The Global Transcription Regulator XooClp Governs Type IV Pili System-Mediated Bacterial Virulence by Directly Binding to TFP-Chp Promoters to Coordinate Virulence Associated Functions.

Type IV pili (TFP) play a crucial role in the sensing of the external environment for several bacteria. This surface sensing is essential for the lifestyle transitions of several bacteria and involvement in pathogenesis. However, the precise mechanisms underlying TFP's integration of environmental cues, particularly in regulating the TFP-Chp system and its effects on Xanthomonas physiology, social behavior, and virulence, remain poorly understood. In this study, we focused on investigating Clp, a global transcriptional regulator similar to CRP-like proteins, in Xanthomonas oryzae pv. oryzae, a plant pathogen. Our findings reveal that Clp integrates environmental cues detected through diffusible signaling factor (DSF) quorum sensing into the TFP-Chp regulatory system. It accomplishes this by directly binding to TFP-Chp promoters in conjunction with intracellular levels of cyclic-di-GMP, a ubiquitous bacterial second messenger, thereby controlling TFP expression. Moreover, Clp-mediated regulation is involved in regulating several cellular processes, including the production of virulence-associated functions. Collectively, these processes contribute to host colonization and disease initiation. Our study elucidates the intricate regulatory network encompassing Clp, environmental cues, and the TFP-Chp system, providing insights into the molecular mechanisms that drive bacterial virulence in Xanthomonas spp. These findings offer valuable knowledge regarding Xanthomonas pathogenicity and present new avenues for innovative strategies aimed at combating plant diseases caused by these bacteria. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

XdfA, a novel membrane-associated DedA family protein of Xanthomonas campestris, is required for optimum virulence, maintenance of magnesium, and membrane homeostasis.

Xanthomonas campestris is an important member of the Xanthomonas group of phytopathogens that causes diseases in crucifers. In X. campestris, several virulence-associated functions, including some belonging to unknown predicted functions, have been implicated in the colonization and disease processes. However, the role of many of these unknown predicted proteins in Xanthomonas-host interaction and their exact physiological function is not clearly known. In this study, we identified a novel membrane-associated protein belonging to the DedA super family, XdfA, which is required for virulence in X. campestris. The DedA family of proteins are generally ubiquitous in bacteria; however, their function and actual physiological role are largely elusive. Characterization of ∆xdfA by homology modeling, membrane localization, and physiological studies indicated that XdfA is a membrane-associated protein that plays a role in the maintenance of membrane integrity. Furthermore, functional homology modeling analysis revealed that the XdfA exhibits structural similarity to a CorA-like magnesium transporter and is required for optimum growth under low magnesium ion concentration. We report for the first time that a putative DedA family of protein in Xanthomonas is required for optimum virulence and plays a role in the maintenance of membrane-associated functions and magnesium homeostasis. IMPORTANCE Bacterial DedA family proteins are involved in a range of cellular processes such as ion transport, signal transduction, and cell division. Here, we have discussed about a novel DedA family protein XdfA in Xanthomonas campestris pv. campestris that has a role in membrane homeostasis, magnesium transport, and virulence. Understanding membrane and magnesium homeostasis will aid in our comprehension of bacterial physiology and eventually will help us devise effective antimicrobial strategies to safeguard horticulturally and agriculturally important crop plants.

DSF-family quorum sensing signal-mediated intraspecies, interspecies, and inter-kingdom communication.

While most bacteria are unicellular microbes they communicate with each other and with their environments to adapt their behaviors. Quorum sensing (QS) is one of the best-studied cell-cell communication modes. QS signaling is not restricted to bacterial cell-to-cell communication - it also allows communication between bacteria and their eukaryotic hosts. The diffusible signal factor (DSF) family represents an intriguing type of QS signal with multiple roles found in diverse Gram-negative bacteria. Over the last decade, extensive progress has been made in understanding DSF-mediated communication among bacteria, fungi, insects, plants, and zebrafish. This review provides an update on these new developments with the aim of building a more comprehensive picture of DSF-mediated intraspecies, interspecies, and inter-kingdom communication.

The diffusible signal factor synthase, RpfF, in Xanthomonas oryzae pv. oryzae is required for the maintenance of membrane integrity and virulence.

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.

Insights into the Cell-to-Cell Signaling and Iron Homeostasis in Xanthomonas Virulence and Lifestyle.

The Xanthomonas group of phytopathogens causes economically important diseases that lead to severe yield loss in major crops. Some Xanthomonas species are known to have an epiphytic and in planta lifestyle that is coordinated by several virulence-associated functions, cell-to-cell signaling (using diffusible signaling factor [DSF]), and environmental conditions, including iron availability. In this review, we described the role of cell-to-cell signaling by the DSF molecule and iron in the regulation of virulence-associated functions. Although DSF and iron are involved in the regulation of several virulence-associated functions, members of the Xanthomonas group of plant pathogens exhibit atypical patterns of regulation. Atypical patterns contribute to the adaptation to different lifestyles. Studies on DSF and iron biology indicate that virulence-associated functions can be regulated in completely contrasting fashions by the same signaling system in closely related xanthomonads.

Interplay between the cyclic di-GMP network and the cell-cell signalling components coordinates virulence-associated functions in Xanthomonas oryzae pv. oryzae.

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.

Bacterial quorum sensing facilitates Xanthomonas campesteris pv. campestris invasion of host tissue to maximize disease symptoms.

Quorum sensing (QS) helps the Xanthomonas group of phytopathogens to infect several crop plants. The vascular phytopathogen Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot disease on Brassicaceae leaves, where a typical v-shaped lesion spans both vascular and mesophyll regions with progressive leaf chlorosis. Recently, the role of QS has been elucidated during Xcc early infection stages. However, a detailed insight into the possible role of QS-regulated bacterial invasion in host chlorophagy during late infection stages remains elusive. In this study, using QS-responsive whole-cell bioreporters of Xcc, we present a detailed chronology of QS-facilitated Xcc colonization in the mesophyll region of cabbage (Brassica oleracea) leaves. We report that QS-enabled localization of Xcc to parenchymal chloroplasts triggers leaf chlorosis and promotion of systemic infection. Our results indicate that the QS response in the Xanthomonas group of vascular phytopathogens maximizes their population fitness across host tissues to trigger stage-specific host chlorophagy and establish a systemic infection.

A Bacteriophytochrome Mediates Interplay between Light Sensing and the Second Messenger Cyclic Di-GMP to Control Social Behavior and Virulence.

Bacteriophytochromes are the most abundant and ubiquitous light-sensing receptors in bacteria and are involved in time-of-day behavior or responses. However, their biological and regulatory role in non-photosynthetic bacteria is poorly understood, and even less is known about how they regulate diverse cellular processes. Here, we show that a bacteriophytochrome (XooBphP) from the plant pathogen Xanthomonas oryzae pv. oryzae perceives light signals and transduces a signal through its EAL-mediated phosphodiesterase activity, modulating the intracellular level of the ubiquitous bacterial second messenger c-di-GMP. We discover that light-mediated fine-tuning of intracellular c-di-GMP levels by XooBphP regulates production of virulence functions, iron metabolism, and transition from a sessile to a free-swimming motile lifestyle, contributing to its colonization of the host and virulence. XooBphP thus plays a crucial role in integrating photo-sensing with intracellular signaling to control the pathogenic lifestyle and social behavior.

Transition of a solitary to a biofilm community life style in bacteria: a survival strategy with division of labour.

Multicellularity is associated with higher eukaryotes having an organized division of labour and a coordinated action of different organs composed of multiple cell types. This division of different cell types and organizations to form a multicellular structure by developmental programming is a key to the multitasking of complex traits that enable higher eukaryotes to cope with fluctuating environmental conditions. Microbes such as bacteria, on the other hand, are unicellular and have flourished in diverse environmental conditions for a much longer time than eukaryotes in evolutionary history. In this review, we will focus on different strategies and functions exhibited by microbes that enable them to adapt to changes in lifestyle associated with transitioning from a unicellular solitary state to a complex community architecture known as a biofilm. We will also discuss various environmental stimuli and signaling processes which bacteria utilize to coordinate their social traits and enable themselves to form complex multicellular-like biofilm structures, and the division of labour operative within such communities driving their diverse social traits. We will also discuss here recent studies from our laboratory using a plant-associated bacterial pathogen as a model organism to elucidate the mechanism of bacterial cell-cell communication and the transition of a bacterial community to a multicellular-like structure driven by the complex regulation of traits influenced by cell density, as well as environmental sensing such as chemotaxis and nutrient availability. These studies are shedding important insights into bacterial developmental transitions and will help us to understand community cooperation and conflict using bacterial cell-cell communication as a model system.

New insight into bacterial social communication in natural host: Evidence for interplay of heterogeneous and unison quorum response.

Many microbes exhibit quorum sensing (QS) to cooperate, share and perform a social task in unison. Recent studies have shown the emergence of reversible phenotypic heterogeneity in the QS-responding pathogenic microbial population under laboratory conditions as a possible bet-hedging survival strategy. However, very little is known about the dynamics of QS-response and the nature of phenotypic heterogeneity in an actual host-pathogen interaction environment. Here, we investigated the dynamics of QS-response of a Gram-negative phytopathogen Xanthomonas pv. campestris (Xcc) inside its natural host cabbage, that communicate through a fatty acid signal molecule called DSF (diffusible signal factor) for coordination of several social traits including virulence functions. In this study, we engineered a novel DSF responsive whole-cell QS dual-bioreporter to measure the DSF mediated QS-response in Xcc at the single cell level inside its natural host plant in vivo. Employing the dual-bioreporter strain of Xcc, we show that QS non-responsive cells coexist with responsive cells in microcolonies at the early stage of the disease; whereas in the late stages, the QS-response is more homogeneous as the QS non-responders exhibit reduced fitness and are out competed by the wild-type. Furthermore, using the wild-type Xcc and its QS mutants in single and mixed infection studies, we show that QS mutants get benefit to some extend at the early stage of disease and contribute to localized colonization. However, the QS-responding cells contribute to spread along xylem vessel. These results contrast with the earlier studies describing that expected cross-induction and cooperative sharing at high cell density in vivo may lead to synchronize QS-response. Our findings suggest that the transition from heterogeneity to homogeneity in QS-response within a bacterial population contributes to its overall virulence efficiency to cause disease in the host plant under natural environment.

Xanthomonas oryzae pv. oryzae chemotaxis components and chemoreceptor Mcp2 are involved in the sensing of constituents of xylem sap and contribute to the regulation of virulence-associated functions and entry into rice.

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.

Low-iron conditions induces the hypersensitive reaction and pathogenicity hrp genes expression in Xanthomonas and is involved in modulation of hypersensitive response and virulence.

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.

Bacterial cyclic ß-(1,2)-glucans sequester iron to protect against iron-induced toxicity.

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 ß-(1,2)-glucans bind iron specifically and promote growth under iron-restricted conditions. Xanthomonas campestris and Escherichia coli mutants blocked in the production of ß-(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.

Xanthoferrin, the α-hydroxycarboxylate-type siderophore of Xanthomonas campestris pv. campestris, is required for optimum virulence and growth inside cabbage.

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 ΔxsuA 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 ΔxssA and ΔxsuA mutants exhibit defects in ferric iron (Fe3+ ) uptake. The ΔxssA mutant is able to utilize and transport the exogenous xanthoferrin-Fe3+ complex; in contrast, the siderophore utilization or uptake mutant ΔxsuA 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 ΔxssA and ΔxsuA 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+ .

Xanthoferrin Siderophore Estimation from the Cell-free Culture Supernatants of Different Xanthomonas Strains by HPLC.

Xanthomonads can scavenge iron from the extracellular environment by secreting the siderophores, which are synthesized by the proteins encoded by xss (Xanthomonas siderophore synthesis) gene cluster. The siderophore production varies among xanthomonads in response to a limited supply of iron where Xanthomonas campestris pv. campestris (Xcc) produces less siderophores than Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc). Siderophore production can be measured by HPLC and with the CAS (Chrome azurol S)-agar plate assay, however HPLC is a more accurate method over CAS-agar plate assay for siderophore quantification in Xanthomonads. Here we describe how to quantify siderophores from xanthomonads using HPLC.

Co-regulation of Iron Metabolism and Virulence Associated Functions by Iron and XibR, a Novel Iron Binding Transcription Factor, in the Plant Pathogen Xanthomonas.

Abilities of bacterial pathogens to adapt to the iron limitation present in hosts is critical to their virulence. Bacterial pathogens have evolved diverse strategies to coordinately regulate iron metabolism and virulence associated functions to maintain iron homeostasis in response to changing iron availability in the environment. In many bacteria the ferric uptake regulator (Fur) functions as transcription factor that utilize ferrous form of iron as cofactor to regulate transcription of iron metabolism and many cellular functions. However, mechanisms of fine-tuning and coordinated regulation of virulence associated function beyond iron and Fur-Fe2+ remain undefined. In this study, we show that a novel transcriptional regulator XibR (named Xanthomonas iron binding regulator) of the NtrC family, is required for fine-tuning and co-coordinately regulating the expression of several iron regulated genes and virulence associated functions in phytopathogen Xanthomonas campestris pv. campestris (Xcc). Genome wide expression analysis of iron-starvation stimulon and XibR regulon, GUS assays, genetic and functional studies of xibR mutant revealed that XibR positively regulates functions involved in iron storage and uptake, chemotaxis, motility and negatively regulates siderophore production, in response to iron. Furthermore, chromatin immunoprecipitation followed by quantitative real-time PCR indicated that iron promoted binding of the XibR to the upstream regulatory sequence of operon's involved in chemotaxis and motility. Circular dichroism spectroscopy showed that purified XibR bound ferric form of iron. Electrophoretic mobility shift assay revealed that iron positively affected the binding of XibR to the upstream regulatory sequences of the target virulence genes, an effect that was reversed by ferric iron chelator deferoxamine. Taken together, these data revealed that how XibR coordinately regulates virulence associated and iron metabolism functions in Xanthomonads in response to iron availability. Our results provide insight of the complex regulatory mechanism of fine-tuning of virulence associated functions with iron availability in this important group of phytopathogen.

Xanthomonas campestris cell-cell signalling molecule DSF (diffusible signal factor) elicits innate immunity in plants and is suppressed by the exopolysaccharide xanthan.

Several secreted and surface-associated conserved microbial molecules are recognized by the host to mount the defence response. One such evolutionarily well-conserved bacterial process is the production of cell-cell signalling molecules which regulate production of multiple virulence functions by a process known as quorum sensing. Here it is shown that a bacterial fatty acid cell-cell signalling molecule, DSF (diffusible signal factor), elicits innate immunity in plants. The DSF family of signalling molecules are highly conserved among many phytopathogenic bacteria belonging to the genus Xanthomonas as well as in opportunistic animal pathogens. Using Arabidopsis, Nicotiana benthamiana, and rice as model systems, it is shown that DSF induces a hypersensitivity reaction (HR)-like response, programmed cell death, the accumulation of autofluorescent compounds, hydrogen peroxide production, and the expression of the PATHOGENESIS-RELATED1 (PR-1) gene. Furthermore, production of the DSF signalling molecule in Pseudomonas syringae, a non-DSF-producing plant pathogen, induces the innate immune response in the N. benthamiana host plant and also affects pathogen growth. By pre- and co-inoculation of DSF, it was demonstrated that the DSF-induced plant defence reduces disease severity and pathogen growth in the host plant. In this study, it was further demonstrated that wild-type Xanthomonas campestris suppresses the DSF-induced innate immunity by secreting xanthan, the main component of extracellular polysaccharide. The results indicate that plants have evolved to recognize a widely conserved bacterial communication system and may have played a role in the co-evolution of host recognition of the pathogen and the communication machinery.

Cell-cell signalling promotes ferric iron uptake in Xanthomonas oryzae pv. oryzicola that contribute to its virulence and growth inside rice.

Cell-cell communication mediated by diffusible signal factor (DSF) plays an important role in virulence of several Xanthomonas group of plant pathogens. In the bacterial pathogen of rice, Xanthomonas 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 Fe(3+) or Fe(2+) form of iron for colonization may vary substantially among closely related members of the Xanthomonas group of plant pathogens.

Reversible non-genetic phenotypic heterogeneity in bacterial quorum sensing.

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 Pseudomonas syringae and Xanthomonas 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.

Production of Xylella fastidiosa diffusible signal factor in transgenic grape causes pathogen confusion and reduction in severity of Pierce's disease.

The rpfF gene from Xylella fastidiosa, encoding the synthase for diffusible signal factor (DSF), was expressed in 'Freedom' grape to reduce the pathogen's growth and mobility within the plant. Symptoms in such plants were restricted to near the point of inoculation and incidence of disease was two- to fivefold lower than in the parental line. Both the longitudinal and lateral movement of X. fastidiosa in the xylem was also much lower. DSF was detected in both leaves and xylem sap of RpfF-expressing plants using biological sensors, and both 2-Z-tetradecenoic acid, previously identified as a component of X. fastidiosa DSF, and cis-11-methyl-2-dodecenoic acid were detected in xylem sap using electrospray ionization mass spectrometry. A higher proportion of X. fastidiosa cells adhered to xylem vessels of the RpfF-expressing line than parental 'Freedom' plants, reflecting a higher adhesiveness of the pathogen in the presence of DSF. Disease incidence in RpfF-expressing plants in field trials in which plants were either mechanically inoculated with X. fastidiosa or subjected to natural inoculation by sharpshooter vectors was two- to fourfold lower in than that of the parental line. The number of symptomatic leaves on infected shoots was reduced proportionally more than the incidence of infection, reflecting a decreased ability of X. fastidiosa to move within DSF-producing plants.