The Screening Of The Pathogenic-Related Mutants Of Magnaporthe Grisea And Determination Of The Insertion Sites Of T-DNA Tags

The genome data from Magnaporthe grisea, the rice blast pathogen, are being rapidly accumulated through BAC bank establishment, ETS and genome sequencing. However, the functions of most sequenced genes and proteins of this fungus so far are still hypothetical due to lack of experimental evidences. It is therefore greatly urgent to elucidate the functional genome of Magnaporthe grisea by means of experimental data.Based on the transformation system established by our laboratory, Magnaporthe grisea strain Y34 mutant bank containing 6855 transformants was constructed through T-DNA insertion mediated by Agrobacterium tumifaciens strain AGL-1 in this study. In average, about 300 transformants could be achieved by transforming 1×106 conidia of the fungus. To test the pathogenicity, 1600 transformants from the mutant bank were inoculated on rice cultivar Nipponbare that was highly susceptible to rice blast and on C101 that was highly resistant as control. The transformants showing difference in pathogenicity from the wild type were secondarily and thirdly inoculated in the same ways to confirm the results of primary assay. Of the 1600 transformants tested, 23 showed completely pathogenic lost and 26 decreased from the normal disease (4-5 grade) to 1-3 disease grade on Nipponbare, accounting for 1.44% and 1.62%, respectively. These 49 transformants were primarily considered as pathogenicity-related mutants.From the 49 pathogenicity-related mutants, 25 were randomly selected for further research, including colony form, conidium production, spore germination and appressorium formation analysis. Some mutants in colony form and conidium production ability were obtained. PCR amplification using the genomic DNAs from these 25 pathogenicity-related as templates were also carried out, and the results showed that all the genomic DNAs contained hph gene.The flanking sequences of the T-DNA inserted sites on the genome DNA from the 25 pathogenicity-related mutants were amplified by TAIL-PCR using part of the T-DNA right...