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Pathogenomics of the RalstoniaSolanacearum Species ComplexStephane Genin1,2,∗ and Timothy P. Denny3

1INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441,F-31326 Castanet-Tolosan, France; email: [email protected], Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR2594,F-31326 Castanet-Tolosan, France3Department of Plant Pathology, The University of Georgia, Athens, Georgia, 30602-7274;email: [email protected]

Annu. Rev. Phytopathol. 2012. 50:4.1–4.23

The Annual Review of Phytopathology is online atphyto.annualreviews.org

This article’s doi:10.1146/annurev-phyto-081211-173000

Copyright c© 2012 by Annual Reviews.All rights reserved

0066-4286/12/0908/0001$20.00

∗Corresponding author

Keywords

bacterial wilt, β-proteobacteria, host specificity, regulatory network,type III effector, virulence

Abstract

Ralstonia solanacearum is a major phytopathogen that attacks many cropsand other plants over a broad geographical range. The extensive geneticdiversity of strains responsible for the various bacterial wilt diseases hasin recent years led to the concept of an R. solanacearum species complex.Genome sequencing of more than 10 strains representative of the mainphylogenetic groups has broadened our knowledge of the evolutionand speciation of this pathogen and led to the identification of novelvirulence-associated functions. Comparative genomic analyses are nowopening the way for refined functional studies. The many moleculardeterminants involved in pathogenicity and host-range specificity aredescribed, and we also summarize current understanding of their rolesin pathogenesis and how their expression is tightly controlled by anintricate virulence regulatory network.

4.1

Review in Advance first posted online on May 1, 2012. (Changes may still occur before final publication online and in print.)

Changes may still occur before final publication online and in print

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Species complex:a group of closelyrelated organismsthought to comprisemore than one species

RSSC: Ralstoniasolanacearum speciescomplex

Blood diseasebacterium (BDB): apathogen of banana inIndonesia

Megaplasmid: thecustomary name forthe smaller essentialreplicon of thebipartite RSSCgenomes

Contig: a region withoverlapping sequencesthat has not beenassembled as part of acomplete genomicsequence

INTRODUCTION

Lethal wilts caused by Ralstonia solanacearumare among the most important bacterialdiseases of plants. This soilborne pathogenis present on all continents and many islandsbetween the Tropics of Cancer and Capricorn(34). Recent pathogen surveys and geneticstudies have proven that, as Buddenhagenopined more than 25 years ago, strains of thispathogen “evolved in widely different placesand they have different capabilities with bothnative flora and introduced hosts” (17). Indeed,because of the extensive diversity revealed byphylogenetic analyses, this group of organismsis now commonly called the R. solanacearumspecies complex (RSSC) (35).

R. solanacearum, a β-proteobacterium, ispathogenic on more than 200 plant speciesbelonging to over 50 different botanicalfamilies (31). The pathogen affects not onlysolanaceous plants, such as tomato and potato,but many weeds, crops, shrubs, and trees inother dicot and monocot families. Unique toIndonesia are Ralstonia syzygii, a pathogen ofclove trees, and the blood disease bacterium(BDB), a banana pathogen. BDB is closelyrelated to R. solanacearum, but its official tax-onomic standing remains unresolved (36, 87).This unusually wide host range is continuouslyexpanding, and descriptions of new hosts arecommon. Although the etiology resulting inwilting symptoms is probably similar for mostsusceptible hosts, different disease names areoften used depending on the crop affected.

R. solanacearum can survive for years inmoist soils or water microcosms (5, 104).When the pathogen encounters a susceptiblehost, it enters the root and colonizes the rootcortex, then invades the xylem vessels, andfinally spreads rapidly to aerial parts of theplant through the vascular system (31). Wiltingsymptoms result from the vascular dysfunc-tion caused by this extensive colonization.The bacterium can also colonize some hostsasymptomatically in latent infections, and thispoorly understood phenomenon is crucial inthe epidemiology of these strains.

This review focuses on advances in researchand understanding made possible in the pastdecade from having genomic sequences forstrains representative of the biodiversity withinthe RSSC. These include insight into the evo-lution and speciation of the pathogen, progressin identifying new virulence and potential host-range determinants, and refinement of the in-tricate regulation networks controlling systemsimportant for pathogenesis.

GENERAL FEATURES OFRALSTONIA SOLANACEARUMSPECIES COMPLEX GENOMES

R. solanacearum genomes are organized in twocircular replicons customarily called the chro-mosome and the megaplasmid, with the latterbeing about one third of the average 5.8 Mbtotal (86, 94). However, because both repli-cons carry essential and pathogenesis-relatedgenes and analyses indicate they have coevolved(22, 55), they comprise a bipartite genome. Al-though it would be technically more correct torename the megaplasmid (e.g., chromosome 2),we will continue using this term for consistencywith the published literature.

Since the publication of the genome se-quence of strain GMI1000 in 2002 (94), 10additional genomes of RSSC strains have beensequenced (as of November 2011). These 11strains are representative of the RSSC and, insome cases, display some host-range selectivity.The main features of these genome sequencesare given in Table 1. Five genomes are notcompletely assembled and have approximately10 (IPO1609, BDB, R. syzygii ) or 40 (Molk2)supercontigs or ∼580 individual contigs(UW551), which precludes determining rear-rangements among replicons. In addition, theunusually low number of tRNAs identified insome of the draft genomes (Table 1) suggeststhat regions of low coverage may have missingor erroneous sequence data.

Functional gene assignments clearly showthat the large majority of housekeeping func-tions are chromosome-borne, whereas themegaplasmid, although carrying some essential

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Tab

le1

Feat

ures

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geno

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spec

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Gen

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726

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553

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www.annualreviews.org • Ralstonia Solanacearum 4.3


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Synteny: preservationof gene order on areplicon

Chemotaxis: theability of a cell tomove in response to achemical or energygradient

HGT: horizontalgene transfer

Type III effector(T3E): a proteinsecreted by the type IIIsecretion system

genes, is enriched in genomic islands and strain-specific genes (86, 87, 94). This observationis also supported by (a) comparative genomichybridization studies on a collection of 18strains, showing that 63% of the genes from themegaplasmid are not conserved (55), and (b) thelevel of synteny is higher on the chromosome(70% to 80% of the coding sequences) thanon the megaplasmid (55% to 65%) (86). Acomparison of the genome replicon sizes alsosuggests that the megaplasmid probably carriesmany genes associated with specific lifestylesbecause its size varies up to 26%, whereasthere is minimal variation for the chromo-some (∼3.5–3.7 Mb, a 6% size variation)(Table 1). Accordingly, most of the functionsinvolved in adaptation to the environment orphytopathogenicity are megaplasmid-borne,including the Type III and Type VI proteinsecretion systems, flagellar motility determi-nants, chemotaxis genes, and the extracellularpolysaccharide (EPS) biosynthesis cluster. Thereduced size of the megaplasmid in R. syzygiiand BDB strains suggests a possible ongoingreductive evolution of this replicon due totheir specific lifestyle as insect-transmitted andhost-specialized parasites (87).

Horizontal Gene TransferEvents and the Contributionof Prophage Elements

Annotation of the GMI1000 genome led tothe definition of alternate codon usage regions(ACUR) that account for 7% of the genome.Because ACUR mostly correspond to lowerguanine plus cytosine content regions and/orprophage-associated regions (94), most con-stitute genomic islands, many of which werepresumably acquired through horizontal genetransfer (HGT). Approximately 70–80 genomicisland regions varying in size from 5 to >100 kbwere defined in several genomes (86) and gen-erally represent the nonsyntenic stretches ob-served after comparing the global gene orderon both replicons. As already demonstratedin other bacterial pathogens (78), it is plausi-ble that such a modular genome organization

distinguishes regions with genes devoted tohousekeeping functions from those more dedi-cated to host adaptation.

Many of R. solanacearum genomic islands ineach of the sequenced genomes correspond toputative prophages or phage-like elements (87)(Table 1). Phages are abundant in soil and arekey factors shaping evolution of soil microbialcommunities (50, 91). Phages are also efficientvectors for HGT (63) that can contributeto the rapid acquisition of novel adaptivefunctions and so play a role in the emergenceof pathogenic variants. A detailed inspectionof R. solanacearum prophage clusters suggeststhat they probably disseminate several genesinvolved in the plant-bacterium interaction,such as genes for type III effectors (T3Es)( popP1, popP2, ripT, etc.), as well as other genesencoding products of unknown function. It isremarkable that in all three strains possessingpopP2 (GMI1000, Po82, and CMR15), thisgene is associated with a prophage insertedat different genomic locations. Several otherR. solanacearum prophages appear to be strain-specific (41, 42, 54), suggesting that theirdistribution may be variable among strains.

Small plasmids were detected in two strains(Table 1) and could also contribute to efficientHGT. No specific biological function could beinferred from their gene content (86), but itseems likely that the CMR15 plasmid was ac-quired from a Xanthomonas sp. because of theapparent conservation of the Type IV secre-tion system genes. Potentially transferable in-tegrative conjugative elements were also foundin strains GMI1000 and Molk2 (see References46, 52).

SYSTEMATICS OF THERALSTONIA SOLANACEARUMSPECIES COMPLEX

Early attempts to codify the diversity in theRSSC resulted in separate race and biovar sys-tems (31). R. solanacearum races are based onhost range at the plant species level and thus,contrary to typical usage, are roughly equivalentto pathovars of other phytopathogenic bacteria

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Phylotype: themajor phylogeneticsubdivisions within theRSSC

Sequevar: a group oftwo or more identicalgene-sequencevariants; usuallydetermined based onthe egl gene sequence

(4, 85). However, our increasing knowledge ofpathogen diversity makes the race system un-wieldy and unreliable (31). The biovar systemgroups strains by their ability to acidify mediacontaining selected carbohydrates, to producenitrite from nitrate, and to produce gas fromnitrate (30). Although useful for many years,the biovar system lacks discriminating powerbecause of its limited genetic basis. Both ofthese systems have now largely been replacedby the widely accepted phylotype-sequevar sys-tem (see below).

A variety of genetic techniques have beenused to investigate the diversity and inter-relatedness within the RSSC (31). Classicalrestriction fragment length polymorphismanalysis initially revealed fundamental hetero-geneity of pathogen strains, but large-scaleDNA sequence analyses more profoundlyinfluenced our understanding of pathogen sys-tematics (19, 31, 85, 107). As the diversity of thestrains examined increased, it became clear thatthe RSSC has four major subdivisions, denotedas phylotypes (35, 85). A multiplex polymerasechain reaction (PCR) can rapidly assign strainsto a phylotype (35). Each phylotype is sub-divided into numbered sequevars and closelyrelated individual strains, which are usuallyidentified by analyzing sequence similarity ofthe egl endoglucanase structural gene.

The phylotypes correspond roughly to thestrains’ geographic origin: Asia (phylotype I),the Americas (II), Africa (III), and Indonesia(IV). Phylotype II has two clearly recognizablesubclusters (IIA and IIB) (19, 36, 85, 107). Boththe BDB and R. syzygii fall within phylotype IV.The same relationships are observed regardlessof the conserved gene examined, suggestingthat they coevolved with the various RSSCgenomes. The same phylotypes also wereobserved when (a) comparative genomic hy-bridization to a microarray was used to generatehierarchical clusters based on gene conserva-tion (55, 86) and (b) the genome sequencesof six representative strains were compared(86, 87).

Multilocus sequence and statistical analysesof a moderately diverse RSSC population

indicated that the phylotypes reflect trueevolutionary lineages that probably arose longago when progenitors became geographicallyseparated (19). More recently, similar analysesof strains that better represent RSSC diver-sity found the same phylotype structure butdetected both inter- and intraphylotype recom-bination in seven of nine genes examined (111).Three methods used to estimate recombinationindicated that (a) phylotype I is freely recom-bining, and (b) although recombination is com-mon in phylotypes IIA, III, and IV, it has notyet eliminated all clonal structure. Wicker et al.(111) also used coalescent genealogy recon-struction to deduce the order in which phylo-types likely evolved, and along with known eco-types they described eight clades superimposedon the phylotypes (Table 2). When combinedwith ancestral state reconstruction, these resultssuggested that the pathogen originated in theOceania/Indonesia region, migrated to Africaand from there to South America (possiblybefore the fragmentation of Gondwana) and toAsia.

Adaptation to different environments andpotential host plants may also be drivingevolution (111). For example, on the island ofMartinique, Wicker et al. (109, 110) docu-mented the appearance and spread of phylotypeIIB sequevar 4 strains that, unlike typical mem-bers of this subgroup, are pathogenic onsolanaceous crops and cucurbits but latentlyinfect banana rather than causing wilt disease.These new strains are exceptionally aggressiveon tomato, as they wilt even the resistant H7996line. How these strains arose is unknown. How-ever, most R. solanacearum strains tested becomenaturally transformation competent in culture,and probably also within plants, and can bothacquire and lose large (30–90 kb) regions oftheir genome when exposed to exogenous DNA(9, 26, 53). Indeed, after strain Psi07 was treatedin vitro with genomic DNA from GMI1000,one transformant was more aggressive ontomato (but not eggplant) (25). Thus, HGTcould contribute to R. solanacearum geneticand pathogenic diversity, and we now have thegenetic tools to determine the molecular



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Table 2 Genetic, geographic, and ecotype diversity within the Ralstonia solanacearum speciescomplex

Phylotypea Origin Cladeb Ecotype informationcProposedspeciesd

I Asia 1 BW of Solanaceae, ginger, mulberry Ralstonia sequeiraeIIA

Americas

2 BW of Solanaceae, Musa Ralstonia solanacearumIIAT 3 BW of Solanaceae (southeastern

United States and Caribbean)

IIB

Americas

4 Moko disease of Musa, new NPB variants Ralstonia solanacearumIIB 5 Brown rot of potato, BW of tomato and

geranium (R3bv2), Moko disease;distributed worldwide

III Africa 6 BW of Solanaceae Ralstonia sequeiraeIV

Indonesia

7 BDB of banana

BW of Solanaceae

Ralstonia haywardii subsp.celebensisR. haywardii subsp.solanacearum

IV 8 R. syzygii, Sumatra disease of clove R. haywardii subsp.syzygii

aThe T indicates that the R. solanacearum type strain K60 is in phylotype IIA.bClades were defined by Wicker et al. (111).cBW, bacterial wilt; R3bv2, previously classified as Race 3 biovar 2; BDB, blood disease bacterium. NPB, not pathogenic tobanana: designates the new pathogenic variants found on Martinique that wilt Anthurium, Heliconia, and cucurbits but notcultivated Musa.dSee Remenant et al. (87) for details. Although genetically similar, R. syzygii is phenotypically quite different from R.solanacearum or other proposed Ralstonia species and subspecies (see Reference 31).

basis underlying such cases of host-rangespecialization.

Analysis of the genomic DNA sequencesof six R. solanacearum strains, and one eachof R. syzygii and BDB strains, representingall the phylotypes provided additional insightinto the phylogeny of the RSSC (54, 86, 87,94). Most significantly, based on pairwisecomparison of average nucleotide identity,Remenant et al. (87) concluded that the RSSChas three groups of strains that exceed theaccepted threshold for speciation. Consideringthese and the extensive genetic results notedabove, they proposed two new species andthree subspecies (Table 2). This conclusionis supported by the limited DNA-DNA hy-bridization data (80, 89, 105) that indicatedthe RSSC is polyphyletic. Unfortunately,the sequence analyses provided no obviousexplanations regarding the molecular basis ofhost preference or ecological niche adaptation.Furthermore, two extensive tests examining thepathogenic potential of diverse R. solanacearumstrains on four solanaceous hosts revealed no

good correlation with phylotype or sequevar(20, 66).

RALSTONIA SOLANACEARUMVIRULENCE DETERMINANTS

General Virulence Factors

R. solanacearum virulence factors enhancethe pathogen’s ability to cause disease. Thesingle most important virulence factor ofR. solanacearum is its high molecular mass EPS,which is produced in massive amounts in cul-ture and in planta (31). This EPS promotesrapid systemic colonization and eps mutantsrarely wilt or kill plants even when bacteria areintroduced directly into the xylem (93). Accu-mulation of EPS is largely responsible for thevascular dysfunction that causes wilt symptomsin susceptible hosts. Surprisingly, EPS triggersenhanced expression of the ethylene and sali-cylic acid defense response pathways in the wilt-resistant H7996 tomato breeding line but notin a susceptible cultivar (74).

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Type II secretionsystem (T2SS):secretes selectedproteins from thebacterial periplasm tothe environment

PCWDE: plant cellwall–degradingenzyme

Twitching motility:the ability of bacteriato propel themselvesover solid surfaces byusing retractable type4 pili

The assemblage of approximately 30 extra-cellular proteins transported across the outermembrane by the Type II secretion system(T2SS) are also essential for colonization andbacterial wilt (68, 83). When tested individu-ally for their contribution to disease of tomato,the four pectolytic enzymes (PehABC and Pme)are generally less important than the two cellu-lolytic enzymes (Egl and CbhA) (31, 68). How-ever, a mutant lacking all six of these plantcell wall–degrading enzymes (PCWDE) is con-siderably more virulent than a mutant with anonfunctional T2SS, which also is severely re-duced in its ability to invade and colonize plants.Therefore, some combination of the other ∼24extracellular proteins, which have diverse pre-dicted and unknown functions, is also quite im-portant for virulence.

Pathogen motility also contributes signif-icantly to disease. Flagella-driven swimmingmotility is growth-phase dependent in culture,but bacteria recovered from within tomatoplants are overwhelmingly nonmotile (99).Both nonmotile, nonflagellated and motile,nonchemotactic mutants are significantlyreduced in virulence when applied as a soildrench of potted tomato plants, but exhibitnormal virulence when inoculated directly intothe xylem via a severed petiole (99, 114). Both ahypermotile motN mutant and an aer2 mutant,which is motile but nonaerotactic, are slightlyreduced in virulence when inoculated bysoil drench (73, 115). These results show thatmotility is required for R. solanacearum to locateand invade roots, although it is dispensablelater during pathogenesis. Twitching motility,which is driven by polar, retractable type 4pili, also contributes to virulence on tomatoregardless of whether plants are inoculatedby soil drench or via severed petioles (61).Type 4 pili also promote autoaggregation andbiofilm formation in broth culture, and polarattachment to tomato roots.

During pathogenesis, R. solanacearumencounters a variety of stressful environmentsand compounds. Among these are a variety ofreactive oxygen species (ROS) made by plantsafter infection (38, 70, 74). The pathogen

appears to have correspondingly redundantmechanisms to detoxify ROS or otherwisetolerate this oxidative environment, and manyof these appear to be expressed in planta (14).Not surprisingly, mutants lacking just the Bcpperoxidase or Dps, a DNA binding proteinthat promotes stress tolerance, are almost fullyvirulent (24, 38), but inactivation of oxyR,which encodes the only identified regulator ofoxidative stress genes, significantly reduces vir-ulence (39). The pathogen may also encountermultiple toxic compounds during pathogen-esis, and inactivation of acrA and dinF, whichlikely encode components of multidrug effluxpumps, reduced tolerance to some compoundsin culture and significantly reduced virulence(15). The pathogen also has multiple cytoplas-mic polyphenol oxidases that may help detoxifyphenolic compounds, but their biological roleshave not been reported (56). Microaerobicconditions may also stress the pathogen, and accb3-type cytochrome c oxidase contributes bothto its survival in very low oxygen conditions andto virulence (23). Some metabolic pathwaysalso appear to be specifically required duringpathogenesis because deletion of the metER me-thionine biosynthesis genes in two phylotypeIIB sequevar 1 strains reduced disease symptomproduction without causing auxotrophy (51).

Bacteria often encounter low concentrationsof soluble iron and therefore produce one ormore siderophores to scavenge this essential el-ement. All R. solanacearum strains probably pro-duce the polycarboxylate siderophore staphylo-ferrin B (10), but it is likely that phylotypes I, III,and IV also make micacocidin, a yersiniabactin-like siderophore (64). However, tomato xylemfluid appears to have sufficient iron to repressexpression of any iron-acquisition system, anda mutant that does not make staphyloferrin Bis fully virulent on tomato (10).

It has long been suspected that phytohor-mones produced by R. solanacearum mightcontribute to virulence. Production of ethylenein culture by GMI1000 is positively regulatedby HrpG (see below), and the pathogenappears to make ethylene in Arabidopsis (103).Nevertheless, ethylene-negative mutants are



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Type III secretionsystem (T3SS):translocates proteinsdirectly from thebacterial to the hostcytoplasm

PhcA: the globalregulator controllingreversible phenotypeconversion in responseto 3-OH PAME

fully virulent (31, 103). Production of auxinis also positively controlled by HrpG, butits role in virulence has not been reported.R. solanacearum can also make cytokinin inculture and there is one report that inactivationof the conserved tzs gene responsible for itsproduction reduces virulence (31).

Host-Range Factors

R. solanacearum strains can strikingly differin their host range, some being obviouslypolyphagic, whereas other strains are charac-terized by a restricted, specialized host range(for a detailed review, see Reference 45).Host-specific interactions of R. solanacearumstrains with certain hosts (18, 66) and at the cul-tivar/ecotype level (33, 49, 65, 102) have beendocumented. In the case of AvrA on tobacco(90), PopP1 on resistant petunia lines (65), andPopP2, which is recognized by the Arabidopsisresistance protein RRS1-R (32), determinantsrestrict host range and correspond to T3E thattrigger incompatibility on resistant plants. In-activation of these bacterial avirulence factorsis generally sufficient to restore pathogenicity(32, 65). However, in tobacco, it was shownthat inactivation of a second gene ( popP1) otherthan avrA is required to restore full pathogenic-ity in some strains because both determinantselicited a hypersensitive response on tobaccoand are involved in host-range restriction (82).In contrast, presence of the gala7 gene extendsthe host range of strain GMI1000 to include thelegume Medicago truncatula (6). The best char-acterized T3E to date is PopP2, which possessesacetyl-transferase activity (100) and interactswith multiple Arabidopsis targets (8), but themolecular functions of most R. solanacearumT3E remain elusive (see Reference 83).

A recent report revealed that R. solanacearumhost specificity is not only determined by T3E.rsa1, a virulence gene from a phylotype IVpotato strain, confers avirulence on pepperwhen introduced into a phylotype I strainnormally able to cause disease on this host(60). Expression of rsa1 is under the transcrip-tional control of the type III secretion system

(T3SS) regulator HrpB (see below). Surpris-ingly, Rsa1 is secreted extracellularly but in aT3SS-independent manner and possesses an as-partic protease activity (60). It will be interest-ing to see how Rsa1 is recognized by peppercells.

VIRULENCE ANDPATHOGENICITYREGULATORY NETWORKS

R. solanacearum pathogenesis functions areregulated by sophisticated, multicomponentinterconnected networks that are sensitive toenvironmental and internal factors. A great dealhas been learned about how these networksfunction in culture, but we only have glimpsesof processes that occur during pathogenesis.

The Phc Confinement-SensingSystem

Spontaneous loss of virulence, EPS, and othertraits by R. solanacearum in culture vexed andperplexed scientists for decades (31). The ge-netics of this phenomenon, called phenotypeconversion (PC), remained unclear until thediscovery of PhcA, a LysR-type transcriptionalregulator that controls expression of manygenes (16, 95). DNA replication errors andtransposition of insertion sequence elementscan inactivate phcA (16, 59, 84), and phcA mu-tants (PC-types) can accumulate because theymultiply preferentially during some stressfulconditions (e.g., prolonged stationary phase inculture or in planta and high salt or low oxygenconcentrations) (31).

PhcA and 3-Hydroxy palmitic acid methylester (3-OH PAME). Traits that are posi-tively regulated directly or indirectly by PhcAare shown in Figure 1a and negatively reg-ulated traits are shown in Figure 1b. ThePhcA regulon almost certainly includes manymore genes than those shown, but transcrip-tome studies have not been reported. ThirtyRSSC strains examined have well-conservedhomologs of phcA, as do R. syzygii, Ralstonia

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3-Hydroxy palmiticacid methyl ester(3-OH PAME): themajor quorum sensingautoinducer in theRSSC

picketii, and three Cupriavidus species (44, 55,86, 87). All these genes are probably functionalbecause one of the most divergent PhcA pro-teins from Cupriavidus metallidurans comple-mented a R. solanacearum phcA mutant (44) (seesidebar, How Unique Is The 3-OH PAME Au-toinducer?).

When cultured in vitro, wild-type PhcA hasa central role because it enables the pathogento cycle between two very different phenotypicstates in response to nutrient availability andcell density (31, 95). Levels of functional PhcAare controlled by a confinement-sensing systemencoded by the phcBSR operon. PhcB appearsto be a methyltransferase that synthesizes3-hydroxy palmitic acid methyl ester (3-OHPAME), an unusual quorum-sensing signal.PhcS and PhcR compose a two-componentregulatory system that responds to thresholdconcentrations of 3-OH PAME by elevatingthe level of functional PhcA (Figure 1). There-fore, cells at low density, such as those in soiland in plants at the leading edge of infection,have little functional PhcA and, like phcAmutants, exhibit a low virulence phenotypethat may be optimized for survival and invasionof plant tissues. In contrast, cells at higher den-sities, such as those in colonized xylem vessels,have abundant functional PhcA and producemultiple virulence factors while suppressingproduction of survival/invasion factors (95).

There are still many aspects of the Phc sens-ing system that we do not understand. For ex-ample, phcA mutants produce almost no 3-OHPAME, but PhcA apparently does not con-trol expression of the phcB synthase. Also, howPhcR, an atypical response regulator that hasa histidine kinase as its output domain, affectsboth expression and function of PhcA has notbeen resolved. Schell (95) presented prelim-inary evidence suggesting that 3-OH PAMEtriggers (auto)phosphorylation of this domain,which prevents it from post-transcriptionallyinhibiting production, stability, or function ofPhcA, but this presumably involves unidenti-fied intermediate regulators. Furthermore, thenutritional status of cells seems to have a crit-ical role in determining the responsiveness of

HOW UNIQUE IS THE 3-OH PAMEAUTOINDUCER?

Genomic sequence analysis suggests that production and sens-ing of 3-OH PAME is restricted to three genera within theβ-proteobacterium Burkholderiaceae family (see Table 3). Inmost Burkholderia spp., the PhcR orthologs lack a kinase out-put domain, and PhcA is absent in all strains. Unexpectedly, Po-laromonas naphthalenivorans, which is in the Comamonadaceaefamily, probably acquired the phcBSR operon by HGT; it alsolacks PhcA. When discovered, 3-OH PAME was the only fattyacid derivative other than the acyl-homoserine lactones and thesimilar γ-butyrolactones to function as an autoinducer. How-ever, this has changed with the discovery that multiple bacteriamake diffusible signal factor family (unsaturated fatty acids firstcharacterized in Xanthomonas campestris) (92) or CAI-1 family(α-hydroxyketones first characterized in Vibrio cholerae) (101) au-toinducers. The RSSC genomes apparently lack genes encodingorthologous synthases for these compounds.

80+% 60–79% 50–59% 40–49%

OrganismPercent identity to GM1000 orthologs

PhcB PhcS PhcR PhcAAll RSSC strains 84–89

Two Ralstonia spp.

Three Cupriavidus spp.

Some Burkholderia spp.

Polaromonas naphthalenivorans

77

64–70

63–68

60

83–95

71–72

50–62

58

50

87–91

76–77

52–53

46–53

41

93–95

86

47–49

absent

absent

wild-type cells to 3-OH PAME because its ad-dition to low-density cultures has no effect un-til the culture reaches >106 cells per ml (21).These observations suggest that, as in otherbacteria (11), additional regulatory factors af-fect quorum sensing in R. solanacearum.

Other regulatory components of the Phcnetwork. There are multiple additional regu-latory proteins that play supporting roles in thisnetwork (Figure 1) (31, 95). Both PhcA andthe VsrAD two-component sensor/responseregulator system are necessary to fully activatetranscription of xpsR, and both XpsR andthe VsrC response regulator bind to the epsoperon’s promoter to enhance expression(43). PhcA indirectly represses production



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phcBRS PhcAphcA

egl

AHL

RpoS

SolI

SolR

PhcS

PhcR

PhcB

3 OH-PAME

VsrA

VsrD

VsrB

VsrC

EpsR

XpsR

pme

EPS

Plant cell wall–

degrading enzymes

exported via T2SS fli genes

Flagellarmotility

Ralfuranone

ralAD

solIR

cbhA

eps genes

a Positively regulated traits

b Negatively regulated traits

xpsR

flhDC

MotN

pehA

Signal?

Signal?

aidA

phcBRS PhcAphcA

PhcS

PhcR

PhcB

via T2SS

Polygalacturonase

fli genes

Flagellarmotility

PrhI

PrhRPrhA

PehR

PehS

HrpGhrpB

HrpB

hdf operon

Metabolicsignal(s)

PrhG

HDF

Ethylene

Type 3secretion

system

prhA

prhIR

PrhJ

prhJ

hrpG

prhG

T3SS & T3E

rsa1

pehSR efe

Type 4 pili

pehA

tfp genes

katE

Catalase

Siderophore

Plant signal

Plant signal?

Protease

3 OH-PAME

Other genesOther genes

Regulatory protein families

Signaling

Extracellular signal perception

Post-transcriptional effect

Transcriptional activation

Transcriptional repression

Culture-specific regulation

Post-translational activation

Gene product

Extracellular secretion

AraC

σ factors

Two-component

LysR

Other

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of PehA as well as swimming and twitchingmotility by reducing the function of thePehSR two-component system that positivelycontrols expression of pehA, fli genes, andpilA (Figure 1b) (31, 61, 73). Production ofPehA is also repressed by VsrC independentlyof PhcA (57). Flagellar motility is positivelycontrolled in culture by VsrC and repressed byMotN, both of which affect expression of theconserved flhDC regulatory protein (73).

The VsrAD two-component systemcontrols multiple other traits, some ofwhich strongly contribute to the ability ofR. solanacearum to colonize tomato stemsand multiply in planta independently of thisregulator’s effect on EPS production (14,95). For example, when strain K60 is grownin culture, VsrAD is required for wild-typebiofilm formation, tolerance of both coldtemperatures and hydrogen peroxide, andacyl-homoserine lactone (AHL) production,but the mechanisms underlying these effectshave not been determined (113). Creation ofgenomic lacZ transcriptional reporter fusionsrecently confirmed that VsrAD positivelycontrols expression of cbhA in strains K60 andAW1 (see Reference 95) but revealed thatthis is not true in GMI1000. In contrast, cbhAis positively controlled by PhcA in all threestrains, although regulation is much strongerin GMI1000 than in the other strains (T.P.Denny, unpublished results). Both VsrADand PhcA positively control production ofthe secondary metabolite ralfuranone [4-phenylfuran-2(5H)-one] (96), but any roleof the latter in pathogenesis has not beenreported. Although halogenated furanonesinhibit AHL-regulated processes in multiplebacteria (72), the LuxIR-type AHL quorumsensing system in R. solanacearum that is

regulated by PhcA (Figure 1a) is dispensablefor virulence (37). VsrAD negatively controlsswimming motility (by strongly repressing ex-pression of flhDC) and twitching motility, butthese effects may be largely independent of itsweak effect on phcA and pehSR (113). Althoughmany of the VsrAD-regulated traits are alsocontrolled by PhcA, the multiple phenotypicand regulatory differences indicate that theseare separate regulons (14, 113). One of themost notable differences is the large effect thatgrowth phase and medium composition haveon some VsrAD-regulated traits.

Type III Secretion System Regulators

R. solanacearum regulates deployment and useof its T3SS with an environmentally respon-sive signal transduction network with at leastseven components, some of which constitute aplant responsive regulatory cascade (from PrhAto HrpB) (Figure 1) (12, 81). The T3SS regu-lation mechanism used by R. solanacearum dif-fers fundamentally from that of Pseudomonassyringae and Erwinia spp. but has commoncomponents (HrpB and HrpG) with the Xan-thomonas spp. T3SS regulatory system (13).

The HrpB regulon. HrpB is an AraC fam-ily transcriptional regulator that controls theT3SS and T3E gene expression (48). HrpB-dependent expression is conferred by a cis-regulatory DNA element named the hrpII box(TTCG-n16-TTCG) that lies just upstream ofthe −35 region in target promoter genes (27).Transcriptomic analyses revealed that, whentested in minimal medium, HrpB influences theexpression of >180 genes (approximately 140positively and 50 negatively) (79). Positivelyregulated genes include the T3SS structural

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 1The major regulatory circuits in Ralstonia solanacearum. The models are organized around the genes and traits directly controlled, either(a) positively or (b) negatively, by the PhcA transcriptional regulator. The network that primarily controls the type III secretion systemand other virulence-associated genes is in b. The metabolic/nutritional signal affecting hrp gene expression is not known. Broken linesindicate presumed indirect regulation. Lines ending with a crossbar indicate repression, whereas lines with an arrowhead indicateactivation. Abbreviations: EPS, exopolysaccharide; HDF, hrpB-dependent diffusible factor (3-hydroxy-oxindole); T2SS, type IIsecretion system.



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genes, T3SS helper proteins (lytic transglyco-sylases, chaperones, etc.) and all known T3E.Given that a subclass of HrpB-dependent T3Epromoters does not possesses an hrpII box (76,79), it is likely that the regulation of some targetgenes is indirect or involves a distinct regula-tory mechanism. However, the HrpB regulonmay be much smaller during pathogenesis be-cause genome-wide expression analysis of cellsrecovered from stems of wilting tomato plantsrevealed that only approximately half of genespositively regulated by HrpB were strongly ex-pressed (58). These results suggest that otherfactors modify expression of this regulon inplanta.

HrpB action extends beyond controllingthe T3SS and T3E genes (60, 79). Forexample, HrpB activates an operon of sixgenes responsible for the synthesis of anhrpB-dependent diffusible factor (HDF),identified as 3-hydroxy-oxindole (29). Becausethis tryptophan derivative induces AHLreceptor-mediated activity, it was hypothe-sized that HDF produced by R. solanacearuminterferes with the quorum-sensing systemsof rival bacteria at early steps of infection(29). Contribution to pathogenicity of theseT3SS-independent functions controlled byHrpB is still unknown, although it was reportedthat an hdf mutant is less competitive than thewild-type strain during mixed infections (69).

The HrpG regulon. HrpG, an OmpR familyresponse regulator, was originally discoveredas positively controlling hrpB expression (13).Transcriptome studies later revealed thatin culture the complete HrpG regulon hasapproximately 180 genes in addition to thoseregulated by HrpB, and that as a group this newset of genes is essential to pathogenesis (103,106). HrpG transcriptionally controls the ex-pression of functions that promote adaptationof the bacterium to life in hosts, as well as pre-viously identified virulence factors (reviewedin Reference 45). The HrpG regulon thereforeincludes some genes encoding PCWDE,detoxifying enzymes, phytohormones, lectins,metabolic enzymes and transporters, some

probable intermediate regulators, and manygenes with unknown functions (103).

HrpG is an orphan response regulator with-out a cognate sensor kinase. How its func-tion is controlled post-transcriptionally re-mains poorly understood, but it appears thatHrpG activity is modulated in response to nu-tritional signals so cross-talk with multiple sen-sor kinases is possible (13, 47, 117). However,mutation of the aspartate residue predicted tobe the phosphorylation site significantly re-duces but does not abolish HrpG activity (117).

Recently, a close paralog of hrpG, namedprhG, was discovered (81). The two cor-responding proteins are very similar (72%identical) and both activate hrpB expression.However, the function of PrhG appearsrestricted to specific environmental conditions(i.e., minimal medium) and is dispensablein planta; accordingly, a prhG mutant is notaltered in pathogenicity. The precise role ofthis hrpG paralog in T3SS activation remainsenigmatic because it does not appear to controladditional genes other than hrpB. Expression ofprhG itself requires the products of a three-geneoperon named prhKLM, none of which aretranscriptional regulators and thereby probablymodulate expression of prhG indirectly (118).

Regulatory ProcessesDuring Pathogenesis

HrpB and HrpG are the downstream compo-nents of the Prh regulatory cascade that alsoincludes the outer-membrane receptor PrhAand the PrhIRJ proteins (Figure 1b). The Prhpathway induces high expression of the T3SSupon plant cell contact (1). The exact natureof the plant signal sensed by the PrhA receptoris unknown, but there is evidence that it is anondiffusible molecule that may be an intrinsicconstituent of the plant cell wall. However,HrpB and HrpG must be activated by multiplesignals in planta because inactivation of thePrhAIRJ components results in partial lossof virulence, whereas hrpB or hrpG mutantsare nonpathogenic (12). Among the knownenvironmental cues that influence T3SS gene

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expression are repressing signals perceivedwhen bacteria are grown in the presence oforganic nitrogen sources and inducing condi-tions when bacteria are grown in a supposedapoplast-mimicking minimal medium.

There is genetic evidence that PhcA impactsthe Prh pathway at different levels. First, PhcArepresses expression of T3SS genes in richmedium, and this effect is probably mediatedthrough HrpG at the post-transcriptional level(47). However, this repression appears to beameliorated after cells enter the stationaryphase or when cultured in minimal medium(113). Second, in cells cultured in rich medium,PhcA binds to the promoter region of prhIand slightly represses its expression (116),thus demonstrating that it may affect the Prhpathway at the transcriptional level. However,the situation likely is more complex in planta.For example, eight hours after cells carryinga hrpB reporter fusion were infiltrated intotobacco leaves, hrpB expression was, contraryto expectations, higher in the wild type thanin a phcA mutant (113). In addition, in plantatranscriptome studies and qRT-PCR tests byJacobs et al. (58) and expression studies inplanta using green fluorescent protein reporterfusions (F. Monteiro, S. Genin, I. van Dijk,M. Valls, unpublished results) found thathrpG, hrpB, T3SS structural genes, and T3Egenes are still strongly expressed when cellsreach high density within tomato stems at theonset of wilting. It seems likely that significantPhcA-dependent repression may be a culturalartifact or is overridden by other regulatoryprocesses during pathogenesis.

Many other questions remain about howthese regulatory systems operate duringpathogenesis. Brown & Allen (14) used invivo expression technology to identify manyR. solanacearum ipx genes that are expressedin tomato stems but not in rich medium andshowed that some are differentially regulated byPhcA, VsrBC, VsrAD, PehR, and FlhDC whentested in minimal medium. Although helpful inidentifying some additional potential virulencegenes, this approach excluded known (and un-known) virulence genes that are expressed both

in culture and in planta and provided no infor-mation about how these regulators function inplanta. Although regulation of the eps operonappears to be similarly cell-density responsivein culture and in tomato stems (62), expressionof pehR and flhDC are reportedly dramaticallyhigher in planta than in culture, and expressionof the fliC flagellin does not require FlhDC inplanta as it does in culture (2, 98). These latterresults again suggest that there are additionalsignals and regulatory circuits functioning inplanta.

In the first transcriptome study to exam-ine gene expression in planta, Jacobs et al. (58)harvested GMI1000 and K60 cells from cul-ture in rich medium or in tomato stems at∼6 × 108 cfu per ml or per gram (at earlywilt symptoms). Of the 285 orthologous genesthat were highly expressed in planta, 250 arein the core genome and included many knownvirulence factors. Among these, the eps operon,the T2SS, and genes encoding PCWDE are ex-pressed similarly (<2.5-fold difference) in cul-ture and in planta. That cbhA was among theten most highly expressed genes in planta helpsto explain its surprisingly large contributionto virulence (68). Unexpectedly, some of thegenes that contribute to stress response (dps,dinF ) were expressed similarly both in cultureand in planta, whereas others (acrA, acrB, bcp)were downregulated in planta. This snapshotof expression is intriguing, but to understandthe regulatory networks as they function inplanta requires studying the transcriptome inwild-type and regulatory mutants at multipletime points during pathogenesis (see sidebar,Unexplored Regulatory Processes).

WHAT HAVE WE LEARNEDFROM COMPARATIVEGENOMICS?

Analyses of eight sequenced strains repre-senting the four phylotypes conducted byRemenant et al. (87) estimated that the coregenome comprises ∼2,850 conserved genes,whereas the variable genome represents∼3,100 genes. These core genes are a smaller



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UNEXPLORED REGULATORY PROCESSES

The application of tiling arrays or next-generation deep RNA se-quencing (RNA-seq) has not been reported for R. solanacearum.These methods, which permit more extensive, sensitive, and ac-curate analyses of transcriptomes, often discover new genes en-coding small peptides or noncoding RNAs, correct gene annota-tion, define untranscribed regions, and reveal operon structuresand plasticity in suboperonic expression (97). Small peptides,which previously were often overlooked, can exhibit regulatoryand other functions. Small noncoding RNAs, such as trans-actingsmall RNA (sRNA) that bind to RNA or protein and cis-actingriboswitches and antisense RNA, have enormous potential to af-fect gene expression (108). For example, sRNAs are known tohelp regulate quorum sensing, virulence, and stress responses—all of which are important for R. solanacearum pathobiology. Inaddition, the contribution of RNA-binding proteins, already wellknown in some plant pathogenic bacteria, is almost completelyunexplored in R. solanacearum (40). Likewise, small moleculeslike cyclic di-GMP and (p)ppGpp are likely to have importantroles in transcription, translation, and replication. Therefore,even though the known regulatory networks in R. solanacearumare already quite complex, you ain’t seen nothin’ yet!

percentage of the total than in some bacteriawith an equivalent genome size, such asP. syringae (∼3,400 core genes) (7), whichemphasizes the greater genetic diversity in theRSSC. It is, therefore, all the more challengingto resolve which genes are responsible forhost-range specialization or other complexbiological traits.

Metabolic Properties

The genomic sequences have revealed ametabolically versatile species complex with theability to thrive on a range of plant metabolitesfrom sugars to phenolic compounds (46). Path-ways for degradation of benzonitrile, benzoate,catechol, vanillate, and other derivatives oflignin were identified, although there are somevariations between strains (86). R. solanacearumstrains also vary in the carbon sources theycan use, and some of these differences may beexplained by the numerous genomic islands

apparently involved in metabolite uptake ordegradation. For example, the differential abil-ity to metabolize the sugar alcohols dulcitol,mannitol, and sorbitol, which is one the criteriaused for classifying R. solanacearum strains intobiovars (31), is due to the presence in somestrains of a 22-kb genomic island carrying thegenes for their utilization (42). Other genomicdifferences concerning the D-galactonatedegradation pathway or the denitrificationpathway have been documented (86), but thisobserved variability in nutrient acquisition andmetabolism has not improved understandingof niche specialization.

T3E Repertoire Dynamics

Post-genomic functional analyses usingregulatory-based approaches and/or T3SS-translocation assays have unraveled the nearlycomplete repertoire of 70–75 T3E in strainGMI1000 and the phylogenetically close strainRS1000 (28, 75–77, 79, 83) (Table 1). Homol-ogy searches combined with criteria used forT3E gene detection (27, 28) in other sequencedgenomes predict ∼110 T3E candidate genefamilies in the RSSC. Each R. solanacearumstrain appears to carry an average of 60–75 po-tential T3E, regardless of whether it has a largeor narrow host range, which is significantlyhigher than for Xanthomonas spp. or P. syringaethat have ∼30–40 T3E. The one exception sofar is R. syzygii, which possesses fewer T3Ethan R. solanacearum or BDB strains (Table 1).

The R. solanacearum T3E repertoire is char-acterized by:

� a large number (>30) of core T3E con-served among all the sequenced strains.This number is approximately 50 whencomparing repertoires from representa-tives of each phylotype and can reachup to 60 within the same phylotype(Figure 2). This contrasts with the pic-ture observed in P. syringae pathovars,where the ratio of conserved versus vari-able T3E is reversed (7). It also im-plies that the R. solanacearum common

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ancestor already possessed a large num-ber of these T3E.

� a strong diversification within the speciesbecause interphylotype T3E repertoirecomparisons generally reveal approxi-mately 25% variation in content.

� the existence of several multigenic fam-ilies because each strain carries severalT3E belonging to five main families, eachcomprising three to seven members (76,83, 88). Ortholog searches reveal thatstrains from the RSSC share some T3Efamilies with other pathogens; for exam-ple, TAL (transcription activator-like)T3E with Xanthomonas spp., AvrPtoB(HopAB) with P. syringae pathovars, andOspD with Shigella flexneri (28, 83).

� the fact that almost half of the T3Erepertoire appear specific to the RSSC.Globally, the most related T3E reper-toires are those of some Acidovorax avenaestrains that are pathogenic on cucurbits.Similarity searches reveal that one thirdof the GMI1000 T3E has at least partialhomology with A. avenae proteins.

T3E Repertoire ComparisonsDo Not Provide Clues onHost-Specificity Factors

Having genome sequences of strains with sim-ilar host-range specificity allows direct com-parison of T3E repertoires (or alternatively,complete proteomes) to tentatively identifyhost-specificity determinants. However, suchcomparisons have not revealed general pat-terns of presence or absence of particular T3Egenes. More genomic sequences probably arerequired to detect robust associations, but it isalso possible that comparisons only based onpresence/absence criteria may not be sensitiveenough to detect host-specificity factors. Forexample, a significant level of sequence diver-gence is present among strains for GALA T3Egenes in the RSSC, which suggests that theymay undergo functional diversification (88). Inaddition, it is also well known that some discrete

48 5

Psi07Phylotype IV

74 T3E

GMI1000

a b

Phylotype I

74 T3E

CMR15Phylotype III

67 T3E

CFBP2957Phylotype II

72 T3E

109

5 72

6

1

12

27

Molk2Phylotype II, banana

75 T3E

Po82Phylotype II, potato

75 T3E

CFBP2957Phylotype II, tomato

72 T3E

10 6

3

59

73

3

Figure 2(a) Number of type III effector genes identified in the sequenced genomes ofstrains representative of the four main phylotypes of the RSSC or (b) in thesequenced genomes of three strains belonging to phylotype II that wereisolated from different host plants.

amino acid changes can alter or modify proteinspecificities.

Because host range–determining factors donot only involve T3E in R. solanacearum (seeabove), comparisons should include additionalclasses of genes. Here again, there is also sig-nificant variation among RSSC strains, both interms of gene distribution or allelic differences.This is true, for example, for the Rsa1 aspar-tic protease family, sugar-binding lectins, andPCWDE. For instance, the gene encoding thepolygalacturonase PehA is disrupted by a trans-posable element in strain CMR15, whereas thisgene underwent duplication at the same lo-cus in strain Psi07. Such genetic differencesare not yet correlated with phenotypic traits.More generally, several classes of genes or loci,such as those encoding nonribosomal pokyke-tide synthases, hemagglutinin-related proteins,or Type VI-secretion system Vgr substrates, arehighly variable among RSSC strains.

PERSPECTIVES ANDFUTURE DIRECTIONS

R. solanacearum was only the third phy-topathogenic bacterium to have its genomecompletely sequenced, and this milestone



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enhanced research progress in many directand indirect ways. However, as this reviewillustrates, our understanding of the complexityand diversity within the RSSC was incompleteuntil gene-specific and genomic sequencesbecame available for multiple strains fromaround the world. The major phylogeneticgroups of the RSSC are now well established,we have a better idea of the ancient andongoing evolution of the pathogen, and thereis a proposal to divide the RSSC into newspecies and subspecies (19, 55, 86, 87, 111).Nevertheless, it is possible that pathogengenetic diversity has still been underestimated.High-throughput, next-generation sequencingmethods will surely expand the known geneticrepertoire of RSSC strains. Most notably, weexpect that examining environmental isolates(e.g., from soil or water courses) will promoteunderstanding of the evolutionary dynamicsof these strains in relation to survival andpathogenicity. Genome sequencing of multipleclosely related strains with different host ranges(such as phylotype IIB strains that differ in theirability to wilt banana) will hopefully identifyconsistent differences in genes encoding T3Eassociated with host preferences and othercandidate host-specificity factors. These resultswill then provide testable hypotheses for futurefunctional and mechanistic studies.

Complete genome resequencing also opensthe way to developing experimental evolutionmethods to unravel determinants of bacterialadaptation. For example, serial inoculationof legume plants with a R. solanacearumstrain carrying a symbiotic plasmid from anitrogen-fixing symbiont resulted in selectionof variants able to reproduce and multiplyintracellularly into root nodules (71). Genomeresequencing of these variants revealed thatadaptive mutations were in T3SS structuralgenes, the hrpG regulator (71), and also otherregulators from the virulence network (C.Masson, personal communication). Similarapproaches could be used to identify moleculartraits associated with the pathogen’s adaptation

to different environments and thereby leadto significant advances in our knowledge ofthe genes conferring selective advantages oradaptability in these conditions.

The genetic resources now available will alsoprovide the foundation upon which advancedtranscriptomic studies can be pursued. It is es-pecially important that future research on generegulation is conducted with pathogen cellsharvested from natural environments ratherthan from cultures. Based on the limited resultsto date, it seems almost certain than our modelof the regulatory networks depicted in Figure 1will be greatly modified once we determine howextracellular and intracellular signals affect thetrue regulons of key regulators (PhcA, VsrAD,HrpG, HrpB, etc.) and how gene expressionvaries with time and location outside and withinplants. These studies will also probably revealcompletely new regulatory processes affectingtranscription, translation, and protein functionand better define how all these systems areinterconnected. Knowing when and wheremany presumed pathogenicity factors (such asmost T3E or T2SS substrates) are producedwill help to decipher how they help to makeR. solanacearum such a successful pathogen onits diverse hosts. Finally, although it is gen-erally assumed that these regulation schemesare conserved among strains of the RSSC, weshould not exclude the possibility that theyhave been modified in some strains to promoteadaptation to specific hosts or environments.

R. solanacearum has been a model forstudying how bacteria cause plant diseases foralmost 60 years, but in many cases we haverelied on research approaches that are akinto shooting in the dark. However, progress inthe genomic analysis of the RSSC has nowshone a bright light on some previously obscureaspects of this pathogen’s inner workings. Thefield now seems prepared to move forwardwith greater assurance toward fulfilling ourdream that basic research on the pathogen canhelp provide innovative tools and strategies tocontrol R. solanacearum wilt diseases.

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DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We apologize to colleagues whose work was not included because of space limitations. The S.G.lab is part of the Laboratoire d’Excellence (LABEX) entitled TULIP (ANR-10-LABX-41). T.D.thanks his students and postdocs for their efforts and the USDA, NSF, Hatch, and state agenciesthat have supported his research. We are grateful to Mark Schell and Christian Boucher for criticalreading of a draft manuscript and thank Caitilyn Allen, Philippe Prior, Emmanuel Wicker, MarcValls, and Catherine Masson for sharing unpublished information, and Laure Plener for hercontribution to Figure 1.

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RELATED RESOURCES

1. French National Institute for Agricultural Research (INRA). 2012. Ralstonia solanacearumGMI1000, MOLK2 and IPO1609: a phytopathogenic bacterium with a wide host range. INRAhttp://iant.toulouse.inra.fr/bacteria/annotation/cgi/ralso.cgi

2. Vallenet D, Engelen S, Mornico D, Cruveiller S, Fleury L, et al. 2009. MicroScope: a plat-form for microbial genome annotation and comparative genomics. Database doi:10.1093/database/bap021.



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http://iant.toulouse.inra.fr/bacteria/annotation/cgi/ralso.cgi

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