The RSc0454-Encoded FAD-Linked Oxidase Is Indispensable for Pathogenicity in Ralstonia solanacearum GMI1000.

Abstract

Ralstonia solanacearum is the causal agent of bacterial wilt disease. Here, we report that a large FAD-linked oxidase encoded by RSc0454 in GMI1000 is required for pathogenicity. The FAD-linked oxidase encoded by RSc0454 is composed of 1,345 amino acids, including DUF3683, lactate dehydrogenase (LDH), and succinate dehydrogenase (SDH) domains. The RSc0454 protein showed both LDH and SDH activities. To investigate its role in pathogenicity, a deletion mutant of the RSc0454 gene was constructed in GMI1000, which was impaired in its ability to cause bacterial wilt disease in tomato. A single DUF3683, LDH, or SDH domain was insufficient to restore bacterial pathogenicity. Mutagenesis of the RSc0454 gene did not affect growth rate but caused cell aggregation at the bottom of the liquid nutrient medium, which was reversed by exogenous applications of lactate, fumarate, pyruvate, and succinate. qRT-PCR and promoter LacZ fusion experiments demonstrated that RSc0454 gene transcription was induced by lactate and fumarate (both substrates of LDH). Compared with the downregulation of the succinate dehydrogenase gene sdhBADC and the lactate dehydrogenase gene ldh, RSc0454 gene transcription was enhanced in planta. This suggests that the oxidase encoded by RSc0454 was involved in a redox balance, which is in line with the different living conditions of R. solanacearum.