| The rice blast fungus (Magnaporthe grisea Barr.) is important both economically and in the study of plant-microbe interaction. Dissection of gene functions at the whole genome level will speed up understanding of the fungal biology and molecular mechanisms regulating the fungal pathogenicity and facilitate the rice breeding for durable resistance and the disease integrated management. Therefore, based on the T-DNA insertional mutagenesis of M, grisea in our lab, we screened for mutants with stable mutated phenotype, and further conducted molecular characterization to confirm the functions of the corresponding genes.T16B-2 is a sporulation-defect mutant. Comparing with the wild type, the mutant produced fewer conidia and formed appressorium, but the ability of incursion on the onion epidermis and the pathogenicity on rice were normal. Southern blot analysis with hph gene insertion showed that the mutant had single-copy insertion. Mating with GUY11, a sexual Fl progenies of the mutant was constructed. Among the progenies, the two traits of hygromycin-resistance and sporulation-defect had a 1: 1 cosegregation. So the phenotype of sporulation maybe be controlled by a single gene. The T-DNA flaking sequnence was amplified by TAIL-PCR, and the T-DNA region inserted in the promoter region of ConLW. Funtional complement analysis of the T-DNA mutant revealed that the complement transformant's sporulation could come back to the level of the wild type.To confirm whether ConLW is a gene regulating sporulation, it was knocked out in the wild type FJ95054B by homologous recombination with hph gene. The KO mutant was defect both in sporulation and in pathogenicity. The defect of mutant's sporulation and pathogenicity recovered by complemented with its native gene. Analysis by using Pfam program indicated that there was a Zn(Ⅱ)2Cys6 domain in ConLW. The Zn(Ⅱ)2Cys6 domain of ConLW could rescue the defect of pathogenicity, but not of sporulation of KO mutants. These data suggest that ConLW is a gene regulating both sporulation and pathogenicity, its Zn(Ⅱ)2Cys6 domain is specific to modulate the pathogenity but not sporulation.Another T-DNA mutant Pi9-1-1 T94A showed pathogenicity-enhanced on the rice with the rice blast resistant gene Pi-9．The T-DNA flaking sequnence was amplified by TAIL-PCR, and the T-DNA region inserted in the region between PthLW and its neighbor gene. PthLW was knocked out in the wild type GUY11 by homologous recombination with hph gene. The KO mutant showed a pathogenicity-enhanced on the rice with the rice blast resistant gene Pi-b (IRBL14) but resistant on Pi-9．The KO mutant and the virulent field isolates showed avirulent on IRBL14 when being complemented with native promoter and ORF regions of PthLW. However their pathogenicity didn't change on other rice varieties. So we conferred that PthLW is a candidate gene of Avr-pib, but not Avr-pi9．Since the bioinformatics analysis suggested that PthLW encodes a putative ATP-NAD kinase, we further tested its ability to use unsaturate fatty acids as the sole carbon source. Results showed the PthLW mutants could not utilize unsaturate fatty acids as carbon source to grow. This preliminarily confirmed that PthLW is a gene encoding ATP-NAD kinase.In summary, functions of the corresponding genes, ConLW and PthLW of two T-DNA mutants were confirmed by gene knock-out and functional complementation. This will provide with a basis of functional analysis of related genes in regulating the sporulation and pathogenecity of M.grisea.
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