Functional Study Of OsAPX1 In Rice Blast Resistance And Identification Of Blast Resistance-related Long (Isi-)RNA

Rice blast disease caused by Magnoporthe oryzae is one of the most serious diseases in many rice-growing regions of the world.Based on the economic essentiality,as the staple food for half of the world current population,and the scientific importance,as a good model for plant-microbe interaction,clarifying the interaction mechanism between rice and M.oryzae and identifying the novel rice defense genes are of extraordinary significance.To investigate the protein profiles during the process of rice response to rice blast and the relevant biological processes,in this study,both the rice blast compatible strain Guy 11 and incompatible strain 2539 were used to infect rice Oryza sativa L.japonica.cv.Nipponbare,followed by extracting protein samples at three time points:Oh(control),24h(early stage),and 96h(late stage).By analyzing the iTRAQ result,combined with the functional analysis of these predicted protein,we narrowed down to individual proteins,such as OsGLO1(B8AKX6)and OsAPX1(Q9FE01),and we studied the preliminary function of these two genes in rice protoplasts.To investigate the role that OsAPXl plays in the rice defense response against the blast disease,we found that callose deposition,which is a wide accepted hallmark of early defense response,was measurably increased in the OsAPX1-OE plants after M.oryzae infection,meanwhile,the elevation of OsAPXl led to an enhanced resistance against M.oryzae.Similar defense indexes were also observed in rice protoplasts.Furthermore,components participating in salicylic acid(SA)synthesis and signaling pathways were activated,whereas jasmonic acid(JA)signaling pathway components were not affected.We also found that the overexpression of OsAPX1 resulted in an increased free SA content in planta.Our results indicate that OsAPX1 is a functional gene mediating rice defense against the rice blast disease,and probably through an SA pathway.To further evaluate the endogenous defense mechanism against rice blast,we used a deep sequencing approach to investigate the global expression of rice long siRNAs after M.oryzae infection.We identified a new class of 25-40 nt-long rice endogenous lsiRNAs(osa-lsiRNAs)which are specifically responsive to M.oryzae infection.For instance,lsiR76113 was reduced after M.oryzae infection,whose distinct suppression profile was further confirmed by Northern blot.Bioinformatic analysis implied that the target gene of lsiR76113 is Os12g28260,which may contribute to the disease resistance.In agreement,the expression of Os12g28260 was induced after M.oryzae infection.Our study indicates that lsiRNA is a class of novel siRNA that may be involved in rice response against the rice blast disease,but the molecular mechanism underlying remains unclear.