Ralstonia solanacearum is regarded as one of the world’s most important bacterial plant pathogens because of its aggressiveness, large host range, broad geographical distribution and long persistence in soil and water environments. This root pathogen is an attractive model to investigate the question of host adaptation as it exhibits a remarkably broad host range, being able to infect numerous plant species belonging to different botanical families. Several effector proteins transiting through the type III secretion system have been shown to restrict or extend specifically the host range of the bacterium. Recent investigations on the mechanisms that coordinate changes in gene expression during the passage between saprophytism and life within host tissues have allowed the identification of other molecular determinants implicated in the adaptation of R. solanacearum to its hosts and pathogenesis. Among these determinants are genes involved in chemotaxis, secondary metabolic pathways and the detoxification of various antimicrobial compounds, and genes directing the biosynthesis of phytohormones or adherence factors. The regulation of many of these genes is coordinated by the master pathogenicity regulator HrpG. These hrpG-dependent genes control major steps during the interaction with plant cells, and probably determine the ecological behaviour of the microorganism, being required for the establishment of pathogenesis or mutualism.
Ralstonia solanacearum, known for most of the 20th century as Pseudomonas solanacearum, was originally described by Erwin F. Smith in 1896 as the causative agent of bacterial wilt of solanaceous plants. It has been intensively studied since then, and is now recognized as a model system for the investigation of the molecular determinants controlling bacterial pathogenicity towards plants. Comprehensive reviews have been written on the biology of R. solanacearum (Hayward, 1991; Denny, 2006), whereas others have focused more on the molecular and genomic analysis of virulence determinants (Schell, 2000; Genin & Boucher, 2004). This review summarizes recent findings on the molecular adaptation of R. solanacearum to its host plants and, more specifically, on the determinants involved in this process beyond the type III secretion system (T3SS) and its type III effector (T3E) substrates.
Ralstonia solanacearum strains: a heterogeneous species’ complex with a broad host range spectrum
Among plant pathogens of major economic importance, R. solanacearum, the causal agent of bacterial wilt, was recognized early as one of the most destructive plant bacterial pathogens. This soil-borne bacterium enters plant roots, invades the xylem vessels and spreads rapidly to aerial parts of the plant through the vascular system (Fig. 1). Typical disease symptoms include browning of the xylem, foliar epinasty and lethal generalized wilting. Wilting symptoms probably result from the extensive bacterial colonization of the xylem and massive exopolysaccharide production, which rapidly induce vascular dysfunction. Huge population numbers of R. solanacearum are reached in susceptible hosts within a few days [up to 1010 colony-forming units (cfu) g−1 ] before the bacterium returns to the soil.
Bacterial wilt is a common disease in tropical and subtropical areas of the globe, and is an emerging disease in some temperate areas, such as North America and Europe (Elphinstone, 2005). Another reason why R. solanacearum is a major constraint to the production of a number of economically important agricultural crops and ornamental plants is its very wide host range. As a whole, this bacterium is able to infect more than 53 botanical families (Hayward, 1991) that represent more than 200 host plant species, including, among many others, tomato, potato, eggplant, banana, groundnut, olive, ginger, Eucalyptus and cultivated geraniums (Pelargonium spp).
The lifestyle of R. solanacearum also includes a saprophytic phase as in many other ‘environmental pathogens’ (Morris et al., 2009): the bacterium can survive for several years in the soil in the absence of a ‘true’ host culture, or in water courses, which are an important means of dissemination of the pathogen. Long-term survival in soil requires moisture and presumably depends on the degradation of plant cell debris through the potential to metabolize derivatives of lignin (Genin & Boucher, 2004), or the ability to colonize weeds or plants that remain asymptomatic (Hayward, 1991). Ralstonia solanacearum is able to survive prolonged nutrient scarcity, as it has been reported to retain the ability to wilt host plants after 132 d of incubation in ultrapure water (van Overbeek et al., 2004). These observations reveal that the bacterium is genetically equipped to respond efficiently to various abiotic stresses, and this property can be correlated with its rather large genome size (5.7 Mb), as with many soil or environmentally versatile bacteria (Salanoubat et al., 2002).
Ralstonia solanacearum is a heterogeneous species composed of many genetic groups. Phylogenetic analyses based on multiple gene sequence loci have revealed that the species is partitioned into four phylotypes (genetic clusters) (Fegan & Prior, 2005), each of which reflects the geographical origin of the strains: phylotype I and II are composed of Asian and American strains, respectively, whereas phylotype III members are African, and phylotype IV isolates are from Indonesia, Japan and Australia (Fig. 2). The robustness of these four main evolutionary lineages is also supported by the hierarchical clustering obtained from comparisons of total gene content among representative strains (Guidot et al., 2007). There is genetic evidence that phylotypes arose from geographical isolation and that populations may have diverged a long time ago, as judged by the amount of fixed polymorphisms observed among phylotypes (Lavie et al., 2004; Castillo & Greenberg, 2007).
The great genetic diversity among the species has led some authors to propose that R. solanacearum belongs to a ‘species’ complex’ (Fegan & Prior, 2005); a species’ complex is defined as a cluster of closely related isolates whose individual members may represent more than one species. In agreement with this view is the fact that the related species R. syzygii and R. celebensis (also named banana blood disease bacterium) strains belong to the R. solanacearum species’ complex within phylotype IV (Fig. 1). It is therefore highly probable that the actual group of R. solanacearum strains will be further subdivided into different distinct species in the future.
Some host range specificity exists
Early classifications of R. solanacearum 40 yr ago divided the species into three major races based on the host range of the strains, and these ‘races’ mostly corresponded to pathovars (Denny, 2006). The phylotype classification scheme then revealed that there were no or few correlations between the host range and phylogenetic relationships of the strains within the species. As R. solanacearum is generally defined as a ‘broad host range pathogen’, it may be difficult for a nonexpert to realize that specialization towards specific hosts exists at the strain level. Whereas some strains, such as GMI1000 (Salanoubat et al., 2002), are able to cause disease over a wide range of hosts distributed in multiple botanical families, other groups of strains have been reported to possess a much more limited host range. This is the case, for example, for those designated as ‘race 3’ strains, responsible for potato brown rot disease, and which were originally described as pathogenic on potato and tomato, but not adapted to other solanaceous crops (Hayward, 1991). In fact, because this pathogen can apparently adapt to many plants and colonize some hosts asymptomatically, the subject of the host range specificity of R. solanacearum is complex, and is hampered by two limitations. First, many strains are poorly characterized in term of host range spectrum and are only referenced on the basis of the host plant from which they were isolated; second, as noted by Denny (2006), artificial inoculations in controlled conditions certainly overestimate the natural host range. This can explain why some strains, originally described as ‘narrow host range’ strains, such as, for example, ‘race 3’ potato strains, have been subsequently reported to possess a significantly broader host range spectrum (including eggplant, geranium, pepper, cabbage and even nonsolanaceous herbaceous weeds) (Elphinstone, 2005; Alvarez et al., 2008).
Host-specific interactions can also be observed with wide host range strains displaying differential ranges of compatible hosts. Host range can sometimes be controlled by a single genetic determinant: this is the case for the avrA T3E gene product, which elicits a defence response from the Nicotiana tabacum host immune system, and therefore determines the incompatibility of some R. solanacearum strains at the host species’ level (Carney & Denny, 1990). A study with strain GMI1000 further revealed that a combination of two T3E genes, avrA and popP1, determined the host range specificity on tobacco, as their double inactivation was sufficient to render GMI1000 fully pathogenic on three tobacco species tested (Poueymiro et al., 2009). popP1 encodes a putative cysteine protease which may be directly or indirectly recognized by plant resistance gene products in distinct Solanaceae, as it also induces specific resistance responses on some Petunia lines. The molecular function of AvrA is unknown, but it has been reported that an avrA disruption mutant is reduced in aggressiveness on Medicago truncatula, thus indicating that this determinant is important for the pathogenic behaviour of R. solanacearum on hosts other than tobacco (Turner et al., 2009).
The first established gene-for-gene-type interaction between R. solanacearum and a host involved the GMI1000 T3E PopP2 and the Arabidopsis thaliana resistance protein RRS1-R. PopP2 is targeted to the plant nucleus and triggers Arabidopsis resistance on physical interaction with RRS1-R (Deslandes et al., 2003). However, recent findings have suggested that the story may be complex, as the resistance gene RPS4, originally identified as conferring resistance against Pseudomonas syringae carrying the avirulence gene avrRPS4, is also required for resistance against R. solanacearum (Narusaka et al., 2009). Interestingly, RPS4 lies just next to RRS1 in the Arabidopsis genome in a head-to-head arrangement. Both loci were also shown to be required for resistance against the fungal pathogen Colletotrichum higginsianum, suggesting that distinct allelic forms of RPS4 and RRS1 probably cooperate to confer resistance to different pathogens (Birker et al., 2009; Narusaka et al., 2009). Concerning PopP2, current models about its function and its interacting partners in plant cells have been discussed elsewhere (Poueymiro & Genin, 2009). popP2 appears to be a bacteriophage-borne T3E gene, as it is surrounded by gene clusters (RSc0852-RSc0884) that most probably correspond to a prophage incorporated into the GMI1000 genome. This certainly explains the irregular and scarce distribution of popP2 within the R. solanacearum species (Lavie et al., 2004; Guidot et al., 2007).
Genotype-specific interactions have also been described between R. solanacearum and the model legume M. truncatula, as both susceptible and resistant lines to specific strains have been identified (Vailleau et al., 2007). Here, the genetic basis of resistance to strain GMI1000 appears to be polygenic, with a major quantitative trait locus mapped on chromosome 5.
Host range extension
A survey of the R. solanacearum literature reveals that new host plants of R. solanacearum strains are being reported continuously (Hayward, 1991; Denny, 2006). For example, the emergence of new strains exhibiting expanded host specificities towards Cucurbitaceae in the French West Indies has been described recently (Wicker et al., 2007). Cantaloupe, watermelon and pumpkin were not considered to be hosts of R. solanacearum until this report. Although the question of how these pathogenic variants arose remains unsolved, such bacterial wilt outbreaks on novel hosts support the view that R. solanacearum has great adaptation potential to plants as a result of its genomic plasticity. It is often considered that the natural competence (i.e. the property to be naturally transformed by exogenous DNA) of many R. solanacearum strains could play a major role in the emergence of new pathotypes by increasing the rate of lateral gene transfer, but this is not supported by any experimental evidence to date. Rather unexpectedly, although some T3E genes, such as popP1 and popP2, have obvious features of genes acquired or transmissible through horizontal gene transfer, their distribution within the species appears to strongly correlate with the phylogenetic position of the strains (Lavie et al., 2004), thus suggesting that unknown but specific selection forces govern the acquisition and evolution of these genes.
The extension of the pathogen’s host range does not proceed solely through the loss or inactivation of avirulence genes. The gala7 T3E gene provides an example of a single bacterial determinant able to extend the host range of the bacterium. Disruption of gala7 specifically impairs the pathogenicity of R. solanacearum GMI1000 on M. truncatula, but not on certain other susceptible hosts tested, indicating that GALA7 acts as a host specificity factor (Angot et al., 2006). gala7 belongs to a seven-gene family that encodes F-box and leucine-rich repeat domain T3Es. GALA effectors are presumed to form composite SCF-type ubiquitin ligase complexes in planta to promote ubiquitination and, possibly, subsequent proteasome-mediated degradation of specific plant targets (Angot et al., 2007). The nature of GALA7’s target(s) in M. truncatula is unknown, but a role of GALA7 in the suppression of specific plant immune responses would fit with this gain-of-function phenotype. However, considering the large repertoire of T3E, which comprises 70–75 proteins in R. solanacearum (Poueymiro & Genin, 2009; Mukaihara et al., 2010), it is probable that the evasion of plant defence responses involves several of these, and rarely relies on only one determinant as in the case of M. truncatula. In addition to a functional overlap among effectors, it is also likely that such functional groups of T3E are required to establish host susceptibility by suppressing immune responses that may vary from one plant to another.
Adaptation to hosts
Because T3SS is essential to R. solanacearum’s pathogenicity, several studies have sought to determine how this secretion machinery and its effector substrates promote the invasion and colonization of different model hosts (Vasse et al., 2000; Cunnac et al., 2004; Turner et al., 2009; Macho et al., 2010). It is tempting to speculate that the huge genetic diversity of T3E among R. solanacearum strains (Poueymiro & Genin, 2009) has developed in response to the intense selective pressures imposed by the coevolutionary arms race with this enlarged range of hosts. Although the action of these effectors in the cytoplasm of plant cells is assumed to suppress basal defence responses and/or facilitate nutrient release, this has not yet been demonstrated for R. solanacearum, and the mechanism of action of these proteins still remains largely unknown.
There are several lines of evidence indicating that adaptation to host plants also involves many determinants beyond type III-dependent pathogenesis (Table 1). Some of these determinants were first identified through an approach aimed at detecting the promoters specifically induced in tomato plants using an in vivo expression technology (IVET) (Brown & Allen, 2004). Approximately 150 genes were recovered from this screen, 44 of which were confirmed to be expressed in planta with an induction ratio ranging from two-fold to over 35-fold. This list comprised relatively few known virulence genes and only one T3E gene (RSc1349), thus suggesting that the approach was nonexhaustive and/or retained genes expressed late in infection. However, this study revealed that, in plant xylem vessels, R. solanacearum adapts its physiology to a stressful and nutritionally poor environment. In particular, the bacterium deploys a vigorous oxidative stress response which involves many reactive oxygen species (ROS)-scavenging enzymes, such as catalases, peroxidases and superoxide dismutases (Flores-Cruz & Allen, 2009). After pathogen recognition, plants produce an oxidative burst and some phenylpropanoid derivatives, which may have direct antimicrobial effects; therefore, the effective production by R. solanacearum of enzymes able to detoxify ROS or phenolic compounds (Hernandez-Romero et al., 2005) is probably important during host infection. Two efflux pumps contributing to expel diverse toxic compounds have also been proposed to protect the pathogen against the effect of host antimicrobials (Brown et al., 2007).
Table 1. A nonexhaustive list of functions potentially involved in the adaptation of Ralstonia solanacearum to its host plants
Role in pathogenic adaptation
Type III secretion effectors
Suppression of pathogen-associated molecular pattern-triggered or type III effector (T3E)-triggered immunity
As apoplastic intercellular spaces and xylem sap fluid are low-nutrient environments, R. solanacearum must cope with limited resources at different steps of infection. The activation of primary or alternate metabolic pathways to overcome nutritional stress, as well as the recycling and/or catabolism of specific intermediates or secondary metabolites from the host, can define a form of ‘metabolic adaptation’ of the pathogen to its hosts. It has been known for many years that several auxotroph mutants are nonpathogenic (Coplin et al., 1974), which results from their inability to multiply within host tissues. With the exception of plant cell wall-degrading enzymes (Schell, 2000) almost nothing is known about the bacterial exploitation of host resources and its contribution to pathogenicity/host specificity. The characterization of an R. solanacearum galacturonate transporter gene, named exuT, showed that the metabolism of galacturonic acid, which is released from plant cell walls by the action of bacterial polygalacturonases, does not contribute significantly to pathogen fitness, as an exuT mutant retained wild-type virulence on tomato (Gonzalez & Allen, 2003). Multiple degradative pathways for a variety of organic substrates have been predicted in the R. solanacearum genome (Genin & Boucher, 2004), and numerous genes identified through IVET have also been predicted to encode metabolic and/or transport functions (Brown & Allen, 2004), but their implication in plant pathogenicity has not been documented. Finally, recent findings have shown that R. solanacearum produces secondary metabolites, such as ralfuranone, a phenyl-furanone derivative (Schneider et al., 2009), and the isatin-related compound Hdf (3-hydroxy-oxindole) (Delaspre et al., 2007). The contribution of these molecules to pathogenesis is unknown, but the fact that their production is under the control of plant-induced virulence regulators is suggestive of a role during the infection process. In the case of Hdf, a recent observation supports this hypothesis, as multiplication in plant leaf tissues of an hdf mutant was shown to be significantly reduced relative to the wild-type strain in mixed infections (Macho et al., 2010).
The attachment of R. solanacearum to plant cell surfaces is essential for pathogenesis. The bacterium can adhere to both host and nonhost plant cells in a polar fashion (i.e. predominantly oriented with one pole towards the plant cell), but T3SS-dependent Hrp pili are not required for this ability (Aldon et al., 2000). It has been shown that type IV pili appendages, which are required for ‘twitching motility’, also promote adherence to plant cell surfaces and virulence on tomato plants (Kang et al., 2002). Virulence was reduced when both unwounded roots and wounded petioles were inoculated; therefore, piliation could contribute to pathogenesis during both the invasion of roots and when the bacterium is inside plants. Because both type IV pili and Hrp pili are polar, this raises the possibility that the lack of polar adherence of type IV pili mutants might impair the ability of T3SS to deliver effector proteins (Kang et al., 2002). Other potential R. solanacearum adherence factors include a large family of filamentous haemagglutinin-like proteins (Salanoubat et al., 2002). These huge proteins are predicted to be exposed to the bacterial cell surface; the expression of at least one member of this gene family is specifically activated in planta (Brown & Allen, 2004), and homologues have been implicated in interactions with host cells in the case of several bacterial pathogens. Finally, it is worth mentioning that surface appendages have also been shown or are presumed to be involved in bacterial autoaggregation and biofilm formation (Kang et al., 2002). The biological significance of biofilm formation by R. solanacearum inside the plant is unknown, but there is evidence that this property is genetically controlled by multiple loci (Vasseur et al., 2005).
Chemotaxis is also an important trait for the parasitic fitness of R. solanacearum on plants. It has been reported that the bacterium is attracted by plant root exudates or plant roots themselves. Interestingly, specific tactile responses vary depending on the nature of the plant, as tomato root exudates are more attractive than rice (a nonhost plant) root exudates (Yao & Allen, 2006), indicating that chemotaxis could be a component of pathogen host selectivity.
Many traits involved in adaptation to plants are controlled by HrpG
Ralstonia solanacearum virulence is controlled by an elaborate sensory and regulatory network (Schell, 2000). In this network, HrpG appears to be more specifically devoted to the control of the genetic programme deployed by the pathogen as soon as it interacts with plant cells and then during the parasitic stage inside the plant. The transcription factor HrpG is a key regulatory node that integrates at least three major environmental signals: physical contact with the host plant, bacterial metabolic status and a quorum-sensing signal. hrpG was originally identified as a response regulator involved in the control of T3SS (Brito et al., 1999), but transcriptomic profiling of a hrpG mutant further revealed that this regulator also controls the expression of a T3SS-independent pathway(s) that includes other virulence determinants and genes more generally involved in the adaptation to life in the host (Valls et al., 2006). Hence, HrpG controls functions involved in plant cell wall degradation (polygalacturonases, endoglucanase), exopolysaccharide biosynthesis, ROS detoxification (a major catalase) and also presumably metabolic adaptation (many transporters and enzymes).
Analysis of the HrpG regulon also revealed functions whose implications in pathogenicity/host adaptation have not yet been demonstrated, but which are presumably ‘guilty by association’ on the basis of apparent coregulation. For example, HrpG controls the expression of genes coding for lectins or involved in the biosynthesis of polyamines and phytohormones, such as ethylene or auxin. The hrpG-dependent lectins have fucose and mannose specificity and bind the plant xyloglucan polysaccharide present in primary cell walls, thus suggesting that they could act as potential adherence factors. Ethylene production by strain GMI1000 has been shown to be sufficient to impact the Arabidopsis ethylene-responsive pathway (Valls et al., 2006). The role of phytohormone production by R. solanacearum remains uncertain, but modulation of plant physiology by tinkering with plant hormone levels has emerged as a major strategy of many bacterial endophytes (Hardoim et al., 2008). It is likely that most of the 184 T3SS-independent genes controlled by HrpG are not direct targets, but there is genetic evidence that these T3SS-independent functions are collectively essential for pathogenesis, as an hrpG mutant strain engineered to express constitutively T3SS is almost unable to cause wilting symptoms on tomato plants (Valls et al., 2006). Many genes of the large hrpG-dependent regulon encode hypothetical proteins with no assigned functions, but the fact that approximately one-third have features of exported or transmembrane proteins raises the possibility of a bacterial camouflage by masking potential pathogen-associated molecular patterns to protect the pathogen from plant defences.
Conversion of R. solanacearum into an intracellular legume symbiont
The most fascinating example illustrating the potential of this pathogen for interaction with plants has been provided recently in a study showing that an ‘evolved’R. solanacearum strain is able to produce root nodules on a legume plant and to infect intracellularly these nodule cells, similarly to a typical bacterial symbiont (Marchetti et al., 2010). The approach developed by the authors exploited the close taxonomical relatedness between R. solanacearum and Cupriavidus taiwanensis, a nitrogen-fixing symbiont that nodulates Mimosa pudica roots. In a first step, the C. tawainensis 550 kb symbiotic plasmid pRalta, which carries the nodulation and nitrogen fixation genes required to establish symbiosis, was introduced into R. solanacearum GMI1000 to generate a chimeric strain able to produce active Nod factors. This chimeric strain was then repeatedly inoculated on seedlings of Mimosa pudica (which is a nonhost for GMI1000) to screen for the production of nodules and to select for nodulation-proficient mutants. Using this experimental evolution approach and genome resequencing, Marchetti and colleagues identified two types of adaptive mutation, which inactivated the T3SS structural gene hrcV and the hrpG master regulator.
Like most rhizobia, C. taiwanensis invades roots by inducing a root hair to curl around and trap a single bacterium, which can then multiply and trigger the formation of infection threads that allow delivery of the bacterium into the plant cells forming the nodule. Contrary to the R. solanacearum chimeric strain, which is unable to initiate infection threads, the hrcV mutant derivative induced nodules and entered root hairs via infection threads, but the bacterium only partially invaded the nodule and remained extracellular. It is therefore likely that one or several T3Es are involved in blocking nodulation and early infection. Remarkably, inactivation of hrpG enabled the chimeric strain to invade intracellularly nodule cells, whereas R. solana-cearum is a strictly extracellular pathogen. This result indicates that one or multiple factors controlled by hrpG independently of T3SS restrict intracellular infection by the R. solanacearum chimeric strain, and further illustrates that this master regulator controls key functions for interaction with eukaryotic cells beyond type III secretion-dependent pathogenesis. The nodulation efficiency of the chimeric hrcV and hrpG mutants was less than that of C. taiwanensis, and they were unable to fix nitrogen, suggesting that additional steps preventing the completion of the symbiotic process remain to be overcome in the hrpG mutant strain. However, the study by Marchetti et al. (2010) reveals how easily, with few genetic changes, a bacterium can acquire two specific symbiotic traits, such as nodulation and intracellular infection, through lateral gene transfer (plasmid acquisition) and under plant selective pressure (hrcV and hrpG mutations).
Because hrpG controls functions essential for resistance to various stresses encountered within the host and for the suppression of plant defence mechanisms, the nodulation-proficient mutant strain should evade Mimosa pudica defence responses by other means, presumably through the signalling action of the Nod nodulation factors. In addition, it cannot be excluded that some other genes among the 802 encoded by the C. tawainensis pRalta plasmid (Amadou et al., 2008) are also required in the adaptation process of the chimeric strain to Mimosa pudica.
In conclusion, an understanding of the evolutionary dynamics of R. solanacearum’s parasitic fitness will certainly be helpful in improving disease control strategies. Gaining an insight into the genes and mechanisms governing adaptation to novel or tolerant hosts, together with the identification of traits that are under selective pressure in disadvantageous host environments, and the molecular nature of the adaptive events involved, will be major goals in this direction. Importantly, in the case of such versatile pathogens, adaptation to one environment (or host) could impact parasitic fitness in other environments, and this knowledge will have important implications for the management of the disease in the field.
I thank Philippe Prior for the generous gift of the phylogenetic tree shown in Fig. 2.