Functional Dissection Of StKH17,a Target Of E3 Ubiquitin Protein StPUB17,in Potato Resistance Against Phytophthora Infestans

Potato is the fourth most important food crop in China.Potato production was suffered by many pests and diseases which seriously reduces potato yield and quality.Late blight caused by Phytophthora infestans is the most devastating disease.Previously,we found that potato U-box E3 ubiquitin ligase StPUB17 is a positive regulator in the potato late blight resistance.StPUB17 was found to be interacted with a potato KH-domain protein,StKH17,a kind of RNA binding protein.StKH17 is a negative regulator in late blight resistance of Nicotiana benthamiana.In this study,we aimed to further dissect the function of StKH17 in potato late blight resistance by over-expressing and RNAi StKH17 in potato;To screen StKH17 interacting target protein by Y2H and to obtain stable expressing StKH17-myc fusion protein transgenic lines for further investigate StKH17 regulatory RNA targets using Chip-seq.The main results are as follows:1.StKH17 over-expression and RNAi vectors were constructed and they were used to transform potato.Two over-expression and six RNAi transgentic lines were obtained.Late blight resistance evaluation results revealed that over-expression StKH17 in potato significantly decreses late blight resistance,however RNAi StKH17 in potato enhances resistance significantly.This results indicated that StKH17 negatively regulates late blight resistance in potato.2.Protein structure analysis showed that StKH17 have 2 conserved domains,one is STAR-domain,another is KH-domain.The auto-activation and pairwise Y2H interaction assay in yeast showed that the interaction between StKH17 and StPUB17 needed STAR-domain and the absence of STAR-domain had no effect on the yeast auto-activation.However KH-domain was important for the auto-activation effect in yeast,but its deletion had no effect on the interaction between StKH17 and StPUB17.3.Three candidate interacting proteins was obtaind by Y2H screen using StKH17^?128-261 mutant as bait.Among them,one was StKH17 itself,another two were StKH17-2 and StKH17-3.Pairwise Y2H interaction assay showed that both StKH17-2 and StKH17-3interact specifically with StKH17^?128-261,but not with StPUB17.4.A StKH17?GDDG?mutant vector which impaired RNA binding capacity were constructed.It was found that StKH17?GDDG?mutant losts the ability to promote the conoloization of P.infestan when it was transiently expressed in N.benthamiana.This result showed that GDDG domain is very important for the function of StKH17.5.StKH17-myc protein tag vector was constructed and it was used to transform potato.11 transgentic lines were obtained.Western blot detection results showed that three lines amonge 11 shows stable expressing of StKH17-3×myc fusion protein.This study provides preliminarily evidence to the hypothesis that StPUB17 might positive regulate the potato late blight resistance by degrading the negative regulatory susceptible factor StKH17.Our results showed that StKH17 may regulate the alternative splicing of defense-related genes therefore negatively regulate the late blight resistance through a complex in potato,which provides a foundation for constructing a StPUB17-StKH17-target protein interaction model,and to clarify the function and molecular mechanism of how StPUB17 malipulats potato late blight resistance.