Functional Studies Of The Type ? Effector AvrXccB In Xanthomonas Campestris Pv.Campestris

Xanthomonas campestris pv.campestris(Xcc)causes black rot,one of the most important diseases of vegetable brassica crops worldwide.The type III effector inventory plays important roles in virulence and pathogenicity of the pathogen.However,little is known about virulence function(s)of individual type III effector in Xcc.Here,we investigated the immune suppression ability of AvrXccB and possible underlying mechanisms through transgenic and biochemical approaches.First,AvrXccB was demonstrated to be secreted in a type III secretion system-dependent manner through a secretion assay.Sequence alignment showed that AvrXccB has a putative N-myristoylation motif with a conserved key residue Gly at the second position.We further demonstrated that green fluorescence was localized to plasma membrane in the transgenic plants expressing AvrXccB-GFP while fluorescence was distributed throughout the cytoplasm in the AvrXccBG2A-GFP-expressing plants.The results indicate that the putative N-myristoylation motif is essential for its subcellular localization.Second,it was shown that AvrXccB functioned in suppressing plant immunity through a series of experiments.We first demonstrated that transiently expressed AvrXccB suppressed flg22-triggered luciferase expression driven by the NHO1 promoter in transfected Arabidopsis protoplsts.The mutant proteins AvrXccBG2A,AvrXccBH182A and AvrXccBC239A with point mutations in the catalytic triad of putative cystease protease/acetyltransferase partially or completely lost the ability to suppress flg22-induced luciferase expression.Subsequently,we demonstrated that chemical-induced expression of AvrXccB suppressed flg22-triggered callose deposition and oxidative burst in transgenic Arabidopsis plants.In addition,ectopic expression of AvrXccB was also found to promote in planta bacterial growth of Xcc and Peudomomas syringae pv.tomato.However,the mutant proteins AvrXccBG2A,AvrXccBH182A and AvrXccBC239A partially or completely lost the ability to suppress plant immunity,indicating that the putative catalytic triad and plasma membrane localization of AvrXccB are required for its immunosuppressive activity.To further explore molecular mechanisms underlying AvrXccB virulence functions,a yeast two-hybrid screening was performed to identify the interacting proteins of AvrXccB in Arabidopsis.An S-adenosyl-L-methionine-dependent methyltransferase,SAM-MT1,was found to interact with AvrXccB in yeast.The interaction of AvrXccB and SAM-MT1 was subsequently confirmed by pull-down and coimmuniprecipitation assays.Furthermore,AvrXccB was demonstrated to interact with SAM-MT2,the closest homolog of SAM-MT1 in Arabidopsis.Interestingly,SAM-MT1 was not only self-associated but also associated with SAM-MT2 in vivo.SAM-MT1 is a putative transmembrane protein.Subcellular localization of the protein was investigated through transient expression of SAM-MT1-GFP in Nicotiana benthamiana.The fusion protein is localized to special punctuate structures,which are mostly associated with plasma membrane,but in the cytoplasm as well.To reveal its potential role in plant immunity,we investigated whether SAM-MT1 expression was induced after the treatment of microbe-associated molecular patterns(MAMPs).SAM-MT1 expression was induced moderately and rapidly upon stimulation of flg22 and elf18 and after Pst DC3000 inoculation,and SAM-MT2 expression was induced dramatically at 48 h after Pst DC3000 inoculation.These results suggest that SAM-MT1 and SAM-MT2 play a potential role in plant immunity.Collectively,the results suggest that the Xcc effector AvrXccB suppresses plant immunity through targeting a putative methyltransferase complex.The findings in this study lay a solid foundation to elucidate molecular mechanisms underlying AvrXccB pathogenicity.