Using the Ralstonia solanacearum Tat Secretome To Identify Bacterial Wilt Virulence Factors

ABSTRACT 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.

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Ralstonia solanacearum Needs Motility for Invasive Virulence on Tomato

Journal of Bacteriology ◽

10.1128/jb.183.12.3597-3605.2001 ◽

2001 ◽

Vol 183 (12) ◽

pp. 3597-3605 ◽

Cited By ~ 201

Author(s):

Julie Tans-Kersten ◽

Huayu Huang ◽

Caitilyn Allen

Keyword(s):

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Soft Agar ◽

Cell Protein ◽

In Planta ◽

Tomato Plants ◽

Bacterial Wilt Disease ◽

Virulence Assay ◽

Flagellar Filament

ABSTRACT 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 thatR. 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 lackingfliC or fliM were created by replacing the genes with marked interrupted constructs. Since fliM is embedded in the fliLMNOPQR operon, the aphAcassette 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.

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Role of Extracellular Polysaccharide and Endoglucanase in Root Invasion and Colonization of Tomato Plants by Ralstonia solanacearum

Phytopathology ◽

10.1094/phyto.1997.87.12.1264 ◽

1997 ◽

Vol 87 (12) ◽

pp. 1264-1271 ◽

Cited By ~ 125

Author(s):

Elke Saile ◽

Jeff A. McGarvey ◽

Mark A. Schell ◽

Timothy P. Denny

Keyword(s):

Ralstonia Solanacearum ◽

Extracellular Polysaccharide ◽

Wild Type ◽

In Planta ◽

Tomato Plants ◽

Potted Plants ◽

Stem Infection ◽

Soil Infestation ◽

Plant Stem

Ralstonia solanacearum is a soilborne plant pathogen that normally invades hosts through their roots and then systemically colonizes aerial tissues. Previous research using wounded stem infection found that the major factor in causing wilt symptoms was the high-molecular-mass acidic extracellular polysaccharide (EPS I), but the β-1,4-endoglucanase (EG) also contributes to virulence. We investigated the importance of EPS I and EG for invasion and colonization of tomato by infesting soil of 4-week-old potted plants with either a wild-type derivative or genetically well-defined mutants lacking EPS I, EG, or EPS I and EG. Bacteria of all strains were recovered from surface-disinfested roots and hypocotyls as soon as 4 h after inoculation; that bacteria were present internally was confirmed using immunofluorescence microscopy. However, the EPS-minus mutants did not colonize stems as rapidly as the wild type and the EG-minus mutant. Inoculations of wounded petioles also showed that, even though the mutants multiplied as well as the wild type in planta, EPS-minus strains did not spread as well throughout the plant stem. We conclude that poor colonization of stems by EPS-minus strains after petiole inoculation or soil infestation is due to reduced bacterial movement within plant stem tissues.

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Ralstonia solanacearum Requires PopS, an Ancient AvrE-Family Effector, for Virulence and To Overcome Salicylic Acid-Mediated Defenses during Tomato Pathogenesis

mBio ◽

10.1128/mbio.00875-13 ◽

2013 ◽

Vol 4 (6) ◽

Cited By ~ 27

Author(s):

Jonathan M. Jacobs ◽

Annett Milling ◽

Raka M. Mitra ◽

Clifford S. Hogan ◽

Florent Ailloud ◽

...

Keyword(s):

Salicylic Acid ◽

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Species Complex ◽

Plant Defenses ◽

Natural Infection ◽

Wild Type ◽

Tomato Plants ◽

Content Type ◽

Type Iii

ABSTRACTDuring bacterial wilt of tomato, the plant pathogen Ralstonia solanacearum upregulates expression ofpopS, which encodes a type III-secreted effector in the AvrE family. PopS is a core effector present in all sequenced strains in theR. solanacearumspecies complex. The phylogeny ofpopSmirrors that of the species complex as a whole, suggesting that this is an ancient, vertically inherited effector needed for association with plants. ApopSmutant ofR. solanacearumUW551 had reduced virulence on agriculturally importantSolanumspp., including potato and tomato plants. However, thepopSmutant had wild-type virulence on a weed host,Solanum dulcamara, suggesting that some species can avoid the effects of PopS. ThepopSmutant was also significantly delayed in colonization of tomato stems compared to the wild type. Some AvrE-type effectors from gammaproteobacteria suppress salicylic acid (SA)-mediated plant defenses, suggesting that PopS, a betaproteobacterial ortholog, has a similar function. Indeed, thepopSmutant induced significantly higher expression of tomato SA-triggered pathogenesis-related (PR) genes than the wild type. Further, pretreatment of roots with SA exacerbated thepopSmutant virulence defect. Finally, thepopSmutant had no colonization defect on SA-deficient NahG transgenic tomato plants. Together, these results indicate that this conserved effector suppresses SA-mediated defenses in tomato roots and stems, which areR. solanacearum’s natural infection sites. Interestingly, PopS did not trigger necrosis when heterologously expressed inNicotianaleaf tissue, unlike the AvrE homolog DspEPccfrom the necrotrophPectobacterium carotovorumsubsp.carotovorum. This is consistent with the differing pathogenesis modes of necrosis-causing gammaproteobacteria and biotrophicR. solanacearum.IMPORTANCEThe type III-secreted AvrE effector family is widely distributed in high-impact plant-pathogenic bacteria and is known to suppress plant defenses for virulence. We characterized the biology of PopS, the only AvrE homolog made by the bacterial wilt pathogenRalstonia solanacearum. To our knowledge, this is the first study ofR. solanacearumeffector function in roots and stems, the natural infection sites of this pathogen. Unlike the functionally redundantR. solanacearumeffectors studied to date, PopS is required for full virulence and wild-type colonization of two natural crop hosts.R. solanacearumis a biotrophic pathogen that causes a nonnecrotic wilt. Consistent with this, PopS suppressed plant defenses but did not elicit cell death, unlike AvrE homologs from necrosis-causing plant pathogens. We propose that AvrE family effectors have functionally diverged to adapt to the necrotic or nonnecrotic lifestyle of their respective pathogens.

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Viability, Stability and Biocontrol Activity in Planta of Specific Ralstonia Solanacearum Bacteriophages After Their Conservation Prior to Commercialization and Use

10.20944/preprints202112.0127.v1 ◽

2021 ◽

Author(s):

Belén Álvarez ◽

Laura Gadea-Pallás ◽

Alejandro Rodríguez ◽

Begonya Vicedo ◽

Àngela Figàs-Segura ◽

...

Keyword(s):

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Reference Strain ◽

Specific Activity ◽

Experimental Period ◽

In Planta ◽

Tomato Plants ◽

Preservation Method ◽

Biocontrol Activity ◽

Susceptible Tomato

Ralstonia solanacearum is a pathogen that causes bacterial wilt producing severe damage in staple solanaceous crops. Traditional control has low efficacy and/or environmental impact. Recently, the bases of a new biotechnological method by lytic bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 with specific activity against R. solanacearum were established. However, some aspects remain unknown, such as the survival and maintenance of the lytic activity after submission to a preservation method as the lyophilization. To this end, viability and stability of lyophilized vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 and their capacity for bacterial wilt biocontrol have been determined against one pathogenic Spanish reference strain of R. solanacearum in susceptible tomato plants in different conditions and making use of various cryoprotectants. The assays carried out have shown satisfactory results with respect to the viability and stability of the bacteriophages after the lyophilization process, maintaining high titres throughout the experimental period, also with respect to the capacity of the bacteriophages for the biological control of bacterial wilt, controlling this disease in more than 50% of the plants. The results offer good prospects for the use of lyophilization as a conservation method for the lytic bacteriophages of R. solanacearum in view of their commercialization as biocontrol agents.

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Efficacy of the Biopesticide NECO in the control of Ralstonia solancearum, causal agent of tomato bacterial wilt in Côte d’Ivoire

JOURNAL OF ADVANCES IN AGRICULTURE ◽

10.24297/jaa.v11i.8813 ◽

2020 ◽

Vol 11 ◽

pp. 138-145

Author(s):

N’guessan Aya Carine ◽

Camara Brahima ◽

Amari Ler N’Ogn Dadé Georges Elisée ◽

Doumbouya Mohamed ◽

Pakora Gilles Alex ◽

...

Keyword(s):

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Control Agent ◽

Soilborne Pathogens ◽

Tomato Crop ◽

Tomato Plants ◽

Control Approach ◽

Bacterial Growth Inhibition

The tomato crop is confronted to numerous soilborne pathogens, including Ralstonia solanacearum, which considerably limits its production. In order to control this bacterium, a biological control approach has been considered by evaluating the efficacy of the NECO biopesticide against this bacteriosis. In vitro confrontations were carried out using a range of five concentrations of the biopesticide. In vivo, NECO solutions of 5 and 10 mL/L were incorporated into soil previously infested with R. solanacearum before transplanting tomato plants. Zones of bacterial growth inhibition were observed after the application of the NECO biopesticide. Results showed that the 20 mL/L concentration resulted in a higher inhibition rate. The biopesticide at the 10 mL/L concentration significantly reduced the incidence of bacterial wilt (54.05%) under in vivo conditions. The NECO biopesticide could be used as a control agent for Ralstonia solanacearum.

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Trehalose Synthesis Contributes to Osmotic Stress Tolerance and Virulence of the Bacterial Wilt Pathogen Ralstonia solanacearum

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-08-19-0218-r ◽

2020 ◽

Vol 33 (3) ◽

pp. 462-473 ◽

Cited By ~ 3

Author(s):

April M. MacIntyre ◽

John X. Barth ◽

Molly C. Pellitteri Hahn ◽

Cameron O. Scarlett ◽

Stéphane Genin ◽

...

Keyword(s):

Osmotic Stress ◽

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Type Strain ◽

Wild Type Strain ◽

Xylem Sap ◽

Wilt Disease ◽

Wild Type ◽

Tomato Plants ◽

Trehalose Synthesis

The xylem-dwelling plant pathogen Ralstonia solanacearum changes the chemical composition of host xylem sap during bacterial wilt disease. The disaccharide trehalose, implicated in stress tolerance across all kingdoms of life, is enriched in sap from R. solanacearum–infected tomato plants. Trehalose in xylem sap could be synthesized by the bacterium, the plant, or both. To investigate the source and role of trehalose metabolism during wilt disease, we evaluated the effects of deleting the three trehalose synthesis pathways in the pathogen: TreYZ, TreS, and OtsAB, as well as its sole trehalase, TreA. A quadruple treY/treS/otsA/treA mutant produced 30-fold less intracellular trehalose than the wild-type strain missing the trehalase enzyme. This trehalose-nonproducing mutant had reduced tolerance to osmotic stress, which the bacterium likely experiences in plant xylem vessels. Following naturalistic soil-soak inoculation of tomato plants, this triple mutant did not cause disease as well as wild-type R. solanacearum. Further, the wild-type strain out-competed the trehalose-nonproducing mutant by over 600-fold when tomato plants were coinoculated with both strains, showing that trehalose biosynthesis helps R. solanacearum overcome environmental stresses during infection. An otsA (trehalose-6-phosphate synthase) single mutant behaved similarly to ΔtreY/treS/otsA in all experimental settings, suggesting that the OtsAB pathway is the dominant trehalose synthesis pathway in R. solanacearum.

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The hrpB and hrpG Regulatory Genes of Ralstonia solanacearum Are Required for Different Stages of the Tomato Root Infection Process

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2000.13.3.259 ◽

2000 ◽

Vol 13 (3) ◽

pp. 259-267 ◽

Cited By ~ 65

Author(s):

Jacques Vasse ◽

Stéphane Genin ◽

Pascal Frey ◽

Christian Boucher ◽

Belen Brito

Keyword(s):

Ralstonia Solanacearum ◽

Infection Process ◽

Regulatory Genes ◽

Wild Type ◽

Root Infection ◽

In Planta ◽

Tomato Root ◽

Phytopathogenic Bacterium ◽

Tomato Plants ◽

Hrp Genes

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.

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Ralstonia solanacearum depends on catabolism of myo-inositol, sucrose, and trehalose for virulence in an infection stage-dependent manner

10.1101/700351 ◽

2019 ◽

Author(s):

Corri D. Hamilton ◽

Olivia Steidl ◽

April M. MacIntyre ◽

Caitilyn Allen

Keyword(s):

Cell Density ◽

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Xylem Sap ◽

Carbon Sources ◽

Dependent Manner ◽

Wild Type ◽

In Planta ◽

Catabolic Pathways ◽

Infection Stage

The soilborne pathogen Ralstonia solanacearum (Rs) causes lethal bacterial wilt disease of tomato and many other crops by infecting host roots and then colonizing the xylem vessels. Tomato xylem sap is nutritionally limiting but it does contain sucrose and trehalose. Transcriptomic analyses revealed that Rs expresses distinct sets of catabolic pathways at low cell density (LCD) and high cell density (HCD). To investigate the links between bacterial catabolism, infection stage, and virulence, we measured the in planta fitness of bacterial mutants lacking carbon catabolic pathways expressed at either LCD or HCD. We hypothesized that the bacterium needs LCD carbon sources early in disease (root infection) while HCD carbon sources are required during late disease (stem colonization). An Rs ΔiolG mutant unable to use the LCD nutrient myo-inositol was defective in root colonization but once it reached the stem, this strain colonized and caused symptoms as well as wild type. In contrast, Rs mutants unable to use sucrose (ΔscrA), trehalose (ΔtreA), or both (ΔscrA/treA), infected roots as well as wild type but were defective in colonization and competitive fitness in tomato mid-stems and were reduced in bacterial wilt virulence. Additionally, xylem sap from tomato plants colonized by ΔscrA, ΔtreA, or ΔscrA/treA contained more sucrose than sap from plants colonized by wild-type Rs. Together, these findings suggest Rs metabolism is specifically adapted for success in the different nutritional environments of plant roots and xylem sap.

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Interactions with Hosts at Cool Temperatures, Not Cold Tolerance, Explain the Unique Epidemiology of Ralstonia solanacearum Race 3 Biovar 2

Phytopathology ◽

10.1094/phyto-99-10-1127 ◽

2009 ◽

Vol 99 (10) ◽

pp. 1127-1134 ◽

Cited By ~ 43

Author(s):

Annett Milling ◽

Fanhong Meng ◽

Timothy P. Denny ◽

Caitilyn Allen

Keyword(s):

North American ◽

Ralstonia Solanacearum ◽

Potato Tubers ◽

In Planta ◽

Plant Host ◽

Tomato Plants ◽

North American Strain ◽

Tropical Highlands ◽

American Strain ◽

Warm Temperate

Most strains of the bacterial wilt pathogen Ralstonia solanacearum are tropical, but race 3 biovar 2 (R3bv2) strains can attack plants in temperate zones and tropical highlands. The basis of this distinctive ecological trait is not understood. We compared the survival of tropical, R3bv2, and warm-temperate North American strains of R. solanacearum under different conditions. In water at 4°C, North American strains remained culturable the longest (up to 90 days), whereas tropical strains remained culturable for the shortest time (≈40 days). However, live/dead staining indicated that cells of representative strains remained viable for >160 days. In contrast, inside potato tubers, R3bv2 strain UW551 survived >4 months at 4°C, whereas North American strain K60 and tropical strain GMI1000 were undetectable after <70 days in tubers. GMI1000 and UW551 grew similarly in minimal medium at 20 and 28°C and, although both strains wilted tomato plants rapidly at 28°C, UW551 was much more virulent at 20°C, killing all inoculated plants under conditions where GMI100 killed just over half. Thus, differences among the strains in the absence of a plant host were not predictive of their behavior in planta at cooler temperatures. These data indicate that interaction with plants is required for expression of the temperate epidemiological trait of R3bv2.

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Identification of a DNA region required for growth of Pseudomonas syringae pv. tomato on tomato plants

Canadian Journal of Microbiology ◽

10.1139/m92-144 ◽

1992 ◽

Vol 38 (9) ◽

pp. 883-890 ◽

Cited By ~ 2

Author(s):

Dennis P. Jackson ◽

Douglas A. Gray ◽

Vincent L. Morris ◽

Diane A. Cuppels

Keyword(s):

Organic Acids ◽

Pseudomonas Syringae ◽

Acid Metabolism ◽

Carbon Sources ◽

Hypersensitive Reaction ◽

Wild Type ◽

In Planta ◽

Tomato Plants ◽

Tartaric Acids ◽

Nonpathogenic Strain

The prototrophic Pseudomonas syringae pv. tomato mutant DC3481, which is the result of a single-site Tn5 insertion, cannot grow and cause disease on tomato plants and cannot use the major organic acids of tomato, i.e., citric, malic, succinic, and tartaric acids, as sole carbon sources. Although nonpathogenic, strain DC3481 can still induce a hypersensitive reaction in nonhost plants. We have identified a 30-kb fragment of P. syringae pv. tomato wild-type DNA that can complement this mutant. EcoRI fragments from this region were subcloned and individually subjected to functional complementation analysis. The 3.8-kb fragment, which was the site of the Tn5 insertion, restored pathogenicity and the ability to use all the major organic acids of tomato as carbon sources. It shares sequence homology with several P. syringae pathovars but not other bacterial tomato pathogens. Our results indicate that sequences on the 3.8-kb EcoRI fragment are required for both the ability to grow on tomato leaves (and thus cause disease) and the utilization of carboxylic acids common to tomato. The 3.8-kb fragment may contain a sequence (or sequences) that regulates both traits. Key words: Pseudomonas syringae pv. tomato, phytopathogenicity, Tn5, tricarboxylic acid metabolism, bacterial speck, growth in planta.

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