Effector Gene xopAE of Xanthomonas euvesicatoria 85-10 Is Part of an Operon and Encodes an E3 Ubiquitin Ligase.

The type III effector XopAE from the Xanthomonas euvesicatoria strain 85-10 was previously shown to inhibit plant immunity and enhance pathogen-induced disease symptoms. Evolutionary analysis of 60 xopAE alleles (AEal) revealed that the xopAE locus is conserved in multiple Xanthomonas species. The majority of xopAE alleles (55 out of 60) comprise a single open reading frame (ORF) (xopAE), while in 5 alleles, including AEal 37 of the X. euvesicatoria 85-10 strain, a frameshift splits the locus into two ORFs (hpaF and a truncated xopAE). To test whether the second ORF of AEal 37 (xopAE85-10 ) is translated, we examined expression of yellow fluorescent protein (YFP) fused downstream to truncated or mutant forms of the locus in Xanthomonas bacteria. 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 the xopAE85-10 ORF but was severely reduced when these elements were abolished. In agreement with the notion that xopAE85-10 is 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 XopAE85-10 contains an E3 ligase XL box domain at the C terminus, and in vitro assays demonstrated that this domain displays monoubiquitination activity. Remarkably, the XL box was essential for XopAE85-10 to inhibit pathogen-associated molecular pattern (PAMP)-induced gene expression in Arabidopsis protoplasts. Together, these results indicate that the xopAE85-10 gene resides in a functional operon, which utilizes the alternative start codon ATT and encodes a novel XL box E3 ligase.IMPORTANCEXanthomonas bacteria 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 of Xanthomonas-host interactions. We establish XopAE as a core effector of seven Xanthomonas species and elucidate the evolution of the Xanthomonas euvesicatoriaxopAE locus, 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 of xopAE85-10 translation provides the first evidence for translation initiation from an ATT codon in Xanthomonas Our data demonstrate that XopAE85-10 is an XL box E3 ubiquitin ligase and provide insights into the structure and function of this effector family.