New Type III Effectors from Xanthomonas campestris pv. vesicatoria Trigger Plant Reactions Dependent on a Conserved N-Myristoylation Motif

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Abstract
Pathogenicity of the gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria depends on a type III secretion system, which translocates bacterial effector proteins into the plant cell. In this study, we identified two novel type III effectors, XopE1 and XopE2 (Xanthomonas outer proteins), using the AvrBs3 effector domain as reporter. XopE1 and XopE2 belong to the HopX family and possess a conserved putative N-myristoylation motif that is also present in the effector XopJ from X. campestris pv. vesicatoria 85-10. XopJ is a member of the YopJ/AvrRxv family of acetyltransferases. Confocal laser scanning microscopy and immunocytochemistry revealed that green fluorescent protein fusions of XopE1, XopE2, and XopJ localized to the plant cell plasma membrane. Targeting to the membrane is probably due to N-myristoylation, because a point mutation in the putative myristoylated glycine residue G2 in XopE1, XopE2, and XopJ resulted in cytoplasmic localization of the mutant proteins. Results of hydroxylamine treatments of XopE2 protein extracts suggest that the proteins are additionally anchored in the host cell plasma membrane by palmitoylation. The membrane localization of the effectors strongly influences the phenotypes they trigger in the plant. Agrobacterium-mediated expression of xopE1 and xopJ in Nicotiana benthamiana led to cell-death reactions that, for xopJ, were dependent on the N-myristoylation motif. In the case of xopE1(G2A), cell death was more pronounced with the mutant than with the wild-type protein. In addition, XopE2 has an avirulence activity in Solanum pseudocapsicum.