Screening And Validation Of Proteins Interaction With Potato Ralstonia Solanacearum Effectors

Bacterial wilt caused by Ralstonia solanacearum is one of the most important devastating bacterial plant disease and is a threat to global potato industry.In order to explore the pathogenic mechanism and interaction mechanism between plant and pathogen,we established the yeast screening system to study the target protein of R.solanacearum effector.The detailed results of this research are as follows:1.The construction of effectors Y2 H library and the screening of interaction proteins with disease-resistant genes of potato.a)Previous proteomic studies have found that compared with type III secretion system mutant,several proteins in potato were significantly down-regulated after inoculation with wild-type R.solanacearum.To study the relationship between down-regulated protein and effectors protein,we constructed a two-hybrid screening library containing 52 effector proteins,.b)We constructed decoy proteins using the potato disease-resistant genes to screening the effector library.We found the interaction between the effector protein Rip17 and the key protein of potato phosphatase StPP1.c)In order to further verify the interaction between effector protein Rip17 and phosphatase StPP1,we used laser confocal microscopy to visualize subcellular localization.When expressed separately,Rip17 is mostly localized in the nucleoplasm,however,Phosphatase StPP1 protein is present in nucleolus and nucleoplasm of nucleus.When Rip17 and StPP1 proteins were co-expressed,localization of StPP1 protein was found to migrated from the nucleolar portion to the nucleoplasmic region.Since the presence of Rip17 has altered the original spatial location of the StPP1 protein,this implicated Rip17 could interact with StPP1.2.Construction of yeast growth inhibition screening library and Rip25 interaction protein screening.a)To study the interaction of effector proteins with virulence function in yeast,we constructed the growth inhibition screening library by using yeast cDNA,aiming to study the interaction protein of these effectors.We will use yeast cDNA library to screen out the endogenous proteins that can restore the growth of yeast.Genetic approaches are used to explore the key proteins or action pathways that effector proteins target in yeast.As a result,we constructed the growth inhibition of yeast selection type of expression library,and the total capacity of the library can reach to 1.25 * 10 ^ 7 CFU.b)We screened the library with Rip25 effector protein and found that the endogenous ScHMO1 of yeast could restore rip25-mediated growth inhibition phenotype of yeast.Meanwhile,degradation experiments in vivo in yeast showed that ScHMO1 protein bands were weakened by after induction of Rip25 effector protein expression.c)We tested whether ScHMO1 and the homologous genes StHMO2 and StHMO7 in potato could restore rip25-mediated cell death in N.benthamiana,after co-expression with rip25 in N.benthamiana.The results showed that ScHMO1 and StHMO2 not only failed to recover rip25-mediated cell death,but also aggravated the degree of cell death.,while StHMO7 could restore cell death to some extent.Suggesting that ScHMO1 and homologous gene coding proteins are involved in rip25-mediated cell death pathways,but the mechanism remains to be verified.A Ralstonia solanacearum effector library for Y2 H system and a yeast cDNA library for screening the yeast growth inhibition effectors had been constructed.We obtained the potential interacting protein of Rip17 in potato by Y2 H,and the interacting protein of Rip25 in yeast by yeast growth inhibition system..It would provide potential gene resourses for bacterial wilt molecular breeding and genetic improvement in potato.