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Review in Advance first posted online on May 1, 2012. (Changes may still occur before final publication online and in print.)
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Pathogenomics of the Ralstonia Solanacearum Species Complex

Annu. Rev. Phytopathol. 2012.50. Downloaded from www.annualreviews.org by INRA Institut National de la Recherche Agronomique on 05/14/12. For personal use only.
St phane Genin1,2, and Timothy P. Denny3 e

1
INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441, F-31326 Castanet-Tolosan, France; email: stephane.genin@toulouse.inra.fr CNRS, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR2594, F-31326 Castanet-Tolosan, France Department of Plant Pathology, The University of Georgia, Athens, Georgia, 30602-7274; email: tdenny@uga.edu
Annu. Rev. Phytopathol. 2012. 50:4.14.23 The Annual Review of Phytopathology is online at phyto.annualreviews.org This articles doi: 10.1146/annurev-phyto-081211-173000 Copyright c 2012 by Annual Reviews. All rights reserved 0066-4286/12/0908/0001$20.00
Keywords
bacterial wilt, -proteobacteria, host specicity, regulatory network, type III effector, virulence
Abstract
Ralstonia solanacearum is a major phytopathogen that attacks many crops and other plants over a broad geographical range. The extensive genetic diversity of strains responsible for the various bacterial wilt diseases has in recent years led to the concept of an R. solanacearum species complex. Genome sequencing of more than 10 strains representative of the main phylogenetic groups has broadened our knowledge of the evolution and speciation of this pathogen and led to the identication of novel virulence-associated functions. Comparative genomic analyses are now opening the way for rened functional studies. The many molecular determinants involved in pathogenicity and host-range specicity are described, and we also summarize current understanding of their roles in pathogenesis and how their expression is tightly controlled by an intricate virulence regulatory network.
Corresponding author
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Changes may still occur before final publication online and in print
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INTRODUCTION
Species complex: a group of closely related organisms thought to comprise more than one species RSSC: Ralstonia solanacearum species complex Blood disease bacterium (BDB): a pathogen of banana in Indonesia Megaplasmid: the customary name for the smaller essential replicon of the bipartite RSSC genomes Contig: a region with overlapping sequences that has not been assembled as part of a complete genomic sequence
Lethal wilts caused by Ralstonia solanacearum are among the most important bacterial diseases of plants. This soilborne pathogen is present on all continents and many islands between the Tropics of Cancer and Capricorn (34). Recent pathogen surveys and genetic studies have proven that, as Buddenhagen opined more than 25 years ago, strains of this pathogen evolved in widely different places and they have different capabilities with both native ora and introduced hosts (17). Indeed, because of the extensive diversity revealed by phylogenetic analyses, this group of organisms is now commonly called the R. solanacearum species complex (RSSC) (35). R. solanacearum, a -proteobacterium, is pathogenic on more than 200 plant species belonging to over 50 different botanical families (31). The pathogen affects not only solanaceous plants, such as tomato and potato, but many weeds, crops, shrubs, and trees in other dicot and monocot families. Unique to Indonesia are Ralstonia syzygii, a pathogen of clove trees, and the blood disease bacterium (BDB), a banana pathogen. BDB is closely related to R. solanacearum, but its ofcial taxonomic standing remains unresolved (36, 87). This unusually wide host range is continuously expanding, and descriptions of new hosts are common. Although the etiology resulting in wilting symptoms is probably similar for most susceptible hosts, different disease names are often used depending on the crop affected. R. solanacearum can survive for years in moist soils or water microcosms (5, 104). When the pathogen encounters a susceptible host, it enters the root and colonizes the root cortex, then invades the xylem vessels, and nally spreads rapidly to aerial parts of the plant through the vascular system (31). Wilting symptoms result from the vascular dysfunction caused by this extensive colonization. The bacterium can also colonize some hosts asymptomatically in latent infections, and this poorly understood phenomenon is crucial in the epidemiology of these strains.
This review focuses on advances in research and understanding made possible in the past decade from having genomic sequences for strains representative of the biodiversity within the RSSC. These include insight into the evolution and speciation of the pathogen, progress in identifying new virulence and potential hostrange determinants, and renement of the intricate regulation networks controlling systems important for pathogenesis.
GENERAL FEATURES OF RALSTONIA SOLANACEARUM SPECIES COMPLEX GENOMES

R. solanacearum genomes are organized in two circular replicons customarily called the chromosome and the megaplasmid, with the latter being about one third of the average 5.8 Mb total (86, 94). However, because both replicons carry essential and pathogenesis-related genes and analyses indicate they have coevolved (22, 55), they comprise a bipartite genome. Although it would be technically more correct to rename the megaplasmid (e.g., chromosome 2), we will continue using this term for consistency with the published literature. Since the publication of the genome sequence of strain GMI1000 in 2002 (94), 10 additional genomes of RSSC strains have been sequenced (as of November 2011). These 11 strains are representative of the RSSC and, in some cases, display some host-range selectivity. The main features of these genome sequences are given in Table 1. Five genomes are not completely assembled and have approximately 10 (IPO1609, BDB, R. syzygii ) or 40 (Molk2) supercontigs or 580 individual contigs (UW551), which precludes determining rearrangements among replicons. In addition, the unusually low number of tRNAs identied in some of the draft genomes (Table 1) suggests that regions of low coverage may have missing or erroneous sequence data. Functional gene assignments clearly show that the large majority of housekeeping functions are chromosome-borne, whereas the megaplasmid, although carrying some essential
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Table 1
Genome Size (kb)b Isolated from CHR 3,716 3,726 3,539 NA NA NA NA 5,961 5,061 1 34 NA 5,895 4,371 1 43 NA 5,313 4,546 1 29 8 ND 5 2,144 5,683 5,310 1 56 11 1,986 5,712 5,496 5 53 ND 2,094 5,811 5,120 4 57 9 74 ND 72 60 58 75 MPL SPL total CDSc tRNAs T3Ee Tomato Tobacco Tomato Potato Geranium Banana IIB(3) draft IIB(1) draft IIB(1) draft IIA(36) complete I draft I(18) complete Phylotypea Sequence status rRNA operons Putative prophaged
Features of sequenced genomes of the Ralstonia solanacearum species complex
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Strains
Origin
Reference (94) (67) (86) (54) (42) (C. Boucher, unpublished data)
GMI1000
French Guyana
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Y45
China
CFBP2957
French West Indies
IPO1609
Netherlands
UW551
Kenya
Molk2
Philippines
Po82 Tomato Tomato Banana Clove IV draft 3,681 1,743 5,424 IV draft 3,574 1,585 5,159 IV(10) complete 3,508 2,085 13 5,606 5,247 4,629 4,867 III(29) complete 3,594 1,963 35 5,593 5,149
Mexico
Potato
IIB(4)
complete
3,481
1,949
5,430
5,019
3 3 1 1 2
54 59 49 45 50
5 10 7 9 ND
75 67 74 57 48
(112) (86) (86) (87) (87)
CMR15
Cameroon
Psi07
Indonesia
BDB R229
Indonesia
Ralstonia syzygii R24
Indonesia
a Sequevar
b CHR,
numbers are in parentheses. chromosome; MPL, megaplasmid; SPL, small plasmid; NA, not assembled. c CDS, number of coding sequences. d Number of prophage or phage-like elements; ND, not determined.
www.annualreviews.org Ralstonia Solanacearum
e Number
of Type III effectors; ND, not determined.
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Synteny: preservation of gene order on a replicon Chemotaxis: the ability of a cell to move in response to a chemical or energy gradient HGT: horizontal gene transfer
Type III effector (T3E): a protein secreted by the type III secretion system
genes, is enriched in genomic islands and strainspecic genes (86, 87, 94). This observation is also supported by (a) comparative genomic hybridization studies on a collection of 18 strains, showing that 63% of the genes from the megaplasmid are not conserved (55), and (b) the level of synteny is higher on the chromosome (70% to 80% of the coding sequences) than on the megaplasmid (55% to 65%) (86). A comparison of the genome replicon sizes also suggests that the megaplasmid probably carries many genes associated with specic lifestyles because its size varies up to 26%, whereas there is minimal variation for the chromosome (3.53.7 Mb, a 6% size variation) (Table 1). Accordingly, most of the functions involved in adaptation to the environment or phytopathogenicity are megaplasmid-borne, including the Type III and Type VI protein secretion systems, agellar motility determinants, chemotaxis genes, and the extracellular polysaccharide (EPS) biosynthesis cluster. The reduced size of the megaplasmid in R. syzygii and BDB strains suggests a possible ongoing reductive evolution of this replicon due to their specic lifestyle as insect-transmitted and host-specialized parasites (87).
Horizontal Gene Transfer Events and the Contribution of Prophage Elements

Annotation of the GMI1000 genome led to the denition of alternate codon usage regions (ACUR) that account for 7% of the genome. Because ACUR mostly correspond to lower guanine plus cytosine content regions and/or prophage-associated regions (94), most constitute genomic islands, many of which were presumably acquired through horizontal gene transfer (HGT). Approximately 7080 genomic island regions varying in size from 5 to >100 kb were dened in several genomes (86) and generally represent the nonsyntenic stretches observed after comparing the global gene order on both replicons. As already demonstrated in other bacterial pathogens (78), it is plausible that such a modular genome organization
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distinguishes regions with genes devoted to housekeeping functions from those more dedicated to host adaptation. Many of R. solanacearum genomic islands in each of the sequenced genomes correspond to putative prophages or phage-like elements (87) (Table 1). Phages are abundant in soil and are key factors shaping evolution of soil microbial communities (50, 91). Phages are also efcient vectors for HGT (63) that can contribute to the rapid acquisition of novel adaptive functions and so play a role in the emergence of pathogenic variants. A detailed inspection of R. solanacearum prophage clusters suggests that they probably disseminate several genes involved in the plant-bacterium interaction, such as genes for type III effectors (T3Es) ( popP1, popP2, ripT, etc.), as well as other genes encoding products of unknown function. It is remarkable that in all three strains possessing popP2 (GMI1000, Po82, and CMR15), this gene is associated with a prophage inserted at different genomic locations. Several other R. solanacearum prophages appear to be strainspecic (41, 42, 54), suggesting that their distribution may be variable among strains. Small plasmids were detected in two strains (Table 1) and could also contribute to efcient HGT. No specic biological function could be inferred from their gene content (86), but it seems likely that the CMR15 plasmid was acquired from a Xanthomonas sp. because of the apparent conservation of the Type IV secretion system genes. Potentially transferable integrative conjugative elements were also found in strains GMI1000 and Molk2 (see References 46, 52).
SYSTEMATICS OF THE RALSTONIA SOLANACEARUM SPECIES COMPLEX

Early attempts to codify the diversity in the RSSC resulted in separate race and biovar systems (31). R. solanacearum races are based on host range at the plant species level and thus, contrary to typical usage, are roughly equivalent to pathovars of other phytopathogenic bacteria
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(4, 85). However, our increasing knowledge of pathogen diversity makes the race system unwieldy and unreliable (31). The biovar system groups strains by their ability to acidify media containing selected carbohydrates, to produce nitrite from nitrate, and to produce gas from nitrate (30). Although useful for many years, the biovar system lacks discriminating power because of its limited genetic basis. Both of these systems have now largely been replaced by the widely accepted phylotype-sequevar system (see below). A variety of genetic techniques have been used to investigate the diversity and interrelatedness within the RSSC (31). Classical restriction fragment length polymorphism analysis initially revealed fundamental heterogeneity of pathogen strains, but large-scale DNA sequence analyses more profoundly inuenced our understanding of pathogen systematics (19, 31, 85, 107). As the diversity of the strains examined increased, it became clear that the RSSC has four major subdivisions, denoted as phylotypes (35, 85). A multiplex polymerase chain reaction (PCR) can rapidly assign strains to a phylotype (35). Each phylotype is subdivided into numbered sequevars and closely related individual strains, which are usually identied by analyzing sequence similarity of the egl endoglucanase structural gene. The phylotypes correspond roughly to the strains geographic origin: Asia (phylotype I), the Americas (II), Africa (III), and Indonesia (IV). Phylotype II has two clearly recognizable subclusters (IIA and IIB) (19, 36, 85, 107). Both the BDB and R. syzygii fall within phylotype IV. The same relationships are observed regardless of the conserved gene examined, suggesting that they coevolved with the various RSSC genomes. The same phylotypes also were observed when (a) comparative genomic hybridization to a microarray was used to generate hierarchical clusters based on gene conservation (55, 86) and (b) the genome sequences of six representative strains were compared (86, 87). Multilocus sequence and statistical analyses of a moderately diverse RSSC population
indicated that the phylotypes reect true evolutionary lineages that probably arose long ago when progenitors became geographically separated (19). More recently, similar analyses of strains that better represent RSSC diversity found the same phylotype structure but detected both inter- and intraphylotype recombination in seven of nine genes examined (111). Three methods used to estimate recombination indicated that (a) phylotype I is freely recombining, and (b) although recombination is common in phylotypes IIA, III, and IV, it has not yet eliminated all clonal structure. Wicker et al. (111) also used coalescent genealogy reconstruction to deduce the order in which phylotypes likely evolved, and along with known ecotypes they described eight clades superimposed on the phylotypes (Table 2). When combined with ancestral state reconstruction, these results suggested that the pathogen originated in the Oceania/Indonesia region, migrated to Africa and from there to South America (possibly before the fragmentation of Gondwana) and to Asia. Adaptation to different environments and potential host plants may also be driving evolution (111). For example, on the island of Martinique, Wicker et al. (109, 110) documented the appearance and spread of phylotype IIB sequevar 4 strains that, unlike typical members of this subgroup, are pathogenic on solanaceous crops and cucurbits but latently infect banana rather than causing wilt disease. These new strains are exceptionally aggressive on tomato, as they wilt even the resistant H7996 line. How these strains arose is unknown. However, most R. solanacearum strains tested become naturally transformation competent in culture, and probably also within plants, and can both acquire and lose large (3090 kb) regions of their genome when exposed to exogenous DNA (9, 26, 53). Indeed, after strain Psi07 was treated in vitro with genomic DNA from GMI1000, one transformant was more aggressive on tomato (but not eggplant) (25). Thus, HGT could contribute to R. solanacearum genetic and pathogenic diversity, and we now have the genetic tools to determine the molecular
Phylotype: the major phylogenetic subdivisions within the RSSC Sequevar: a group of two or more identical gene-sequence variants; usually determined based on the egl gene sequence
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Table 2 complex
Genetic, geographic, and ecotype diversity within the Ralstonia solanacearum species
Phylotypea
I IIA IIAT IIB IIB
Origin
Asia Americas
Cladeb
1 2 3 4 5
Ecotype informationc
BW of Solanaceae, ginger, mulberry BW of Solanaceae, Musa BW of Solanaceae (southeastern United States and Caribbean) Moko disease of Musa, new NPB variants Brown rot of potato, BW of tomato and geranium (R3bv2), Moko disease; distributed worldwide BW of Solanaceae BDB of banana BW of Solanaceae
Proposed speciesd
Ralstonia sequeirae Ralstonia solanacearum
Ralstonia solanacearum
Americas Africa
III IV
6 7
Ralstonia sequeirae Ralstonia haywardii subsp. celebensis R. haywardii subsp. solanacearum R. haywardii subsp. syzygii
Indonesia IV 8
R. syzygii, Sumatra disease of clove
The T indicates that the R. solanacearum type strain K60 is in phylotype IIA. Clades were dened by Wicker et al. (111). c BW, bacterial wilt; R3bv2, previously classied as Race 3 biovar 2; BDB, blood disease bacterium. NPB, not pathogenic to banana: designates the new pathogenic variants found on Martinique that wilt Anthurium, Heliconia, and cucurbits but not cultivated Musa. d See 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).
b
basis underlying such cases of host-range specialization. Analysis of the genomic DNA sequences of six R. solanacearum strains, and one each of R. syzygii and BDB strains, representing all the phylotypes provided additional insight into the phylogeny of the RSSC (54, 86, 87, 94). Most signicantly, based on pairwise comparison of average nucleotide identity, Remenant et al. (87) concluded that the RSSC has three groups of strains that exceed the accepted threshold for speciation. Considering these and the extensive genetic results noted above, they proposed two new species and three subspecies (Table 2). This conclusion is supported by the limited DNA-DNA hybridization data (80, 89, 105) that indicated the RSSC is polyphyletic. Unfortunately, the sequence analyses provided no obvious explanations regarding the molecular basis of host preference or ecological niche adaptation. Furthermore, two extensive tests examining the pathogenic potential of diverse R. solanacearum strains on four solanaceous hosts revealed no
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good correlation with phylotype or sequevar (20, 66).
RALSTONIA SOLANACEARUM VIRULENCE DETERMINANTS General Virulence Factors

R. solanacearum virulence factors enhance the pathogens ability to cause disease. The single most important virulence factor of R. solanacearum is its high molecular mass EPS, which is produced in massive amounts in culture and in planta (31). This EPS promotes rapid systemic colonization and eps mutants rarely wilt or kill plants even when bacteria are introduced directly into the xylem (93). Accumulation of EPS is largely responsible for the vascular dysfunction that causes wilt symptoms in susceptible hosts. Surprisingly, EPS triggers enhanced expression of the ethylene and salicylic acid defense response pathways in the wiltresistant H7996 tomato breeding line but not in a susceptible cultivar (74).
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The assemblage of approximately 30 extracellular proteins transported across the outer membrane by the Type II secretion system (T2SS) are also essential for colonization and bacterial wilt (68, 83). When tested individually for their contribution to disease of tomato, the four pectolytic enzymes (PehABC and Pme) are generally less important than the two cellulolytic enzymes (Egl and CbhA) (31, 68). However, a mutant lacking all six of these plant cell walldegrading enzymes (PCWDE) is considerably more virulent than a mutant with a nonfunctional T2SS, which also is severely reduced in its ability to invade and colonize plants. Therefore, some combination of the other 24 extracellular proteins, which have diverse predicted and unknown functions, is also quite important for virulence. Pathogen motility also contributes significantly to disease. Flagella-driven swimming motility is growth-phase dependent in culture, but bacteria recovered from within tomato plants are overwhelmingly nonmotile (99). Both nonmotile, nonagellated and motile, nonchemotactic mutants are signicantly reduced in virulence when applied as a soil drench of potted tomato plants, but exhibit normal virulence when inoculated directly into the xylem via a severed petiole (99, 114). Both a hypermotile motN mutant and an aer2 mutant, which is motile but nonaerotactic, are slightly reduced in virulence when inoculated by soil drench (73, 115). These results show that motility is required for R. solanacearum to locate and invade roots, although it is dispensable later during pathogenesis. Twitching motility, which is driven by polar, retractable type 4 pili, also contributes to virulence on tomato regardless of whether plants are inoculated by soil drench or via severed petioles (61). Type 4 pili also promote autoaggregation and biolm formation in broth culture, and polar attachment to tomato roots. During pathogenesis, R. solanacearum encounters a variety of stressful environments and compounds. Among these are a variety of reactive oxygen species (ROS) made by plants after infection (38, 70, 74). The pathogen
appears to have correspondingly redundant mechanisms to detoxify ROS or otherwise tolerate this oxidative environment, and many of these appear to be expressed in planta (14). Not surprisingly, mutants lacking just the Bcp peroxidase or Dps, a DNA binding protein that promotes stress tolerance, are almost fully virulent (24, 38), but inactivation of oxyR, which encodes the only identied regulator of oxidative stress genes, signicantly reduces virulence (39). The pathogen may also encounter multiple toxic compounds during pathogenesis, and inactivation of acrA and dinF, which likely encode components of multidrug efux pumps, reduced tolerance to some compounds in culture and signicantly reduced virulence (15). The pathogen also has multiple cytoplasmic polyphenol oxidases that may help detoxify phenolic compounds, but their biological roles have not been reported (56). Microaerobic conditions may also stress the pathogen, and a ccb3 -type cytochrome c oxidase contributes both to its survival in very low oxygen conditions and to virulence (23). Some metabolic pathways also appear to be specically required during pathogenesis because deletion of the metER methionine biosynthesis genes in two phylotype IIB sequevar 1 strains reduced disease symptom production without causing auxotrophy (51). Bacteria often encounter low concentrations of soluble iron and therefore produce one or more siderophores to scavenge this essential element. All R. solanacearum strains probably produce the polycarboxylate siderophore staphyloferrin B (10), but it is likely that phylotypes I, III, and IV also make micacocidin, a yersiniabactinlike siderophore (64). However, tomato xylem uid appears to have sufcient iron to repress expression of any iron-acquisition system, and a mutant that does not make staphyloferrin B is fully virulent on tomato (10). It has long been suspected that phytohormones produced by R. solanacearum might contribute to virulence. Production of ethylene in culture by GMI1000 is positively regulated by HrpG (see below), and the pathogen appears to make ethylene in Arabidopsis (103). Nevertheless, ethylene-negative mutants are
Type II secretion system (T2SS): secretes selected proteins from the bacterial periplasm to the environment PCWDE: plant cell walldegrading enzyme Twitching motility: the ability of bacteria to propel themselves over solid surfaces by using retractable type 4 pili
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Type III secretion system (T3SS): translocates proteins directly from the bacterial to the host cytoplasm PhcA: the global regulator controlling reversible phenotype conversion in response to 3-OH PAME
fully virulent (31, 103). Production of auxin is also positively controlled by HrpG, but its role in virulence has not been reported. R. solanacearum can also make cytokinin in culture and there is one report that inactivation of the conserved tzs gene responsible for its production reduces virulence (31).
(T3SS) regulator HrpB (see below). Surprisingly, Rsa1 is secreted extracellularly but in a T3SS-independent manner and possesses an aspartic protease activity (60). It will be interesting to see how Rsa1 is recognized by pepper cells.
Host-Range Factors
R. solanacearum strains can strikingly differ in their host range, some being obviously polyphagic, whereas other strains are characterized by a restricted, specialized host range (for a detailed review, see Reference 45). Host-specic interactions of R. solanacearum strains with certain hosts (18, 66) and at the cultivar/ecotype level (33, 49, 65, 102) have been documented. In the case of AvrA on tobacco (90), PopP1 on resistant petunia lines (65), and PopP2, which is recognized by the Arabidopsis resistance protein RRS1-R (32), determinants restrict host range and correspond to T3E that trigger incompatibility on resistant plants. Inactivation of these bacterial avirulence factors is generally sufcient to restore pathogenicity (32, 65). However, in tobacco, it was shown that inactivation of a second gene ( popP1) other than avrA is required to restore full pathogenicity in some strains because both determinants elicited a hypersensitive response on tobacco and are involved in host-range restriction (82). In contrast, presence of the gala7 gene extends the host range of strain GMI1000 to include the legume Medicago truncatula (6). The best characterized T3E to date is PopP2, which possesses acetyl-transferase activity (100) and interacts with multiple Arabidopsis targets (8), but the molecular functions of most R. solanacearum T3E remain elusive (see Reference 83). A recent report revealed that R. solanacearum host specicity is not only determined by T3E. rsa1, a virulence gene from a phylotype IV potato strain, confers avirulence on pepper when introduced into a phylotype I strain normally able to cause disease on this host (60). Expression of rsa1 is under the transcriptional control of the type III secretion system
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VIRULENCE AND PATHOGENICITY REGULATORY NETWORKS

R. solanacearum pathogenesis functions are regulated by sophisticated, multicomponent interconnected networks that are sensitive to environmental and internal factors. A great deal has been learned about how these networks function in culture, but we only have glimpses of processes that occur during pathogenesis.
The Phc Connement-Sensing System

Spontaneous loss of virulence, EPS, and other traits by R. solanacearum in culture vexed and perplexed scientists for decades (31). The genetics of this phenomenon, called phenotype conversion (PC), remained unclear until the discovery of PhcA, a LysR-type transcriptional regulator that controls expression of many genes (16, 95). DNA replication errors and transposition of insertion sequence elements can inactivate phcA (16, 59, 84), and phcA mutants (PC-types) can accumulate because they multiply preferentially during some stressful conditions (e.g., prolonged stationary phase in culture or in planta and high salt or low oxygen concentrations) (31). PhcA and 3-Hydroxy palmitic acid methyl ester (3-OH PAME). Traits that are positively regulated directly or indirectly by PhcA are shown in Figure 1a and negatively regulated traits are shown in Figure 1b. The PhcA regulon almost certainly includes many more genes than those shown, but transcriptome studies have not been reported. Thirty RSSC strains examined have well-conserved homologs of phcA, as do R. syzygii, Ralstonia
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picketii, and three Cupriavidus species (44, 55, 86, 87). All these genes are probably functional because one of the most divergent PhcA proteins from Cupriavidus metallidurans complemented a R. solanacearum phcA mutant (44) (see sidebar, How Unique Is The 3-OH PAME Autoinducer?). When cultured in vitro, wild-type PhcA has a central role because it enables the pathogen to cycle between two very different phenotypic states in response to nutrient availability and cell density (31, 95). Levels of functional PhcA are controlled by a connement-sensing system encoded by the phcBSR operon. PhcB appears to be a methyltransferase that synthesizes 3-hydroxy palmitic acid methyl ester (3-OH PAME), an unusual quorum-sensing signal. PhcS and PhcR compose a two-component regulatory system that responds to threshold concentrations of 3-OH PAME by elevating the level of functional PhcA (Figure 1). Therefore, cells at low density, such as those in soil and in plants at the leading edge of infection, have little functional PhcA and, like phcA mutants, exhibit a low virulence phenotype that may be optimized for survival and invasion of plant tissues. In contrast, cells at higher densities, such as those in colonized xylem vessels, have abundant functional PhcA and produce multiple virulence factors while suppressing production of survival/invasion factors (95). There are still many aspects of the Phc sensing system that we do not understand. For example, phcA mutants produce almost no 3-OH PAME, but PhcA apparently does not control expression of the phcB synthase. Also, how PhcR, an atypical response regulator that has a histidine kinase as its output domain, affects both expression and function of PhcA has not been resolved. Schell (95) presented preliminary evidence suggesting that 3-OH PAME triggers (auto)phosphorylation of this domain, which prevents it from post-transcriptionally inhibiting production, stability, or function of PhcA, but this presumably involves unidentied intermediate regulators. Furthermore, the nutritional status of cells seems to have a critical role in determining the responsiveness of
HOW UNIQUE IS THE 3-OH PAME AUTOINDUCER?

Genomic sequence analysis suggests that production and sensing of 3-OH PAME is restricted to three genera within the -proteobacterium Burkholderiaceae family (see Table 3). In most Burkholderia spp., the PhcR orthologs lack a kinase output domain, and PhcA is absent in all strains. Unexpectedly, Polaromonas naphthalenivorans, which is in the Comamonadaceae family, probably acquired the phcBSR operon by HGT; it also lacks PhcA. When discovered, 3-OH PAME was the only fatty acid derivative other than the acyl-homoserine lactones and the similar -butyrolactones to function as an autoinducer. However, this has changed with the discovery that multiple bacteria make diffusible signal factor family (unsaturated fatty acids rst characterized in Xanthomonas campestris) (92) or CAI-1 family (-hydroxyketones rst characterized in Vibrio cholerae) (101) autoinducers. The RSSC genomes apparently lack genes encoding orthologous synthases for these compounds.
Percent identity to GM1000 orthologs Organism
All RSSC strains Two Ralstonia spp. Three Cupriavidus spp. Some Burkholderia spp. Polaromonas naphthalenivorans 80+% PhcB 8489 77 6470 6368 60 PhcS 8395 7172 5062 58 50 5059% PhcR 8791 7677 5253 4653 41 4049% PhcA 9395 86 4749 absent absent
6079%
wild-type cells to 3-OH PAME because its addition to low-density cultures has no effect until the culture reaches >106 cells per ml (21). These observations suggest that, as in other bacteria (11), additional regulatory factors affect quorum sensing in R. solanacearum. Other regulatory components of the Phc network. There are multiple additional regulatory proteins that play supporting roles in this network (Figure 1) (31, 95). Both PhcA and the VsrAD two-component sensor/response regulator system are necessary to fully activate transcription of xpsR, and both XpsR and the VsrC response regulator bind to the eps operons promoter to enhance expression (43). PhcA indirectly represses production
3-Hydroxy palmitic acid methyl ester (3-OH PAME): the major quorum sensing autoinducer in the RSSC
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Positively regulated traits

AHL EPS SolR RpoS SolI 3 OH-PAME aidA solIR EpsR PhcB eps genes
PhcS
phcBRS PhcR
phcA
PhcA
xpsR
XpsR VsrB VsrC Signal?
egl Plant cell wall degrading enzymes exported via T2SS
pme
flhDC
pehA fli genes
cbhA VsrD MotN ralAD
Flagellar motility
Ralfuranone Signal?
Regulatory protein families

VsrA
Negatively regulated traits

Plant signal PrhA PrhR prhA prhIR 3 OH-PAME PrhJ hrpG PhcB Siderophore HrpG PhcS phcBRS PhcR phcA PrhG PrhI prhJ prhG Metabolic signal(s)
AraC LysR factors Two-component Other Signaling Transcriptional activation Post-transcriptional effect Post-translational activation Extracellular signal perception Transcriptional repression Culture-specific regulation Gene product Extracellular secretion
PhcA
Other genes
hrpB HrpB
Other genes
katE
Catalase Polygalacturonase
T3SS & T3E
pehSR Plant signal? PehS fli genes Flagellar motility PehR
pehA via T2SS
efe hdf operon rsa1

Protease
tfp genes
Type 3 secretion system
HDF
Type 4 pili
Ethylene
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of PehA as well as swimming and twitching motility by reducing the function of the PehSR two-component system that positively controls expression of pehA, i genes, and pilA (Figure 1b) (31, 61, 73). Production of PehA is also repressed by VsrC independently of PhcA (57). Flagellar motility is positively controlled in culture by VsrC and repressed by MotN, both of which affect expression of the conserved hDC regulatory protein (73). The VsrAD two-component system controls multiple other traits, some of which strongly contribute to the ability of R. solanacearum to colonize tomato stems and multiply in planta independently of this regulators effect on EPS production (14, 95). For example, when strain K60 is grown in culture, VsrAD is required for wild-type biolm formation, tolerance of both cold temperatures and hydrogen peroxide, and acyl-homoserine lactone (AHL) production, but the mechanisms underlying these effects have not been determined (113). Creation of genomic lacZ transcriptional reporter fusions recently conrmed that VsrAD positively controls expression of cbhA in strains K60 and AW1 (see Reference 95) but revealed that this is not true in GMI1000. In contrast, cbhA is positively controlled by PhcA in all three strains, although regulation is much stronger in GMI1000 than in the other strains (T.P. Denny, unpublished results). Both VsrAD and PhcA positively control production of the secondary metabolite ralfuranone [4phenylfuran-2(5H)-one] (96), but any role of the latter in pathogenesis has not been reported. Although halogenated furanones inhibit AHL-regulated processes in multiple bacteria (72), the LuxIR-type AHL quorum sensing system in R. solanacearum that is
regulated by PhcA (Figure 1a) is dispensable for virulence (37). VsrAD negatively controls swimming motility (by strongly repressing expression of hDC) and twitching motility, but these effects may be largely independent of its weak effect on phcA and pehSR (113). Although many of the VsrAD-regulated traits are also controlled by PhcA, the multiple phenotypic and regulatory differences indicate that these are separate regulons (14, 113). One of the most notable differences is the large effect that growth phase and medium composition have on some VsrAD-regulated traits.
Type III Secretion System Regulators

R. solanacearum regulates deployment and use of its T3SS with an environmentally responsive signal transduction network with at least seven components, some of which constitute a plant responsive regulatory cascade (from PrhA to HrpB) (Figure 1) (12, 81). The T3SS regulation mechanism used by R. solanacearum differs fundamentally from that of Pseudomonas syringae and Erwinia spp. but has common components (HrpB and HrpG) with the Xanthomonas spp. T3SS regulatory system (13). The HrpB regulon. HrpB is an AraC family transcriptional regulator that controls the T3SS and T3E gene expression (48). HrpBdependent expression is conferred by a cisregulatory DNA element named the hrpII box (TTCG-n16-TTCG) that lies just upstream of the 35 region in target promoter genes (27). Transcriptomic analyses revealed that, when tested in minimal medium, HrpB inuences the expression of >180 genes (approximately 140 positively and 50 negatively) (79). Positively regulated genes include the T3SS structural

Figure 1 The 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 system and other virulence-associated genes is in b. The metabolic/nutritional signal affecting hrp gene expression is not known. Broken lines indicate presumed indirect regulation. Lines ending with a crossbar indicate repression, whereas lines with an arrowhead indicate activation. Abbreviations: EPS, exopolysaccharide; HDF, hrpB-dependent diffusible factor (3-hydroxy-oxindole); T2SS, type II secretion system.
www.annualreviews.org Ralstonia Solanacearum 4.11
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genes, T3SS helper proteins (lytic transglycosylases, chaperones, etc.) and all known T3E. Given that a subclass of HrpB-dependent T3E promoters does not possesses an hrpII box (76, 79), it is likely that the regulation of some target genes is indirect or involves a distinct regulatory mechanism. However, the HrpB regulon may be much smaller during pathogenesis because genome-wide expression analysis of cells recovered from stems of wilting tomato plants revealed that only approximately half of genes positively regulated by HrpB were strongly expressed (58). These results suggest that other factors modify expression of this regulon in planta. HrpB action extends beyond controlling the T3SS and T3E genes (60, 79). For example, HrpB activates an operon of six genes responsible for the synthesis of an hrpB-dependent diffusible factor (HDF), identied as 3-hydroxy-oxindole (29). Because this tryptophan derivative induces AHL receptor-mediated activity, it was hypothesized that HDF produced by R. solanacearum interferes with the quorum-sensing systems of rival bacteria at early steps of infection (29). Contribution to pathogenicity of these T3SS-independent functions controlled by HrpB is still unknown, although it was reported that an hdf mutant is less competitive than the wild-type strain during mixed infections (69). The HrpG regulon. HrpG, an OmpR family response regulator, was originally discovered as positively controlling hrpB expression (13). Transcriptome studies later revealed that in culture the complete HrpG regulon has approximately 180 genes in addition to those regulated by HrpB, and that as a group this new set of genes is essential to pathogenesis (103, 106). HrpG transcriptionally controls the expression of functions that promote adaptation of the bacterium to life in hosts, as well as previously identied virulence factors (reviewed in Reference 45). The HrpG regulon therefore includes some genes encoding PCWDE, detoxifying enzymes, phytohormones, lectins, metabolic enzymes and transporters, some
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probable intermediate regulators, and many genes with unknown functions (103). HrpG is an orphan response regulator without a cognate sensor kinase. How its function is controlled post-transcriptionally remains poorly understood, but it appears that HrpG activity is modulated in response to nutritional signals so cross-talk with multiple sensor kinases is possible (13, 47, 117). However, mutation of the aspartate residue predicted to be the phosphorylation site signicantly reduces but does not abolish HrpG activity (117). Recently, a close paralog of hrpG, named prhG, was discovered (81). The two corresponding proteins are very similar (72% identical) and both activate hrpB expression. However, the function of PrhG appears restricted to specic environmental conditions (i.e., minimal medium) and is dispensable in planta; accordingly, a prhG mutant is not altered in pathogenicity. The precise role of this hrpG paralog in T3SS activation remains enigmatic because it does not appear to control additional genes other than hrpB. Expression of prhG itself requires the products of a three-gene operon named prhKLM, none of which are transcriptional regulators and thereby probably modulate expression of prhG indirectly (118).
Regulatory Processes During Pathogenesis

HrpB and HrpG are the downstream components of the Prh regulatory cascade that also includes the outer-membrane receptor PrhA and the PrhIRJ proteins (Figure 1b). The Prh pathway induces high expression of the T3SS upon plant cell contact (1). The exact nature of the plant signal sensed by the PrhA receptor is unknown, but there is evidence that it is a nondiffusible molecule that may be an intrinsic constituent of the plant cell wall. However, HrpB and HrpG must be activated by multiple signals in planta because inactivation of the PrhAIRJ components results in partial loss of virulence, whereas hrpB or hrpG mutants are nonpathogenic (12). Among the known environmental cues that inuence T3SS gene
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expression are repressing signals perceived when bacteria are grown in the presence of organic nitrogen sources and inducing conditions when bacteria are grown in a supposed apoplast-mimicking minimal medium. There is genetic evidence that PhcA impacts the Prh pathway at different levels. First, PhcA represses expression of T3SS genes in rich medium, and this effect is probably mediated through HrpG at the post-transcriptional level (47). However, this repression appears to be ameliorated after cells enter the stationary phase or when cultured in minimal medium (113). Second, in cells cultured in rich medium, PhcA binds to the promoter region of prhI and slightly represses its expression (116), thus demonstrating that it may affect the Prh pathway at the transcriptional level. However, the situation likely is more complex in planta. For example, eight hours after cells carrying a hrpB reporter fusion were inltrated into tobacco leaves, hrpB expression was, contrary to expectations, higher in the wild type than in a phcA mutant (113). In addition, in planta transcriptome studies and qRT-PCR tests by Jacobs et al. (58) and expression studies in planta using green uorescent protein reporter fusions (F. Monteiro, S. Genin, I. van Dijk, M. Valls, unpublished results) found that hrpG, hrpB, T3SS structural genes, and T3E genes are still strongly expressed when cells reach high density within tomato stems at the onset of wilting. It seems likely that signicant PhcA-dependent repression may be a cultural artifact or is overridden by other regulatory processes during pathogenesis. Many other questions remain about how these regulatory systems operate during pathogenesis. Brown & Allen (14) used in vivo expression technology to identify many R. solanacearum ipx genes that are expressed in tomato stems but not in rich medium and showed that some are differentially regulated by PhcA, VsrBC, VsrAD, PehR, and FlhDC when tested in minimal medium. Although helpful in identifying some additional potential virulence genes, this approach excluded known (and unknown) virulence genes that are expressed both
in culture and in planta and provided no information about how these regulators function in planta. Although regulation of the eps operon appears to be similarly cell-density responsive in culture and in tomato stems (62), expression of pehR and hDC are reportedly dramatically higher in planta than in culture, and expression of the iC agellin does not require FlhDC in planta as it does in culture (2, 98). These latter results again suggest that there are additional signals and regulatory circuits functioning in planta. In the rst transcriptome study to examine gene expression in planta, Jacobs et al. (58) harvested GMI1000 and K60 cells from culture in rich medium or in tomato stems at 6 108 cfu per ml or per gram (at early wilt symptoms). Of the 285 orthologous genes that were highly expressed in planta, 250 are in the core genome and included many known virulence factors. Among these, the eps operon, the T2SS, and genes encoding PCWDE are expressed similarly (<2.5-fold difference) in culture and in planta. That cbhA was among the ten most highly expressed genes in planta helps to explain its surprisingly large contribution to virulence (68). Unexpectedly, some of the genes that contribute to stress response (dps, dinF ) were expressed similarly both in culture and in planta, whereas others (acrA, acrB, bcp) were downregulated in planta. This snapshot of expression is intriguing, but to understand the regulatory networks as they function in planta requires studying the transcriptome in wild-type and regulatory mutants at multiple time points during pathogenesis (see sidebar, Unexplored Regulatory Processes).
WHAT HAVE WE LEARNED FROM COMPARATIVE GENOMICS?

Analyses of eight sequenced strains representing the four phylotypes conducted by Remenant et al. (87) estimated that the core genome 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 sequencing (RNA-seq) has not been reported for R. solanacearum. These methods, which permit more extensive, sensitive, and accurate analyses of transcriptomes, often discover new genes encoding small peptides or noncoding RNAs, correct gene annotation, dene untranscribed regions, and reveal operon structures and plasticity in suboperonic expression (97). Small peptides, which previously were often overlooked, can exhibit regulatory and other functions. Small noncoding RNAs, such as trans-acting small RNA (sRNA) that bind to RNA or protein and cis-acting riboswitches and antisense RNA, have enormous potential to affect gene expression (108). For example, sRNAs are known to help regulate quorum sensing, virulence, and stress responses all of which are important for R. solanacearum pathobiology. In addition, the contribution of RNA-binding proteins, already well known in some plant pathogenic bacteria, is almost completely unexplored in R. solanacearum (40). Likewise, small molecules like cyclic di-GMP and (p)ppGpp are likely to have important roles in transcription, translation, and replication. Therefore, even though the known regulatory networks in R. solanacearum are already quite complex, you aint seen nothin yet!
apparently involved in metabolite uptake or degradation. For example, the differential ability to metabolize the sugar alcohols dulcitol, mannitol, and sorbitol, which is one the criteria used for classifying R. solanacearum strains into biovars (31), is due to the presence in some strains of a 22-kb genomic island carrying the genes for their utilization (42). Other genomic differences concerning the D-galactonate degradation pathway or the denitrication pathway have been documented (86), but this observed variability in nutrient acquisition and metabolism has not improved understanding of niche specialization.
T3E Repertoire Dynamics

Post-genomic functional analyses using regulatory-based approaches and/or T3SStranslocation assays have unraveled the nearly complete repertoire of 7075 T3E in strain GMI1000 and the phylogenetically close strain RS1000 (28, 7577, 79, 83) (Table 1). Homology searches combined with criteria used for T3E gene detection (27, 28) in other sequenced genomes predict 110 T3E candidate gene families in the RSSC. Each R. solanacearum strain appears to carry an average of 6075 potential T3E, regardless of whether it has a large or narrow host range, which is signicantly higher than for Xanthomonas spp. or P. syringae that have 3040 T3E. The one exception so far is R. syzygii, which possesses fewer T3E than R. solanacearum or BDB strains (Table 1). The R. solanacearum T3E repertoire is characterized by: a large number (>30) of core T3E conserved among all the sequenced strains. This number is approximately 50 when comparing repertoires from representatives of each phylotype and can reach up to 60 within the same phylotype (Figure 2). This contrasts with the picture observed in P. syringae pathovars, where the ratio of conserved versus variable T3E is reversed (7). It also implies that the R. solanacearum common
percentage of the total than in some bacteria with an equivalent genome size, such as P. syringae (3,400 core genes) (7), which emphasizes the greater genetic diversity in the RSSC. It is, therefore, all the more challenging to resolve which genes are responsible for host-range specialization or other complex biological traits.
Metabolic Properties
The genomic sequences have revealed a metabolically versatile species complex with the ability to thrive on a range of plant metabolites from sugars to phenolic compounds (46). Pathways for degradation of benzonitrile, benzoate, catechol, vanillate, and other derivatives of lignin were identied, although there are some variations between strains (86). R. solanacearum strains also vary in the carbon sources they can use, and some of these differences may be explained by the numerous genomic islands
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ancestor already possessed a large number of these T3E. a strong diversication within the species because interphylotype T3E repertoire comparisons generally reveal approximately 25% variation in content. the existence of several multigenic families because each strain carries several T3E belonging to ve main families, each comprising three to seven members (76, 83, 88). Ortholog searches reveal that strains from the RSSC share some T3E families with other pathogens; for example, TAL (transcription activator-like) T3E with Xanthomonas spp., AvrPtoB (HopAB) with P. syringae pathovars, and OspD with Shigella exneri (28, 83). the fact that almost half of the T3E repertoire appear specic to the RSSC. Globally, the most related T3E repertoires are those of some Acidovorax avenae strains that are pathogenic on cucurbits. Similarity searches reveal that one third of the GMI1000 T3E has at least partial homology with A. avenae proteins.
a
GMI1000
Phylotype I 74 T3E
b
Psi07
Phylotype IV 74 T3E
Molk2
Phylotype II, banana 75 T3E
Po82
Phylotype II, potato 75 T3E
9 2 1 7 5
6 1 48 2 2
10 5
10 3
3 59 7 3
CMR15
Phylotype III 67 T3E
CFBP2957
Phylotype II 72 T3E
CFBP2957
Phylotype II, tomato 72 T3E
Figure 2 (a) Number of type III effector genes identied in the sequenced genomes of strains representative of the four main phylotypes of the RSSC or (b) in the sequenced genomes of three strains belonging to phylotype II that were isolated from different host plants.
T3E Repertoire Comparisons Do Not Provide Clues on Host-Specicity Factors

Having genome sequences of strains with similar host-range specicity allows direct comparison of T3E repertoires (or alternatively, complete proteomes) to tentatively identify host-specicity determinants. However, such comparisons have not revealed general patterns of presence or absence of particular T3E genes. More genomic sequences probably are required to detect robust associations, but it is also possible that comparisons only based on presence/absence criteria may not be sensitive enough to detect host-specicity factors. For example, a signicant level of sequence divergence is present among strains for GALA T3E genes in the RSSC, which suggests that they may undergo functional diversication (88). In addition, it is also well known that some discrete
amino acid changes can alter or modify protein specicities. Because host rangedetermining factors do not only involve T3E in R. solanacearum (see above), comparisons should include additional classes of genes. Here again, there is also signicant variation among RSSC strains, both in terms of gene distribution or allelic differences. This is true, for example, for the Rsa1 aspartic protease family, sugar-binding lectins, and PCWDE. For instance, the gene encoding the polygalacturonase PehA is disrupted by a transposable element in strain CMR15, whereas this gene underwent duplication at the same locus in strain Psi07. Such genetic differences are not yet correlated with phenotypic traits. More generally, several classes of genes or loci, such as those encoding nonribosomal pokyketide synthases, hemagglutinin-related proteins, or Type VI-secretion system Vgr substrates, are highly variable among RSSC strains.
PERSPECTIVES AND FUTURE DIRECTIONS

R. solanacearum was only the third phytopathogenic bacterium to have its genome completely sequenced, and this milestone
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enhanced research progress in many direct and indirect ways. However, as this review illustrates, our understanding of the complexity and diversity within the RSSC was incomplete until gene-specic and genomic sequences became available for multiple strains from around the world. The major phylogenetic groups of the RSSC are now well established, we have a better idea of the ancient and ongoing evolution of the pathogen, and there is a proposal to divide the RSSC into new species and subspecies (19, 55, 86, 87, 111). Nevertheless, it is possible that pathogen genetic diversity has still been underestimated. High-throughput, next-generation sequencing methods will surely expand the known genetic repertoire of RSSC strains. Most notably, we expect that examining environmental isolates (e.g., from soil or water courses) will promote understanding of the evolutionary dynamics of these strains in relation to survival and pathogenicity. Genome sequencing of multiple closely related strains with different host ranges (such as phylotype IIB strains that differ in their ability to wilt banana) will hopefully identify consistent differences in genes encoding T3E associated with host preferences and other candidate host-specicity factors. These results will then provide testable hypotheses for future functional and mechanistic studies. Complete genome resequencing also opens the way to developing experimental evolution methods to unravel determinants of bacterial adaptation. For example, serial inoculation of legume plants with a R. solanacearum strain carrying a symbiotic plasmid from a nitrogen-xing symbiont resulted in selection of variants able to reproduce and multiply intracellularly into root nodules (71). Genome resequencing of these variants revealed that adaptive mutations were in T3SS structural genes, the hrpG regulator (71), and also other regulators from the virulence network (C. Masson, personal communication). Similar approaches could be used to identify molecular traits associated with the pathogens adaptation
to different environments and thereby lead to signicant advances in our knowledge of the genes conferring selective advantages or adaptability in these conditions. The genetic resources now available will also provide the foundation upon which advanced transcriptomic studies can be pursued. It is especially important that future research on gene regulation is conducted with pathogen cells harvested from natural environments rather than from cultures. Based on the limited results to date, it seems almost certain than our model of the regulatory networks depicted in Figure 1 will be greatly modied once we determine how extracellular and intracellular signals affect the true regulons of key regulators (PhcA, VsrAD, HrpG, HrpB, etc.) and how gene expression varies with time and location outside and within plants. These studies will also probably reveal completely new regulatory processes affecting transcription, translation, and protein function and better dene how all these systems are interconnected. Knowing when and where many presumed pathogenicity factors (such as most T3E or T2SS substrates) are produced will help to decipher how they help to make R. solanacearum such a successful pathogen on its diverse hosts. Finally, although it is generally assumed that these regulation schemes are conserved among strains of the RSSC, we should not exclude the possibility that they have been modied in some strains to promote adaptation to specic hosts or environments. R. solanacearum has been a model for studying how bacteria cause plant diseases for almost 60 years, but in many cases we have relied on research approaches that are akin to shooting in the dark. However, progress in the genomic analysis of the RSSC has now shone a bright light on some previously obscure aspects of this pathogens inner workings. The eld now seems prepared to move forward with greater assurance toward fullling our dream that basic research on the pathogen can help provide innovative tools and strategies to control R. solanacearum wilt diseases.
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DISCLOSURE STATEMENT
The authors are not aware of any afliations, memberships, funding, or nancial holdings that might 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 dExcellence (LABEX) entitled TULIP (ANR-10-LABX-41). T.D. thanks his students and postdocs for their efforts and the USDA, NSF, Hatch, and state agencies that have supported his research. We are grateful to Mark Schell and Christian Boucher for critical reading of a draft manuscript and thank Caitilyn Allen, Philippe Prior, Emmanuel Wicker, Marc Valls, and Catherine Masson for sharing unpublished information, and Laure Plener for her contribution to Figure 1.
LITERATURE CITED
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15. Brown DG, Swanson JK, Allen C. 2007. Two host-induced Ralstonia solanacearum genes, acrA and dinF, encode multidrug efux pumps and contribute to bacterial wilt virulence. Appl. Environ. Microbiol. 73:277786 16. Brumbley SM, Carney BF, Denny TP. 1993. Phenotype conversion in Pseudomonas solanacearum due to spontaneous inactivation of PhcA, a putative LysR transcriptional regulator. J. Bacteriol. 175:5477 87 17. Buddenhagen I. 1986. Bacterial wilt revisited. In Bacterial Wilt Disease in Asia and the South Pacic, ed. GJ Persley, pp. 12643. Canberra, Australia: Aust. Cent. Int. Agric. Res. 18. Carney B, Denny T. 1990. A cloned avirulence gene from Pseudomonas solanacearum determines incompatibility on Nicotiana tabacum at the host species level. J. Bacteriol. 172:483643 19. Castillo JA, Greenberg JT. 2007. Evolutionary dynamics of Ralstonia solanacearum. Appl. Environ. Microbiol. 73:122538 20. Cellier G, Prior P. 2010. Deciphering phenotypic diversity of Ralstonia solanacearum strains pathogenic to potato. Phytopathology 100:125061 21. Clough SJ, Flavier AB, Schell MA, Denny TP. 1997. Differential expression of virulence genes and motility in Ralstonia (Pseudomonas) solanacearum during exponential growth. Appl. Environ. Microbiol. 63:84450 22. Coenye T, Vandamme P. 2003. Simple sequence repeats and compositional bias in the bipartite Ralstonia solanacearum GMI1000 genome. BMC Genomics 4:10 23. Colburn-Clifford J, Allen C. 2010. A cbb3 -type cytochrome c oxidase contributes to Ralstonia solanacearum R3bv2 growth in microaerobic environments and to bacterial wilt disease development in tomato. Mol. Plant-Microbe Interact. 23:104252 24. Colburn-Clifford JM, Scherf JM, Allen C. 2010. Ralstonia solanacearum Dps contributes to oxidative stress tolerance and to colonization of and virulence on tomato plants. Appl. Environ. Microbiol. 76:7392 99 25. Coupat B, Chaumeille-Dole F, Fall S, Prior P, Simonet P, et al. 2008. Natural transformation in the Ralstonia solanacearum species complex: number and size of DNA that can be transferred. FEMS Microbiol. Ecol. 66:1424 26. Coupat-Goutaland B, Bernillon D, Guidot A, Prior P, Nesme X, Bertolla F. 2011. Ralstonia solanacearum virulence increased following large interstrain gene transfers by natural transformation. Mol. PlantMicrobe Interact. 24:497505 27. Cunnac S, Boucher C, Genin S. 2004. Characterization of the cis-acting regulatory element controlling HrpB-mediated activation of the type III secretion system and effector genes in Ralstonia solanacearum. J. Bacteriol. 186:230918 28. Cunnac S, Occhialini A, Barberis P, Boucher C, Genin S. 2004. Inventory and functional analysis of the large Hrp regulon in Ralstonia solanacearum: identication of novel effector proteins translocated to plant host cells through the type III secretion system. Mol. Microbiol. 53:11528 29. Delaspre F, Nieto Penalver CG, Saurel O, Kiefer P, Gras E, et al. 2007. The Ralstonia solanacearum pathogenicity regulator HrpB induces 3-hydroxy-oxindole synthesis. Proc. Natl. Acad. Sci. USA 104:1587075 30. Denny T, Hayward AC. 2001. Ralstonia. In Laboratory Guide for Identication of Plant Pathogenic Bacteria, 3rd Ed., ed. N Schaad, JB Jones, W Chun, pp. 16589. St. Paul, MN: APS Press 31. Denny TP. 2006. Plant pathogenic Ralstonia species. In Plant-Associated Bacteria, ed. SS Gnanamanickam, pp. 573644. Dordrecht, The Netherlands: Springer 32. Deslandes L, Olivier J, Peeters N, Feng DX, Khounlotham M, et al. 2003. Physical interaction between RRS1-R, a protein conferring resistance to bacterial wilt, and PopP2, a type III effector targeted to the plant nucleus. Proc. Natl. Acad. Sci. USA 100:802429 33. Deslandes L, Pileur F, Liaubet L, Camut S, Can C, et al. 1998. Genetic characterization of RRS1, a recessive locus in Arabidopsis thaliana that confers resistance to the bacterial soilborne pathogen Ralstonia solanacearum. Mol. Plant-Microbe Interact. 11:65967 34. Elphinstone J. 2005. The current bacterial wilt situation: a global view. See Reference 3, pp. 928 35. Fegan M, Prior P. 2005. How complex is the Ralstonia solanacearum species complex? See Reference 3, pp. 44961
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RELATED RESOURCES
1. French National Institute for Agricultural Research (INRA). 2012. Ralstonia solanacearum GMI1000, MOLK2 and IPO1609: a phytopathogenic bacterium with a wide host range. INRA http://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 platform for microbial genome annotation and comparative genomics. Database doi:10.1093/ database/bap021.
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Pathogenomics of the Ralstonia Solanacearum Species Complex

St phane Genin1,2, and Timothy P. Denny3 e

GENERAL FEATURES OF RALSTONIA SOLANACEARUM SPECIES COMPLEX GENOMES

Features of sequenced genomes of the Ralstonia solanacearum species complex

French West Indies

(112) (86) (86) (87) (87)

Ralstonia syzygii R24

www.annualreviews.org Ralstonia Solanacearum

of Type III effectors; ND, not determined.

Horizontal Gene Transfer Events and the Contribution of Prophage Elements

SYSTEMATICS OF THE RALSTONIA SOLANACEARUM SPECIES COMPLEX

R. syzygii, Sumatra disease of clove

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

RALSTONIA SOLANACEARUM VIRULENCE DETERMINANTS General Virulence Factors

VIRULENCE AND PATHOGENICITY REGULATORY NETWORKS

The Phc Connement-Sensing System

HOW UNIQUE IS THE 3-OH PAME AUTOINDUCER?

Positively regulated traits

XpsR VsrB VsrC Signal?

egl Plant cell wall degrading enzymes exported via T2SS

pehA fli genes

cbhA VsrD MotN ralAD

Regulatory protein families

Negatively regulated traits

T3SS & T3E

pehSR Plant signal? PehS fli genes Flagellar motility PehR

pehA via T2SS

efe hdf operon rsa1

Type 3 secretion system

Type III Secretion System Regulators

Regulatory Processes During Pathogenesis

WHAT HAVE WE LEARNED FROM COMPARATIVE GENOMICS?

UNEXPLORED REGULATORY PROCESSES

T3E Repertoire Dynamics

T3E Repertoire Comparisons Do Not Provide Clues on Host-Specicity Factors

PERSPECTIVES AND FUTURE DIRECTIONS

www.annualreviews.org Ralstonia Solanacearum

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