ABSTRACTThe type III effector XopAE from theXanthomonas euvesicatoriastrain 85-10 was previously shown to inhibit plant immunity and enhance pathogen-induced disease symptoms. Evolutionary analysis of 60xopAEalleles (AEal) revealed that thexopAElocus is conserved in multipleXanthomonasspecies. The majority ofxopAEalleles (55 out of 60) comprise a single open reading frame (ORF) (xopAE), while in 5 alleles, includingAEal 37of theX. euvesicatoria85-10 strain, a frameshift splits the locus into two ORFs (hpaFand a truncatedxopAE). To test whether the second ORF ofAEal 37(xopAE85-10) is translated, we examined expression of yellow fluorescent protein (YFP) fused downstream to truncated or mutant forms of the locus inXanthomonasbacteria. YFP fluorescence was detected at maximal levels when the reporter was in proximity to an internal ribosome binding site upstream of a rare ATT start codon in thexopAE85-10ORF but was severely reduced when these elements were abolished. In agreement with the notion thatxopAE85-10is a functional gene, its protein product was translocated into plant cells by the type III secretion system, and translocation was dependent on its upstream ORF,hpaF. Homology modeling predicted that XopAE85-10contains an E3 ligase XL box domain at the C terminus, andin vitroassays demonstrated that this domain displays monoubiquitination activity. Remarkably, the XL box was essential for XopAE85-10to inhibit pathogen-associated molecular pattern (PAMP)-induced gene expression inArabidopsisprotoplasts. Together, these results indicate that thexopAE85-10gene resides in a functional operon, which utilizes the alternative start codon ATT and encodes a novel XL box E3 ligase.IMPORTANCEXanthomonasbacteria utilize a type III secretion system to cause disease in many crops. This study provides insights into the evolution, translocation, and biochemical function of the XopAE type III secreted effector, contributing to the understanding ofXanthomonas-host interactions. We establish XopAE as a core effector of sevenXanthomonasspecies and elucidate the evolution of theXanthomonas euvesicatoriaxopAElocus, which contains an operon encoding a truncated effector. Our findings indicate that this operon evolved from the split of a multidomain gene into two ORFs that conserved the original domain function. Analysis ofxopAE85-10translation provides the first evidence for translation initiation from an ATT codon inXanthomonas. Our data demonstrate that XopAE85-10is an XL box E3 ubiquitin ligase and provide insights into the structure and function of this effector family.
A Pathogen Type III Effector with a Novel E3 Ubiquitin Ligase Architecture