T-DNA Insertional Mutagenesis Of Magnaporthe Grisea

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 mangament. Therefore, we studied on large scale T-DNA insertional mutagenesis of the fungus by Agrobacterium fumefaciens-mediated transformation (ATMT) and achieved the following results.The conditions for ATMT in the rice blast fungus was further optimized based on the basic procedures established in Fusarium by Mullins and co-workers, including the optimal concentration of hydromycin B in transformant selection, the ratio of cefotaxime to carbenicillin for Agrobacterium inhibition, the efficient medium in the different stage of transformation,etc.In average, about 385 transformants could be achieved by transforming 1*106 conidia of the fungus. Polymerase chain reaction (PCR) and Southern blot analysis showed that all transformants had hph gene insertion but only 85% had T-DNA insertion, among which there were one to three copies of T-DNA insertion with 50% having single-copy insertion.Morphology and development were observed with 120 randonly selected mutants. Thirteen mutants were found mutated in colony color, 5 in growth rate, 14 in conidium morphology and sporulation, 2 in spore germination and 10 in appressorium development.Virulence analysis of 160 mutants was conducted on rice variety C101LACPi-1(t), C101A51Pi-20, C104PKTPi-3(t), C101PKTPi-4a, C105TTP-4L-23Pi*46, BL22Pi-9, Duoxi . No.1, CO39, and LTH. Fifteen mutants were weakened and 40 were enhanced in pathogenicity. Seventeen out of 40 pathogenicity-enhanced mutants changed from avirulent to virulent.Thermal asymmetric interlaced PCR (TAIL-PCR) was efficiently used to amplify the T-DNA flanking regions. AD9 was the most suitable arbitrary primer among 9 primers used.Among 10 specific tertiary TAIL-PCR products sequenced, 6 mutants were found with T-DNA insertion within gene regions and 4 with T-DNA insertion in the region between two genes. The sequences of T-DNA flanking regions were submitted to GenBank (Accession numbers AY295779-AY295788).It was found that the hypothetical protein MG03076.1, MG09558.1 and MG08181.1 were tagged respectively in mutant T940001001 T940003102 and T940002001 which showed pathogenicity-enhanced on Pi-l(t), pathogenicity-decreased on CO39 and pathogenicity-enhanced both on Pi-1(t) and Pi-4b, which suggested that these be pathogenicity related.genes.It was also found that mutant T940000302 was enhanced in pathogenicity on Pi-1(t) and Pi-4b, T940001001 changed from avirulent to virulent on Pi-1(t), and T940001604 was enhanced in pathogenicity on Pi-1(t) and Pi-9. However, the T-DNA was found to insert respectively in between hypothetical proteins MG02674.1 and MG02675.1.MG09558.1 and MG09559.1, MG08092.1 and MG08093.1, which suggested that T-DNA insert at the promoting regions regulating the pathogenicity.Mutant T940004402 had T-DNA inertion at the hypothetical protein NCU03146.1 which homologs were found both in Saccharomyces cerevisiae and in Schizosaccharomyces pombe, but no known functions and mutated phenotypes were observed.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.