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.

In Xylella fastidiosa the fatty acid signal molecule diffusible signaling factor (DSF) is produced and sensed by components of the regulation of pathogenicity factors (rpf) cluster; lack of DSF production in RpfF mutants results in a non-vector-transmissible phenotype yet cells are hypervirulent to grape. rpfB has not been characterized in Xylella fastidiosa, although its homolog has been suggested to be required for DSF synthesis in Xanthomonas campestris pv. campestris. We show that RpfB is involved in DSF processing in both Xylella fastidiosa and Xanthomonas campestris, affecting the profile of DSF-like fatty acids observed in thin-layer chromatography. Although three fatty acids whose production is dependent on RpfF were detected in Xylella fastidiosa and Xanthomonas campestris wild-type strains, their respective rpfB mutants accumulated primarily one chemical species. Although no quantifiable effect of rpfB on plant colonization by Xylella fastidiosa was found, insect colonization and transmission was reduced. Thus, RpfB apparently is involved in DSF processing, and like Xanthomonas campestris, Xylella fastidiosa also produces multiple DSF molecules. It is possible that Xylella fastidiosa coordinates host vector and plant colonization by varying the proportions of different forms of DSF signals via RpfB.

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.

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.

Although it is known that Xylella fastidiosa (Xf) is located on the foregut of infected insects (infective nymphs loose transmissibility after molting and there is no latent period required for transmission), the exact location in the foregut from which the bacterium Xf is transmitted by sharpshooter leafhoppers is not known. We examined the spatial distribution of Xf in the precibarium of vectors that had been fed on infected grapevines. Xf cells attached on end to the insect's cuticle and were distributed throughout the precibarium, with few exceptions, on both pharynges. Xf was not present on top of the precibarial valve, but interestingly cells were observed within the valve's pit. In a transmission experiment with long inoculation access period, all infective insects transmitted Xf to healthy grapevines; individuals free of Xf on the precibarium did not vector the pathogen. An ancillary objective was to determine if the relatively low transmission rates (5-15% daily) by the glassy-winged sharpshooter (GWSS) were influenced by the plant tissue inoculated: stem or leaf. On grape, adult GWSS feed mainly on stems. In an initial experiment with low transmission rates (less than 7% per GWSS adult) there were no differences among treatments (stem only, leaf only, stem and leaf). This will be repeated with more insects per plant and longer exposure for inoculation, but these initial results suggest that feeding site on green tissues are not a major factor.

Reconstructing synthetic metabolic pathways in microbes holds great promise for the production of pharmaceuticals in large-scale fermentations. By recreating biosynthetic pathways in bacteria, complex molecules traditionally harvested from scarce natural resources can be produced in microbial cultures. Here we report on a strain of Escherichia coli containing a heterologous, nine-gene biosynthetic pathway for the production of the terpene amorpha-4,11-diene, a precursor to the anti-malarial drug artemisinin. Previous reports have underestimated the productivity of this strain due to the volatility of amorphadiene. Here we show that amorphadiene evaporates from a fermentor with a half-life of about 50 min. Using a condenser, we take advantage of this volatility by trapping the amorphadiene in the off-gas. Amorphadiene was positively identified using nuclear magnetic resonance spectroscopy and determined to be 89% pure as collected. We captured amorphadiene as it was produced in situ by employing a two-phase partitioning bioreactor with a dodecane organic phase. Using a previously characterized caryophyllene standard to calibrate amorphadiene production and capture, the concentration of amorphadiene produced was determined to be 0.5 g/L of culture medium. A standard of amorphadiene collected from the off-gas showed that the caryophyllene standard overestimated amorphadiene production by approximately 30%. © 2006 Wiley Periodicals, Inc.

Battling malaria Drug-resistant strains of the malaria parasite are widespread, and as a result mortality due to malaria has increased significantly in recent years. Artemisinin, isolated from the herb Artemisia annua (sweet wormwood), is one drug that shows a high efficacy in killing multi-resistant strains of the parasite. The drug is extremely expensive, and high demand has led to a shortage of artemisinin, available only by extraction from the plant source. Ro et al . now report the development of a yeast strain engineered to carry a cytochrome P450 monooxygenase from A. annua that can produce the drug precursor, artemisinic acid. Artemisinin can be synthesized from this precursor. If the efficiency of this process can be improved, this engineered yeast strain has the potential to alleviate the drug shortage.

Xylella fastidiosa, which causes Pierce's disease of grapevine and other important plant diseases, is a xylem-limited bacterium that depends on insect vectors for transmission. Although many studies have addressed disease symptom development and transmission of the pathogen by vectors, little is known about the bacterial mechanisms driving these processes. Recently available X. fastidiosa genomic sequences and molecular tools have provided new routes for investigation. Here, we show that a diffusible signal molecule is required for biofilm formation in the vector and for vector transmission to plants. We constructed strains of X. fastidiosa mutated in the rpfF gene and determined that they are unable to produce the signal activity. In addition, rpfF mutants are more virulent than the wild type when mechanically inoculated into plants. This signal therefore directs interaction of X. fastidiosa with both its insect vector and plant host. Interestingly, rpfF mutants can still form in planta biofilms, which differ architecturally from biofilms in insects, suggesting that biofilm architecture, rather than a passive response to the environment, is actively determined by X. fastidiosa gene expression. This article reports a cell-cell signaling requirement for vector transmission. Identification of the genes regulated by rpfF should elucidate bacterial factors involved in transmission and biofilm formation in the insect.

Xylella fastidiosa, which causes Pierce's disease of grapevine and other important plant diseases, is a xylem-limited bacterium that depends on insect vectors for transmission. Although many studies have addressed disease symptom development and transmission of the pathogen by vectors, little is known about the bacterial mechanisms driving these processes. Recently availableX. fastidiosagenomic sequences and molecular tools have provided new routes for investigation. Here, we show that a diffusible signal molecule is required for biofilm formation in the vector and for vector transmission to plants. We constructed strains ofX. fastidiosamutated in therpfFgene and determined that they are unable to produce the signal activity. In addition,rpfFmutants are more virulent than the wild type when mechanically inoculated into plants. This signal therefore directs interaction ofX. fastidiosawith both its insect vector and plant host. Interestingly,rpfFmutants can still formin plantabiofilms, which differ architecturally from biofilms in insects, suggesting that biofilm architecture, rather than a passive response to the environment, is actively determined byX. fastidiosagene expression. This article reports a cell-cell signaling requirement for vector transmission. Identification of the genes regulated byrpfFshould elucidate bacterial factors involved in transmission and biofilm formation in the insect.

Xylella fastidiosa (Xf) is an endophyte that is restricted to the xylem, a network of vessels for water transport, in which it forms an aggregated biofilm. It is transmitted from plant to plant by xylem sap-feeding insects, and forms a polar biofilm in these insects' foreguts. In other systems, biofilms are characterized by community behavior under the control of cell density- dependent gene expression, which requires cell-cell signaling. Xf has homologs of the cell-cell signaling genes found in the important plant pathogen Xanthomonas campestris pathovar campestris (Xcc) and probably shares a similar means of coordinating gene expression in a community (2, 7). Using the Xcc paradigm as a guide, we have investigated cell-cell signaling in Xf with the aim of developing cell-cell signaling disruption as a means of controlling Pierce's disease. We have determined that Xf produces a cell-cell signal and that the rpfF gene is necessary and sufficient for signal synthesis. We compared rpfF mutants to the wild type and found they are hypervirulent and non-transmissible. Lack of transmissibility was linked to an inability of the rpfF mutant to form a biofilm in the insect foregut. We are in the process of investigating the mechanism of hypervirulence. To further elucidate the behavior of Xf in planta, we created a green fluorescent strain of Xf and used confocal laser scanning microscopy to observe this strain of Xf within the xylem of plants. We found that vessel plugging is the colonization feature most tightly correlated with disease symptom expression, providing strong evidence that vessel plugging causes disease. We screened several collections of bacterial strains isolated from plants and identified bacterial strains that can interfere with Xf signaling. We are in the process of testing how these strains interact with Xf in the xylem, identifying to which species they belong and isolating the genes responsible for signal interference activity.

ABSTRACT Xylella fastidiosa causes Pierce's disease of grapevine as well as several other major agricultural diseases but is a benign endophyte in most host plants. X. fastidiosa colonizes the xylem vessels of host plants and is transmitted by xylem sap-feeding insect vectors. To understand better the pattern of host colonization and its relationship to disease, we engineered X. fastidiosa to express a green fluorescent protein (Gfp) constitutively and performed confocal laser-scanning microscopic analysis of colonization in a susceptible host, Vitis vinifera. In symptomatic leaves, the fraction of vessels colonized by X. fastidiosa was fivefold higher than in nearby asymptomatic leaves. The fraction of vessels completely blocked by X. fastidiosa colonies increased 40-fold in symptomatic leaves and was the feature of colonization most dramatically linked to symptoms. Therefore, the extent of vessel blockage by bacterial colonization is highly likely to be a crucial variable in symptom expression. Intriguingly, a high proportion (>80%) of colonized vessels were not blocked in infected leaves and instead had small colonies or solitary cells, suggesting that vessel blockage is not a colonization strategy employed by the pathogen but, rather, a by-product of endophytic colonization. We present evidence for X. fastidiosa movement through bordered pits to neighboring vessels and propose that vessel-to-vessel movement is a key colonization strategy whose failure results in vessel plugging and disease.

Plants produce proximal-distal growth axes with two types of growth potential: they can be indeterminate, in which case growth continues indefinitely, or they can be determinate, in which case growth is limited to the production of a single organ or a discrete set of organs. The indeterminate shoot axes of Arabidopsis pinhead/zwille mutants frequently are transformed to a determinate state. PINHEAD (PNH) is expressed in the central domain of the developing plant: the provascular tissue, the shoot apical meristem, and the adaxial (upper) sides of lateral organ primordia. Here, we show that ectopic expression of PNH on the abaxial (lower) sides of lateral organs results in upward curling of leaf blades. This phenotype correlates with a loss of cell number coordination between the two surfaces of the blade, indicating that ectopic PNH can cause changes in cell division rates. More strikingly, moving PNH expression from the central to the peripheral domain of the embryo causes transformation of the determinate cotyledon axis to an indeterminate state. We propose that growth axes are specified as determinate versus indeterminate in a PNH-mediated step. Our results add to a growing body of evidence that radial positional information is important in meristem formation. These results also indicate that genes regulating cell division and axis determinacy are likely to be among PNH targets.

Although many studies have addressed systemic movement of Xylella fastidiosa in the plant (4), we still lack a basic understanding of the mechanism by which X. fastidiosa cells, which initially are inoculated into relatively few xylem vessels, are able to spread throughout the xylem network of the host plant. Furthermore, we lack an understanding of why systemic movement by X. fastidiosa occurs in some host plant species but not in others. In other plant pathogens, including Xanthomonas campestris, which is closely related to X. fastidiosa, the expression of virulence genes such as those encoding gum synthesis, endoglucanase and pectinase activities, is required for systemic movement in planta. The genomic similarities between X. fastidiosa and Xanthomonas strongly suggest that similar virulence genes are required by X. fastidiosa for symptom formation in the plant (2). In some plant pathogens expression of virulence genes is dependent on certain plant factors. In this study we aim to determine the expression pattern of these virulence genes by X. fastidiosa during colonization of grapevines susceptible and resistant to Pierce's disease. Reporter genes will be placed under the control of regulatory regions of the genes to be studied, and these promoter-reporter constructs will be integrated into the X. fastidiosa genome to allow their stable expression in planta. Reporter gene expression will be monitored during different phases of disease progress to elucidate when expression of each gene occurs during disease progression. These data will enable us to construct a fairly accurate description of the course of virulence events leading to Pierce's disease after infection and to detect plant factors that affect virulence of X. fastidiosa.