Screening For Magnaporthe Grisea Mutants With Nitrate Utilization Defection And Study On The Related Gene

The rice blast fungus (Magnaporthe grisea Barr.) is important both in economy and in the study of plant-microbe interaction. Dissection of gene functions at the whole genome level will improve understanding the fungal biology and molecular mechanisms regulating the fungal pathogenicity in order to facilitate the rice breeding for durable resistance and manage the disease integratedly. Therefore, we screened four hundred and fifty T-DNA insertional mutants of the fungus established by Agrobacterium tumefaciens-mediated transformation (ATMT) in our lab with nitrate utilization defection. Six such mutants were obtained. We focused on one of such mutants and achieved the following results:T-DNA flanking sequences of M.grisea by TAIL-PCR were amplified and sequenced.34 progeies were obtained after Guy11 crossed with the mutant of T940002002-1-2 (5) .The rate between Hypmycin B resistance and Hypmycin B sensitivity was according to 1:1.That showed that there may be only one copy insertion of T-DNA.Based on the T-DNA flanking sequences , T-DNA was presumed to insert into the position 318bp far from the promoting site ATG of MGG₀6042.5 .Then MGG₀6042.5 was appeared to be effected. appressoriumThe form of appressoriums and the invasion of the cuticle of onion had not significantly different with that of wild type. However, the quantities of spores decreaed.The mutant of T940002002-1-2 (5) had strong pathogenicity on the rice C101LACPi-1(t) C101PKTPi-4a.The result demonstrate that the inserted gene may relate to the pathogenicity of Magnaporthe grisea.Spores collected from the culture of 29 progenies were inoculated on the rice C101LACPi-1(t) C101PKTPi-4a,repectively; the two rice all product disease symptom,so as the parents ,Guy11and T940002002-1-2 (5) .That showed the gene inserted by T-DNA may affect the expression of avr-genes. It is also suggested that T940002002-1-2 (5) had really strong pathogenicity on the rice C101LACPi-1(t) C101PKTPi-4a and could transmit steadily .Several sequences for knock-out tests were also amplied. The segment for complement test was cloned to the cloning vector pMD18-T.In summary, T-DNA was successfully inserted into genome of the rice blast fungus by ATMT technique, a large scale of T-DNA insertional mutants were generated, and several genes related to important biological functions were found to be tagged by T-DNA in this study. This is a good start for functional genomics of the fungus and will have important impact in science and future application.