Functional Characterization Of OsMKS1 And OsMKS2 In Rice Disease Resistance And Analysis Of OsVQ Family Expression In Responses To Pathogens

During the long-term struggle with pathogens,plants have evolved a complex and clever response system.They can choose different resistant mechanisms to respond to the infection of various kinds of pathogens.Plant immune systems were considered to have three procedures:the recognition of pathogen,the transduction of defense signal and the regulation of downstream defense genes.Transduction of defense signal is the most flexible part among them.According to the recognition of pathogens,plants can activate and combine different signal transduction pathways,to regulate the expression of specific defense genes.Rice is not only staple food of the worldwide population,but also an important monocot model plant for functional genomics research.Therefore,it is of great theoretical and practical value to elucidate the processes of defense signal transduction,and explore new resistance gene resources of rice.Mitogen-activated protein kinase(MAPK)cascades play important roles in defense signal transduction,they make great contribution during the interactions of rice with Xanthomonas oryzae pv.oryzae(Xoo)and Magnaporthe oryzae(M.oryzae),that cause bacterial blight and fungal blast,respectively.As a plant-specific transcription factor family,WRKY transcription factors also involve in rice-pathogen interactions.In Arabidopsis,AtMKSl(Arabidopsis thaliana mitogen-activated protein kinase 4 substrate 1),substrate of AtMPK4,contributed to MAPK defense signal pathway by coupling the kinase to specific WRKY transcription factors.Here we reported two AtMKS1 homologs in rice,named OsMKS1 and OsMKS2,which maybe pass the defense signal from OsMPK6-regulated defense pathway to downstream WRKY transcription factors.OsMKSl and OsMKS2 are two members of OsVQ gene family,encode two small nucleus proteins.Both of them can interact with OsMPK6 in nucleus by N-terminal domains,OsMKS1 also can form dimer with itself or OsMKS2 by C-terminals.Phosphorylation results indicated that OsMKS1 and OsMKS2 were substrates of OsMPK6 in vitro,and most of the phosphorylation sites were located at C-terminals of these two MKS proteins.We generated OsMKS1-overexpression and OsMKS2-overexpression plants(OsMKS2-oe)in Zhonghua 11,found both of them show enhanced resistance to Xoo,and the enhanced resistance was clearly associated with expression level of OsMKS1 and OsMKS2.Besides,the OsMKS2-oe plants also showed dwarf phenotype.Resistant spectrum analysis showed that overexpression of OsMKSl or OsMKS2 could induce a broad spectrum of resistance to Xoo.We generated gene deletion mutants of these two genes by a modified CRISPR/CRISPR-associated(Cas)system,and found the plants,which had smaller transcripts of OsMKS1,showed more susceptible to Xoo.These results indicated that:as the two substrates of OsMPK6,OsMKS1 and OsMKS2 probably implemented their funtion in the form of dimer to positively regulate rice resistance to Xoo.Our findings of this founctional mechanism are different from Arabidopsis.Furthermore,our results showed that OsMKS2 also played a role in rice growth and development.To identify downstream proteins in the signaling pathway(s)mediated by OsMKS1 and OsMKS2,we screened Arabidopsis transcription factors(TFs)yeast two-hybrid library with these two proteins.Combining with sequence analysis,we found 5 homologous WRKY transcription factors in rice,and then confirmed both of two MKSs could interact with these WRKY proteins by their VQ domain in yeast.So we inferred here OsMKSl and OsMKS2 may be functional as "bridge" between OsMPK6 and specific WRKY transcription factors.In addition,the results of yeast one-hybrid and expression analysis suggested that these two MKSs may negatively feed-back OsMPK6 expression by interacting with WRKY proteins.As a plant-specific protein family,VQ family members have been shown to influence various physiological processes by interacting with specific WRKY proteins in Arabidopsis recent year.Since both OsMKS1 and OsMKS2 belong to rice VQ family,we have systematically investigated the transcription level of VQ genes during rice defense resistance.We found that some of the rice VQ genes showed quite different expression level when inoculated with different kinds of pathogens(Xoo,Xoc and M oryzae),and some of them were found to share co-expression patterns with defense-responsive WRKY genes.These results suggested that rice VQ proteins,at least some of them,may have functions in rice-pathogen interactions by cooperating with WRKY proteins.