Mechanisms Of Mitogen-activated Protein Kinase Kinase StMKK2 In Regulation Of Potato Immunity

Potato（Solanum tuberosum L.）is an important vegetable crop in China.Potato late blight disease was caused by Phytophthora infestans seriously threatens the development of the potato industry.Therefore,it is urgent to cultivate high-quality and disease-resistant potato varieties for green control of late blight disease in production.Mining resistant and susceptible genes and analyzing their regulatory mechanisms can provide useful gene resources and theoretical basis for disease-resistant breeding.MAPK cascade plays an important role throughout plant growth.However,there is currently limited research on MAPKs regulating potato resistance to late blight.In previous research,it was found that StMKK2 can rapidly respond to P.infestans,but the downstream signaling components of StMKK2 are unclear.To unravel the downstream signaling component of StMKK2,this study identified the function of StMKK2 and used StMKK2 as bait protein to screen for interaction with the cloned 13 potato MAPKs（StMPKs）through yeast two-hybrid system.The candidate downstream MAPKs were verified by phosphorylation experiments to test whether them can be phosphorylated by StMKK2.The major results are as follows:1.Bioinformatics analysis showed that StMKK2 belongs to group A StMKKs and located in cytoplasm and nucleus.2.Overexpression of StMKK2 significantly promotes the infection of P.infestans on N.benthamiana,while silencing of Nb MKK2 significantly inhibits the infection of plant by P.infestans.3.Overexpression of StMKK2 constitutively active mutant StMKK2^S219DT225Dsignificantly promotes the infection of P.infestans,while overexpression of its kinase-dead mutants StMKK2^K98Rand StMKK2^R190AD190Acannot promote the infection of P.infestans,indicating that the function of StMKK2 depends on its kinase activity.4.StMKK2 inhibits the plant PTI response by suppressing the transcription level of PTI marker gene Nb WRKY33 and flg22-induced reactive ROS burst.5.StMKK2 inhibits the expression of SA pathway marker genes,indicating that StMKK2 participates in the regulation of plant immune responses via the SA signaling pathway.6.Using yeast two-hybrid（Y2H）assay we identified StMPK13 as the downstream signaling target of StMKK2.CO-IP assay and Firefly Luciferase assay further confirmed the interaction between StMKK2 and StMPK13.7.StMPK13 inhibits the PTI response in plants and can be phosphorylated by StMKK2.Moreover,StMPK13 is phosphorylated in planta by flg22 treatment,the process of which can stabilize StMPK13 protein.