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The Xanthomonas group of phytopathogens communicate with a fatty acid‐like cell–cell signalling molecule, cis‐11‐2‐methyl‐dodecenoic acid, also known as diffusible signal factor (DSF). In the pathogen of rice, Xanthomonas oryzae pv. oryzae, DSF is involved in the regulation of several virulence‐associated functions, including production and secretion of several cell wall hydrolysing type II secretion effectors. To understand the role of DSF in the secretion of type II effectors, we characterized DSF synthase‐deficient (rpfF) and DSF‐deficient, type II secretion (xpsE) double mutants. Mutant analysis by expression analysis, secretion assay, fatty acid analysis, and physiological studies indicated that rpfF mutants exhibit hypersecretion of several type II effectors due to a perturbed membrane and DSF is required for maintaining membrane integrity. The rpfF mutants exhibited significantly higher uptake of 1‐N‐phenylnapthylamine and ethidium bromide, and up‐regulation of rpoE (σE). Increasing the osmolarity of the medium could rescue the hypersecretion phenotype of the rpfF mutant. The rpfF mutant exhibited highly reduced virulence. We report for the first time that in X. oryzae pv. oryzae RpfF is involved in the maintenance of membrane integrity by playing a regulatory role in the fatty acid synthesis pathway. [ABSTRACT FROM AUTHOR]

Summary: Xanthomonas oryzae pv. oryzae (Xoo) causes a serious disease of rice known as bacterial leaf blight. Several virulence‐associated functions have been characterized in Xoo. However, the role of important second messenger c‐di‐GMP signalling in the regulation of virulence‐associated functions still remains elusive in this phytopathogen. In this study we have performed an investigation of 13 c‐di‐GMP modulating deletion mutants to understand their contribution in Xoo virulence and lifestyle transition. We show that four Xoo proteins, Xoo2331, Xoo2563, Xoo2860 and Xoo2616, are involved in fine‐tuning the in vivo c‐di‐GMP abundance and also play a role in the regulation of virulence‐associated functions. We have further established the importance of the GGDEF domain of Xoo2563, a previously characterized c‐di‐GMP phosphodiesterase, in the virulence‐associated functions of Xoo. Interestingly the strain harbouring the GGDEF domain deletion (ΔXoo2563GGDEF) exhibited EPS deficiency and hypersensitivity to streptonigrin, indicative of altered iron metabolism. This is in contrast to the phenotype exhibited by an EAL overexpression strain wherein, the ΔXoo2563GGDEF exhibited other phenotypes, similar to the strain overexpressing the EAL domain. Taken together, our results indicate a complex interplay of c‐di‐GMP signalling with the cell–cell signalling to coordinate virulence‐associated function in Xoo. [ABSTRACT FROM AUTHOR]

Summary: The Xanthomonas group of phytopathogens causes several economically important diseases in crops. In the bacterial pathogen of rice, Xanthomonas oryzae pv. oryzae (Xoo), it has been proposed that chemotaxis may play a role in the entry and colonization of the pathogen inside the host. However, components of the chemotaxis system, including the chemoreceptors involved, and their role in entry and virulence, are not well defined. In this study, we show that Xoo displays a positive chemotaxis response to components of rice xylem sap—glutamine, xylose and methionine. In order to understand the role of chemotaxis components involved in the promotion of chemotaxis, entry and virulence, we performed detailed deletion mutant analysis. Analysis of mutants defective in chemotaxis components, flagellar biogenesis, expression analysis and assays of virulence‐associated functions indicated that chemotaxis‐mediated signalling in Xoo is involved in the regulation of several virulence‐associated functions, such as motility, attachment and iron homeostasis. The ∆cheY1 mutant of Xoo exhibited a reduced expression of genes involved in motility, adhesins, and iron uptake and metabolism. We show that the expression of Xoo chemotaxis and motility components is induced under in planta conditions and is required for entry, colonization and virulence. Furthermore, deletion analysis of a putative chemoreceptor mcp2 gene revealed that chemoreceptor Mcp2 is involved in the sensing of xylem sap and constituents of xylem exudate, including methionine, serine and histidine, and plays an important role in epiphytic entry and virulence. This is the first report of the role of chemotaxis in the virulence of this important group of phytopathogens. [ABSTRACT FROM AUTHOR]

Summary: Expression of hrp (hypersensitive reaction and pathogenicity) genes inside the host is crucial for virulence of phytopathogenic bacteria. The hrp genes encode components of type3 secretion system (T3SS), HR elicitors and several regulators, which are involved in the co‐ordinated expression of hrp genes in the host environment and in hrp inducing chemically defined medium. However, little is known about specific host or environmental factors which may play a role in the induction of hrp gene expression. In this study, we show that iron‐limiting condition elicits induced expression of hrp genes, including type3 secretion system (T3SS) and effectors (T3E). Expression analysis using qRT‐PCR and promoter probe strains suggest significant induction in the expression of Hrp and T3S‐associated genes of Xanthomonas campestris pv. campestris (Xcc) under low‐iron condition, and is suppressed by exogenous supplementation of iron. Furthermore, we show that with exogenous iron supplementation, wild type Xcc exhibited reduced disease symptoms in host‐plant, and exhibited significant reduction in HR and callose deposition in the non‐host plants. Xanthomonas oryzae and oryzicola pathovars also exhibited the iron affect, albeit to a lesser extend compared with the Xcc. Overall, our results suggest that low‐iron condition inside the host may play a crucial role in pathogenicity. [ABSTRACT FROM AUTHOR]

Abstract: Cellular iron homeostasis is critical for survival and growth. Bacteria employ a variety of strategies to sequester iron from the environment and to store intracellular iron surplus that can be utilized in iron‐restricted conditions while also limiting the potential for the production of iron‐induced reactive oxygen species (ROS). Here, we report that membrane‐derived oligosaccharide (mdo) glucan, an intrinsic component of Gram‐negative bacteria, sequesters the ferrous form of iron. Iron‐binding, uptake, and localization experiments indicated that both secreted and periplasmic β‐(1,2)‐glucans bind iron specifically and promote growth under iron‐restricted conditions. Xanthomonas campestris and Escherichia coli mutants blocked in the production of β‐(1,2)‐glucan accumulate low amounts of intracellular iron under iron‐restricted conditions, whereas they exhibit elevated ROS production and sensitivity under iron‐replete conditions. Our results reveal a critical role of glucan in intracellular iron homeostasis conserved in Gram‐negative bacteria. [ABSTRACT FROM AUTHOR]

Xanthomonas campestris pv. campestris causes black rot, a serious disease of crucifers. Xanthomonads encode a siderophore biosynthesis and uptake gene cluster xss ( Xanthomonas siderophore synthesis) involved in the production of a vibrioferrin-type siderophore. However, little is known about the role of the siderophore in the iron uptake and virulence of X. campestris pv. campestris. In this study, we show that X. campestris pv. campestris produces an α-hydroxycarboxylate-type siderophore (named xanthoferrin), which is required for growth under low-iron conditions and for optimum virulence. A mutation in the siderophore synthesis xssA gene causes deficiency in siderophore production and growth under low-iron conditions. In contrast, the siderophore utilization Δ xsuA mutant is able to produce siderophore, but exhibits a defect in the utilization of the siderophore-iron complex. Our radiolabelled iron uptake studies confirm that the Δ xssA and Δ xsuA mutants exhibit defects in ferric iron (Fe3+) uptake. The Δ xssA mutant is able to utilize and transport the exogenous xanthoferrin-Fe3+ complex; in contrast, the siderophore utilization or uptake mutant Δ xsuA exhibits defects in siderophore uptake. Expression analysis of the xss operon using a chromosomal gusA fusion indicates that the xss operon is expressed during in planta growth and under low-iron conditions. Furthermore, exogenous iron supplementation in cabbage leaves rescues the in planta growth deficiency of Δ xssA and Δ xsuA mutants. Our study reveals that the siderophore xanthoferrin is an important virulence factor of X. campestris pv. campestris which promotes in planta growth by the sequestration of Fe3+. [ABSTRACT FROM AUTHOR]

Cell-cell communication mediated by diffusible signal factor ( DSF) plays an important role in virulence of several X anthomonas group of plant pathogens. In the bacterial pathogen of rice, X anthomonas oryzae pv. oryzicola, DSF is required for virulence and in planta growth. In order to understand the role of DSF in promoting in planta growth and virulence, we have characterized the DSF deficient mutant of X. oryzae pv. oryzicola. Mutant analysis by expression analysis, radiolabelled iron uptake studies and growth under low-iron conditions indicated that DSF positively regulates ferric iron uptake. Further, the DSF deficient mutant of X. oryzae pv. oryzicola exhibited a reduced capacity to use ferric form of iron for growth under low-iron conditions. Exogenous iron supplementation in the rice leaves rescued the in planta growth deficiency of the DSF deficient mutant. These data suggest that DSF promotes in planta growth of X . oryzae pv. oryzicola by positively regulating functions involved in ferric iron uptake which is important for its virulence. Our results also indicate that requirement of iron uptake strategies to utilize either Fe3+ or Fe2+ form of iron for colonization may vary substantially among closely related members of the X anthomonas group of plant pathogens. [ABSTRACT FROM AUTHOR]

Bacteria co-ordinate their social behaviour in a density-dependent manner by production of diffusible signal molecules by a process known as quorum sensing ( QS). It is generally assumed that in homogenous environments and at high cell density, QS synchronizes cells in the population to perform collective social tasks in unison which maximize the benefit at the inclusive fitness of individuals. However, evolutionary theory predicts that maintaining phenotypic heterogeneity in performing social tasks is advantageous as it can serve as a bet-hedging survival strategy. Using P seudomonas syringae and X anthomonas campestris as model organisms, which use two diverse classes of QS signals, we show that two distinct subpopulations of QS-responsive and non-responsive cells exist in the QS-activated population. Addition of excess exogenous QS signal does not significantly alter the distribution of QS-responsive and non-responsive cells in the population. We further show that progeny of cells derived from these subpopulations also exhibited heterogeneous distribution patterns similar to their respective parental strains. Overall, these results support the model that bacteria maintain QS-responsive and non-responsive subpopulations at high cell densities in a bet-hedging strategy to simultaneously perform functions that are both positively and negatively regulated by QS to improve their fitness in fluctuating environments. [ABSTRACT FROM AUTHOR]