Proteomics Analysis Of Potato Response To Infection By Ralstonia Solanacearum And Functional Study Of The StPP1

The phloytype ?B clade of Ralstonia solanacearum are widely distributed in China and gives rise to bacterial wilt disease via colonization of potato roots.R.solanacearum interferes potato immune system through using the type ? secretion system(T3SS)to inject the secretion proteins(T3Es)into plant cells.However,there are fewer reports about the mechanism for T3 Es of potato R.solanacearum interfering plant immunity.This study employed a set of inoculation and phenotype identification system on potato with R.solanacearum and used iTRAQ proteomic to screen the potato proteins responding to the invasion of R.solanacearum.The function identification of one protein StPP1 was performed,which provides a light of theory on bacterial wilt resistance mechanism of potato.The main results are as follows:1.The screen of potato interaction protein responding to the invasion of R.solanacearumThe experiments of potato inoculation and R.solanacearum root colonization showed T3 SS mutant strain lost its pathogenicity totally and was limited the ability of colonization.Base on the phenotype difference,we used iTRAQ proteomics to measure the differences in potato root protein expression after inoculation with UW551 and its T3 SS mutant(UW551?Hrc V).Ten potato root proteins showed significantly lower abundance in plants inoculated with UW551 than that done with UW551?Hrc V,while 11 proteins showed significantly higher abundance;q PCR analysis showed the correlation between the levels of proteins and transcriptions was low for these DAPs.Notably,none of four downregulated proteins(HBP2,PP1,HSP22,and TOM20)were changed at the transcriptional level,suggesting that they were significantly downregulated at the posttranscriptional level.We further coexpressed those four proteins with 33 core T3 Es,and we found that multiple effectors were able to significantly decrease the target proteins' abundance.We used VIGS system to test the resistance function of the silenced plants of these 10 downregulated proteins to the pathogen.4 genes with stable phenotype difference were identified.Results showed that miraculin,HBP2,and TOM20 contribute to immunity to R.solanacearum.In contrast,PP1 contributes to susceptibility.So,these four genes were preliminarily proved to involve the regulation of plant immunity.Based on above experimental results,we found that the three proteins HBP2,TOM20 and PP1 screened by the proteome are related to resistance and have no change at the transcription level.2.The identification of the interaction mechanism between StPP1 and RipASThrough bioinformatics comparison,StPP1 is a protein phosphatase with a conserved phosphatase active site.Further in vitro enzyme activity verification shows that StPP1 has phosphatase activity,and the enzyme activity disappears after the enzyme active site was mutated.The results of subcellular localization showed that StPP1 accumulated in the cytoplasm,nucleoplasm and nucleolus of the nucleus,and its nucleolus localization was dependent on the active site of the phosphatase enzyme;Further genetic transformation results showed that StPP1 expression was negatively correlated with the resistance to bacterial wilt;In addition,through yeast and BiFc experimental verifications,we also found that StPP1 can directly interact with MAPK3,MAPK9,and MAPK16 in potato,suggesting that StPP1 function in MAPK mediated signaling pathways and negatively regulates potato resistance to bacterial wilt.Using StPP1 as the bait protein,a yeast library consisting of 56 effector proteins of R.solanacearum UW551 was screened and the effector protein RipAS was found,and StPP1 interacted with RipAS was also confirmed through Co-IP.Moreover,it was proved that the interaction between StPP1 and RipAS depends on the active site of the phosphatase.Searching and analyzing the similarity of the structure of RipAS protein,it is found that RipAS has a certain structural similarity with protein phosphatase PP2 A,but the enzyme activity test results show that RipAS does not have protein phosphatase activity;Moreover,RipAS was proved playing a negative regulatory role on the pathogenicity of the strain through being mutated and showing the increased pathogenicity.The subcellular localization results of RipAS showed that it was mainly expressed in the nucleoplasm and has a common localization with StPP1;When StPP1 and RipAS were co-expressed in tobacco,the nucleolar localization signal of StPP1 disappeared,which was consistent with the single localization result of StPP1 m.We speculate that RipAS may affect the enzymatic activity of StPP1,but RipAS can't inhibit StPP1's phosphatase activity,nor did it affect the protein abundance of StPP1.Based on the above results,we speculate that RipAS enters plants as a structural analogue of protein phosphatase,which affects the accumulation of StPP1 in the nucleus and makes it unable to function normally.