The plant pathogen Ralstonia solanacearum needs aerotaxis for normal biofilm formation and interactions with its tomato host

Abstract

Ralstonia solanacearum is a soilborne pathogen that causes bacterial wilt of diverse plant species. To locate and infect host plant roots R. solanacearum needs taxis, the ability to move toward more favorable conditions. However, the specific signals that attract this pathogen were unknown. One candidate is aerotaxis, or energy taxis, which guides bacteria toward optimal intracellular energy levels. The R. solanacearum genome encodes two putative aerotaxis transducers. Cloned R. solanacearum aer1 and aer2 genes restored aerotaxis to an Escherichia coli aer mutant, demonstrating that both genes encode heterologously functional aerotaxis transducers. Site-directed mutants lacking aer1, aer2, or both aer1 and aer2 were significantly less able to move up an oxygen gradient than the wild-type parent strain; in fact, the aerotaxis of the aer mutants was indistinguishable from that of a completely nonmotile strain. Tomato plants inoculated with either the aer2 or the aer1/aer2 mutant had slightly delayed wilt disease development. Furthermore, the aer1/aer2 double mutant was significantly impaired in the ability to rapidly localize on tomato roots compared to its wild-type parent. Unexpectedly, all nonaerotactic mutants formed thicker biofilms on abiotic surfaces than the wild type. These results indicate that energy taxis contributes significantly to the ability of R. solanacearum to locate and effectively interact with its host plants.

FIG. 1.

Two probable aerotaxis transducers in the R. solanacearum strain K60 genome. (A) The genomic regions of aerotaxis transducer genes aer1 and aer2 in R. solanacearum strain K60. The arrows indicate the direction of transcription, the solid arrows represent the intact predicted gene, and the dotted arrows represent partial predicted genes. Two aer mutants were created by inserting a gentamicin resistance cassette (accC1, solid triangle) into aer1 (to make strain K770) or a kanamycin resistance cassette (nptII, open triangle) into aer2 (to make strain K773). B, BamHI; Bs, BstEII; Bg, BglII; EI, EcoRI; EV, EcoRV; N, NdeI; No, NotI; S, SacI; Sa, SacII; Sm, SmaI; Sp, SphI. (B) Predicted protein domain architectures of Aer1 and Aer2 as defined by the SMART database (37, 52): PAS and PAC domains are for FAD binding and signal transduction (10, 49, 57), and the HAMP domain is for transmitting signals (5). T, transmembrane domain, which was further analyzed by the DAS program (16); MA, methyl-accepting chemotaxis-like domains, which are required for response to stimuli during bacterial taxis (24).

FIG. 2.

Cloned R. solanacearum Aer proteins can function as taxis transducers in E. coli. (A) Colony morphology of E. coli aer mutant UU1117 on soft-agar plate containing 40 mM succinate; (B) colony morphology of E. coli multiple MCP knockout BT3388 on soft-agar plate containing 10 mM glycerol. The strains harbor the following plasmid constructs: pTrc99A, empty vector as a negative control; pGH1, vector expressing E. coli Aer as a positive control; pTJYaer1, vector expressing R. solanacearum Aer1; and pTJYaer2, vector expressing R. solanacearum Aer2. The plates were photographed after incubation at 32°C for 24 h. The assays were replicated at least four times, and the images shown are typical.

FIG. 3.

Both aer1 and aer2 genes are required for aerotaxis in R. solanacearum. A modified nested chemotaxis well method was used to quantify R. solanacearum aerotaxis. Aerotactic ability is represented as the proportion of bacterial cells in the lower well that migrated through an 8-μm-pore-size filter into the upper well in response to an oxygen gradient. (A) R. solanacearum aer mutants had significantly reduced aerotactic ability; (B) The aer mutations were complemented with a functional copy of the relevant gene in trans. Each column represents the mean of three individual experiments with two replicates per treatment. The error bars represent the standard error of the mean. Columns with different letters above are significantly different according to the Fisher least significant difference test (P < 0.05).

FIG. 4.

Disease progress of R. solanacearum aer mutants on tomato. Unwounded 16-day-old tomato plants (cv. Bonny Best) were inoculated by pouring bacterial suspensions near the crown to a final concentration of about 3 × 10⁷ CFU/g of potting mix. Plants were rated daily on a disease index scale from 0 to 4 (see Materials and Methods for details). Each point represents the mean disease index of four individual experiments each containing 16 plants per treatment. For the time points marked with asterisks, wild-type strain K60 and aer1/aer2 mutant K774 are significantly different according to ANOVA (P < 0.05).

FIG. 5.

An R. solanacearum aer1/aer2 double mutant is impaired in rapid localization of tomato seedling roots. Aseptic tomato seedling roots were incubated with 5 ml of the different GFP-tagged R. solanacearum strains at a cell density of about 10⁷ CFU/ml. After 30 min at room temperature, the seedling roots were rinsed with sterile water, blotted dry on tissues, excised, and either directly examined under a laser scanning confocal microscope or ground and dilution plated on TZC medium. (A to C) Seedling root surfaces were visualized under the microscope with the indicated strain. Each strain constitutively expressed GFP, visible as a green color on the root surface. Assays were repeated three times; the images shown are typical results. (D) Number of bacterial CFU recovered from tomato seedling roots. Each column is the mean of three individual experiments with two replicates per treatment. The error bars represent the standard error of the mean. Columns with different letters are significantly different according to Fisher least significant difference test (P < 0.05).

FIG. 6.

R. solanacearum aer mutants overproduce biofilm on PVC surfaces. PVC microtiter plate biofilm assays were performed as previously described (46). (A) R. solanacearum formed biofilms on PVC surfaces, visible as purple crystal violet stains. The assays were repeated at least three times and photos shown are typical. (B) R. solanacearum biofilm formation was quantified by measuring A₅₃₀ of crystal violet-stained wells rinsed with ethanol. Each column is the mean of three individual experiments with two replicates per treatment. The error bars represent the standard error of the mean. Columns with different letters are significantly different according to the Fisher least significant difference test (P < 0.05).