Diseases caused by Xylella fastidiosa have attained great importance worldwide as the pathogen and its insect vectors have been disseminated. Since this is the first plant pathogenic bacterium for which a complete genome sequence was determined, much progress has been made in understanding the process by which it spreads within the xylem vessels of susceptible plants as well as the traits that contribute to its acquisition and transmission by sharpshooter vectors. Although this pathogen shares many similarities with Xanthomonas species, such as its use of a small fatty acid signal molecule to coordinate virulence gene expression, the traits that it utilizes to cause disease and the manner in which they are regulated differ substantially from those of related plant pathogens. Its complex lifestyle as both a plant and insect colonist involves traits that are in conflict with these stages, thus apparently necessitating the use of a gene regulatory scheme that allows cells expressing different traits to co-occur in the plant.

Cell-cell signaling in Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, mediated by a fatty acid Diffusible Signaling Factor (DSF), is required to colonize insect vectors and to suppress virulence to grape. Here, we show that a hybrid twocomponent regulatory protein RpfC is involved in negative regulation of DSF synthesis by RpfF in X. fastidiosa. X. fastidiosa rpfC mutants hyperexpress rpfF and overproduce DSF and are deficient in virulence and movement in the xylem vessels of grape. The expression of the genes encoding the adhesins FimA, HxfA, and HxfB is much higher in rpfC mutants, which also exhibit a hyperattachment phenotype in culture that is associated with their inability to migrate in xylem vessels and cause disease. rpfF mutants deficient in DSF production have the opposite phenotypes for all of these traits. RpfC is also involved in the regulation of other signaling components including rpfG, rpfB, a GGDEF domain protein that may be involved in intracellular signaling by modulating the levels of cyclic-di-GMP, and the virulence factors tolC and pglA required for disease. rpfC mutants are able to colonize the mouthparts of insect vectors and wild-type strains but are not transmitted as efficiently to new host plants, apparently because of their high levels of adhesiveness. Because of the conflicting contributions of adhesiveness and other traits to movement within plants and vectoring to new host plants, X. fastidiosa apparently coordinates these traits in a population-size-dependent fashion involving accumulation of DSF.biofilm ͉ endophytes ͉ Pierce's disease ͉ quorum sensing ͉ adhesins

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. In the related bacterium Xanthomonas campestris pv. campestris, the rpfF gene is involved in production of a diffusible extracellular factor (DSF) that positively regulates synthesis of virulence-associated functions like extracellular polysaccharide (EPS) and extracellular enzymes. Transposon insertions in the rpfF homolog of X. oryzae pv. oryzae are deficient for virulence and production of a DSF but are proficient for EPS and extracellular enzyme production. The rpfF X. oryzae pv. oryzae mutants exhibit an unusual tetracycline susceptibility phenotype in which exogenous iron supplementation is required for phenotypic expression of a tetracycline resistance determinant that is encoded on an introduced plasmid. The rpfF X. oryzae pv. oryzae mutants also overproduce one or more siderophores and exhibit a growth deficiency under low iron conditions as well as in the presence of reducing agents that are expected to promote the conversion of Fe+3 to Fe+2. Exogenous iron supplementation promotes migration of rpfF X. oryzae pv. oryzae mutants in rice leaves. The results suggest that rpfF may be involved in controlling an iron-uptake system of X. oryzae pv. oryzae and that an inability to cope with the conditions of low iron availability in the host may be the reason for the virulence deficiency of the rpfF X. oryzae pv. oryzae mutants.

Xanthomonas oryzae pv. oryzicola, the causal agent of bacterial leaf streak in the model plant rice, possesses a hypersensitive response and pathogenicity (hrp), hrp-conserved (hrc), hrp-associated (hpa) cluster (hrp-hrc-hpa) that encodes a type III secretion system (T3SS) through which T3SS effectors are injected into host cells to cause disease or trigger plant defenses. Mutations in this cluster usually abolish the bacterial ability to cause hypersensitive response in nonhost tobacco and pathogenicity in host rice. In Xanthomonas spp., these genes are generally assumed to be regulated by the key master regulators HrpG and HrpX. However, we present evidence that, apart from HrpG and HrpX, HrpD6 is also involved in regulating the expression of hrp genes. Interestingly, the expression of hpa2, hpa1, hpaB, hrcC, and hrcT is positively controlled by HrpD6. Transcriptional expression assays demonstrated that the expression of the hrcC, hrpD5, hrpE, and hpa3 genes was not completely abolished by hrpG and hrpX mutations. As observed in analysis of their corresponding mutants, HrpG and HrpX exhibit contrasting gene regulation, particularly for hpa2 and hrcT. Other two-component system regulators (Zur, LrpX, ColR/S, and Trh) did not completely inhibit the expression of hrcC, hrpD5, hrpE, and hpa3. Immunoblotting assays showed that the secretion of HrpF, which is an HpaB-independent translocator, is not affected by the mutation in hrpD6. However, the mutation in hrpD6 affects the secretion of an HpaB-dependent TAL effector, AvrXa27. These novel findings suggest that, apart from HrpG and HrpX, HrpD6 plays important roles not only in the regulation of hrp genes but also in the secretion of TAL effectors.

Diffusible signal factor (DSF) is a fatty acid signal molecule involved in regulation of virulence in several Xanthomonas species as well as Xylella fastidiosa. In this study, we identified a variety of bacteria that could disrupt DSF-mediated induction of virulence factors in Xanthomonas campestris pv. campestris. While many bacteria had the ability to degrade DSF, several bacterial strains belonging to genera Bacillus, Paenibacillus, Microbacterium, Staphylococcus, and Pseudomonas were identified that were capable of particularly rapid degradation of DSF. The molecular determinants for rapid degradation of DSF in Pseudomonas spp. strain G were elucidated. Random transposon mutants of strain G lacking the ability to degrade DSF were isolated. Cloning and characterization of disrupted genes in these strains revealed that carAB, required for the synthesis of carbamoylphosphate, a precursor for pyrimidine and arginine biosynthesis is required for rapid degradation of DSF in strain G. Complementation of carAB mutants restored both pyrimidine prototrophy and DSF degradation ability of the strain G mutant. An Escherichia coli strain harboring carAB of Pseudomonas spp. strain G degrades DSF more rapidly than the parental strain, and overexpression of carAB in trans increased the ability of Pseudomonas spp. strain G to degrade as compared with the parental strain. Coinoculation of X. campestris pv. campestris with DSF-degrading bacteria into mustard and cabbage leaves reduced disease severity up to twofold compared with plants inoculated only with the pathogen. Likewise, disease incidence and severity in grape stems coinoculated with Xylella fastidiosa and DSF-degrading strains were significantly reduced compared with plants inoculated with the pathogen alone. Coinoculation of grape plants with a carAB mutant of Pseudomonas spp. strain G complemented with carAB in trans reduced disease severity as well or better than the parental strain. These results indicate that overexpression of carAB in other endophytes could be a useful strategy of biocontrol for the control of diseases caused by plant pathogens that produce DSF.

Cell-to-cell signaling mediated by a fatty acid diffusible signaling factor (DSF) is central to the regulation of the virulence of Xylella fastidiosa. DSF production by X. fastidiosa is dependent on rpfF and, although required for insect colonization, appears to reduce its virulence to grape. To understand what aspects of colonization of grape are controlled by DSF in X. fastidiosa and, thus, those factors that contribute to virulence, we assessed the colonization of grape by a green fluorescent protein-marked rpfF-deficient mutant. The rpfF-deficient mutant was detected at a greater distance from the point of inoculation than the wild-type strain at a given sampling time, and also attained a population size that was up to 100-fold larger than that of the wild-type strain at a given distance from the point of inoculation. Confocal laser-scanning microscopy revealed that approximately 10-fold more vessels in petioles of symptomatic leaves harbored at least some cells of either the wild type or rpfF mutant when compared with asymptomatic leaves and, thus, that disease symptoms were associated with the extent of vessel colonization. Importantly, the rpfF mutant colonized approximately threefold more vessels than the wild-type strain. Although a wide range of colony sizes were observed in vessels colonized by both the wild type and rpfF mutant, the proportion of colonized vessels harboring large numbers of cells was significantly higher in plants inoculated with the rpfF mutant than with the wild-type strain. These studies indicated that the hypervirulence phenotype of the rpfF mutant is due to both a more extensive spread of the pathogen to xylem vessels and unrestrained multiplication within vessels leading to blockage. These results suggest that movement and multiplication of X. fastidiosa in plants are linked, perhaps because cell wall degradation products are a major source of nutrients. Thus, DSF-mediated cell-to-cell signaling, which restricts movement and colonization of X. fastidiosa, may be an adaptation to endophytic growth of the pathogen that prevents the excessive growth of cells in vessels.

In Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, a secreted fatty acid signaling molecule known as diffusible signal factor (DSF) is required for virulence and growth on low-iron medium. To identify other virulence-associated traits that are regulated by DSF in this pathogen, we have performed microarray analysis of transcriptional changes between the wild type and DSF-deficient mutants of X. oryzae pv. oryzae. Expression of genes that encode secreted hydrolytic enzymes, motility, and chemotaxis functions are negatively regulated by DSF while functions involved in adhesion and biofilm formation are positively regulated. Enzymatic assays for hydrolytic enzymes as well as assays for chemotaxis, motility, attachment, and biofilm formation corroborate these findings. These results demonstrate that, in X. oryzae pv. oryzae, DSF-mediated cell-to-cell signaling coordinates transition from solitary to biofilm lifestyle by promoting expression of attachment functions and negatively regulating expression of motility functions. This is in contrast to X. campestris pv. campestris, a pathogen of crucifers, wherein the DSF system positively regulates motility functions and negatively regulates biofilm formation. These results indicate that virulence-associated functions can be regulated in a completely contrasting fashion by the same signaling system in very closely related bacteria.

Cell-cell signaling in Xylella fastidiosa has been implicated in the coordination of traits enabling colonization in plant hosts as well as insect vectors. This cell density-dependent signaling has been attributed to a diffusible signaling factor (DSF) produced by the DSF synthase RpfF. DSF produced by related bacterial species are unsaturated fatty acids, but that of X. fastidiosa was thought to be different from those of other taxa. We describe here the isolation and characterization of an X. fastidiosa DSF (XfDSF) as 2(Z)-tetradecenoic acid. This compound was isolated both from recombinant Erwinia herbicola expressing X. fastidiosa rpfF and from an X. fastidiosa rpfC deletion mutant that overproduces DSF. Since an rpfF mutant is impaired in biofilm formation and underexpresses the hemagglutinin-like protein-encoding genes hxfA and hxfB, we demonstrate that these traits can be restored by ca. 0.5 µM XfDSF but not by myristic acid, the fully saturated tetradecenoic acid. A phoA-based X. fastidiosa biosensor that assesses DSF-dependent expression of hxfA or hxfB revealed a high level of molecular specificity of DSF signaling.