NorA, HmpX, and NorB cooperate to reduce NO toxicity during denitrification and plant pathogenesis in Ralstonia solanacearum

Abstract

Ralstonia solanacearum, which causes bacterial wilt disease of many crops, needs denitrifying respiration to succeed inside its plant host. In the hypoxic environment of plant xylem vessels this pathogen confronts toxic oxidative radicals like nitric oxide (NO), which is generated by both bacterial denitrification and host defenses. R. solanacearum has multiple distinct mechanisms that could mitigate this stress, including Repair of Iron Cluster (RIC) homolog NorA, nitric oxide reductase NorB, and flavohaemoglobin HmpX. During denitrification and tomato pathogenesis and in response to exogenous NO, R. solanacearum upregulated norA, norB, and hmpX. Single mutants lacking{Delta} norB, {Delta}norA, or{Delta} hmpX increased expression of many iron and sulfur metabolism genes, suggesting that losing even one NO detoxification system demands metabolic compensation. Single mutants suffered only moderate fitness reductions in host plants, possibly because they upregulated their remaining detoxification genes. However,{Delta} norA/norB, {Delta}norB/hmpX, and{Delta} norA/hmpX double mutants grew poorly in denitrifying culture and in planta. Loss of norA, norB, and hmpX may be lethal, since the methods used to construct the double mutants did not generate a triple mutant. Aconitase activity assays showed that NorA, HmpX and especially NorB are important for maintaining iron-sulfur cluster proteins. Additionally, plant defense genes were upregulated in tomatoes infected with the NO-overproducing{Delta} norB mutant, suggesting that bacterial detoxification of NO reduces pathogen visibility. Thus, R. solanacearums three NO detoxification systems each contribute to and are collectively essential for overcoming metabolic oxidative stress during denitrification, for virulence and growth in tomato, and for evading host plant defenses. ImportanceThe soilborne plant pathogen Ralstonia solanacearum (Rs) causes bacterial wilt, a serious and widespread threat to global food security. Rs is metabolically adapted to low oxygen conditions, using denitrifying respiration to survive in the host and cause disease. However, bacterial denitrification and host defenses generate nitric oxide (NO), which is toxic and also alters signaling pathways in both plants and the pathogen. Rs mitigates NO with a trio of mechanistically distinct proteins: NO-reductase NorB, Repair of Iron Centers NorA, and oxidoreductase HmpX. This redundancy, together with analysis of mutants and in-planta dual transcriptomes, indicates that maintaining low NO levels is integral to Rs fitness in tomatoes (because NO damages iron-cluster proteins) and to evading host recognition (because bacterially produced NO can trigger plant defenses).