Diversification of the Function of Cell-to-Cell Signaling in Regulation of Virulence Within Plant Pathogenic Xanthomonads

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Science Signaling  27 May 2008:
Vol. 1, Issue 21, pp. pe23
DOI: 10.1126/stke.121pe23


The virulence of plant pathogenic bacteria belonging to the genera Xanthomonas and Xylella depends upon cell-to-cell signaling mediated by the diffusible signal molecule DSF (Diffusible Signaling Factor). Synthesis and perception of the DSF signal require products of the rpf gene cluster. The synthesis of DSF depends on RpfF, whereas the RpfC/RpfG two-component system is implicated in DSF perception and signal transduction. The sensor RpfC acts to negatively regulate synthesis of DSF. In Xanthomonas campestris, mutation of rpfF or rpfC leads to a coordinate down-regulation in synthesis of virulence factors and a reduction in virulence. In contrast, in Xylella fastidiosa, the causal agent of Pierce’s disease of grape, mutation of rpfF and rpfC have opposite effects on virulence, with rpfF mutants exhibiting a hypervirulent phenotype. The findings suggest that different xanthomonads have adapted the perception and function of similar types of signaling molecule to fit the specific needs for colonization of different hosts.

Plant pathogenic bacteria belonging to the related genera Xanthomonas and Xylella cause diseases in many economically important plants throughout the world. The virulence of these bacteria depends on cell-to-cell signaling mediated by the diffusible signal molecule DSF (for Diffusible Signaling Factor) (15). DSF from the crucifer pathogen Xanthomonas campestris pv. campestris (Xcc) has been characterized as the unsaturated fatty acid cis-11-methyl-dodecenoic acid (6). Work in Xcc has established that synthesis and perception of the DSF signal require products of the rpf gene cluster (for regulation of pathogenicity factors) (1, 2, 7). A recent publication by Chatterjee and colleagues (8) addresses the role of the Rpf/DSF system in Xylella fastidiosa, the causal agent of Pierce’s disease of grape and citrus variegated chlorosis. This report, together with previous work from the same laboratory (4), shows that DSF and the components of the cell-cell signaling system in Xylella fastidiosa have substantially different actions in regulation of virulence from those described in Xcc. These exciting findings suggest that different xanthomonads have adapted the perception and function of similar types of signaling molecule to fit the specific needs for colonization of different hosts.

The role of DSF signaling in the regulation of synthesis of virulence factors and in virulence has been most extensively studied in Xcc. The synthesis of DSF is dependent on RpfF, which has some amino acid sequence similarity to enoyl CoA hydratases, whereas the two-component system comprising the sensor kinase RpfC and regulator RpfG is implicated in DSF perception (1, 2, 7, 9). RpfC is a complex sensor kinase with a predicted membrane-associated sensory input domain as well as histidine kinase, CheY-like receiver (REC), and C-terminal histidine phosphotransfer (HPt) domains. RpfG is a regulator with a REC domain attached to an HD-GYP domain (named for the conserved histidine, aspartic acid, glycine, tyrosine, and proline residues) (2), which acts to degrade the second messenger bis(3′,5′)-cyclic diguanylic acid (cyclic di-GMP) (7). Mutation of rpfF, rpfG, and rpfC in Xcc leads to a coordinate reduction in the synthesis of virulence factors, including extracellular protease, endogluconase, and endomannanase enzymes and the extracellular polysaccharide xanthan; alterations in biofilm formation; and a reduction in virulence (1, 2, 9). Addition of DSF can restore virulence factor synthesis to rpfF mutants but not to Xcc strains with mutations in rpfG or rpfC. These findings are consistent with a role for RpfC/RpfG in perception and transduction of the DSF signal in Xcc. Further support for this model has come from experiments in which the RpfC/RpfG two-component system was reconstructed in Pseudomonas aeruginosa and shown to confer responsiveness to exogenously added DSF as seen through effects on swarming motility (7).

In addition to positive regulation of virulence factor synthesis, RpfC acts to negatively regulate DSF synthesis, a function that does not involve RpfG (2, 6). These dual signaling functions of RpfC are achieved by different mechanisms (10). Phosphorelay via the REC and HPt domain to RpfG is essential for activation of the production of extracellular enzymes and xanthan. In contrast, repression of DSF biosynthesis may be exerted through a protein-protein interaction between RpfF and the REC domain of RpfC. In this view, sequestration of RpfF renders it inactive in DSF synthesis, but structural changes in RpfC, perhaps as a result of DSF binding and autophosphorylation, allow release of RpfF, resulting in increased DSF synthesis. In this fashion, perception of DSF would promote its own synthesis, but not through a mechanism that involves changes in expression of the rpfF gene. Notably, in an rpfC mutant of Xcc in which DSF abundance is considerably higher than in wild type, rpfF transcripts are only modestly increased (2).

Although there is a high degree of sequence conservation of the RpfF, RpfC, and RpfG proteins of Xylella fastidiosa with those of Xcc (11), there appear to be substantial differences between the two bacteria in the action of these components in regulation of virulence and virulence factor synthesis (4, 8). Xylella fastidiosa produces a DSF molecule that is different from, although structurally related to, that of Xcc. The molecule 12-methyl-tetradecanoic acid has been identified in culture supernatants of Xylella fastidiosa as the putative DSF signal (12). More important, mutation of rpfF in Xylella fastidiosa gives rise to enhanced virulence to grape and prevents colonization of the insect vector (4). In contrast, rpfC mutants are deficient in virulence and movement in xylem vessels, and although they can colonize the insect vector, they are not transmitted to the host plant. As in Xcc, mutation of rpfC leads to an overproduction of DSF (8), but in contrast to Xcc this is associated with increased expression of rpfF. Mutation of rpfC also leads to an enhanced expression of genes encoding the adhesins HxfA, HxfB, and FimA, which is correlated with an enhanced attachment of the mutant to glass at the air-liquid interface of shaken cultures. Conversely, mutation of rpfF leads to a reduced expression of adhesin genes and a severely reduced attachment phenotype. Mutation of hxfA, hxfB, or fimA leads to hypervirulence on grape and deficiency in biofilm formation (13, 14), which provides a ready explanation for the divergent phenotypes of rpfC and rpfF mutants in virulence and insect transmission. Nevertheless mutations of rpfF and rpfC have similar effects on the expression of genes encoding other virulence determinants; expression of tolC, which encodes a protein involved in Type I secretion (15), and pglA, which encodes a polygalacturonase (16), is increased in both rpfF and rpfC mutants (8).

The virulence of an rpfCrpfF double mutant is similar to that of the wild type and greater than that of the rpfC mutant. Analysis of the pattern of gene transcription in these different strains is consistent with the notion that DSF negatively influences virulence and movement in xylem vessels by promoting expression of genes encoding adhesins, thus increasing attachment. Effects on expression of tolC and pglA are more complex, however, because the rpfCrpfF double mutant expresses substantially less transcript for these genes than do either of the single mutants. Chatterjee and colleagues envisage that this is due to the action of a repressor, which can be sequestered by protein-protein interaction with either RpfC or RpfF (8). Because the effects of DSF on positive regulation of adhesin genes are seen in an rpfC mutant but not in the rpfCrpfF strain, Chatterjee and colleagues further propose the existence of two distinct pathways for DSF perception in Xylella fastidiosa (8). In the first pathway, in which RpfC acts as the sensor, DSF negatively regulates expression of rpfF, tolC, and pglA independently of RpfG and activates genes involved in biofilm formation through RpfG. The second proposed pathway involves a separate (perhaps intracellular) sensor and RpfG to positively regulate expression of genes linked to biofilm formation (Fig. 1).

Fig. 1.

Model for signal transduction by the Rpf/DSF signaling system of xanthomonads. RpfF is the enzyme responsible for DSF synthesis. RpfC is a complex sensor kinase responsible for DSF perception. RpfC sequesters RpfF and perhaps other regulators that may be released after structural changes that occur upon DSF binding to the RpfC sensory input domain. The consequences are auto-induction of DSF synthesis and alteration in expression of particular subsets of genes. Binding of DSF also triggers autophosphorylation of RpfC and phosphorelay to RpfG (white arrow), thereby activating cyclic di-GMP degradation by the HD-GYP domain. RpfG may also physically interact with GGDEF domain (cyclic di-GMP synthase) proteins to influence cyclic di-GMP concentrations and with other regulators, modulating their activity in transcription (dotted lines). In X. campestris, RpfG influences expression of Clp by an unknown mechanism, leading to activation of a downstream signaling cascade involving other transcriptional regulators. It is not known whether this occurs in Xylella fastidiosa. In Xylella fastidiosa, a second sensor may phosphorylate RpfG in response to DSF. Although shown as a membrane-bound protein, this sensor may equally be located in the cytoplasm. Terms in italics represent functional descriptions.

The current understanding of the signal transduction pathways downstream of RpfC/RpfG is fragmentary. The DSF/Rpf system of Xcc has been shown to activate transcription of the gene encoding the adenosine 3′,5′-monophosphate (cyclic-AMP) receptor-like protein Clp (17), although the mechanism remains obscure. In Xcc, Clp regulates many functions, including the expression of genes encoding extracellular enzymes and enzymes involved in xanthan synthesis, and those encoding the regulators Zur and FhrR. In turn, Zur regulates genes involved in functions such as iron uptake, the tricarboxylic acid cycle, multidrug resistance, and detoxification, whereas FhrR regulates expression of genes involved in flagellar synthesis and type III secretion (17). Not all of the regulatory effects of RpfG are exerted through the action of Clp. For example, Clp is not apparently involved in regulation of biofilm dynamics (17, 18). Yeast two-hybrid analysis reveals interactions of the HD-GYP domain of RpfG of X. axonopodis pv. citri with the sigma factor σ54, NtrBC (both of which are involved in nitrogen-regulated transcription) and other regulatory proteins, although it is not yet clear whether this has biological relevance (19). Whether Clp is involved in the signaling pathways in Xylella fastidiosa is currently not known.

The demonstration that the HD-GYP domain of RpfG is a cyclic di-GMP phosphodiesterase implicates cyclic di-GMP as a second messenger in DSF signal transduction (7, 20). Furthermore the HD-GYP domain of RpfG has been shown by yeast two-hybrid analysis to interact with a subset of GGDEF (named for the conserved glycine, glycine, aspartic acid, glutamatic acid, and phenylalanine residues) domain-containing proteins (19), which are cyclic di-GMP synthases (2123). This may suggest an action of RpfG in modulating the activity of specific cyclic di-GMP generating systems. In many bacteria, larger amounts of cyclic di-GMP promote biofilm formation, whereas smaller amounts promote motility (2123). Consonantly, mutation of rpfG leads to enhanced biofilm formation and reduced motility (9, 24). Although it is not known how cyclic di-GMP exerts its effects in xanthomonads, it is important to note that in other bacteria this nucleotide can act at the posttranslational level—for example, in allosteric activation of polysaccharide synthesis (21, 22). Thus, analysis of the roles of the DSF/Rpf system through transcriptional profiling alone may not explain all phenotypic variations seen in rpf mutants.

The next few years should see an increase in our understanding of DSF signal perception in different xanthomonads to include identification of proposed alternative sensors and dissection of downstream signaling cascades. Such studies may have practical implications because interference or confusion of pathogen signaling may be a strategy for disease control (25). Intriguingly, there may be impact beyond pathogens of agronomic significance because DSF has been implicated in interspecies signaling between Stenotrophomonas maltophilia and Pseudomonas aeruginosa (26), and a signal molecule related to DSF has been characterized from Burkholderia cenocepacia, which like P. aeruginosa is a major opportunistic human pathogen (27).


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