Quantitative Immunofluorescence of Regulated eps Gene Expression in Single Cells of Ralstonia solanacearum

Ralstonia solanacearum, a phytopathogenic bacterium, uses an environmentally sensitive and complex regulatory network to control expression of multiple virulence genes. Part of this network is an unusual autoregulatory system that produces and senses 3-hydroxypalmitic acid methyl ester. In culture, this autoregulatory system ensures that expression of virulence genes, such as those of the eps operon encoding biosynthesis of the acidic extracellular polysaccharide, occurs only at high cell density (>10⁷ cells/ml). To determine if regulation follows a similar pattern within tomato plants, we first developed a quantitative immunofluorescence (QIF) method that measures the relative amount of a target protein within individual bacterial cells. For R. solanacearum, QIF was used to determine the amount of β-galactosidase protein within wild-type cells containing a stable eps-lacZ reporter allele. When cultured cells were examined to test the method, QIF accurately detected both low and high levels of eps gene expression. QIF analysis of R. solanacearum cells recovered from stems of infected tomato plants showed that expression of eps during pathogenesis was similar to that in culture. These results suggest that there are no special signals or conditions within plants that override or short-circuit the regulatory processes observed in R. solanacearum in culture. Because QIF is a robust, relatively simple procedure that uses generally accessible equipment, it should be useful in many situations where gene expression in single bacterial cells must be determined.     

Ralstonia solanacearum extracellular polysaccharide is a specific elicitor of defense responses in wilt-resistant tomato plants

Ralstonia solanacearum, which causes bacterial wilt of diverse plants, produces copious extracellular polysaccharide (EPS), a major virulence factor. The function of EPS in wilt disease is uncertain. Leading hypotheses are that EPS physically obstructs plant water transport, or that EPS cloaks the bacterium from host plant recognition and subsequent defense. Tomato plants infected with R. solanacearum race 3 biovar 2 strain UW551 and tropical strain GMI1000 upregulated genes in both the ethylene (ET) and salicylic acid (SA) defense signal transduction pathways. The horizontally wilt-resistant tomato line Hawaii7996 activated expression of these defense genes faster and to a greater degree in response to R. solanacearum infection than did susceptible cultivar Bonny Best. However, EPS played different roles in resistant and susceptible host responses to R. solanacearum. In susceptible plants the wild-type and eps(-) mutant strains induced generally similar defense responses. But in resistant Hawaii7996 tomato plants, the wild-type pathogens induced significantly greater defense responses than the eps(-) mutants, suggesting that the resistant host recognizes R. solanacearum EPS. Consistent with this idea, purified EPS triggered significant SA pathway defense gene expression in resistant, but not in susceptible, tomato plants. In addition, the eps(-) mutant triggered noticeably less production of defense-associated reactive oxygen species in resistant tomato stems and leaves, despite attaining similar cell densities in planta. Collectively, these data suggest that bacterial wilt-resistant plants can specifically recognize EPS from R. solanacearum.     

Ralstonia solanacearum genes induced during growth in tomato: an inside view of bacterial wilt

The phytopathogen Ralstonia solanacearum has over 5000 genes, many of which probably facilitate bacterial wilt disease development. Using in vivo expression technology (IVET), we screened a library of 133 200 R. solanacearum strain K60 promoter fusions and isolated approximately 900 fusions expressed during bacterial growth in tomato plants. Sequence analysis of 307 fusions revealed 153 unique in planta-expressed (ipx) genes. These genes included seven previously identified virulence genes (pehR, vsrB, vsrD, rpoS, hrcC, pme and gspK) as well as seven additional putative virulence factors. A significant number of ipx genes may reflect adaptation to the host xylem environment; 19.6%ipx genes are predicted to encode proteins with metabolic and/or transport functions, and 9.8%ipx genes encode proteins possibly involved in stress responses. Many ipx genes (18%) encode putative transmembrane proteins. A majority of ipx genes isolated encode proteins of unknown function, and 13% were unique to R. solanacearum. The ipx genes were variably induced in planta; beta-glucuronidase reporter gene expression analysis of a subset of 44 ipx fusions revealed that in planta expression levels were between two- and 37-fold higher than in culture. The expression of many ipx genes was subject to known R. solanacearum virulence regulators. Of 32 fusions tested, 28 were affected by at least one virulence regulator; several fusions were controlled by multiple regulators. Two ipx fusion strains isolated in this screen were reduced in virulence on tomato, indicating that gene(s) important for bacterial wilt pathogenesis were interrupted by the IVET insertion; mutations in other ipx genes are necessary to determine their roles in virulence and in planta growth. Collectively, this profile of ipx genes suggests that in its host, R. solanacearum confronts and overcomes a stressful and nutrient-poor environment.     

Identification of 3-hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum

Expression of virulence genes in Ralstonia solanacearum , a phytopathogenic bacterium, is controlled by a complex regulatory network that integrates multiple signal inputs. Production of several virulence determinants is co-ordinately reduced by inactivation of phcB , but is restored by growth in the presence of a volatile extracellular factor (VEF) produced by wild-type strains of R. solanacearum . The VEF was purified from spent culture broth by distillation, solvent extraction, and liquid chromatography. Gas chromatography and mass spectroscopy identified 3-hydroxypalmitic acid methyl ester (3-OH PAME) as the major component in the single peak of VEF activity. Authentic 3-OH PAME and the purified VEF were active at ≤1 nM, and had nearly equivalent specific activities for stimulating the expression of eps (the biosynthetic locus for extracellular polysaccharide) in a phcB mutant. Authentic 3-OH PAME also increased the production of three virulence factors by a phcB mutant over 20-fold to wild-type levels, restored normal cell density-associated expression of eps and increased expression of eps when delivered via the vapour phase. Reanalysis of the PhcB amino acid sequence suggested that it is a small-molecule S -adenosylmethionine-dependent methyltransferase, which might catalyse synthesis of 3-OH PAME from a naturally occurring fatty acid. Biologically active concentrations of extracellular 3-OH PAME were detected before the onset of eps expression, suggesting that it is an intercellular signal that autoregulates virulence gene expression in wild-type R. solanacearum . Other than acyl-homoserine lactones, 3-OH PAME is the only endogenous fatty acid derivative shown to be an autoregulator and may be the first example of a new family of compounds that can mediate long-distance intercellular communication.     

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.     

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.     

Ralstonia solanacearum needs motility for invasive virulence on tomato

Ralstonia solanacearum, a widely distributed and economically important plant pathogen, invades the roots of diverse plant hosts from the soil and aggressively colonizes the xylem vessels, causing a lethal wilting known as bacterial wilt disease. By examining bacteria from the xylem vessels of infected plants, we found that R. solanacearum is essentially nonmotile in planta, although it can be highly motile in culture. To determine the role of pathogen motility in this disease, we cloned, characterized, and mutated two genes in the R. solanacearum flagellar biosynthetic pathway. The genes for flagellin, the subunit of the flagellar filament (fliC), and for the flagellar motor switch protein (fliM) were isolated based on their resemblance to these proteins in other bacteria. As is typical for flagellins, the predicted FliC protein had well-conserved N- and C-terminal regions, separated by a divergent central domain. The predicted R. solanacearum FliM closely resembled motor switch proteins from other proteobacteria. Chromosomal mutants lacking fliC or fliM were created by replacing the genes with marked interrupted constructs. Since fliM is embedded in the fliLMNOPQR operon, the aphA cassette was used to make a nonpolar fliM mutation. Both mutants were completely nonmotile on soft agar plates, in minimal broth, and in tomato plants. The fliC mutant lacked flagella altogether; moreover, sheared-cell protein preparations from the fliC mutant lacked a 30-kDa band corresponding to flagellin. The fliM mutant was usually aflagellate, but about 10% of cells had abnormal truncated flagella. In a biologically representative soil-soak inoculation virulence assay, both nonmotile mutants were significantly reduced in the ability to cause disease on tomato plants. However, the fliC mutant had wild-type virulence when it was inoculated directly onto cut tomato petioles, an inoculation method that did not require bacteria to enter the intact host from the soil. These results suggest that swimming motility makes its most important contribution to bacterial wilt virulence in the early stages of host plant invasion and colonization.     

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.     

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.     

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.     

Inventory and functional analysis of the large Hrp regulon in Ralstonia solanacearum: identification of novel effector proteins translocated to plant host cells through the type III secretion system

The ability of Ralstonia solanacearum strain GMI1000 to cause disease on a wide range of host plants (including most Solanaceae and Arabidopsis thaliana) depends on genes activated by the regulatory gene hrpB. HrpB controls the expression of the type III secretion system (TTSS) and pathogenicity effectors transiting through this pathway. In order to establish the complete repertoire of TTSS-dependent effectors belonging to the Hrp regulon and to start their functional analysis, we developed a rapid method for insertional mutagenesis, which was used to monitor the expression of 71 candidate genes and disrupt 56 of them. This analysis yielded a total of 48 novel hrpB-regulated genes. Using the Bordetella pertussis calmodulin-dependent adenylate cyclase reporter fusion system, we provide direct biochemical evidence that five R. solanacearum effector proteins are translocated into plant host cells through the TTSS. Among these novel TTSS effectors, RipA and RipG both belong to multigenic families, RipG defining a novel class of leucine-rich-repeats harbouring proteins. The members of these multigenic families are differentially regulated, being composed of genes expressed in either an hrpB-dependent or an hrpB-independent manner. Pathogenicity assays of the 56 mutant strains on two host plants indicate that, with two exceptions, mutations in individual effectors have no effect on virulence, a probable consequence of genetic and functional redundancy. This large repertoire of HrpB-regulated genes, which comprises > 20 probable TTSS effector genes with no counterparts in other bacterial species, represents an important step towards a full-genome understanding of R. solanacearum virulence.     

The hrpB and hrpG regulatory genes of Ralstonia solanacearum are required for different stages of the tomato root infection process

hrp genes, encoding type III secretion machinery, have been shown to be key determinants for pathogenicity in the vascular phytopathogenic bacterium Ralstonia solanacearum GMI1000. Here, we show phenotypes of R. solanacearum mutant strains disrupted in the prhJ, hrpG, or hrpB regulatory genes with respect to root infection and vascular colonization in tomato plants. Tests of bacterial colonization and enumeration in tomato plants, together with microscopic observations of tomato root sections, revealed that these strains display different phenotypes in planta. The phenotype of a prhJ mutant resembles that of the wild-type strain. An hrpB mutant shows reduced infection, colonization, and multiplication ability in planta, and induces a defense reaction similar to a vascular hypersensitive response at one protoxylem pole of invaded plants. In contrast, the hrpG mutant exhibited a wild-type level of infection at secondary root axils, but the ability of the infecting bacteria to penetrate into the vascular cylinder was significantly impaired. This indicates that bacterial multiplication at root infection sites and transit through the endodermis constitute critical stages in the infection process, in which hrpB and hrpG genes are involved. Moreover, our results suggest that the hrpG gene might control, in addition to hrp genes, other functions required for vascular colonization.     

Two host-induced Ralstonia solanacearum genes, acrA and dinF, encode multidrug efflux pumps and contribute to bacterial wilt virulence

Multidrug efflux pumps (MDRs) are hypothesized to protect pathogenic bacteria from toxic host defense compounds. We created mutations in the Ralstonia solanacearum acrA and dinF genes, which encode putative MDRs in the broad-host-range plant pathogen. Both mutations reduced the ability of R. solanacearum to grow in the presence of various toxic compounds, including antibiotics, phytoalexins, and detergents. Both acrAB and dinF mutants were significantly less virulent on the tomato plant than the wild-type strain. Complementation restored near-wild-type levels of virulence to both mutants. Addition of either dinF or acrAB to Escherichia coli MDR mutants KAM3 and KAM32 restored the resistance of these strains to several toxins, demonstrating that the R. solanacearum genes can function heterologously to complement known MDR mutations. Toxic and DNA-damaging compounds induced expression of acrA and dinF, as did growth in both susceptible and resistant tomato plants. Carbon limitation also increased expression of acrA and dinF, while the stress-related sigma factor RpoS was required at a high cell density (>10(7) CFU/ml) to obtain wild-type levels of acrA expression both in minimal medium and in planta. The type III secretion system regulator HrpB negatively regulated dinF expression in culture at high cell densities. Together, these results show that acrAB and dinF encode MDRs in R. solanacearum and that they contribute to the overall aggressiveness of this phytopathogen, probably by protecting the bacterium from the toxic effects of host antimicrobial compounds.     

Mutations in the lrpE gene of Ralstonia solanacearum affects Hrp pili production and virulence

The Ralstonia solanacearum hrpB-regulated gene lrpE (hpx5/brg24) encodes a PopC-like leucine-rich repeat (LRR) protein that carries 11 tandem LRR in the central region. Defects in the lrpE gene slightly reduced the virulence of R. solanacearum on host plants and changed the bacterial morphology leading to the formation of large aggregates in a minimal medium. The aggregation in the deltalrpE background required the presence of a functional Hrp type III secretion system. In wild-type R. solanacearum, Hrp pili disappeared from the bacterial surface at the end of the exponential growth phase, when the pili form into long bundles. However, even in the late growth phase, bundled Hrp pili were still observed on the cell surface of the deltalrpE mutant. Such bundles were entangled and anchored the mutant cells in the aggregates. In contrast to PopC, LrpE accumulated in bacterial cells and did not translocate into plant cells as an effector protein. The expression levels of hrp genes increased three- to fivefold in the deltalrpE background compared with those in the wild type. We propose that LrpE may negatively regulate the production of Hrp pili on the cell surface of R. solanacearum to disperse bacterial cells from aggregates. In turn, dispersal may contribute to the movement of the pathogen in the plant vascular system and, as a consequence, the pathogenicity of R. solanacearum.     

The awr gene family encodes a novel class of Ralstonia solanacearum type III effectors displaying virulence and avirulence activities

We present here the characterization of a new gene family, awr, found in all sequenced Ralstonia solanacearum strains and in other bacterial pathogens. We demonstrate that the five paralogues in strain GMI1000 encode type III-secreted effectors and that deletion of all awr genes severely impairs its capacity to multiply in natural host plants. Complementation studies show that the AWR (alanine-tryptophan-arginine tryad) effectors display some functional redundancy, although AWR2 is the major contributor to virulence. In contrast, the strain devoid of all awr genes (Δawr1-5) exhibits enhanced pathogenicity on Arabidopsis plants. A gain-of-function approach expressing AWR in Pseudomonas syringae pv. tomato DC3000 proves that this is likely due to effector recognition, because AWR5 and AWR4 restrict growth of this bacterium in Arabidopsis. Transient overexpression of AWR in nonhost tobacco species caused macroscopic cell death to varying extents, which, in the case of AWR5, shows characteristics of a typical hypersensitive response. Our work demonstrates that AWR, which show no similarity to any protein with known function, can specify either virulence or avirulence in the interaction of R. solanacearum with its plant hosts.