from those of the Guy-11and rescued strain. The mutants showed less stress tolerance generated by NaCl, H2O2and several chemicals. Mutants showed reduced growth on MM, MM-C and MM-N media at8days post-inoculation. In cell wall integrity test, the mutants although showed reduced growth but halo degradation was similar to that in the Guy-11strain. It was also investigated that MoFIS1gene did not play any role in surface hydrophobicity. In mating experiment, the mutants formed numerous perithecia at the junctions with opposite mating2539strain showing that MoFIS1gene is not required for sexual reproduction in M. oryzae. The MoFIS1-GFP expressing conidia and hyphae displayed bright fluorescence under a laser confocal microscope indicating the MoFIS1localization in the conidia and hypha cells mitochondria.However, mutants deleted for the genes MoYPL1, MoNUC1, MoAIF1, MoZAP1, MoSTM1, MoCAPN1, MoCAPN3and MoCAPN4showed normal phenotypes such as full pathogenicity, normal culture colony growth, un-altered conidiation, conidial germination, appressorium formation and mating with2539strain were almost similar to those of the Guy-11indicating that these genes are not involved in rice blast disease. However, reduction in conidiation can be observed in the mutants deleted for the MoYPLl, MoNUCl and MoCAPN1genes with non-significant impact on pathogenicity.We conclude that MoFIS1, an important pathogenicity-involved gene may encode an important protein that is required for culture growth, conidiation and full virulence in M. oryzae, however, genes including MoYPL1, MoNUC1, MoAIF1, MoZAP1, MoSTM1, MoCAPNl, MoCAPN3and MoCAPN4are not essential for the blast fungus to cause pathogenicity. Further researches at molecular basis that are necessary for understanding the molecular machinery of this fungal pathogen will result in more durable control strategies of this disease.