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A novel Xanthomonas citri subsp. citri NADPH quinone reductase involved in salt stress response and virulence.
Biochimica et Biophysica Acta (BBA) - General Subjects ( IF 2.8 ) Pub Date : 2020-01-03 , DOI: 10.1016/j.bbagen.2020.129514 Maria Victoria Barcarolo 1 , Betiana S Garavaglia 1 , Natalia Gottig 1 , Eduardo A Ceccarelli 1 , Daniela L Catalano-Dupuy 1 , Jorgelina Ottado 1
Biochimica et Biophysica Acta (BBA) - General Subjects ( IF 2.8 ) Pub Date : 2020-01-03 , DOI: 10.1016/j.bbagen.2020.129514 Maria Victoria Barcarolo 1 , Betiana S Garavaglia 1 , Natalia Gottig 1 , Eduardo A Ceccarelli 1 , Daniela L Catalano-Dupuy 1 , Jorgelina Ottado 1
Affiliation
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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.
中文翻译：
一种新颖的柠檬黄单胞菌。柠檬NADPH醌还原酶参与盐胁迫反应和毒力。
背景技术黄单胞菌柠檬亚种。柑橘溃疡病的病原体柠檬（Xcc）一直作为柑橘叶片上的附生植物维持，直到进入植物组织。在附生生存期间，细菌可能会遇到水分利用率低的问题，这会挑战感染过程。Xcc在盐胁迫下的蛋白质组学分析模仿了在附生生存期间发现的条件，显示出XAC2229编码的假定NAD（P）H脱氢酶的丰度增加。方法分析盐和氧化胁迫下以及植物与病原体相互作用过程中XAC2229和XAC突变体在XAC2229中的表达水平。获得了表达XAC2229的大肠杆菌，并研究了该蛋白质在抗氧化应激和产生活性氧方面的作用。最后，纯化Xac2229蛋白，进行了分光光度法和辅助因子分析，并确定了酶活性。结果XAC2229在盐胁迫下和植物-病原体相互作用期间表达。与野生型菌株相比，ΔXAC2229突变体显示出溃疡病的数量减少，附生存活受损。与野生型菌株相比，ΔXAC2229在H2O2存在下存活时间短，并产生更多的活性氧和对硫代巴比妥酸有反应的物质。对于表达XAC2229的大肠杆菌观察到相似的结果。Xac2229是一种含有黄素蛋白的FAD，在最适pH值为6.0时显示出最适合心肌黄递酶的活性，并且使用NADPH作为电子供体时具有醌还原酶的活性。结论含有Xcc的黄素蛋白的FAD是一种新的NADPH醌还原酶，对于细菌的毒性，特别是柑橘叶片上Xcc的附生生存而言，是必需的。
更新日期：2020-01-04
中文翻译：
一种新颖的柠檬黄单胞菌。柠檬NADPH醌还原酶参与盐胁迫反应和毒力。
背景技术黄单胞菌柠檬亚种。柑橘溃疡病的病原体柠檬（Xcc）一直作为柑橘叶片上的附生植物维持，直到进入植物组织。在附生生存期间，细菌可能会遇到水分利用率低的问题，这会挑战感染过程。Xcc在盐胁迫下的蛋白质组学分析模仿了在附生生存期间发现的条件，显示出XAC2229编码的假定NAD（P）H脱氢酶的丰度增加。方法分析盐和氧化胁迫下以及植物与病原体相互作用过程中XAC2229和XAC突变体在XAC2229中的表达水平。获得了表达XAC2229的大肠杆菌，并研究了该蛋白质在抗氧化应激和产生活性氧方面的作用。最后，纯化Xac2229蛋白，进行了分光光度法和辅助因子分析，并确定了酶活性。结果XAC2229在盐胁迫下和植物-病原体相互作用期间表达。与野生型菌株相比，ΔXAC2229突变体显示出溃疡病的数量减少，附生存活受损。与野生型菌株相比，ΔXAC2229在H2O2存在下存活时间短，并产生更多的活性氧和对硫代巴比妥酸有反应的物质。对于表达XAC2229的大肠杆菌观察到相似的结果。Xac2229是一种含有黄素蛋白的FAD，在最适pH值为6.0时显示出最适合心肌黄递酶的活性，并且使用NADPH作为电子供体时具有醌还原酶的活性。结论含有Xcc的黄素蛋白的FAD是一种新的NADPH醌还原酶，对于细菌的毒性，特别是柑橘叶片上Xcc的附生生存而言，是必需的。
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