Functional Study Of Osa-miR164a And Osa-miR1436 In Rice Immunity Against Magnaporthe Oryzae

Rice is one of the most important staple food,which contributes to more than 30%energy uptake of the world.The rice blast disease caused by the fungal pathogen Magnaporthe oryzae is one of the three most devastating diseases threatening rice quality and production.In the past,prevention and treatment of the rice blast mainly depend on the growth of resistant varieties and application of chemicals.However,due to the rapidness of mutation of M.resistant varieties are very vulnerable,especially when a single variety is continuously used in a regione.Similarly,using of single chemical for a long time,leads to resistance to chemicals.Therefore,how to improve the resistance to the rice blast fungus,is a tremendous problem to food security in our nation.In plant-fungal interaction,increasing evidence demonstrates that small RNAs(sRNAs)are involved in the regulation of plant defense response.In rice,a large number of sRNAs are reported being involved in the rice immunity against rice blast fungus,although the immune mechanism is largely unknown.Understanding the mechanism of resistance to this disease is of great significance both scientifically and economically.In our study,rice seedlings were inoculated with M oryzae Guy11 spore suspension.sRNA libraries were constructed using four total RNA samples extracted from infected rice at 0,24,48,and 120 hours post inoculation.By comparing expression patterns of miRNA,identifying miRNA involved in rice immunity,and analysing predicted targets,we explored the molecular mechanism of rice endogenous sRNAs involved in resistance to the blast disease.The major discoveries are summarized as follows:1.We focused on the sRNAs with significantly changes in libraries.We discovered that osa-miR164a expression reduced upon Guy11 infection at both early and late stages,which was perfectly associated with the induced expression of its target gene,OsNAC60.OsNAC60 encodes a transcription factor,over-expression of which enhanced defense responses such as increased programmed cell death,greater ion leakage,more ROS accumulation and callose deposition,and up-regulation of defense-related genes,such as NbERF1,NbAcre31,NbPR1b.By using transgenic rice over-expressing osa-miR164a,and a Tos 17 transposon insertion mutant of OsNAC60,we showed that when the miR 164a/OsNAC60 regulatory module was dysfunctional,rice developed significant susceptibility to Guy11 infection.The OsNAC60 activity was abolished by the co-expression of OsNAC60 and osa-miR164a,but not its synonymous mutant.Our results demonstrate that the miR164a/OsNAC60 regulatory module manipulates rice defense responses to M.oryzae infection.We further demonstrated that this regulatory module was conserved in plant resistance to multiple pathogens,such as M oryzae,Rhizoctonia solani,Phytophthora infestans,Phytophthora sojae.This discovery is of great potential for resistant variety breeding and disease control to a broad spectrum of pathogens in the future.2.We also found another interesting sRNA,miR1436,which has never been reported.We showed that miR1436 expression increased upon Guy11 infection at both early and late stages,which was perfectly associated with the suppressed expression of its predicted target gene,OsMLO11(at 3' UTR).By using transgenic rice lines expressing 35S:miR1436/35S:OsMLO11 and Tos 17 transposon insertion mutant of osmloll,we showed that transgenic rice lines expressing 35S:miR1436 and osmlo11 mutant exhibited enhance resistance to the rice blast fungus.At the same time,overexpression of OsMLO11 was more susceptible to the rice blast fungus.Mainly in disease classification,the burst of reactive oxygen species,the accumulation of the callose,the free salicylic content,and the expression of defense-related genes.At microscopic observation level,transgenic rice overexpressing miR1436 or osmloll mutation line can suppress spore germination and invading hyphae growth.Compared to the wide type rice,overexpressing miR1436 and osmlo11 mutation line have appearant ROS accumulation and callose deposition in the leaf sheath cells after Guy11 infection.We also tested the expression of defense related genes,such as OsPAL1,OsNPR1,OsPR1a,OsPR1b,OsPBZ1.Compared to the wide type,transgenice rice expressing miR1436 and osmlo11 mutation can significantly induce the exprssion of SA-related genes.These results suggested that miR1436 regulates rice resistance response against M.oryzae through affecting the SA signaling pathway.In the absence of pathogen infection,OsMLO11 is expressed without miR1436 restrain.OsMLO11 is perhaps a susceptible gene(as deduced from its barley homolog)that inhibits the accumulation of salicylic acid.Inactivation of the salicylic acid(SA)signaling pathway benefits plants for growth and development.Upon infection,the expression of miR1436 is induced,so does the SA signaling pathway,which leads to enhance resistance.Taken together,our results demonstrate that miR1436 is a positive factor that modulates expression of OsMLO11,and regulates rice resistance to the blast fungus.