Functional Analysis Of Effector Protein AopK And AopD In Acidovorax Citrulli

Bacterial fruit blotch(BFB)is a seed-borne bacterial disease that seriously damages watermelon,muskmelon and other cucurbit crops,causing significant economic losses to the watermelon industry in various countries.The pathogen of BFB is Acidovorax citrulli.The typeⅢ secreted effectors(T3Es)play important roles in the pathogenesis of A.citrulli.However,there are few studies on T3 Es in A.citrulli.Therefore,in this experiment,the effector proteins AopK and AopD were identified and functionally studied in AAC00-1.The results are as follows:1.Identification and functional study of effector protein AopKXopK is a T3 E with E3 ubiquitin ligase activity in Xanthomonas spp..The homolog of XopK,designated as aopK,in A.citrulli has not been investigated.The aopK deletion mutant and complementary strain were constructed in A.citrulli wild type strain AAC00-1.The pathogenicity assay showed that the deletion of the gene aopK significantly reduces the virulence of A.citrulli on watermelon seedlings and attenuate the ability of AAC00-1 to stimulate HR in non-host tobacco.The transcriptional expression level,secretory and BAX co-expression analyses showed that the expression of aopK was positively regulated by Hrp X and Hrp G.AopK was secreted into extracellular matrix through the Type Ⅲ secretion system,and played impacts on virulence via inhibiting plant defense responses.Subcellular localization analysis of AopK in Nicotiana benthamiana and the PTI reaction assays such as reactive oxygen species burst,callose deposition,and cell necrosis were further carried out.Results revealed that AopK was delivered to the cytoplasmic membrane of N.benthamiana,suggesting that AopK could interfere the host’s immune response via inhibiting the PTI.In summary,our data indicated that AopK was a T3 E which inhibit the basic defense response of the host in A.citrulli.2.Identification and functional study of effector protein AopDXanthomonas effector protein XopD has SUMO protease activity.Through bioinformatics analysis,it was found that AopD was homologous to the effector protein XopD in A.citrulli.The aopD mutant strain was constructed,as well as aopD-complementation strain.It was found that AopD was closely related to the pathogenicity of A.citrulli,and the aopD mutant strain attenuated the ability of AAC00-1 to induce HR in tobacco.Subsequent q RT-PCR,Western blot and Agrobacterium-mediated transient expression in tobacco were performed on aopD.The results showed that AopD is a T3 E.aopD was induced to express in T3 SS induction medium.The expression of AopD is negatively regulated by Hrp X and Hrp G.AopD was able to inhibit PCD induced by BAX in the Agrobacterium-mediated transient expression system of tobacco.Through the subcellular localization experiment of AopD in N.benthamiana and the detection experiment of PTI reaction such as reactive oxygen species burst,callose deposition,cell necrosis,it is shown that AopD can be localized in the cytoplasm of plants,and it is speculated that it has the function of inhibiting plant PTI.