Functional Analysis Of Some Serine Proteases In Magnaporthe Grisea

Serine protease,which exists widely in viruses, bacteria,and eukaryotes,has proteolytic activity.The serine proteases secreted during the rice blast fungus infection are refered to as latent pathogenicity factor. To further analysis the funtion of serine protease during the rice blast fungus growth and infection, the gene function of some serine proteases in this fungus were investigated in this paper.According to bioinformatics prediction, MGSP2(MGG₀2863.6), highly homologous with a known serine protease --spm1, has a relatively high protein secretory activity. Knockout mutantΔkosp2 and overexpressed mutantΔ863oe have been generated by transforming Knockout and overexpression vector to the protoplast of wide type strain KU70. Phenotype analysis of mutants was performed to observe their phenotypic changes, including colony morphology, sporulation, pathogenicity. The result showed that the knockout mutantΔkosp2 exibited no apparent difference with wide type in growth rate, mycelial growth, sporulation, penetration of appressorium, and pathogenicity, which indicated that there have function redundancy of MGG₀2863.6 gene in M. grisea.Overexpressed mutantΔ863oe showed no apparent difference with wide type in pathogenicity,but its growth rate was slightly higer than that of wide type. We used SDS stress to obersve the integrality of cell wall, and found thatΔ863oe had a relatively high resistance to SDS, which indicated its cell wall integrity was increased, and therefore, MGG₀2863.6 may be a positive regulatory factor of cell integrality.In the meantime, conservative nucleotide sequences of serine proteases was found from Magnaporthe grisea Database to construct RNA interference vector p733R., After transformation of KU70 protoplasts, the RNAi mutants ofΔ733r-30 andΔ733r-37 were generated. Phenotype analysis showed that although the growth rate of mutants exibited no difference with wild type strain, the mutants showed reduced airal mycelia , darkened colony color, declined sporulation, decreased germination rate of conidia and slowed appressorium formation . The rice pathogenicity tests showed that the mutants have less infection ability with much less disease spots. These results indicated that serine proteases in rice blast fungus are related to rice blast fungus growth, as well as pathogenicity.