Twitching and Swimming Motility Play a Role in Ralstonia solanacearum Pathogenicity

Abstract

Ralstonia solanacearum is a bacterial plant pathogen causing important economic losses worldwide. In addition to the polar flagella responsible for swimming motility, this pathogen produces type IV pili (TFP) that govern twitching motility, a flagellum-independent movement on solid surfaces. The implication of chemotaxis in plant colonization, through the control flagellar rotation by the proteins CheW and CheA, has been previously reported in R. solanacearum In this work, we have identified in this bacterium homologues of the Pseudomonas aeruginosapilI and chpA genes, suggested to play roles in TFP-associated motility analogous to those played by the cheW and cheA genes, respectively. We demonstrate that R. solanacearum strains with a deletion of the pilI or the chpA coding region show normal swimming and chemotaxis but altered biofilm formation and reduced twitching motility, transformation efficiency, and root attachment. Furthermore, these mutants displayed wild-type growth in planta and impaired virulence on tomato plants after soil-drench inoculations but not when directly applied to the xylem. Comparison with deletion mutants for pilA and fliC-encoding the major pilin and flagellin subunits, respectively-showed that both twitching and swimming are required for plant colonization and full virulence. This work proves for the first time the functionality of a pilus-mediated pathway encoded by pil-chp genes in R. solanacearum, demonstrating that pilI and chpA genes are bona fide motility regulators controlling twitching motility and its three related phenotypes: virulence, natural transformation, and biofilm formation.IMPORTANCE Twitching and swimming are two bacterial movements governed by pili and flagella. The present work identifies for the first time in the Gram-negative plant pathogen Ralstonia solanacearum a pilus-mediated chemotaxis pathway analogous to that governing flagellum-mediated chemotaxis. We show that regulatory genes in this pathway control all of the phenotypes related to pili, including twitching motility, natural transformation, and biofilm formation, and are also directly implicated in virulence, mainly during the first steps of the plant infection. Our results show that pili have a higher impact than flagella on the interaction of R. solanacearum with tomato plants and reveal new types of cross-talk between the swimming and twitching motility phenotypes: enhanced swimming in bacteria lacking pili and a role for the flagellum in root attachment.

Keywords: Ralstonia solanacearum; chpA; fliC; pilA; pilI.

FIG 1

Characterization of the pil-chp operon. (A) Representation of flagellin-dependent (left) and pilus-dependent (right) pathways based on protein homology data from Sampedro et al. (11). (B) Schematic diagram of the pil-chp operon gene cluster in the R. solanacearum GMI1000 genome. Small arrows represent the oligonucleotides used in the RT-PCR. Spacings between protein-coding sequences in the pil-chp operon are as follows: pilG-pilH 31 bp, pilH-pilI 58 bp, pilI-pilJ 245 bp, pilJ-chpA 79 bp. (C) RT-PCRs of the indicated pil-chp intergenic regions. Each primer pair (Table S1) was used to prepare a PCR mixture with cDNA, RNA, or DNA from the GMI1000 strain as a template. V Ladder NzyTech was used as the DNA marker (M).

FIG 2

Motility assays. (A) Representative optical microscope images (×100 magnification) of three independent twitching motility assays. (B) Representative images of three independent swimming motility assays. (C) Representation of the swimming halo diameters measured in three independent assays with three replicates each. Error bars represent standard deviations of the means, and significant (P < 0.05) differences from the R. solanacearum WT strain are represented as single or double asterisks for a bacterial halo of smaller or larger diameter, respectively.

FIG 3

Chemotaxis capillarity assays. Data represent fold change (CFU) between viable bacteria counted in capillaries containing chemoattractant CA (Casamino Acids) divided by the CFU counted in control capillary CB (chemotaxis buffer). Error bars represent standard deviations of the means of results from five replicates per strain, and the asterisk denotes a significant (P < 0.05) difference from the R. solanacearum WT strain. The assay was performed three times. The results of a representative experiment are shown.

FIG 4

Biofilm and root attachment quantification. (A) Biofilm assay in which y-axis data represent biofilm absorbance (OD₅₈₀) divided by the biomass (OD₆₀₀). The error bars represent standard deviations of the means of results from 16 replicates per strain. Significant (P < 0.05) differences from the R. solanacearum WT strain are represented as single or double asterisks for lower or higher absorbance ratio values, respectively. The assay was performed three times. The results of a representative experiment are shown. (B) Representative root attachment assay showing means of logarithms of counts of viable bacteria (CFU). Error bars represent standard errors of the means of data from five 1-week-old tomato roots per assay, and asterisks denote significant (P < 0.05) differences from the R. solanacearum WT strain. The assay was performed three times. The results of a representative experiment are shown.

FIG 5

Drenching assays. (A) Disease index scaled from 0 (no wilt) to 4 (death), with levels measured daily after soil soaking of 4-week-old tomato plants by the use of a naturalistic inoculation method. Error bars represent standard errors of the means of results from 20 replicates per strain. According to their wilting reduction (P < 0.05), strains are classified in four groups (labeled a through d). The assay was performed three times. The results of a representative experiment are shown. (B) Logarithm of counts of viable bacteria (CFU per milliliter) after soil soaking of 4-week-old tomato plants by the use of a naturalistic soil soak inoculation method at 4, 8, and 12 days postinoculation. Error bars represent standard errors of the means of results from 20 replicates per strain. Asterisks denote significant (P < 0.05) differences from the R. solanacearum WT strain. The assay was performed three times. The results of a representative experiment are shown.

FIG 6

Petiole inoculation assays. (A) Disease index scaled from 0 (no wilt) to 4 (death), measured daily in 4-week-old tomato plants after the use of a direct inoculation method. Error bars represent standard errors of the means of results from 20 replicates per strain. According to statistically significant differences (P < 0.05), strains were classified in two groups (labeled a and b). The assay was performed three times. The results of a representative experiment are shown. (B) Logarithms of viable bacteria (CFU per milliliter) of the indicated R. solanacearum strains counted by the use of a direct inoculation method using 4-week-old tomato plants at 3, 6, and 9 dpi. Error bars represent standard errors of the means of results from 20 replicates per strain, and asterisks denote significant (P < 0.05) differences from the R. solanacearum WT strain. The assay was performed three times. The results of a representative experiment are shown.

FIG 7

Bacterial spread in plant tissue. Luminescence detection of the indicated R. solanacearum strains prepared by the use of a direct inoculation method was performed using 4-week-old tomato plants at 3 and 6 dpi. x-axis data represent means of luminescence (RLU/s) data from 10 replicates per strain. Error bars represent standard errors of the means, and asterisks denote significant (P < 0.05) differences from the R. solanacearum WT strain. The assay was performed three times. The results of a representative experiment are shown.