Xanthomonas Campestris Effector Proteins XopD_Xcc8004 Interferes Host Plant Immunity By Interacting With NAC Transcriptional Factor ATAF2

Plants are engaged in a continuous co-evolutionary struggle for dominance with their pathogens. The outcomes of these interactions are of particular importance. The recent convergence of molecular studies of plant immunity and pathogen infection strategies is revealing an integrated picture of the plant-pathogen interaction from the perspective of both organisms. Plants have an amazing capacity to recognize pathogens through strategies involving both conserved and variable pathogen elicitors, and pathogens manipulate the defence response through secretion of virulence effector molecules.Recognition of invariant microbial surface patterns (pathogen-associated molecular patterns, PAMP) through plant pattern recognition receptors is referred to as PAMP-triggered immunity (PTI) and is the basis for broad-spectrum resistance of plants against host non-adapted microbial pathogens. A hallmark of many virulent phytopathogenic bacteria is their ability to deliver a plethora of effector proteins into host cell by conversed type Ⅲ secretion system to promote parasitism. Suppression of PTI by microbial effectors (effector-triggered susceptibility, ETS) is a prerequisite for plant infection by adapted pathogens and is likely the cause for susceptibility of many crops to virulent microbial pathogens. Co-evolution of susceptible plant hosts and host-adapted pathogens has shaped immune receptors (resistance proteins) that guard microbial effector-mediated perturbations of host cell functions and thereby trigger plant immune responses (effector-triggered immunity, ETI) While a number of effector proteins have been shown to inhibit host immunity, the biochemical function and molecular basis remains unknown for the vast majority of these effectors. Therefore, identification of plant targets and research of their interactions will make us know more about the mechanisms of bacterial virulence and plant resistance.Xanthomonas campestris pv. campestris (Xcc) is a vascular pathogen that causes black rot disease on a large number of crucifer plants.The interaction between Xcc and Arabidopsis has been studied as a model in microbe-plant interactions. Xanthomonas type Ⅲ secretion system (TTSS) is a powerful apparatus to secret effector proteins into the plant cell, and it is also the most important weapon for bacterial virulence. Little is known about the biochemical function of these effector proteins and their host targets. In this study, biochemical analysis was carried out on effector proteins XopDXcc8004of Xcc8004to uncover its virulence or avirulence function in Arabidopsis. XopDXcc8004encodes a putative48kD protein. Using the Arabidopsis-Xcc model system, our research demonstrates that XopDXcc8004plays avirulence role in mesophyll tissues of Arabidopsis. XopDXcc8004can inhibit plant innate immunity PTI but strongly activate SA and JA signal pathway and restrict Xcc8004virulence in Arabidopsis. XopDxcc8004suppresses flg22induced FRK1(flg22-induced receptor-like kinase) gene expression, oxidative burst and callose deposition. Although XopDxcc8004does not suppresses flg22induced MAPKs, it suppresses MKK5DD (constitutively active form of MKK5) induced FRK1gene expression. These results suggest that XopDXcc8004suppressed FRK1gene expression is downstream of MAPKs signaling or MAPK-independent.To find XopDXcc8004target and identify new components in PTI signal pathway, the interaction partners of XopDXccgoo4was screened by yeast two hybrid system using an Arabidopsis cDNA library. One of the interactors encoding NAC transcription factor ATAF2was isolated. Arabidopsis thaliana NAC transcription factors, one of the largest families of transcriptional regulators in plants, act as key components in plant disease resistance. However, the specific contribution of NAC family transcription factors to the regulation of PTI is not completely defined. The transcription factor ATAF2, a member of NAC family transcription factors, was found to repress FRK1gene expression in Arabidopsis protoplast. However, little is known about how FRK1gene was repressed by ATAF2at the molecular level.In this work, Our results showed ATAF2interact with Arabidopsis thaliana MPK3and MPK6in vitro. MPK3and MPK6, two mitogen-activated protein kinases (MAPKs or MPKs), play critical roles in plant innate immune PTI(PAMP-triggered immunity) by regulating multiple defense responses. Analysis of single mapk mutants revealed that lack of MPK3decreases flg22induced FRK1expression. ATAF2can be phosphorylated by MPK3in Arabidopsis protoplast and MKK5DD-Induced phosphorylation of ATAF2Is prevented by the mutation of serine67. ATAF2S67abolish the ability of inhibiting flg22-induced FRK1expression. Based on these data, we conclude that serine67of ATAF2plays important roles downstream of MPK3cascade in regulating FRK1expression.Further experiments shown that ATAF2is a positive regulator of SA and JA signal pathway and the stabilization of ATAF2by XopDXcc8004in Arabidopsis protoplast is26S proteasome dependent. To further identify new components in PTI and find the protein responsible for the degradation of ATAF2, the interaction partners of ATAF2NAC domain was screened by yeast two hybrid system using an Arabidopsis cDNA library. A bait plasrnid pGBKT7-ATAF2was constructed and transformed to yeast (Saccharomyces cerevisiae) Y2HGold strain, pGADT7-cDNA Arabidopsis library were transformed to yeast (Saccharomyces cerevisiae) Y187strain. After mating and selection of HIS3and ADE2auxotroph, several interaction proteins were found. A RING (Really Interesting New Gene) domain E3ubiquitin ligase, named RHA2a, were occurred in Y2H several times. GST-pull down and in planta immunoprecipitation assays were applied to confirm the interactions between ATAF2and RHA2a in vitro and in vivo in E.coli and tobacco (Nicotiana benthamiana). We also find ATAF2protein levels are highly related with RHA2a. However, the regulatory mechanism of RHA2a in PAMP-triggered immunity is poorly understood. Here, we show that Arabidopsis thaliana RING-H2E3ubiquitin ligase RHA2a, previously known to positively regulates ABA-mediated control of seed germination and early seedling development, negatively regulate PAMP-triggered immunity—flg22-induced ROS, callose deposits, root growth inhibition and seedling growth inhibition.