RNA-seq analysis of Ralstonia solanacearum treated with nitrosative and oxidative stress

Ralstonia solancearum causes bacterial wilt disease on diverse plant hosts. R. solanacearum cells enter a host from soil or infested water through the roots, then multiply and spread in the water-transporting xylem vessels. Despite the low nutrient content of xylem sap, R. solanacearum grows extremely well inside the host, using denitrification to respire in this hypoxic environment. R. solanacearum growth in planta also depends on the successful deployment of protein effectors into host cells using a Type III Secretion System (T3SS). The T3SS is absolutely required for R. solanacearum virulence, but it is metabolically costly and can trigger host defenses. Thus, the pathogen’s success depends on optimized regulation of the T3SS. We found that a byproduct of denitrification, the toxic free-radical nitric oxide (NO), positively regulates the R. solanacearum T3SS both in vitro and in planta. Using chemical treatments and R. solanacearum mutants with altered NO levels, we show that the expression of a key T3SS regulator is induced by NO in culture. Analyzing the transcriptome of R. solanacearum responding to varying levels of NO both in culture and in planta revealed that the T3SS and effectors were broadly upregulated with increasing levels of NO. This regulation was specific to the T3SS and was not shared by other stressors. Our results suggest that R. solanacearum experiences an NO-rich environment in the plant host and may use this NO as a signal to activate T3SS during infection. We analyze gene expression of WT R. solanacearum strain GMI1000 grown in denitrification-promoting low oxygen conditions in culture and when grown in tomato stems. Two mutant R. solanacearum strains, ∆narG, which cannot denitrify, and ∆norB, which accumulates nitric oxide (NO), were also analyzed both in culture and in tomato stems. In addition, we included two culture treatments, one with 100 uM hydrogen peroxide to induce oxidative stress, and one with the NO donor CysNO (1mM) to induce nitrosative stress. Each treatment or mutant included three biological replicates from separate overnight R. solanacearum cultures.