The Xanthomonadaceae family consists of species of non-pathogenic and pathogenic γ-proteobacteria that infect different hosts, including humans and plants. In this study, we performed a comparative analysis using 69 fully sequenced genomes belonging to this family, with a focus on identifying proteins enriched in phytopathogens that could explain the lifestyle and the ability to infect plants. Using a computational approach, we identified seven phytopathogen-enriched protein families putatively secreted by type II secretory system: PheA (CM-sec), LipA/LesA, VirK, and four families involved in N-glycan degradation, NixE, NixF, NixL, and FucA1. In silico and phylogenetic analyses of these protein families revealed they all have orthologs in other phytopathogenic or symbiotic bacteria, and are involved in the modulation and evasion of the immune system. As a proof of concept, we performed a biochemical characterization of LipA from Xac306 and verified that the mutant strain lost most of its lipase and esterase activities and displayed reduced virulence in citrus. Since this study includes closely related organisms with distinct lifestyles and highlights proteins directly related to adaptation inside plant tissues, novel approaches might use these proteins as biotechnological targets for disease control, and contribute to our understanding of the coevolution of plant-associated bacteria.

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LesA, from the citrus canker pathogen Xanthomonas axonopodis pv. citri (Xac), is an α/β hydrolase fold protein with lipase/esterase function encoded by the gene XAC0501. In silico analysis revealed that LesA gene encodes a lipase/esterase orthologous to the LipA enzyme, which is present in several species of Xanthomonas. In this study, we aimed to biochemically characterize LesA, and to determine the importance of this protein in the virulence of Xac. A LesA mutant was generated, and lipase and esterase activities showed that it was significantly diminished in the lesA mutant compared with wild-type str. 306 (Xac306wt). If LesA is partially responsible for citrus canker symptoms, there should be spatial association of LesA with the leaf symptom. As a demonstration of the ability of LesA to induce symptoms, ΔXac-LesA and Xac306wt cells were pressure-infiltrated into citrus leaves. The mutants were able to induce local necrosis, but not as much as the Xac306wt. The ΔXac-LesA mutants may be deficient in pathogenesis due to the lack of LesA. Furthermore, with the aim of confirming the importance of the LesA protein on the virulence of Xac, we proceeded with a secretome analysis in planta (citrus leaf infected with Xac306wt) as well as under three different in vitro growth conditions. For the cultivation of bacteria, we used a virulence noninductive medium, NB, and two virulence-inductive media XAM1 and XAM1-Ex (XAM1 plus 30% of citrus leaf extract). Unfortunately, we could not identify the presence of the lipase/esterase LesA from the secretomes. The secretome analysis presented here offers, nonetheless, new insights into the pathobiology of this pathogen. This secretome enabled us to identify a series of putative virulence factors that might play important roles in the development of citrus canker. We proceeded with the biochemical characterization of Xac306wt grown under different in vitro conditions. Results showed that the lipase activity was higher in the cells grown under virulence-inductive medium than the non-inductive medium. Nevertheless, the esterase activity did not demonstrate the same result. We performed the recombinant expression of LesA protein in E. coli cells. The results confirmed the lipase/esterase activity of this enzyme. In addition, LesA was able to activate a hypersensitive response in Nicotiana tabacum. In conclusion, we propose that LesA may play an important role in the virulence of Xac.