Ralstonia solanacearum Dps Contributes to Oxidative Stress Tolerance and to Colonization of and Virulence on Tomato Plants

Ralstonia solanacearum, an economically important soilborne plant pathogen, infects host roots to cause bacterial wilt disease. However, little is known about this pathogen''s behavior in the rhizosphere and early in pathogenesis. In response to root exudates from tomato, R. solanacearum strain UW551 upregulated a gene resembling Dps, a nonspecific DNA binding protein from starved cells that is critical for stress survival in other bacteria. An R. solanacearum dps mutant had increased hydrogen peroxide sensitivity and mutation rate under starvation. Furthermore, dps expression was positively regulated by the oxidative stress response regulator OxyR. These functional results are consistent with a Dps annotation. The dps mutant caused slightly delayed bacterial wilt disease in tomato after a naturalistic soil soak inoculation. However, the dps mutant had a more pronounced reduction in virulence when bacteria were inoculated directly into host stems, suggesting that Dps helps R. solanacearum adapt to conditions inside plants. Passage through a tomato plant conferred transient increased hydrogen peroxide tolerance on both wild-type and dps mutant strains, demonstrating that R. solanacearum acquires Dps-independent oxidative stress tolerance during adaptation to the host environment. The dps mutant strain was also reduced in adhesion to tomato roots and tomato stem colonization. These results indicate that Dps is important when cells are starved or in stationary phase and that Dps contributes quantitatively to host plant colonization and bacterial wilt virulence. They further suggest that R. solanacearum must overcome oxidative stress during the bacterial wilt disease cycle.Bacterial wilt caused by Ralstonia solanacearum is a lethal disease affecting diverse economically important crops worldwide (20). The pathogen attacks over 200 species in more than 50 plant families (21). Although known primarily as a soilborne plant pathogen, R. solanacearum also survives in soil, water, and latently infected plants (20). The bacterium typically invades its host through natural or mechanical root wounds, multiplies in the root cortex, and then rapidly colonizes the xylem, where it reaches high cell densities. Once wilt symptoms develop, plants usually die, releasing the pathogen back into the soil (42).R. solanacearum is a tropical bacterium adapted to warmer climates, with the exception of a clonal group belonging to phylotype II, sequevar 1, of the R. solanacearum species complex (13). This group, historically and for regulatory purposes known as race 3 biovar 2 (R3bv2), causes brown rot of potato and bacterial wilt of tomato in tropical highlands and some temperate zones (11, 41, 45, 46). Because of its virulence at relatively cool temperatures, R3bv2 is a quarantine pest in Europe and Canada and a select agent pathogen in the United States (27).R. solanacearum virulence is quantitative and complex, with many contributing factors such as type II-secreted proteins, type III-secreted effectors, extracellular polysaccharide, and several plant cell wall-degrading enzymes (16, 17, 36, 38). Much of what is known about R. solanacearum comes from studies focusing on mid- or end-stage disease caused by tropical or warm-temperate strains (8, 30). A few virulence factors are known to function early in disease development: motility, energy taxis, and type IV pili, which collectively direct the bacterium toward and facilitate attachment to the host root (26, 44, 49, 50). However, R. solanacearum traits that contribute to fitness and virulence in the rhizosphere are not well understood for either tropical or R3bv2 strains.In soil, bacteria experience environmental stressors, such as pH and temperature extremes and water and oxygen limitation, as well as competition for nutrients (47). Plant roots release exudates and sloughed-off cells, supplying sufficient energy to sustain large microbial communities, provided other nutrients such as N, P, and Fe are present (19, 47). While rhizosphere bacteria can enjoy rapid growth in this relatively rich environment, fluctuating nutrient availability means that soil-dwelling microbes must survive periods of starvation (47).R. solanacearum also encounters oxidative stress in the rhizosphere. Plant roots produce reactive oxygen species (ROS) in response to many stimuli (25, 32). Several studies implicate ROS in root development and in interactions between roots and microbes (5, 24). We previously found that during plant colonization R. solanacearum is exposed to host-derived ROS, which triggers a bacterial oxidative stress response that adapts the pathogen to the xylem environment and is necessary for full virulence (8, 14).We previously described an in vivo expression technology (IVET)-like screen that identified R. solanacearum genes upregulated in the tomato rhizosphere (12). These genes encoded several known bacterial wilt virulence factors, such as the type 3 secretion regulator HrpG, the type IV pilus assembly protein PilP, global virulence regulator VsrA, and early virulence regulator PehR. The screen further identified a high-affinity cytochrome c oxidase necessary for R. solanacearum growth in microaerobic conditions (12). This paper presents our analysis of another rhizosphere-induced gene that encodes Dps, a DNA binding protein from starved cells originally described in Escherichia coli (2). Dps belongs to a family of ferritin-like stress-induced proteins that bind nonspecifically to DNA in stationary-phase bacteria (2, 29, 40). In E. coli, Dps helps maintain DNA integrity under environmentally challenging conditions, including starvation, oxidative damage, pH shock, and thermal stress (2, 10, 18, 29, 33). Dps also protects the soilborne plant-associated bacteria Agrobacterium tumefaciens and Pseudomonas putida from oxidative stress (9, 37).Traits that adapt R. solanacearum to detrimental conditions in the rhizosphere are likely to be important for pathogenic success. We hypothesized that Dps is required for adaptation to nutrient and oxidative stress and, thus, for bacterial wilt disease development. We found that Dps was highly expressed after starvation and contributed to oxidative stress tolerance in starved R. solanacearum cells. Furthermore, this protein was necessary for wild-type bacterial wilt disease development and for colonization of tomato xylem, suggesting that the bacterium must overcome a nutrient-poor and/or oxidative environment in the rhizosphere and xylem of host plants.