Functional Dissection Of WIP Genes In Immune Response In Arabidopsis

WIP(wound-induced protein)family proteins consist mainly of a signal peptide at the N terminus and two hydrophobic transmembrane domains.To date,little is known about their characteristics and functions in plant.By searching public database,we found that Arabidopsis genome harbors 6 AtWIP genes,AtWIP1-AtWIP6,which belong to a gene family.Some members in the WIP family are affected by pathogen infection.Therefore,we speculate that WIP genes are involved in plant immune response.Through gene expression pattern assay,we found that AtWIP1 and AtWIP2 are ubiquitiously expressed in Arabidopsis,with the highest expression levels in leaves.To study the relationship of WIP genes and immune response in Arabidopsis,we first treated Arabidopsis with several conditions.When Arabidopsis leaves were infected with pathogenic bacterium Pst DC3000,expression of AtWIP2 gene was significantly down-regulated;and this suppression was also observed when treated with PAMP elicitor flg22.However,when treated with plant immunity relative hormones,such as JA,SA,and ACC(ethylene precursor),no alteration of AtWIP expressions was observed.On the other hand,expression of At WIP1 gene was induced by low temperature.To study the subcellular localization of WIP proteins,we constructed 35S:AtWIP1/2-GFP vectors and introduced into tobacco leaves and onion epidermal cells.In these two transient expression systems,both WIP1-GFP and WIP2-GFP fusion proteins were localized at the cytomembrane,indicative of their functions at cell face.To dissect the function of WIP genes in Arabidopsis immunity,we bought two putative T-DNA insertion mutants of At WIP2.Unfortunately,neither showed loss of function of this gene.We then constructed 35S:AtWIP1/2 overexpression vectors and transformed into Arabidopsis,however,elevated expression levels of AtWIP1/2 genes were not observed.We supposed that AtWIP genes expression istightly controlled in Arabidopsis.Then,we chose soybean WIP homologous genes,GmWIP1 and GmWIP2,and overexpressed in Arabidopsis.This heterologous overexpression succeeded.Meanwihle,we used RNAi technology to suppress the expression of AtWIP1/2,and results showed that both these genes were significantly down-regulated.When these plants were infected with Pst DC3000,the GmWIP overexpression lines exhibited a weak phenotype of local yellow leaves;while the RNAi lines severely browned and even died.Therefore,overexpression of WIP genes significantly enhanced plant resistance to pathogenic bacteria,and on the contrary,suppression of WIP genes significantly impaired the resistance.Our results demonstrate that WIP genes play an important role during plant immune response.