Participation of two general stress response proteins from Xanthomonas citri subsp. citri in environmental stress adaptation and virulence.

Xanthomonas citri subsp. citri (Xcc) is the bacteria responsible for citrus canker. During its life cycle Xcc is found on leaves as epiphyte, where desiccation conditions may occur. In this work, two Xcc genes, XAC0100 and XAC4007, predicted in silico to be involved in general stress response, were studied under salt, osmotic, desiccation, oxidative and freezing stress, and during plant-pathogen interaction. Expression of XAC0100 and XAC4007 genes was induced under these stress conditions. Disruption of both genes in Xcc caused decreased bacterial culturability under desiccation, freezing, osmotic and oxidative stress. Importantly, the lack of these genes impaired Xcc epiphytic fitness. Both Xac0100 and Xac4007 recombinant proteins showed protective effects on Xanthomonas cells subjected to drought stress. Also, Escherichia coli overexpressing Xac4007 showed a better performance under standard culture, saline and osmotic stress and were more tolerant to freezing and oxidative stress than wild type E. coli. Moreover, both Xac0100 and Xac4007 recombinant proteins were able to prevent the freeze-thaw-induced inactivation of L-Lactate dehydrogenase. In conclusion, Xac0100 and Xac4007 have a relevant role as bacteria and protein protectors; and these proteins are crucial to bacterial pathogens that must face environmental stressful conditions that compromise the accomplishment of the complete virulence process.

A novel Xanthomonas citri subsp. citri NADPH quinone reductase involved in salt stress response and virulence.

BACKGROUND: Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker is maintained as an epiphyte on citrus leaves until entering the plant tissue. During epiphytic survival, bacteria may encounter low water availability that challenges the infection process. Proteomics analyses of Xcc under saline stress, mimicking the conditions found during epiphytic survival, showed increased abundance of a putative NAD(P)H dehydrogenase encoded by XAC2229. METHODS: Expression levels of XAC2229 and a Xcc mutant in XAC2229 were analyzed in salt and oxidative stress and during plant-pathogen interaction. An Escherichia coli expressing XAC2229 was obtained, and the role of this protein in oxidative stress resistance and in reactive oxygen species production was studied. Finally, Xac2229 protein was purified, spectrophotometric and cofactor analyses were done and enzymatic activities determined. RESULTS: XAC2229 was expressed under salt stress and during plant-pathogen interaction. ΔXAC2229 mutant showed less number of cankers and impaired epiphytic survival than the wild type strain. ΔXAC2229 survived less in the presence of H2O2 and produced more reactive oxygen species and thiobarbituric acid-reactive substances than the wild type strain. Similar results were observed for E. coli expressing XAC2229. Xac2229 is a FAD containing flavoprotein, displays diaphorase activity with an optimum at pH 6.0 and has quinone reductase activity using NADPH as an electron donor. CONCLUSIONS: A FAD containing flavoprotein from Xcc is a new NADPH quinone reductase required for bacterial virulence, particularly in Xcc epiphytic survival on citrus leaves. GENERAL SIGNIFICANCE: A novel protein involved in the worldwide disease citrus canker was characterized.

Proteome changes and physiological adaptations of the phytopathogen Xanthomonas citri subsp. citri under salt stress and their implications for virulence.

The bacterium Xanthomonas citri subsp. citri (Xcc) is responsible for the widely distributed disease citrus canker. In the last years, Xcc has become a model for the study of plant pathogens, and here we used this bacterium to examine stress on the pathogen during adaptions required for leaf colonization. In the first stages of citrus canker cycle, bacteria encounter low water availability and osmotic stress that can affect their maintenance on plant surfaces. To examine such conditions, we conducted a proteome analysis of Xcc grown in culture medium supplemented with 0.25 M sodium chloride and compared it to control conditions. We found that salt stress induced changes in known stress-induced proteins and also revealed novel stress response proteins. Moreover, some of the bacterial processes associated with bacterial fitness and virulence were modified under salt stress conditions. In particular, swimming, twitching and surface motilities were decreased, while biofilm formation was increased under salt stress. Other adaptations to high salt included reduced bacterial size and increased survival of bacteria exposed to oxidative stress. Furthermore, expression of type III protein secretion system related genes were augmented under salt stress condition. Our results offer new insight into molecular mechanisms that govern phytopathogen adaptation to harsh environments. These adaptations affect life cycle progression which in turn influences virulence.