Functional Analysis Of OsMKK1 In Rice Disease Resistance

The highly conserved Mitogen Activated Protein Kinase（MAPK）cascade signaling system in eukaryotes is a phosphorylation cascade composed of MAPKs,MAPK kinases（MAPKKs）and MAPKK kinases（MAPKKKs）which regulate plant growth and development and response to changes in the external environment.Early studies have shown that there are two classic signaling MEKK1-MKK1/2-MPK4 and pathways-MAPKKK3/MAPKKK5-MKK4/5-MPK3/6 in Arabidopsis,which are closely related to the disease resistance.For the previous researches of MAPK,we mainly focused on MAPK families in Arabidopsis.As a monocot model plant,rice is also an important food crop in the world.Due to abiotic and biotic stresses,the rice yield is reduced by more than 20%annually all over the world.Therefore,researches on the function analysis of the OsMAPK cascade in disease resistance is of great significance and application value.Compared with MEKK1-MKK1/2-MPK4 in Arabidopsis,it is speculated that there may be a similar signaling pathway in rice.In order to study the function of this cascade in plant disease resistance,we have focused our research on the candidate upstream kinase OsMKK1 of OsMPK4 in the disease resistance pathway.In Arabidopsis,functions of MKK1 and MKK2 are redundant,and they co-regulate the downstream MPK4 to participate in plant disease resistance.In rice,the related researches on OsMKK1 mainly focused on salt resistance and other abiotic stress processes.There is no relevant report on the function of OsMKK1 in plant disease resistance.Therefore,research on the function of OsMKK1 in rice disease resistance is helpful to understand the disease resistance mechanism of monocotyledonous plants.We constructed a knockout mutant Osmkk1 by CRISPR-Cas9 technology,and obtained the inducible gain of function mutant GVG-OsMKK1^DDwhich can expressOsMKK1^DDprotein with sustainable function in phosphorylation downstream OsMAPKs after Dex induction.After further screening,homozygous Osmkk1（-16bp）mutant plant without Cas9 and GVG-OsMKK1^DD#11 and GVG-OsMKK1^DD#13 transgenic lines with higher induced protein expression were obtained.After DEX treatment,the non-specific up-regulation of phytoalexin genes in OsMKK^1DDplants was inhibited,while the PR gene was significantly up-regulated.In order to study the function of OsMKK1 in biotic and abiotic stresses,the GVG-OsMKK1^DD,Osmkk1 and NIP lines were treated by Chitin salt and inoculation of rice bacterial blight pathogen PXO99 respectively,the following experimental results were obtained:Chitin treatment can activate phosphorylation of OsMPK3 and OsMPK6.After treatment,the phosphorylation level of OsMPK3 was initially slightly lower than that of wild type,and then there was no significant difference between NIP and mutant.Therefore,OsMKK1 may be involved in the initial PTI response induced by fungi,and it is speculated that OsMKK1 plays a negative regulatory role in disease-resistance.After salt treatment,seedlings of Osmkk1 were sensitive to salt,thus OsMKK1 can positively regulate the salt resistance in rice.After inoculation with PXO99,the Osmkk1 mutant exhibited a disease-resistant phenotype.We speculate that OsMKK1 negatively regulates the Xanthomonas oryzae blight mediated plant immunity.After injection of X.oryzae pv.oryzae strain PXO99,leaves of Osmkk1 and NIP were taken at 0h,24h,and 48h after inoculation.The qRT-PCR results showed that the expression levels of disease resistance-related genes were higher in Osmkk1 in contrast to wild type.In Osmkk1,the expression of the cytochrome P450 gene family changed a lot and in which the expression of Os CYP71Z2 was increased by one hundred times.It indicates that OsMKK1 has effect on the synthesis and metabolism of phytoalexin.In summary,experiments above prove that OsMKK1 is involved in negative regulation of plant immunity,and its downstream kinases and substrates need to be further studied and revealed.