Analysis On The Mechanism Of Different Sensitivity Of Magnaporthe Grisea And MG-CYP51 To Triazoles

Magnaporthe grisea is the fungal pathogen of rice blast which caused millions of economic loss every year in the world.14a-demethylase(CYP51)is an important antifungal target which is an important enzyme in fungal sterol biosynthesis.Triazole drugs as a sterol 14a-demethylase inhibitors(DMIS)in their daily lives are widely used as the fungicide.However,due to its single acting site more and more azole-resistant strains are appearing.It is great significant to design the novel,effective,specific and low-toxic DMIs.In this study,experiments were excuted to study the mechanism of the difference in sensitivity of M.grisea and its CYP51(MG-CYP51)to triazole drugs.The results would provide more information to the design of effective and specific DMIs of M.grisea.On the one hand,the mechanisms of difference in sensitivity of M.grisea to the triazoles were analysized.We collect 49 isolates of M.grisea from Hubei.,Hunan,Jiangsu and Heilongjiang Province.The inhibition rates of diniconazole,tebuconazole,triadimenol,triadimefon and propiconazole on M.grisea were measured.The inhibition rate of Tebuconazole on CY8,YA10,101301 and Triadimefon on 101301 all reached 100%.In addition,by comparing the inhibition rates of five drugs,the 3 strains(CY8,YA10,101301)and other 4 stains(1-1-6,HM2,CY3,CY6)are found to be divide into the sensitive and non-sensitive group respectively.The results of REP-PCR and real-time fluorescence quantitative PCR of MG-CYP51 indicated that the genetic background and expression of MG-CYP51 of different strain were not related to the difference in sensitivity of M.grisea to the triazoles.However,the difference in the DNA sequences of CYP51,the mutation of Ser into Leu at site 312,could make some contribution.On the other hand,the binding of MG-CYP51 mutants to the Diniconazole were carried out to elucidate the mechanism of difference in sensitivity of MG-CYP51 to the triazoles.The 38 MG-CYP51 mutants in B’helix-BC loop(SRS1),N-terminal of C helix,K/β1-4 loop-β1-4(SRS5),β4 hairpin(SRS6)and F helix were heterologosly over-expressed in BL21(DE3)rossta strain.The results of binding spectrum showed that all these mutants could bond to the Diniconazole and presented type II spectrum.The Kd of 38 mutants have increased slightly or significantly,which meaned their ability binding to the diniconazole reduced.According to Kd value of different mutants binding to Diniconazole,P222 in the F helix,I367,K373,V374 in the SRS5 and S497,R498,M500 in the SRS6 may be the critical sites of binding to the Diniconazole and the hydrophobic interaction could play a major role in the binding of MG-C YP51 to the Diniconazole.