野油菜黄单胞菌AvrXccE1是一个毒性/无毒性双功能Ⅲ型效应蛋白

The bifunctional type Ⅲ effector AvrXcc E1 of Xanthom onas cam pestris pv. campestris triggers virulence/avirulence in host plants

植物病原细菌通过III型分泌系统将大量的效应蛋白分泌到宿主细胞内,从而抑制宿主的先天免疫。效应蛋白Avr Xcc E1广泛存在于黄单胞菌中,然而关于Avr Xcc E1具体的作用机理尚不清楚。本研究利用野油菜黄单胞菌野油菜致病变种(Xanthomonas campestris pv.campestris,Xcc)和拟南芥互作模式系统,发现来自Xcc8004菌株的效应蛋白Avr Xcc E1对于Xcc8004在拟南芥上的全毒性是必须的,并且Avr Xcc E1可以抑制病原细菌鞭毛蛋白Flg22诱导的FRK1(Flg22-induced receptor-like kinase)基因表达,但并不抑制Flg22诱导的M AP激酶活性。通过Avr Xcc E1与GFP蛋白的融合表达,证实效应蛋白Avr Xcc E1定位在细胞膜上,并且细胞膜定位对于Avr Xcc E1在拟南芥上发挥毒性功能和在大白菜品种"中白83"上发挥无毒功能是必须的。

Xanthomonas delivers many effector proteins into the host cell through the type Ⅲ secretion system to inhibit host innate immunity. Avr Xcc E1 is an effector protein of Xanthomonas campestris pv. campestris strain 8004,and its homologues exist in many strains of Xanthomonas. Previous study has show n that Avr Xcc E1 mediates avirulence function in Chinese cabbage cultivar zhongbai 83,how ever,the virulence function of Avr Xcc E1 remains unclear. In this study,an avr Xcc E1 knock-out strain（ Xcc8004 △avr Xcc E1） w as generated,and competitive index assays w ere performed on Arabidopsis plants. The ratios of Xcc8004 △avr Xcc E1 to Xcc8004 indicated that Avr Xcc E1 w as required for full virulence of Xcc8004 on Arabidopsis. Well-know n bacterial flagellar peptide Flg22 is recognized by pattern recognition receptors FLS2 to induce the expression of a PAM P-responsive reporter gene FRK1∷LUC. Here,it w as found that Avr Xcc E1 could largely inhibit FRK1∷LUC induction in protoplasts,but not suppress Flg22-induced M APKs activity,suggesting that Avr Xcc E1 w as a potent suppressor of the PTI signaling pathw ay,and the PTI suppression mediated by Avr Xcc E1 w as M APKs independent. In addition,the confocal microscopy revealed that green fluorescent protein（ GFP）-Avr Xcc E1 fusions localized on plant cell plasma membrane in Arabidopsis protoplasts. Protein sequence analysis show s that Avr Xcc E1 contained a putative myristoylated glycine residue G2,and this site is know n to be required for the plasma membrane location. To test w hether the G2 w as also involved in the Avr Xcc E1 localization,the point mutant construct of GFP-Avr Xcc E1（ G2A） w as generated. The results show ed that in contrast w ith WT GFP-Avr Xcc E1,the point mutant GFP-Avr-Xcc E1（ G2A） mainly localized on cytoplasm and nucleus in Arabidopsis proto-plast,which is consistent w ith previous notions. Furthermore,the role of G2 of Avr Xcc E1 in the inhibition of PTI signaling and virulence or avirulence function w as generated. The point mutant Avr Xcc E1（ G2A） w as unable to inhibit FRK1 ∷LUC expression. Only the WT Avr Xcc E1 but not the point mutant Avr Xcc E1（ G2A）w as able to promote Xcc8004 △avr Xcc E1 bacterial grow th on Arabidopsis or recover Avr Xcc E1 avirulence function on Chinese cabbage cultivar zhongbai 83. Together these results indicated that Avr Xcc E1 w as a bifunctional effector protein,and the putative myristoylated glycine residue G2 w as required for Avr Xcc E1 virulence or avirulence on host plants.