Ralstonia solanacearum needs Flp pili for virulence on potato

Type IV pili are virulence factors in various bacteria. Several subclasses of type IV pili have been described according to the characteristics of the structural prepilin subunit. Although type IVa pili have been implicated in the virulence of Ralstonia solanacearum, type IVb pili have not previously been described in this plant pathogen. Here, we report the characterization of two distinct tad loci in the R. solanacearum genome. The tad genes encode functions necessary for biogenesis of the Flp subfamily of type IVb pili initially described for the periodontal pathogen Aggregatibacter actinomycetemcomitans. To determine the role of the tad loci in R. solanacearum virulence, we mutated the tadA2 gene located in the megaplasmid that encodes a predicted NTPase previously reported to function as the energizer for Flp pilus biogenesis. Characterization of the tadA2 mutant revealed that it was not growth impaired in vitro or in planta, produced wild-type levels of exopolysaccharide galactosamine, and exhibited swimming and twitching motility comparable with the wild-type strain. However, the tadA2 mutant was impaired in its ability to cause wilting of potato plants. This is the first report where type IVb pili in a phytopathogenic bacterium contribute significantly to plant pathogenesis.     

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

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.     

Ralstonia solanacearum colonization of tomato roots infected by Meloidogyne incognita

Bacterial wilt, caused by Ralstonia solanacearum, is one of the most serious diseases of tomato (Solanum lycopersicum). Concomitant infection of R. solanacearum and root‐knot nematode Meloidogyne incognita increases the severity of bacterial wilt in tomato, but the role of this nematode in disease complexes involving bacterial pathogens is not completely elucidated. Although root wounding by root‐knot nematode infection seems to play an important role, it might not entirely explain the increased susceptibility of plants to R. solanacearum. In the present study, green fluorescent protein (GFP)‐labelled R. solanacearum distribution was observed in the root systems of the tomato cultivar Momotaro preinoculated with root‐knot nematode or mock‐inoculated with tap water. Fluorescence microscopy revealed that GFP‐labelled R. solanacearum mainly colonized root‐knot nematode galls, and little or no green fluorescence was observed in nematode‐uninfected roots. These results suggest that the gall induced by the nematode is a suitable location for the growth of R. solanacearum. Thus, it is crucial to control both R. solanacearum and root‐knot nematode in tomato production fields to reduce bacterial wilt disease incidence and effects.     

Transposon mutagenesis reveals differential pathogenesis of Ralstonia solanacearum on tomato and Arabidopsis

Ralstonia solanacearum causes a deadly wilting disease on a wide range of crops. To elucidate pathogenesis of this bacterium in different host plants, we set out to identify R. solanacearum genes involved in pathogenesis by screening random transposon insertion mutants of a highly virulent strain, Pss190, on tomato and Arabidopsis thaliana. Mutants exhibiting various decreased virulence levels on these two hosts were identified. Sequence analysis showed that most, but not all, of the identified pathogenesis genes are conserved among distinct R. solanacearum strains. A few of the disrupted loci were not reported previously as being involved in R. solanacearum pathogenesis. Notably, a group of mutants exhibited differential pathogenesis on tomato and Arabidopsis. These results were confirmed by characterizing allelic mutants in one other R. solanacearum strain of the same phylotype. The significantly decreased mutants' colonization in Arabidopsis was found to be correlated with differential pathogenesis on these two plants. Differential requirement of virulence genes suggests adaptation of this bacterium in different host environments. Together, this study reveals commonalities and differences of R. solanacearum pathogenesis on single solanaceous and nonsolanaceous hosts, and provides important new insights into interactions between R. solanacearum and different host plants.     

Swimming motility, a virulence trait of Ralstonia solanacearum, is regulated by FlhDC and the plant host environment

Swimming motility allows the bacterial wilt pathogen Ralstonia solanacearum to efficiently invade and colonize host plants. However, the bacteria are essentially nonmotile once inside plant xylem vessels. To determine how and when motility genes are expressed, we cloned and mutated flhDC, which encodes a major regulator of flagellar biosynthesis and bacterial motility. An flhDC mutant was nonmotile and less virulent than its wild-type parent on both tomato and Arabidopsis; on Arabidopsis, the flhDC mutant also was less virulent than a nonmotile fliC flagellin mutant. Genes in the R. solanacearum motility regulon had strikingly different expression patterns in culture and in the plant. In culture, as expected, flhDC expression depended on PehSR, a regulator of early virulence factors; and, in turn, FlhDC was required for fliC (flagellin) expression. However, when bacteria grew in tomato plants, flhDC was expressed in both wild-type and pehR mutant backgrounds, although PehSR is necessary for motility both in culture and in planta. Both flhDC and pehSR were significantly induced in planta relative to expression levels in culture. Unexpectedly, the fliC gene was expressed in planta at cell densities where motile bacteria were not observed, as well as in a nonmotile flhDC mutant. Thus, expression of flhDC and flagellin itself are uncoupled from bacterial motility in the host environment, indicating that additional signals and regulatory circuits repress motility during plant pathogenesis.     

Identification of QTLs for Ralstonia solanacearum race 3-phylotype II resistance in tomato

Resistance against a Ralstonia solanacearum race 3-phylotype II strain JT516 was assessed in a F_2:3 and a population of inbred lines (RIL), both derived from a cross between L. esculentum cv. Hawaii 7996 (partially resistant) and L. pimpinellifolium WVa700 (susceptible). Resistance criteria used were the percentage of wilted plants to calculate the AUDPC value, and bacterial colonization scores in roots and stem (hypocotyl and epicotyl) assessed in two independent greenhouse experiments conducted during the cool and hot seasons in Réunion Island, France. Symptoms were more severe during the cool season trials. Heritability estimates in individual seasons ranged from 0.82 to 0.88, depending on resistance criterion. A set of 76 molecular markers was used for quantitative trait loci (QTL) mapping using the single- and composite- interval mapping methods, as well as ANOVA. Four QTLs, named Bwr- followed by a number indicating their map location, were identified. They explained from 3.2 to 29.8% of the phenotypic variation, depending on the resistance criterion and the season. A major QTL, Bwr-6, and a minor one, Bwr-3, were detected in each season for all resistance criteria. Both QTLs showed stronger effects in the hot season than in the cool one. Their role in resistance to R. solanacearum race 3-phylotype II was subsequently confirmed in the RIL population derived from the same cross. Two other QTLs, Bwr-4 and Bwr-8, with intermediate and minor effects, respectively, were only detected in the hot season, demonstrating that environmental factors may strongly influence the expression of resistance against the race 3-phylotype II strain JT516. These QTLs were compared with those detected in the RIL population against race 1-phylotype I strain JT519 as well as those detected in other previous studies in the same genetic background against other race 1-phylotype I and II strains. This comparison revealed the possible occurrence of some phylotype-specific resistance QTLs in Hawaii 7996.     

An exo-poly-alpha-D-galacturonosidase, PehB, is required for wild-type virulence of Ralstonia solanacearum.

Ralstonia solanacearum, which causes bacterial wilt disease of many plant species, produces several extracellular plant cell wall-degrading enzymes that are suspected virulence factors. These include a previously described endopolygalacturonase (PG), PehA, and two exo-PGs. A gene encoding one of the exo-PGs, pehB, was cloned from R. solanacearum K60. The DNA fragment specifying PehB contained a 2,103-bp open reading frame that encodes a protein of 74.2 kDa with a typical N-terminal signal sequence. The cloned pehB gene product cleaves polygalacturonic acid into digalacturonic acid units. The amino acid sequence of pehB resembles that of pehX, an exo-PG gene from Erwinia chrysanthemi, with 47.2% identity at the amino acid level. PehB also has limited similarity to plant exo-PGs from Zea mays and Arabidopsis thaliana. The chromosomal pehB genes in R. solanacearum wild-type strain K60 and in an endo-PG PehA- strain were replaced with an insertionally inactivated copy of pehB. The resulting mutants were deficient in the production of PehB and of both PehA and PehB, respectively. The pehB mutant was significantly less virulent than the wild-type strain in eggplant virulence assays using a soil inoculation method. However, the pehA mutant was even less virulent, and the pehA pehB double mutant was the least virulent of all. These results suggest that PehB is required for a wild-type level of virulence in R. solanacearum although its individual role in wilt disease development may be minor. Together with endo-PG PehA, however, PehB contributes substantially to the virulence of R. solanacearum.     

Ralstonia solanacearum virulence increased following large interstrain gene transfers by natural transformation

Horizontal gene transfer (HGT) is a major driving force of evolution and is also likely to play an important role in the threatening emergence of novel pathogens, especially if it involves distantly related strains with substantially different pathogenicity. In this study, the impact of natural transformation on pathogenicity in six strains belonging to the four phylotypes of the plant-pathogenic bacterium Ralstonia solanacearum was investigated. The study focused on genomic regions that vary between donor and recipient strains and that carry genes involved in pathogenicity such as type III effectors. First, strains from R. solanacearum species complex were naturally transformed with heterologous genomic DNA. Transferred DNA regions were then determined by comparative genomic hybridization and polymerase chain reaction sequencing. We identified three transformant strains that acquired large DNA regions of up to 80 kb. In one case, strain Psi07 (phylotype IV tomato isolate) acquired 39.4 kb from GMI1000 (phylotype I tomato isolate). Investigations revealed that i) 24.4 kb of the acquired region contained 20 new genes, ii) an allelic exchange of 12 genes occurred, and iii) 27 genes (33.4 kb) formerly present in Psi07 were lost. Virulence tests with the three transformants revealed a significant increase in the aggressiveness of BCG20 over its Psi07 parent on tomato. These findings demonstrate the potential importance of HGT in the pathogenic evolution of R. solanacearum strains and open new avenues for studying pathogen emergence.     

Using the Ralstonia solanacearum Tat secretome to identify bacterial wilt virulence factors

To identify secreted virulence factors involved in bacterial wilt disease caused by the phytopathogen Ralstonia solanacearum, we mutated tatC, a key component of the twin-arginine translocation (Tat) secretion system. The R. solanacearum tatC mutation was pleiotropic; its phenotypes included defects in cell division, nitrate utilization, polygalacturonase activity, membrane stability, and growth in plant tissue. Bioinformatic analysis of the R. solanacearum strain GMI1000 genome predicted that this pathogen secretes 70 proteins via the Tat system. The R. solanacearum tatC strain was severely attenuated in its ability to cause disease, killing just over 50% of tomato plants in a naturalistic soil soak assay where the wild-type parent killed 100% of the plants. This result suggested that elements of the Tat secretome may be novel bacterial wilt virulence factors. To identify contributors to R. solanacearum virulence, we cloned and mutated three genes whose products are predicted to be secreted by the Tat system: RSp1521, encoding a predicted AcvB-like protein, and two genes, RSc1651 and RSp1575, that were identified as upregulated in planta by an in vivo expression technology screen. The RSc1651 mutant had wild-type virulence on tomato plants. However, mutants lacking either RSp1521, which appears to be involved in acid tolerance, or RSp1575, which encodes a possible amino acid binding protein, were significantly reduced in virulence on tomato plants. Additional bacterial wilt virulence factors may be found in the Tat secretome.     

Effects of high grafting on tomato plants infected by Meloidogyne incognita and Ralstonia solanacearum

The root‐knot nematode Meloidogyne incognita is known to increase the severity of bacterial wilt in many solanaceous crops. In Japan, several bacterial wilt‐resistant rootstocks that have the M. incognita resistance (Mi) gene in their genome have been developed for tomatoes. In this study, we aimed to examine whether the presence of Mi gene‐breaking M. incognita population affects the development of bacterial wilt in bacterial‐wilt‐resistant tomato rootstocks with Mi in their genetic background. We also aimed to examine the possibility of using high‐grafted tomatoes to control bacterial wilt in plants infected by M. incognita. Our results indicate that the resistance to bacterial wilt was easy to break in usual‐grafted tomato plants infected with M. incognita and that M. incognita enhanced the vertical movement of Ralstonia solanacearum in the bacterial‐wilt‐resistant tomato rootstocks. In addition, our results suggest that high grafting led to significantly less wilting in the plants infected by M. incognita than did usual grafting.     

The vascular plant‐pathogenic bacterium Ralstonia solanacearum produces biofilms required for its virulence on the surfaces of tomato cells adjacent to intercellular spaces

The mechanism of colonization of intercellular spaces by the soil‐borne and vascular plant‐pathogenic bacterium Ralstonia solanacearum strain OE1‐1 after invasion into host plants remains unclear. To analyse the behaviour of OE1‐1 cells in intercellular spaces, tomato leaves with the lower epidermis layers excised after infiltration with OE1‐1 were observed under a scanning electron microscope. OE1‐1 cells formed microcolonies on the surfaces of tomato cells adjacent to intercellular spaces, and then aggregated surrounded by an extracellular matrix, forming mature biofilm structures. Furthermore, OE1‐1 cells produced mushroom‐type biofilms when incubated in fluids of apoplasts including intercellular spaces, but not xylem fluids from tomato plants. This is the first report of biofilm formation by R. solanacearum on host plant cells after invasion into intercellular spaces and mushroom‐type biofilms produced by R. solanacearum in vitro. Sugar application led to enhanced biofilm formation by OE1‐1. Mutation of lecM encoding a lectin, RS‐IIL, which reportedly exhibits affinity for these sugars, led to a significant decrease in biofilm formation. Colonization in intercellular spaces was significantly decreased in the lecM mutant, leading to a loss of virulence on tomato plants. Complementation of the lecM mutant with native lecM resulted in the recovery of mushroom‐type biofilms and virulence on tomato plants. Together, our findings indicate that OE1‐1 produces mature biofilms on the surfaces of tomato cells after invasion into intercellular spaces. RS‐IIL may contribute to biofilm formation by OE1‐1, which is required for OE1‐1 virulence.     

Improved biovar test for Ralstonia solanacearum

Race 3, biovar 2 strains of Ralstonia solanacearum are quarantined pathogens in Europe and Canada and Select Agent pathogens in the United States. The biovar classification of R. solanacearum strains is based on their biochemical abilities to utilize a carbohydrate panel. The standard biovar test uses bromothymol blue as a pH indicator in 15 ml culture tubes containing 3 to 5 ml of test media, and takes weeks to complete at 24 or 28 °C. We improved the biovar test by using phenol red as a pH indicator that changes color at a higher pH when a carbohydrate is utilized. We also conducted the test at 32 °C in 0.2 ml of 8-tube strips that reduced the medium needed by at least 20 fold. Using the improved test, biovars of R. solanacearum strains can be determined in 4 days when a panel of seven carbohydrates is used including glucose, trehalose, mannitol, sorbitol, dulcitol, maltose and cellobiose. To differentiate biovars 1, 2, 3 and 4, the test can be further simplified and completed in 3 days using a panel of four carbohydrates containing glucose, trehalose, maltose and dulcitol, significantly saving money, space and time.     

Necessity of OxyR for the hydrogen peroxide stress response and full virulence in Ralstonia solanacearum

The plant pathogen Ralstonia solanacearum, which causes bacterial wilt disease, is exposed to reactive oxygen species (ROS) during tomato infection and expresses diverse oxidative stress response (OSR) genes during midstage disease on tomato. The R. solanacearum genome predicts that the bacterium produces multiple and redundant ROS-scavenging enzymes but only one known oxidative stress response regulator, OxyR. An R. solanacearum oxyR mutant had no detectable catalase activity, did not grow in the presence of 250 μM hydrogen peroxide, and grew poorly in the oxidative environment of solid rich media. This phenotype was rescued by the addition of exogenous catalase, suggesting that oxyR is essential for the hydrogen peroxide stress response. Unexpectedly, the oxyR mutant strain grew better than the wild type in the presence of the superoxide generator paraquat. Gene expression studies indicated that katE, kaG, ahpC1, grxC, and oxyR itself were each differentially expressed in the oxyR mutant background and in response to hydrogen peroxide, suggesting that oxyR is necessary for hydrogen peroxide-inducible gene expression. Additional OSR genes were differentially regulated in response to hydrogen peroxide alone. The virulence of the oxyR mutant strain was significantly reduced in both tomato and tobacco host plants, demonstrating that R. solanacearum is exposed to inhibitory concentrations of ROS in planta and that OxyR-mediated responses to ROS during plant pathogenesis are important for R. solanacearum host adaptation and virulence.     

Chemotaxis is required for virulence and competitive fitness of the bacterial wilt pathogen Ralstonia solanacearum

Ralstonia solanacearum, a soilborne plant pathogen of considerable economic importance, invades host plant roots from the soil. Qualitative and quantitative chemotaxis assays revealed that this bacterium is specifically attracted to diverse amino acids and organic acids, and especially to root exudates from the host plant tomato. Exudates from rice, a nonhost plant, were less attractive. Eight different strains from this heterogeneous species complex varied significantly in their attraction to a panel of carbohydrate stimuli, raising the possibility that chemotactic responses may be differentially selected traits that confer adaptation to various hosts or ecological conditions. Previous studies found that an aflagellate mutant lacking swimming motility is significantly reduced in virulence, but the role of directed motility mediated by the chemotaxis system was not known. Two site-directed R. solanacearum mutants lacking either CheA or CheW, which are core chemotaxis signal transduction proteins, were completely nonchemotactic but retained normal swimming motility. In biologically realistic soil soak virulence assays on tomato plants, both nonchemotactic mutants had significantly reduced virulence indistinguishable from that of a nonmotile mutant, demonstrating that directed motility, not simply random motion, is required for full virulence. In contrast, nontactic strains were as virulent as the wild-type strain was when bacteria were introduced directly into the plant stem through a cut petiole, indicating that taxis makes its contribution to virulence in the early stages of host invasion and colonization. When inoculated individually by soaking the soil, both nontactic mutants reached the same population sizes as the wild type did in the stems of tomato plants just beginning to wilt. However, when tomato plants were coinoculated with a 1:1 mixture of a nontactic mutant and its wild-type parent, the wild-type strain outcompeted both nontactic mutants by 100-fold. Together, these results indicate that chemotaxis is an important trait for virulence and pathogenic fitness in this plant pathogen.     

Unravelling physiological signatures of tomato bacterial wilt and xylem metabolites exploited by Ralstonia solanacearum

The plant pathogen Ralstonia solanacearum uses plant resources to intensely proliferate in xylem vessels and provoke plant wilting. We combined automatic phenotyping and tissue/xylem quantitative metabolomics of infected tomato plants to decipher the dynamics of bacterial wilt. Daily acquisition of physiological parameters such as transpiration and growth were performed. Measurements allowed us to identify a tipping point in bacterial wilt dynamics. At this tipping point, the reached bacterial density brutally disrupts plant physiology and rapidly induces its death. We compared the metabolic and physiological signatures of the infection with drought stress, and found that similar changes occur. Quantitative dynamics of xylem content enabled us to identify glutamine (and asparagine) as primary resources R. solanacearum consumed during its colonization phase. An abundant production of putrescine was also observed during the infection process and was strongly correlated with in planta bacterial growth. Dynamic profiling of xylem metabolites confirmed that glutamine is the favoured substrate of R. solanacearum. On the other hand, a triple mutant strain unable to metabolize glucose, sucrose and fructose appears to be only weakly reduced for in planta growth and pathogenicity.