Functional Characterization Of MAPK Cascade Genes And MiR395 In Rice-pathogen Interactions

As one of the most important food crops in the world,the yields of rice has always been the focus of different countries.In recent years,the impact of various diseases on rice yields is becoming more and more serious.Therefore,it is very important to study the mechanism of rice resistance to pathogen.Mitogen-activated protein kinase(MAPK)cascades,with each cascade consisting of a MAPK kinase kinase(MAPKKK),a MAPK kinase(MAPKK),and a MAPK,play important roles in dicot plant responses to pathogen infection.However,no single MAP kinase cascade has been identified in rice-pathogen interaction,and the functions of MAP kinase cascades in disease resistance process are unknown.To explore the contribution of MAP kinase cascade in rice in response to Xanthomonas oryzae pv.oryzae(Xoo),the most devastating bacterial disease of rice worldwide,we performed a comprehensive expression analysis of rice MAP kinase cascade genes after Xoo infection.We transcriptionally analyzed all the 74 MAPKKK genes,8 MAPKK,and 17 MAPK genes in two pairs of susceptible and resistant rice lines,with each pair having the same genetic background,to determine the rice response to Xoo infection.The expression of a large number of MAP kinase cascade genes changed in response to infection,and some of the genes also showed different expression in resistant and susceptible reactions.In addition,some MAPKKK genes co-expressed with MAPKK and/or MAPK genes,and MAPKK genes co-expressed with MAPK genes in rice-Xoo interactions.These results suggest that transcriptome-based analysis of MAP kinase cascades may be a starting point to identify potential candidates in a MAP kinase cascade in rice–Xoo interactions.Based on the above studies,we analyzed two resistance-related MAPK signal cascade genes,MPKK5 and MPKK6.Both proteins interact with the MPK6,which has been reported to have a disease resistance function,in the yeast,and are located in the nucleus as well.In vitro phosphorylation showed that MPKK5 and MPKK6 phosphorylated MPK6 and increased the ability of it to phosphorylate downstream substrates.Among them,MPKK5 could enhance the resistance of rice to Xoo by positively regulating the accumulation of reactive oxygen species in plant.MicroRNAs(miRNAs)play important roles in plant physiological activities.However,their roles in rice innate immunity are largely unknown.Here we show that a mi RNA,mi R395,positively regulates rice resistance to Xoo.Overexpressing mi R395 enhanced rice resistance to a broad spectrum of Xoo,which was associated with suppressed expression of rice Os APS1 gene,an ATP sulfurylase,which encoding protein is predicted to be involved in rice sulfate assimilation pathway.By using 5' RACE,we found the cleavage site of Os APS1 targeted by mi R395.Suppressing Os APS1 by RNA interference(RNAi)enhanced rice resistance to Xoo.Sulfur is a vital element during plant growth.Both mi R395-overexpressing and Os APS1-RNAi plants had increased concentration of sulfate in leaves.In addition,increasing sulfate concentration inhibited the growth of Xoo in culture.These results suggest that mi R395 positively regulates rice disease resistance mainly through improving the accumulation of sulfate in leaves,which could significantly inhibit the growth of Xoo,by directing cleavage of OsAPS1.