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L 8 J J 9b L L L L !T 9b 9b x J l ZH ^ , i 0 $ ] 9b ] X 9b r r # 8 d r r 8 Final Report for CDFA Contract Number 06-0222
Project title: Biology of the Xylella fastidiosa-vector interface
Final report: July 2006 to June 2010.
Principal Investigator: Rodrigo P.P. Almeida,137 Mulford Hall, Dept. Environmental Science, Policy and Management, University of California, Berkeley, CA, 94720, rodrigoalmeida@berkeley.edu
Researcher: Nabil Killiny, Dept. Environmental Science, Policy and Management, University of California, Berkeley, CA, 94720, nabilkilliny@berkeley.edu
Summary
The goal of this project was to generate information on how Xylella fastidiosa interacts with its insect vectors. In addition to the general characteristics of X. fastidiosa transmission by vectors, reviewed by Almeida et al. (2005), little was known about the X. fastidiosa-vector interface. Information existed showing that this bacterium colonized the foregut of vectors (Purcell et al. 1979) and that the presence of cells in a narrow canal termed precibarium was associated with infection of plants (Almeida and Purcell 2006). In addition, Newman et al. (2004) observed that a cell-cell signaling mutant, deficient in producing the signal molecule, was not capable of colonizing vectors and was not transmissible to plants. Initially this project focused on determining the nature of the interactions in this system; we found that carbohydrate-binding proteins on the surface of X. fastidiosa are important in cell adhesion to the cuticle of leafhoppers and chitin. In addition to protein-carbohydrate interactions, this research also determined the role of adhesins and a cell signaling system on pathogen-vector interactions. To further test hypotheses generated through this initial characterization we developed a protocol to deliver vector-transmissible cells to leafhoppers through an artificial diet system. In addition to this technological advancement, this work generated new insights on the regulation of X. fastidiosa genes and its interactions with host structural polysaccharides. Lastly, proof-of-concept experiments were conducted to test if knowledge generated in the initial phase of this project would lead to alternative strategies to control Pierces disease spread. In this report we focus on the results of our research, methodological details can be found in peer-reviewed manuscripts listed at the end of this report. Many figures and large portions of text from previously published/accepted material were used here, as those were obtained through this project and best describe the results. For general information on X. fastidiosa transmission biology we refer the reader to Almeida et al. (2005) and Chatterjee et al. (2008). Lastly, we would be glad to provide any clarifications and further information if requested.
Objectives
. Characterize the X. fastidiosa-vector molecular interactions
. Develop an in vitro protocol to deliver transmissible X. fastidiosa cells to vectors
. Identify molecules that block X. fastidiosa transmission
The nature of Xylella fastidiosa-vector interactions
Summary
We used different approaches to determine how X. fastidiosa cells interact with the cuticular surface of the foregut of vectors. We demonstrate that X. fastidiosa binds to different polysaccharides with variable affinities, and that these interactions are mediated by cell surface carbohydrate-binding proteins. In addition, competition assays showed that N-acetylglucosamine inhibited bacterial adhesion to vector foregut extracts and intact wings, demonstrating that attachment to leafhopper surfaces is affected in the presence of specific polysaccharides. In vitro experiments with several X. fastidiosa knockout mutants indicated that hemagglutinin-like proteins are associated with cell adhesion to polysaccharides. These results were confirmed with biological experiments, when
hemagglutinin-like proteins mutants were transmitted to plants by vectors at lower rates when compared to the wild type. Furthermore, although these mutants were defective in adhesion to the cuticle of vectors, their growth rate once attached to leafhoppers was similar to the wild type, suggesting that these proteins are important for X. fastidiosa initial adhesion to leafhoppers. We propose that X. fastidiosa colonization of leafhopper vectors is a complex, stepwise process, similar to the formation of biofilms on surfaces.
The interaction of X. fastidiosa with the foregut cuticle differs from other xylem-limited bacteria such as Leifsonia xyli which can be acquired from plants but are not transmitted by insects (Barbehen and Purcell 1993). Only two studies with X. fastidiosa knockout mutants have addressed aspects of vector transmission (Chatterjee et al. 2008, Newman et al. 2004). However, both studies focused on X. fastidiosas cell-cell signaling system, which regulates cascades of genes and pathways, thus allowing the identification of target genes, but not identifying specific interactions between vector and pathogen. The rpfF gene (Regulation of Pathogenicity Factors F) encodes an enzyme that synthesizes the signaling molecule DSF (diffusible signaling factor), whereas rpfC is part of a hybrid two-component DSF sensor (Chatterjee et al. 2008). An rpfF- mutant is not transmissible by insects because it does not colonize the foregut of vectors (Newman et al. 2004), while rpfC- colonizes insects foregut but is transmitted at lower rates compared to the wild type (Chatterjee et al. 2008). In vitro adhesion assays indicated that rpfF- did not form biofilms, while rpfC- adhered to surfaces more strongly than did the wild type. Targeted gene expression analyses of X. fastidiosa adhesins indicated that hemagglutinin-like proteins (hxf, afimbrial adhesins) and type I pili (fimbrial adhesin) were associated with adhesion of these knockout strains to glass surfaces, but type IV pili were not (Chatterjee et al. 2008). Thus, indirect evidence allowed us to hypothesize that some adhesins are important for X. fastidiosa attachment to and colonization of vectors, and subsequent inoculation into susceptible hosts, while other adhesins have little or no role in this process. In this study we sought to determine the nature of the X. fastidiosa-vector interactions using biochemical, molecular and biological assays.
X. fastidiosa surface proteins bind to polysaccharides in vitro. Because the foregut cuticle of leafhoppers should be rich in polysaccharides, we hypothesized that carbohydrates mediate the attachment of X. fastidiosa to vectors. We incubated X. fastidiosa cells with polysaccharide-coated nitrocellulose membrane (NCM) pieces, determining cell adhesion with an amino acid stain. The lectin, wheat germ agglutinin (WGA) was used as a positive control because of its carbohydrate-binding profile. WGA was detected bound to all polysaccharide-coated NCM. We found X. fastidiosa cells bound to NCM coated with chitosan, chitin, carboxymethylcellulose, cellulose, and dextran sulphate; similar to what we observed with WGA. As expected, bovine serum albumin (not a carbohydrate-binding protein) did not bind to any of these compounds, except for some background binding to chitosan (Fig. A). To test whether X. fastidiosa surface proteins were implicated in attachment to polysaccharides, we washed cells to remove the secreted proteins and loosely bound extracellular polysaccharides (EPS, extracellular polymeric substances or gum). Washed cells attached to polysaccharide-coated NCM similarly to unwashed cells (Fig. B), suggesting that molecules responsible for cell adhesion properties are not secreted in the environment. We also determined that cells treated with proteases attached to polysaccharides less than untreated cells (Fig. B). These observations indicate the presence of carbohydrate-binding proteins on the outer surface of X. fastidiosa cells.
Components of the bacterial surface have affinity to glucose and mannose but not to galactose. To determine the affinity of X. fastidiosa cells to different sugars we used a competition assay based on the concept that polysaccharide-binding proteins on the surface of X. fastidiosa can be saturated by exogenous molecules, reducing overall cell attachment to leafhopper foregut extracts. D(+)-galactose did not interfere with the binding of X. fastidiosa to foregut extracts while D(+)-mannose had a small effect (Fig A). However the monomeric moiety of chitin, N-acetylglucosamine, as well as its dimmer chitobiose and its trimer chitotriose, and its core molecule glucose, blocked cell adhesion to leafhopper foregut extracts (Fig A). The affinity of X. fastidiosa cells to sugars was also tested using synthetic copolymers (9), which eliminate potential sources of error compared to the competition assays, as the later was performed with leafhopper extracts mimicking in vivo conditions that were certainly complex in composition. We also used GFP (green fluorescent protein)-labeled X. fastidiosa (Newman et al. 2003) to limit sample processing. We determined that cells specifically bound to the glucosyl ligand poly (O-(-D-glucopyranosylacrylamide) copolymer. A negligible interaction was obtained with the galactosyl ligand, while binding of X. fastidiosa to the mannosylated copolymer was detected half way through the dilution series used (Fig. B).
In order to compare our in vitro observations to in vivo cell adhesion to leafhoppers we used the hindwings of insect vectors to mimic the cuticular surface of the foregut canal that X. fastidiosa colonizes. The entire exoskeleton of insects is generally assumed to have similar chemical composition, although details are lacking for this specific system. We used N-acetylglucosamine as competitor molecule in assays testing for GFP-labeled X. fastidiosa cell attachment to hindwings. Attachment diminished as N-acetylglucosamine concentration increased in the dilution series (Fig. C). These results indicate that X. fastidiosa binding to polysaccharides in vitro is similar in its characteristics to its binding to the cuticle of leafhoppers. Lastly, in order to test the specificity of bacterial adhesion to leafhopper hindwings, we tested if other GFP-labeled bacteria, including the plant pathogens Pseudomonas syringae, Xanthomonas campestris, and Erwinia herbicola, and Escherichia coli attached to that surface (Fig. D). Interestingly, only X. fastidiosa cells attached to the wings. Thus, X. fastidiosa cells have surface proteins with affinity to polysaccharides on the surface of insects wings and glucosylated molecules, which can be saturated by N-acetylglucosamine and similar molecules.
Role of hemagglutinin-like proteins on cell adhesion. We used several X. fastidiosa knockout mutants to identify proteins associated with cell adhesion to leafhopper foregut extracts and other substrates spotted on NCM strips. Adhesion to leafhopper foregut (G. atropunctata) and wing (H. vitripennis) extracts, in addition to crab shell chitin, was observed for the wild type (Fig. below). We found that only the rpfF-, rpfF-/rpfC-, hxfA- and hxfB- showed less attachment than the wild type. The cell-cell signaling mutants regulate a cascade of pathways and genes, including hxfA and hxfB. To confirm our biochemical observations we quantified the expression of five genes of putative importance in cell adhesion (afimbrial and fimbrial adhesins and gum) for the same mutants used in these assays, compared to the wild type. Our results showed that hxfA and hxfB were downregulated in rpfF- and rpfF-/rpfC- mutants, which showed reduced attachment in the biochemical study. Thus, of the genes tested in our study, the afimbrial adhesins hxfA and hxfB were implicated on X. fastidiosa cell adhesion to the substrates tested.
Hemagglutinin-like proteins are important for X. fastidiosa early attachment to vectors and transmission to plants. We conducted two experiments to determine the role of hxfA and hxfB in X. fastidiosa transmission by sharpshooters to plants (Fig A). In the first experiment, we confined non-infected G. atropunctata on plants mechanically inoculated with the wild type, hxfA- and hxfB- cells, after which groups of 2 individuals were moved to healthy plants for 4 days as an inoculation access period. Transmission occurred in all treatments, with hxfA- and hxfB- being transmitted less than the wild type (70, 80 and 100%, respectively), albeit not with any statistical difference (X2 test, P = 0.1864). In a second experiment we used individuals instead of pairs to more precisely estimate single insect transmission efficiency. With this more discriminating approach we found that hxfA- and hxfB- mutants were transmitted at lower rates than the wild type (36, 46 and 88%, respectively X2 test, df = 1, P <0.001). Because X. fastidiosa transmission rates are correlated with bacterial population in plants, we quantified the infection level in plants used in these tests. Plants infected with hxfA- and hxfB- mutants used for the transmission tests had populations ~10-fold higher than the wild type (data not shown, results similar to Guilhabert and Kirkpatrick 2005), suggesting that hxfA- and hxfB- mutants were transmitted less than the wild type because of their impaired interactions with insects rather than because of lower populations in source plants.
We hypothesized that the reduced transmission rate of hxfA- and hxfB- mutants was due to limited colonization of vectors early in biofilm formation. In order to test this hypothesis we conducted another experiment and quantified the number of X. fastidiosa cells in the head of vectors over time after a 12-hour pathogen acquisition access period. Overall, 80% of insects that fed on grapevines infected with the wild type were positive for X. fastidiosa, whereas only 38% and 42% of those fed on hxfA- and hxfB- mutants were infected, respectively (X2 test, df = 2, P <0.001). We quantified the number of cells of these strains within vectors (positive samples only). There were significant effects of strain (F2,54=23.229, P<0.0001), time (F1,54=803.341, P<0.0001), and an strain by time interaction (F2,54=5.362, P=0.0075). Populations of the two mutants soon after leafhopper access to infected plants were similar to each other but statistically different from the wild-type (Fig. B). Twelve hours after acquisition we found insects fed on the wild type averaged 415 detectable cells, whereas average of 96 and 120 cells were detected in leafhoppers fed on hxfA- and hxfB- plants respectively. However, after 96 hours the bacterial populations of all three strains were similar to each other (Fig B). Thus, the knockouts were impaired in early attachment to insects, but after initial adhesion, their patterns of foregut colonization (i.e. population growth) were similar to the wild type (slope of regressions), suggesting that hxfA and hxfB may have redundant roles in relation to vector transmission.
Discussion
Molecular interactions between insects and pathogens are often neglected in the study of vector-borne diseases. The characterization of vector-pathogen interactions has been especially poor for bacterial plant pathogens, although a large number of plant viruses have been studied in detail in relation to their transmission biology. X. fastidiosa is not an exception; despite the fact that several genome sequences of this organism are available and much of its biology and ecology are well understood, no studies have determined the molecular basis of cell attachment to leafhopper vectors. We conducted a series of experiments that allowed us to conclude that specific X. fastidiosa surface proteins bind to chitin-like polysaccharides on the foregut cuticular lining of leafhoppers. In addition, we demonstrated that these proteins are required for initial cell adhesion to vectors, but that once adhered, mutants with reduced attachment abilities colonized insects similarly to the wild type. In fact, several adhesins may be necessary for the first steps in leafhopper colonization; those may also have different or complementary functions in the first stages. These results suggest that X. fastidiosa colonization of surfaces on vectors is a complex multi-step process similar to that of other biofilm-forming bacteria (Davey and OToole 2000).
The cuticular lining of the foregut of insects is part of the exoskeleton. However, little is known about insect foregut ultrastructure, particularly for sap-sucking insects. In addition, the physical and chemical properties of the cuticle of insects vary within individuals and may change during development. In another study, the foregut cuticle of the cockroach Periplaneta americana was found to be different from that of bodys cuticle, with Mallorys stain indicating the presence of chitin in the foreguts cuticle surface (Murthy 1975). Accordingly, we assumed the presence of carbohydrate moieties in leafhopper foregut surfaces colonized by X. fastidiosa. We demonstrated that X. fastidiosa has affinity for polysaccharides, acting similarly to lectins, and that proteins on the cell surface are responsible for such activity. Specificity tests indicated that X. fastidiosa cells bind to chitin and similar carbohydrates, but not to galactose. These biochemical observations, coupled with our finding that X. fastidiosa attaches to leafhopper wings, but other plant pathogenic bacteria do not, suggest that adhesion to these surfaces is a specific process. Although we did not characterize the surface of the insects foregut, we used vector hindwings as proxies for the foregut to mimic the cellintact insect surface interactions. In our study we found that the leafhopper wings can be stained with Alcian blue 8GX, which is specific for HYPERLINK "http://www.google.com/search?hl=en&client=firefox-a&channel=s&rls=org.mozilla:en-US:official&hs=iCE&sa=X&oi=spell&resnum=0&ct=result&cd=1&q=glycosaminoglycan&spell=1" glycosaminoglycans that are similar to the polysaccharides we found that X. fastidiosa cells can bind to (data not shown). This finding suggests the presence of a layer of polysaccharides on the surface of leafhoppers wings cuticle. To determine which cell surface proteins were associated with adhesion to vectors, we tested a series of site-directed knockouts of genes of putative importance in vector colonization. X. fastidiosa has a limited number of annotated candidate adhesins, including type I and IV pili (short and long fimbriae, respectively) and hemagglutinin-like proteins (HxfA and HxfB). We found that hxfA- and hxfB- mutant were deficient in cell adhesion in vitro, as were two cell-cell signaling mutants (rpfF- and rpfC-/rpfF-). rpfF- is not transmissible to plants and does not colonize the foregut of vectors (Newman et al. 2004); rpfC-/rpfF- is also non-transmissible (unpublished data).
To determine if the levels of hxfA and hxfB expression were reduced in these adhesion deficient mutants, we compared the transcription level of these afimbrial adhesins, two pili and one gum gene for all mutants tested biochemically. hxfA and hxfB expression levels were consistently lower than the wild type only in the knockouts that did not adhere to leafhopper foregut extracts. Thus, although we only focused our study on a limited number of candidate proteins, our results demonstrate that X. fastidiosa adhesion to carbohydrates is mediated by HxfA and HxfB. Other afimbrial adhesin homologs have been identified in X. fastidiosa genomes (Simpson et al. 2000) and may also be important in cell-carbohydrate interactions. No studies have been conducted on those proteins. Furthermore, X. fastidiosa genomes still have a large number of hypothetical open reading frames, and screens for carbohydrate-binding proteins may identify new adhesins of importance in this system.
Except for the observation that X. fastidiosas cell-cell signaling system controls genes associated with vector transmission (Chatterjee et al. 2008, Newman et al. 2004), our previous knowledge of how interactions in this system were based on indirect data and assumptions. Chatterjee et al. (2008) summarized a hypothesis suggesting that X. fastidiosa has separate plant colonization and insect acquisition phases, which are controlled by accumulation of the signaling molecule DSF. Indeed, hxfA and hxfB are upregulated in the presence of DSF, supporting Chatterjee et al.s hypothesis and corroborating work done with the cell-cell signaling deficient mutant rpfF-. However, our results show that both HxfA and HxfB are required for optimal initial adhesion to vectors, but their importance for biofilm maturation seems limited. In the future, a double knockout mutant for these genes will need to be tested to determine if these proteins have redundant roles in mediating cell adhesion to carbohydrates (hxfA and hxfB share high sequence similarity, with exception of two deletions in hxfB in relation to hxfA).
We propose that X. fastidiosa colonization of vectors is similar to the formation of biofilms on surfaces. Scanning electron microscopy observations we have made previously (Almeida and Purcell 2006) support this hypothesis. We hypothesize that cells initially adhere laterally to the foregut cuticle via carbohydrate-binding proteins, such as HxfA and HxfB (Fig. A,B). As these proteins are assumed to occur throughout cells, adhering laterally increases the cell surface area in contact with the substrate and streamline the bacteria to the flow of xylem sap ingested by the insect vector. After initial adhesion, cells may produce large quantities of EPS that can result in the concentration of resources and DSF in microcolonies. As the colony size increases, cells at the center of the biofilm become polarly attached to the foregut surface (Fig C), potentially through polar short type I pili, increasing surface area for nutrient absorption. Cell-cell attachment may be mediated by hemagglutinin-like or other cell surface proteins. Lastly, a typical mature X. fastidiosa biofilm within vectors is formed, with all cells polarly attached (Fig D). At this stage, newly divided cells are not anchored on the cuticle of insects and may be occasionally detached from vectors and inoculated into plants. This hypothesis may be useful to guide future studies on this system by providing testable questions, as up until know no data on these interactions, with the exception of microscopy observations, were available.
Development of an artificial diet system to deliver X. fastidiosa to vectors
Summary
We hypothesized that factors of host origin such as plant structural polysaccharides are important in regulating X. fastidiosa gene expression and mediating vector transmission of this pathogen. The addition of pectin and glucan to a simple defined medium resulted in dramatic changes in X. fastidiosas phenotype and gene expression profile. Cells grown in the presence of pectin became more adhesive than in other media tested. In addition, the presence of pectin and glucan in media resulted in significant changes in the expression of several genes previously identified as important for X. fastidiosas pathogenicity in plants. Furthermore, vector transmission of X. fastidiosa was induced in the presence of both polysaccharides. Our data show that host structural polysaccharides mediate gene regulation in X. fastidiosa, which results in phenotypic changes required for vector transmission.
One of X. fastidiosas unique characteristics is that it interacts with polysaccharide-coated surfaces in both host plant and vector (Chatterjee et al. 2008) ADDIN EN.CITE Chatterjee200844417Chatterjee, S.Almeida, R. P. P.Lindow, S.Chatterjee, S
Univ Calif Berkeley, Dept Plant & Microbial Biol, Berkeley, CA 94720 USA
Univ Calif Berkeley, Dept Plant & Microbial Biol, Berkeley, CA 94720 USA
Univ Calif Berkeley, Dept Environm Sci Policy & Management, Berkeley, CA 94720 USAAnnual Review of PhytopathologyAnnual Review of Phytopathology243-27146pierce's disease of grapesharpshootersxylem vesselscell-cell signalingvector transmissionxanthomonasoryzae pv. oryzaecell-cell communicationhd-gyp domainpierces disease bacteriumcyclic di-gmpcitrus variegated chlorosisglassy-winged sharpshootervitis-vinifera grapevinesheat-shock responsexanthomonas-campestris20080066-4286ISI:000259199300011English. In plants, as a xylem-limited organism, X. fastidiosa is confined in vessels composed of polysaccharides, such as cellulose and pectin. Within its leafhopper vectors this bacterium colonizes the foregut surface (Almeida and Purcell 2006, which is part of the insects exoskeleton and is composed primarily of chitin and chitin-like polysaccharides. Xylella fastidiosa transmission is dependent on cell-cell signaling (i.e. quorum sensing), as a signal-production mutant is not transmissible by vectors and does not colonize vectors (Newman et al. 2004). In addition, this bacterium requires afimbrial adhesins to colonize leafhoppers (Killiny and Almeida 2009a) ADDIN EN.CITE Killiny200911117Killiny, N.Almeida, R. P. P.Almeida, RPP
Univ Calif Berkeley, Dept Environm Sci Policy & Management, Berkeley, CA 94720 USA
Univ Calif Berkeley, Dept Environm Sci Policy & Management, Berkeley, CA 94720 USAApplied and Environmental MicrobiologyApplied and Environmental Microbiology521-528752mannose-sensitive hemagglutininpierces disease bacteriumvibrio-choleraetwitching motilityvitis-viniferachitincolonizationvirulencegrapevinesattachment2009Jan 150099-2240ISI:000262197700029English, which are under control of a cell-cell signaling regulatory system. Therefore, it is expected that cells grown in vitro to high densities should be transmitted by vectors if provided to insects through an artificial diet system. However, X. fastidiosa grown in commonly used media are not transmissible by vectors, despite the fact they reach high cell densities in culture and are acquired by vectors in large numbers through artificial diet systems (Almeida et al. 2005). Those observations suggest that biotic interactions impacting gene regulation in plant hosts, but not in vitro, or an unidentified plant factor, are required for X. fastidiosa to transition into a vector transmissible phenotype.
Pectin degradation is required for X. fastidiosa pathogenicity and movement within plants because it is the major component of pit membranes between xylem vessels (Roper et al. 2007) ADDIN EN.CITE Roper200715151517Roper, M. C.Greve, L. C.Warren, J. G.Labavitch, J. M.Kirkpatrick, B. C.Kirkpatrick, BC
Univ Calif Davis, Dept Plant Pathol, Davis, CA 95616 USA
Univ Calif Davis, Dept Plant Pathol, Davis, CA 95616 USA
Univ Calif Davis, Dept Plant Sci, Davis, CA 95616 USAMolecular Plant-Microbe InteractionsMolecular Plant-Microbe Interactions411-419204pectinasepierces disease bacteriumcampestris pv campestrisralstonia-solanacearumgenome sequencewater-stresscell wallsplantsvirulencestrainsgene2007Apr0894-0282ISI:000244970000008English. Pit membrane degradation may also expose cell wall polysaccharides that could then be digested by other extracellular enzymes, such as glucanases (Chatterjee et al. 2008). Thus, it is possible that such compounds of host origin, specifically the polysaccharides pectin and glucan, induce a state change in X. fastidiosa gene expression that leads to vector colonization and also transmission. We tested the hypothesis that X. fastidiosa responds to environmental cues in a context-dependent fashion so that the balance between host plant colonization and dispersal can be optimized through phenotypic transitions. We show for the first time that host plant structural carbohydrates, especially pectin, induce regulons that are required for plant pathogen vector transmission.
Carbohydrate-driven phenotypic changes in X. fastidiosa.
The growth of X. fastidiosa was compared in four different media, a rich complex medium (PWG), a simple defined medium (XFM), ADDIN EN.CITE Almeida200419191917Almeida, R. P. P.Mann, R.Purcell, A. H.Almeida, RPP
Univ Calif Berkeley, Dept Environm Sci Policy & Management, Berkeley, CA 94720 USA
Univ Calif Berkeley, Dept Environm Sci Policy & Management, Berkeley, CA 94720 USA
Univ Hawaii Manoa, Dept Plant & Environm Protect Sci, Honolulu, HI 96822 USACurrent MicrobiologyCurrent Microbiology368-372485plum leaf scaldpierces-diseasechemical-compositionaxenic culturegrapevinesbacteriumgrowthpathogenicitytemperaturestrain2004May0343-8651ISI:000220678600009Englishand XFM supplemented with either glucan or pectin. Cell growth was similar in XFM and the supplemented media but lower in PWG when compared to all other media (P<0.0001, Fig A). However, the ratio of planktonic/attached cells found in the various media was variable. In XFM we found more planktonic than attached cells, while that proportion was equal for XFM-glucan; in XFM-pectin, more cells were attached to the surface of vials than in suspension (Fig B). These results were visually confirmed by staining cells forming biofilms in the air/broth interface of flasks maintained in a shaker (Fig C). We used an immunological assay to relatively quantify the amount of exopolysaccharides (EPS, gum) produced by cells grown in the different media and found that all XFM based media had approximately twice as much EPS present on cells compared to PWG (Fig D). Bacterial lawns grown on solid XFM and XFM-polysaccharides had a glossy appearance when compared to PWG (Fig E). Afimbrial adhesins (Hxfs) have been shown to be important for X. fastidiosa adhesion to insect vectors (Killiny and Almeida 2009a) and pathogenicity to plants (Guilhabert and Kirkpatrick 2005). We used a dot-blot assay based on polyclonal antibodies produced against a domain of X. fastidiosas Hxfs to show that cells in XFM-pectin produced more Hxfs than in other media (Fig F), indicating that expression of these adhesins are regulated by polysaccharides and associated with the bacterial adhesion phenotypes observed. Altogether our results show several phenotypic changes in X. fastidiosa based on media composition; pectin and, to a lesser extent glucan, induced phenotypic changes in X. fastidiosa.
Host polysaccharides induce changes in gene expression profiles. To better explore the phenotypic changes observed, we compared transcription levels of 2,036 X. fastidiosa genes through gene expression microarrays. Gene expression profiles were different among cells grown in the 4 media tested, with XFM-pectin inducing a larger number of changes to gene transcription levels. A large number of genes (133) were differentially regulated only in the presence of pectin. Those up-regulated included type I fimbriae (fimA), afimbrial adhesins (hxfs), EPS (gumJ) and phage-related genes in addition to a considerable number of hypothetical proteins. On the other hand, down-regulated genes were more diverse in function.
We used real-time quantitative PCR to estimate the level of gene expression of several genes previously shown to affect X. fastidiosas pathogenicity and adhesion to surfaces. All genes tested had higher transcription levels on XFM, XFM-glucan and XFM-pectin when compared to the rich medium PWG (Fig A). To analyze the role of polysaccharides on gene regulation, gene transcription rate on XFM-glucan and XFM-pectin in relation to XFM alone was compared (Fig B). The cell-cell signaling molecule synthase gene (rpfF) was up-regulated by pectin and glucan. This may partly explain results observed for some of the genes tested as it would affect cell-cell signaling-dependent genes; exceptions are the afimbrial adhesins hxfA and hxfB that were up-regulated over 3 orders of magnitude, and the induction of engxcA (an endoglucanase) in the presence of its substrate (glucan). Accordingly, one gene associated with pathogen movement within plants (pilY1, a component of type IV pili) was down-regulated. These results suggest that polysaccharides differentially mediate gene expression in X. fastidiosa by i) affecting rpfF expression levels and, thus, cell-signaling dependent genes, ii) substrate dependent induction of enzymes, and iii) possibly directly inducing genes associated with pathogenicity (i.e. afimbrial adhesins). These results are consistent with our phenotypic observations.
Vector transmission of cells grown in pectin-supplemented medium. Artificial diet systems are useful to study how insect-borne pathogens interact with their respective vectors. Through these systems, pathogen cells (or virus particles) are delivered to vectors under controlled conditions that allow for experimental manipulation in the absence of host plants and host plant-vector interactions. We tested whether cells grown in each of our four different media were transmissible by leafhopper vectors when an artificial diet system was used. Cells in PWG were not transmissible. However, cells in the basal medium XFM, XFM-glucan, and XFM-pectin were transmissible by vectors, albeit with different efficiencies. It is notable that genes with high transcription levels (>1,000-fold) in the presence of pectin include hxfA and hxfB, which were directly implicated in the initial colonization of vectors mouthparts (Killiny and Almeida 2009a).
Pectin is not directly responsible for gene regulation. Because pectin is a large molecule, we tested if it was directly involved in the phenotype observed in our bioassays by using a polygalacturonase (pglA) mutant, which is the only X. fastidiosa gene expected to degrade pectin. We found that the phenotypic changes observed required pectin degradation, as the pglA mutant had the same phenotype on XFM-pectin as on XFM. The pglA bacterial lawns grown on solid XFM-polysaccharides had a glossy appearance when compared to PWG; and an immunological assay showed that the amount of EPS produced by the pglA mutant was similar in both the XFM-pectin medium and XFM. These data show that pectin degradation into its subunits is required for the phenotypic changes observed.
Pectin is primarily composed of galacturonic acid with rhamnose side chains; the ratio of these sugars is host plant species dependent. We compared the phenotype and gene expression level of wild type cells and a pglA mutant grown in XFM, and XFM-pectin, -Na-galacturonate (in place of galacturonic acid) and rhamnose. Biofilm formation on glass vials was more pronounced for wild type cells in XFM-pectin and XFM-Na-galacturonate. A similar biofilm ring was observed in XFM-Na-galacturonate but not XFM-pectin for the pglA mutant, suggesting that pectin is indirectly mediating gene expression of adhesins (Fig A). These results were confirmed by comparing the ratio of planktonic cells to those adhered to glass under the same conditions (Fig B). As EPS production and glossy colonies were correlated to growth conditions, a comparison was made between bacterial lawns formed by cells grown in these different media. Again, glossy colonies were only observed for the pglA mutant grown in XFM supplemented with Na-galacturonate, indicating that pectin degradation is required for the phenotypic changes observed (Fig C). We also tested transcription levels of one gene up-regulated in the presence of pectin (hxfB) and another down regulated (pilY1). Expression patterns support the phenotype-based findings that Na-galacturonate, not pectin, is responsible for gene induction and subsequent phenotypic changes in X. fastidiosa (Fig D). Lastly, the presence of pectin or Na-galacturonate in XFM induced the formation of small wild type colonies on solid medium rather than a diffuse lawn. However, this was only observed for the pglA mutant when Na-galacturonate was added to XFM. This phenotype may be a consequence of increased cell-cell adhesion in the presence of pectin, as it up-regulates the afimbrial adhesins Hxfs.
Discussion
The transmission of X. fastidiosa was initially thought to be the consequence of simple insect-pathogen interactions due to the fact that a large group of insects (sharpshooter leafhoppers, and spittlebugs) are vectors of this pathogen, apparently without any specificity (Almeida et al. 2005). However, the finding that transmission is regulated by cell-cell signaling (Newman et al. 2004) indicated that X. fastidiosa colonization of vectors was not a trivial mechanical process. Our results show that two host structural polysaccharides, pectin and glucan, induced regulons that affect the phenotype of this pathogen. The role of pectin in up-regulating genes associated with cell adhesion to surfaces was of particular significance, as observed through in vitro phenotypic results, and the fact that cells grown in XFM-pectin were transmissible by vectors more than in any other treatment tested here. Because pectin is a complex and large polysaccharide, we hypothesized that one of its building blocks was responsible for gene induction. We found that Na-galacturonate, not pectin itself or another one of its subunits, rhamnose, was in fact responsible for the molecular, biochemical, and biological results obtained.
Chatterjee et al. (2008) proposed that X. fastidiosa has a dual lifestyle controlled by cell-cell signaling, particularly during plant colonization and initial attachment to insects. In low cell densities X. fastidiosa moves within the xylem network of plants by up-regulating genes required for degradation of pit membranes and movement, such as type IV pilus. At higher densities cells have adhesins induced, some of which have been associated with reduced movement within plants and insect colonization (Killiny and Almeida 2009a, Guilhabert and Kirkpatrick 2005, Meng et al. 2005) ADDIN EN.CITE ADDIN EN.CITE.DATA . Our quantitative PCR results showed that the signal synthase gene (rpfF) is up-regulated in the presence of pectin. Although cell density reached similar levels in all XFM media tested, induction of genes such as adhesins was only observed in XFM supplemented with polysaccharides. It is possible that alternative regulatory pathways independent of, or functioning in tandem with, the cell-cell signaling cascade exist in X. fastidiosa. Future work with knockout mutants and different media conditions are needed to explore this possibility. It is also possible that carbohydrate utilization pathways are linked to virulence in X. fastidiosa, as has been observed in Group A Streptococcus (Shelburne et al. 2008). Although mechanistically unclear, our results support the general hypothesis of Chatterjee et al. (2008), but we provide evidence that environmental cues affect gene regulation of X. fastidiosa, apparently inducing an overdrive of the expected high cell density-derived phenotype.
Induction of the dispersal state in X. fastidiosa is controlled by cell-cell signaling (Newman et al. 2004), but it is also regulated by the presence of host structural carbohydrates. In this context, cells prepare for dispersal once they reach high densities within a vessel, which only occurs after other vessels in the xylem network have already been colonized (Newman et al. 2003). Because X. fastidiosa colonization of plants is heterogeneous, some vessels may harbor cells in a non-transmissible state while others that are fully colonized have transitioned into a vector-transmissible phenotype. This dichotomy might be a mechanism used to optimize dispersal opportunities and increase fitness within plants, as both occur in the presence of host structural carbohydrates. At low cell densities the probability of being acquired by vectors is likely lower than at high cell densities and within-plant movement would increase the probability of eventually being acquired by vectors; at high cell densities colonies confined to vessels may eventually die and not result in new vessel infections (Chatterjee et al. 2008). Therefore, X. fastidiosa cells capable of colonizing vectors might originate primarily from microcolonies permanently restricted to individual xylem vessels and no longer involved in plant colonization.
It is possible that phenotypic transitions associated with dispersal or increased fitness in new environments, regulated in a host-factor and cell density-dependent fashion, are common for bacterial pathogens that explore multiple environments. Investigation of such potential state switches may yield valuable information on the biology of pathogens with a complex life history. Similar processes, but in a different context, may also be relevant to other plant pathogens such as Xanthomonas spp., which have a distinct biology such as vector-independent dispersal, but have a regulatory system dependent on cell-cell signaling homologous to X. fastidiosa.
Blocking of Xylella fastidiosa transmission
Summary
The work described above allowed us to test specific hypotheses on how to block X. fastidiosa transmission by vectors. The initial characterization showed that important interactions were of a protein-carbohydrate nature, suggesting that carbohydrate binding molecules, or carbohydrates themselves, should be able to block transmission in competition assays. The artificial diet system permitted us to deliver cells with specific molecules to leafhoppers to test if any of those would reduce the frequency with which X. fastidiosa was transmitted to plants. We tested if molecules that would bind to the cuticle of vectors or surface of X. fastidiosa could result in a decrease in transmission efficiency of this pathogen to plants. As these experiments were primarily focused on demonstrating the feasibility of this approach, generic rather than system-specific molecules were used. We showed that both approaches significantly reduced X. fastidiosa vector transmission efficiency. Ongoing work is now aimed at identifying and testing molecules that are target specific, i.e. would only block X. fastidiosa adhesion to vectors.
The broader goal of this project is to develop a strategy to block vector transmission of Xylella fastidiosa by disrupting insect-pathogen interactions. Our research has used approaches that build on each other to i) generate information on the interactions in this system and ii) test ideas on how to block transmission. First, we need to understand how X. fastidiosa colonizes sharpshooters, more specifically, how it interacts with the chitinous surface of the foregut of vectors. Although there is still much to be studied about this system, we now have a hypothesis for X. fastidiosa colonization of vectors, from a cellular and molecular perspective, that was proposed based on molecular, biochemical and biological data (Killiny and Almeida 2009a,b). In this hypothesis, cell initial adhesion to the cuticle of sharpshooters is mediated by afimbrial adhesins. These proteins have affinity for N-acetylglucosamine, which can be used to block this interaction in vitro. Although we are continuing research on all the steps of colonization present in our hypothesis, we believe that this initial step is a good target for disruption because mature biofilms still have not formed within insects.
In the laboratory we were able to block X. fastidiosa adhesion to leafhopper foregut extracts by saturating solutions with N-acetylglucosamine, its dimer and trimer. Essentially, cells bound to these carbohydrates in solution rather than to the foregut extracts. However, to test this approach with transmission experiments we needed to develop an artificial diet system to deliver cells in a transmissible state to leafhoppers, so that normal adhesion to vectors would occur and similar competition tests performed. We modified a medium for cell growth developed earlier and found that X. fastidiosa grown in this new environment was hyperadhesive and transmissible by vectors (Killiny and Almeida 2009b). In fact, transmission rates were close to 100% for one medium (XFM-pectin), which is interesting when compared to the commonly used medium PWG, for which no transmission was observed. A byproduct of this effort was that X. fastidiosas gene expression profile was dramatically different from cells grown in traditionally used media, and that is now being explored by the research community in a varied of manners.
We now have i) some information on how X. fastidiosa interacts with insect vectors, and ii) a protocol to deliver vectors transmissible cells and, importantly, other compounds of interest that may block transmission. We used this approach to test if two groups of generic molecules reduced X. fastidiosa transmission. In these experiments, cells and delivered to vectors through a diet system (figure on right) together with candidate transmission-blocking agents. After that insects are transferred to healthy grape plants for a pathogen inoculation access period. First, we tested if lectins, which bind to carbohydrates, reduced transmission by out-competing X. fastidiosa for adhesion sites in the foregut of vectors. We showed that two lectins that have affinity for N-acetylglucosamine and similar carbohydrates reduced transmission rates by half compared to the control. We also tested if transmission could be blocked by saturating adhesins on the surface of X. fastidiosa with molecules they have affinity for, like N-acetylglucosamine, which would lower the frequency of cell adhesion to vectors as the proteins responsible for that activity would be already bound to other molecules. Below are the experimental results of these tests, together with hypothetical models describing the mechanisms proposed to explain our data.
We are also using this artificial diet system to test the role of different X. fastidiosa mutants on vector colonization and transmission to plants. Although our work has provided enough information to initiate trials that have been successful in reducing pathogen transmission, we believe that a better understanding of X. fastidiosa colonization of vectors will permit the identification of new targets for disruption of these interactions. Below is a list of different mutants we have tested, are testing, or hope to test in the near future, and their proposed role in vector transmission.
GeneGene productHypothetical role in transmissionTest statushxfA1Afimbrial adhesinsInitial attachmentCompletedhxfB1Initial attachmentCompletedfimA2Type I pilusPolar attachmentCompletedpilB2Type IV pilusNo roleCompletedfimA/pilB2Type I/IV piliCompletedpglA1PectinaseGene regulation cascadeCompletedgumD3Exopolysaccharides Biofilm structure and protectionCompletedgumH3CompletedrpfF4
Cell-cell signaling system
Gene regulationCompletedrpfC4CompletedrpfF/rpfC4CompletedrpfB4OngoingcgsA4Ongoingeal4in 2010eal/rpfF4in 2010tolC5Secretion systemsSecreted proteins may be associated with transmissionin 2010tps1OngoingPD02186
AT-1 serine proteases
To be determinedin 2010PD3136in 2010PD09506in 2010PD0218/PD09506in 2010PD0218/PD03136in 2010PD313/PD09506in 2010PD0218/PD0313/PD09506in 2010PD05286
Membrane proteinsOngoingPD13796OngoingPD0528/PD13796Ongoingin vitro Evolution strains7---------------------------in 20101: Kirkpatrick, UC-Davis 2: Hoch and Burr, Cornell University 3: Cooksey, UC-Riverside 4: Lindow, UC-Berkeley 5: Gabriel, University of Florida 6: Igo, UC-Davis 7: Our lab
Results for selected genes are shown below to illustrate experimental data.
In summary, we now have a protocol to test X. fastidiosa transmission blocking agents. Furthermore, data obtained allowed the development of a hypothesis for X. fastidiosa-vector interactions and using this paradigm we successfully tested candidate molecules that disrupt transmission. Our results show that this is a promising approach to limit the spread of Pierces disease that is system-specific.
Publications (actual and pending) resulting from the work
Killiny, N. and Almeida, R.P.P. 2009. Xylella fastidiosa afimbrial adhesins mediate cell transmission to plants by leafhopper vectors. Applied and Environmental Microbiology 75: 521-528.
Killiny, N. and Almeida, R.P.P. 2009. Host structural carbohydrate induces vector transmission of a bacterial plant pathogen. Proc. Natl. Acad. Sci. USA 106: 22416-22420.
Chatterjee, S.*, Killiny, N.*, Almeida, R.P.P. and Lindow, S.E. 2010. Role of cyclic di-GMP in Xylella fastidiosa biofilm formation, plant virulence and insect transmission. Molecular Plant-Microbe Interactions accepted. *Authors contributed equally to this work.
Killiny, N., Rashed, A., Almeida, R.P.P. Blocking transmission of a vector-borne plant pathogen. in preparation
Intellectual property issues you are aware of
We are not aware of any intellectual property issues.
How this work and its results will help contribute to solving the PD problem in California
This work addressed half of X. fastidiosas life cycle, which occurs within its sharpshooter vectors, and is responsible for pathogen spread between plants. Our goals were to generate information on how X. fastidiosa colonizes its vectors to develop approaches to block its transmission between plants. The experimental results obtained show that it is possible to significantly reduce X. fastidiosa transmission with generic molecules that compete with cells for binding sites in vectors, or that bind directly to adhesins on the cell surface. In addition, the protocol used to induce a vector transmissible state in X. fastidiosa resulted dramatic changes in gene expression of this pathogen, which apparently better mimic its profile while colonizing plants. This finding is now being widely used by the X. fastidiosa community for a variety of tests, from gene regulation studies to tests of enzymatic activity. Thus, generating knowledge about vector-X. fastidiosa interactions is also leading to advances in our understanding of X. fastidiosa-plant interactions. Altogether, in addition to generating new information on X. fastidiosas biology, this project demonstrated that disease spread could be blocked using molecules that disrupt vector-pathogen interactions. This alternative approach to manage Pierces disease is very system-specific and could be developed as a new control strategy that is environmentally friendly and sustainable.
References
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Almeida, R.P.P. and A.H. Purcell. 2006. Patterns of Xylella fastidiosa colonization on the precibarium of leafhopper vectors relative to transmission to plants. HYPERLINK "http://www.entsoc.org/Pubs/Periodicals/Ann/Index.htm" Annals of the Entomological Society of America 99: 884-890.
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Chatterjee, S., C. Wistrom, and S.E. Lindow. 2008. A cell-cell signaling sensor is required for virulence and insect transmission of Xylella fastidiosa. Proceedings of the National Academy of Sciences of the United States of America 105: 2670-2675.
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Guilhabert, M.R., and B.C. Kirkpatrick. 2005. Identification of Xylella fastidiosa antivirulence genes: hemagglutinin adhesins contribute a biofilm maturation to X. fastidiosa and colonization and attenuate virulence. Molecular Plant-Microbe Interactions 18: 856-868.
Killiny, N. and Almeida, R.P.P. 2009a. Xylella fastidiosa afimbrial adhesins mediate cell transmission to plants by leafhopper vectors. Applied and Environmental Microbiology 75: 521-528.
Killiny, N. and Almeida, R.P.P. 2009b. Host structural carbohydrate induces vector transmission of a bacterial plant pathogen. Proc. Natl. Acad. Sci. USA 106: 22416-22420.
Meng Y, et al. 2005. Upstream migration of Xylella fastidiosa via pilus-driven twitching motility Journal of Bacteriology 187:5560-5567.
Murthy, R.C. 1975. Structure of the foregut cuticle of Periplaneta americana. Entomologia Experimentalis et Applicata 32: 312-317
Newman, K.L., R.P.P. Almeida, A.H. Purcell, and S.E. Lindow. 2003. Use of a green fluorescent strain for analysis of Xylella fastidiosa colonization of Vitis vinifera. Applied and Environmental Microbiology 69: 7319-7327.
Newman, K.L., R.P.P. Almeida, A.H. Purcell, and S.E. Lindow. 2004. Cell-cell signaling controls Xylella fastidiosa interactions with both insects and plants. HYPERLINK "http://www.pnas.org/" Proceedings of the National Academy of Sciences of the United States of America 101: 1737-1742.
Purcell, A. H., A.H. Finlay, and D.L. Mclean. 1979. Pierce's disease bacterium: Mechanism of transmission by leafhopper vectors. Science 206: 839-841.
Roper MC, Greve LC, Labavitch JA, Kirkpatrick BC. 2007, Detection and visualization of an exopolysaccharide produced by Xylella fastidiosa in vitro and in planta. Applied and Environmental Microbiology 73:7252-7258.
Simpson, A.J.G., et al. 2000. The genome sequence of the plant pathogen Xylella fastidiosa. Nature 406: 151-157.
Shelburne SA, et al. 2008. A direct link between carbohydrate utilization and virulence in the major human pathogen group A Streptococcus. Proceedings of the National Academy of Sciences of the United States of America 105:1698-1703.
Transmission rate of fimbrial and afimbrial adhesins, and DSF production mutants; two inoculation access periods were used (n=20/IAP).
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