The Functional Analysis Of Effector Proteins AvrXccE1on The Interactions Of Xanthomonas Campestris With Arabidopsis

Xanthomonas campestris pv. campestris (Xcc) is a causal agent of black rot diseases on numerous crucifer plants. Xcc delivers many effector proteins into the host cell through the type Ⅲ secretion system to inhibit host innate immunity. AvrXccE1is an effector protein of Xcc strain8004, and its homologues exist in many strains of Xanthomonas.In this study, we generated an avrXccEl knock-out strain (Xcc8004△avrXccE1) and performed competitive index assays on Arabidopsis plants. The ratios of AavrXccEl to Xcc8004indicated that avrXccEl is required for full virulence of Xcc8004on Arabidopsis. Well-known bacterial flagellar peptide flg22is recognized by Pattern Recognition Receptors FLS2to induce the expression of a PAMP-responsive reporter gene FRK1::LUC. We found AvrXccE1can largely inhibit FRK1::LUC induction in protoplasts, but not suppress flg22-induced MAPKs activity, suggesting that AvrXccEl is a potent inhibitor of the PTI signaling pathway, and the PTI inhibition mediated by AvrXccE1is MAPKs-independent.In addition, the confocal microscopy revealed that green fluorescent protein GFP-AvrXccE1fusions localized to plant cell plasma membrane in Arabidopsis protoplasts. Protein sequence analysis shows that AvrXccE1contains a putative myristoylated glycine residue G2, and this site is known to be required for the plasma membrane location.To test whether the G2is also involved in the AvrXccE1localization, we generated the point mutation construct of GFP-AvrXccE1(G2A). The results showed that in contrast with WT GFP-AvrXccEl, the point mutant GFP-AvrXccE1(G2A) mainly localized to cytoplasm and nucleus in Arabidopsis protoplast, which is consistent with previous notions. Furthermore, we determined the role of G2of AvrXccEl in the inhibition of PTI signaling and virulence or avirulence function. The mutant protein AvrXccE1(G2A) was unable to inhibit FRK1::LUC expression. Only the WT AvrXccE1but not the AvrXccE1(G2A) was able to promote Xcc8004AavrXccE1bacterial growth on Arabidopsis or recover avrXccEl avirulence function on Chinese cabbage cultivar zhongbai83.