Preliminary Study On The Resistance Mechanism Of Magnaporthe Oryzae To Isoprothiolane

Rice blast is the most important diseases on rice which is caused by Magnaporthe oryzae. It causes a great loss in rice production, and significantly influences the production level of the rice in the world. Isoprothiolane which was introduced in1970s is an effective fungicide in controlling rice blast. It suppresses the transmethylation before phosphatidylcholine’s synthesis by inhibiting the activity of methyltransferase. Isolates of M. oryzae from fields are sensitive or low level of resistance to isoprothiolane. However in the lab, it’s not difficult to obtain the resistant mutants by fungicide adaption/selection. Based on three pairs of resistant mutants and their corresponding sensitive parents, we explored the difference of DNA sequences and expression level of genes, especially the methyltransferases in the biosynthesis process of choline between the resistant mutants and sensitive parents, so that the molecular mechanism of resistance to isoprothiolane in M. oryzae could be clarified and demonstrated.The biological characteristics e.g. mycelial growth and sporulation were investigated. Results showed that no difference has been observed for these characteristics among the resistant mutants and sensitive parents, indicating no fitness penalty in resistant mutants. On the other side, no any resistant isolate has been found even more than400field isolates were investigated.In present study, we analyzed the polymorphism of DNA sequences between the resistant mutant and sensitive parental isolate by RAPD and ISSR which have been widely used in surveying the genetic diversity of many organisms. Unfortunately, we did not find the difference in primer binding sequences between the resistant and sensitive isolates.Based on the RNA-seq analysis, there were755genes showing difference in expression level between the resistant and sensitive isolates which were treated by isoprothiolane for4h before RNA extraction. Among them,142and613genes of H08-la_mut are up-regulated（UR） and down-regulated（DR） compared to that of H08-la. Only23genes are involved in known biological process and others are unknown. Among these differential expression genes, MGG₀9793has the most remarkable change in expression level between the resistant and sensitive isolates. And then MGG₀9793were knocked out in H08-la_mut and H08-la by constructing a knocking out vector. Several positive transformants were obtained, there was no significant change in sensitivity to isoprothiolane, as well as the mycelial growth rate and sporulation compared to their initial isolates. These results suggested that MGG₀9793is not the gene conferring resistance to isoprothiolane.Results above are preliminary but essential for demonstrating the molecular mechanism of resistance to isoprothiolane in M. oryzae.