NorA, HmpX, and NorB Cooperate to Reduce NO Toxicity during Denitrification and Plant Pathogenesis in Ralstonia solanacearum

Abstract

ABSTRACT Ralstonia solanacearum, which causes bacterial wilt disease of many crops, requires denitrifying respiration to survive in 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 putative NO-binding protein (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 ΔnorB, ΔnorA, or ΔhmpX increased expression of many iron and sulfur metabolism genes, suggesting that the loss of even one NO detoxification system demands metabolic compensation. Single mutants suffered only moderate fitness reductions in host plants, possibly because they upregulated their remaining protective genes. However, ΔnorA/norB, ΔnorB/hmpX, and ΔnorA/hmpX double mutants grew poorly in denitrifying culture and in planta. It is likely that the loss of norA, norB, and hmpX is lethal, since the methods used to construct the double mutants could not generate a triple mutant. Functional 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 ΔnorB mutant, suggesting that bacterial detoxification of NO reduces the ability of the plant host to perceive the presence of the pathogen. Thus, R. solanacearum’s three NO detoxification systems each contribute to and are collectively essential for overcoming metabolic nitrosative stress during denitrification, for virulence and growth in the tomato, and for evading host plant defenses. IMPORTANCE The 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 the pathogen and its plant hosts. Rs mitigates NO with a trio of mechanistically distinct proteins: NO-reductase (NorB), predicted iron-binding (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).

Keywords

• denitrifying respiration
• iron metabolism
• nitrosative stress
• oxidative stress
• plant defenses
• plant pathogen