Maria Victoria Barcarolo1, Betiana S Garavaglia1, Natalia Gottig1, Eduardo A Ceccarelli1, Daniela L Catalano-Dupuy2, Jorgelina Ottado3. 1. Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas (IBR-CONICET) and Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Ocampo y Esmeralda, Rosario 2000, Argentina. 2. Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas (IBR-CONICET) and Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Ocampo y Esmeralda, Rosario 2000, Argentina. Electronic address: catalano@ibr-conicet.gov.ar. 3. Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas (IBR-CONICET) and Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Ocampo y Esmeralda, Rosario 2000, Argentina. Electronic address: ottado@ibr-conicet.gov.ar.
Abstract
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.
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.