Analysis On Pathogenicity Mutants Of Magnaporthe Grisea Generated By T-DNA Insertion

The rice blast disease ,caused by the rice blast fungus (Magnaporthe grisea (Hebert) Barr. , Pyricularia gmea(Cooke)Sacc.) ,is the dominant rice disease worldwide. Due to the varieties of pathogenicity, the varieties of races and volatility of the races, the resistant rice variety will decrease in the resistance after application about 3 to 5 years. In order to cultivate the persistent resistant rice varieties and make out more powerful control strategy, we should understand the pathogenicity of rice blast fungus and the mechanism of variance deeply, especially we should find out the function of avirulent genes in the pathogenicity of blast.In the study, we constructed the T-DNA-inserted mutant library of the rice blast fungus by Agrobacterium tumefaciens-mediated transformation (ATMT), and got 6,000 transformants. In average, about 300 transformants could be achieved by transforming 1*106 conidia of the fungus. 80 transformants selected randomly were cultured them in the JM liquid media containing hygromycin, and 72 transformants could grow well. 14 of these transformants were positive transformant after being tested by PCR. So we confer that we can get 5400 positive T-DNA-inserted mutants.We selected 300 mutants randomly for virulence analysis on rice variety C101LACPi-1(t), C101A51Pi-2(t), C104PKTPi-3(t) , ClOlPKTPi-4a, C101TTP-4L-23Pi-4b , 75-1-127Pi-9(t) and CO39. Among them, 32 pathogenicity mutants were screened, 3 mutants were decreased in pathogenicity on CO39,19 mutants decreased on Pi-4b, and 1 was decreased on Pi-3(t). 19 mutants enhanced pathogenicity on C101LACPi-1 (t) ,and 17 mutants were compatible with C101LACPi-1 (t); 11 mutants' pathogenicity were enhanced on C101A51Pi-2(t), and 8 mutants were compatible to C101A51Pi-2(t); 6 mutants were compatible with C101PKTPi-4a; 8 mutants' pathogenicity were enhanced on 75-1-127Pi-9(t), and 3 mutants were compatible with 75-1-127Pi-9(t).The PCR results showed that pathogenecity mutants all had hph gene. Thesouthern-blot result showed that 4 pathogenecity mutants were only inserted by single copy of T-DNA, which is in favour of further analysis. Moreover, we analyzed the pathogenecity mutants by TAIL-PCR. We amplified certain fragment among 6 mutants. One in six mutants, T940009401, was sequenced, and its sequence was the same as contig 2.1003 of rice blast fungus from 26462 to 27034 bp; T-DNA was inserted in the end of clone 09D12, it was 1.2 kb away from the unknown protein MG05451.1; if T-DNA in mutant was integrated by double exchange, the promoter region of Protein MG05451.4 might be exchanged by T-DNA, and the protein would not express. The T-DNA-franking sequence of mutant Pi2-T940009701-l-2 was also sequenced, and its sequence is the same as contig 2.1354 of rice blast fungus from 11630 to 12304 bp; the T-DNA inserted site was in front of two close ESTs' sequence, mgns007xL21f. b 28 561: (12825 - 13461) BLAT_SPAN and mgns007xN17f. b 28 504: (13049 - 13460) BLAT_SPAN, so may lead both ESTs not to express.