Caracterização funcional parcial de dois genes de Xanthomonas citri potencialmente relacionados com a patogenicidade: XAC1006 (malato desidrogenase) e XAC0223 (proteína hipotética conservada)

Abstract

The bacterium Xanthomonas citri subsp. citri (XAC) is the cause of citrus canker, a disease that affects many species of citric plants, causing drop in productivity and losses to producers. Many proteins have been detected by proteomic analysis and related to the invasion and dissemination of the bacterium in the citrus host. In the present work, biochemical techniques were applied for the obtaining and partial functional characterization of two XAC genes: XAC1006 (malate dehydrogenase - MDH) and XAC0223 (conserved hypothetical protein), both detected in previous proteomics studies performed at the Laboratory of Biochemistry and Applied Molecular Biology (LBBMA)/UFSCar as potentially involved in XAC´s pathogenicity. The conserved hypothetical protein (XAC0223) does not have a described function in literature for XAC, but its gene sequence analysis by bioinformatics tools shows similarities with membrane proteins and outer membrane vesicles (OMVs) of known function in other species. It was possible to characterize it partially by pathogenicity test with a deletion mutant of this ORF, previously obtained, which apparently presented greater virulence than the wild XAC, which may corroborate the hypothesis that it may be a protein detected by the host plant which can trigger their defense responses. MDH was described as a signature protein in XAC cells surface under in vivo infectious conditions, which has also been described for other pathogenic organisms, as a determinant protein in their infectious processes. In this work, a recombinant MDH was obtained in the native form with confirmed enzymatic activity, and anti-MDH antibodies were also produced and used in Western blot, providing more evidence of its possible presence on XAC´s surface under infectious conditions. It is hoped, through the obtained results, to contribute to the knowledge of the pathogenicity mechanisms of Xanthomonas sp., confirming potential targets of biotechnological interest against the disease and helping in its future combat.