A phage display-selected peptide inhibitor of Agrobacterium vitis polygalacturonase.

Agrobacterium vitis, the causal agent of crown gall of grapevine, is a threat to viticulture worldwide. A major virulence factor of this pathogen is polygalacturonase, an enzyme that degrades pectin components of the xylem cell wall. A single gene encodes for the polygalacturonase gene. Disruption of the polygalacturonase gene results in a mutant that is less pathogenic and produces significantly fewer root lesions on grapevines. Thus, the identification of peptides or proteins that could inhibit the activity of polygalacturonase could be part of a strategy for the protection of plants against this pathogen. A phage-displayed combinatorial peptide library was used to isolate peptides with a high binding affinity to A. vitis polygalacturonase. These peptides showed sequence similarity to regions of Oryza sativa (EMS66324, Japonica) and Triticum urartu (NP_001054402, wild wheat) polygalacturonase-inhibiting proteins (PGIPs). Furthermore, these panning experiments identified a peptide, SVTIHHLGGGS, which was able to reduce A. vitis polygalacturonase activity by 35% in vitro. Truncation studies showed that the IHHL motif alone is sufficient to inhibit A. vitis polygalacturonase activity.

Insights into the Activity and Substrate Binding of Xylella fastidiosa Polygalacturonase by Modification of a Unique QMK Amino Acid Motif Using Protein Chimeras.

Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or, alternatively, it has a different mechanism of substrate binding than other polygalacturonases characterized to date.

The Xylella fastidiosa PD1063 protein is secreted in association with outer membrane vesicles.

Xylella fastidiosa is a gram-negative, xylem-limited plant pathogenic bacterium that causes disease in a variety of economically important agricultural crops including Pierce's disease of grapevines. Xylella fastidiosa biofilms formed in the xylem vessels of plants play a key role in early colonization and pathogenicity by providing a protected niche and enhanced cell survival. Here we investigate the role of Xylella fastidiosa PD1063, the predicted ortholog of Xanthomonas oryzae pv. oryzae PXO_03968, which encodes an outer membrane protein. To assess the function of the Xylella fastidiosa ortholog, we created Xylella fastidiosa mutants deleted for PD1063 and then assessed biofilm formation, cell-cell aggregation and cell growth in vitro. We also assessed disease severity and pathogen titers in grapevines mechanically inoculated with the Xylella fastidiosa PD1063 mutant. We found a significant decrease in cell-cell aggregation among PD1063 mutants but no differences in cell growth, biofilm formation, disease severity or titers in planta. Based on the demonstration that Xanthomonas oryzae pv. oryzae PXO_03968 encodes an outer membrane protein, secreted in association with outer membrane vesicles, we predicted that PD1063 would also be secreted in a similar manner. Using anti-PD1063 antibodies, we found PD1063 in the supernatant and secreted in association with outer membrane vesicles. PD1063 purified from the supernatant, outer membrane fractions and outer membrane vesicles was 19.2 kD, corresponding to the predicted size of the processed protein. Our findings suggest Xylella fastidiosa PD1063 is not essential for development of Pierce's disease in Vitis vinifera grapevines although further research is required to determine the function of the PD1063 outer membrane protein in Xylella fastidiosa.

Genome wide sequence analysis grants unbiased definition of species boundaries in "Candidatus Phytoplasma".

The phytoplasmas are currently named using the Candidatus category, as the inability to grow them in vitro prevented (i) the performance of tests, such as DNA-DNA hybridization, that are regarded as necessary to establish species boundaries, and (ii) the deposition of type strains in culture collections. The recent accession to complete or nearly complete genome sequence information disclosed the opportunity to apply to the uncultivable phytoplasmas the same taxonomic approaches used for other bacteria. In this work, the genomes of 14 strains, belonging to the 16SrI, 16SrIII, 16SrV and 16SrX groups, including the species "Ca. P. asteris", "Ca. P. mali", "Ca. P. pyri", "Ca. P. pruni", and "Ca. P. australiense" were analyzed along with Acholeplasma laidlawi, to determine their taxonomic relatedness. Average nucleotide index (ANIm), tetranucleotide signature frequency correlation index (Tetra), and multilocus sequence analysis of 107 shared genes using both phylogenetic inference of concatenated (DNA and amino acid) sequences and consensus networks, were carried out. The results were in large agreement with the previously established 16S rDNA based classification schemes. Moreover, the taxonomic relationships within the 16SrI, 16SrIII and 16SrX groups, that represent clusters of strains whose relatedness could not be determined by 16SrDNA analysis, could be comparatively evaluated with non-subjective criteria. "Ca. P. mali" and "Ca. P. pyri" were found to meet the genome characteristics for the retention into two different, yet strictly related species; representatives of subgroups 16SrI-A and 16SrI-B were also found to meet the standards used in other bacteria to distinguish separate species; the genomes of the strains belonging to 16SrIII were found more closely related, suggesting that their subdivision into Candidatus species should be approached with caution.

Almond Leaf Scorch Disease Development on Almond Branches High-Grafted on Peach Rootstock.

Development of almond leaf scorch (ALS) disease was monitored on young almond (Prunus dulcis 'NePlus' and 'Peerless') shoots produced from almond scion wood that was high-grafted on peach rootstocks (P. persica 'Queencrest/Nemaguard'), after the almond shoots were mechanically inoculated with Xylella fastidiosa. The objective of this study was to evaluate the potential movement of X. fastidiosa through almond-peach graft unions. ALS symptoms developed on both X. fastidiosa-inoculated and uninoculated almond shoots that were high-grafted on different peach limbs of the same tree in September following inoculations with X. fastidiosa made in June and July 2002, respectively, when the average distance in peach rootstock between the two almond-peach graft unions was 30.5 cm or shorter. No ALS symptoms were observed on uninoculated almond shoots late in the growing season of the second year. The incidence of ALS-affected leaves on shoots inoculated with X. fastidiosa decreased in the second year on the inoculated shoots of Peerless as determined by the number of inoculated shoots showing ALS symptoms in 2002 versus 2003. No visible ALS symptoms were observed in NePlus late in the growing season of the second year, suggesting that survival of X. fastidiosa in NePlus was less than in Peerless. These data demonstrate that movement of X. fastidiosa through two almond-peach graft unions was possible during the year of inoculation when the peach rootstock stem distance between the two almond-peach graft unions was minimal. However, X. fastidiosa may not survive the winter season or survived so poorly as not to be able to produce ALS symptoms in the second growing season on the uninoculated shoots that previously showed ALS symptoms in the fall of the year when an adjacent shoot was inoculated. The results suggest that high-grafting almond scion wood on multiple peach rootstock limbs may provide a means to limit movement of X. fastidiosa from one almond limb to another on the same tree.

Identification of a response regulator involved in surface attachment, cell-cell aggregation, exopolysaccharide production and virulence in the plant pathogen Xylella fastidiosa.

Xylella fastidiosa, the causal agent of Pierce's disease of grapevine, possesses several two-component signal transduction systems that allow the bacterium to sense and respond to changes in its environment. Signals are perceived by sensor kinases that autophosphorylate and transfer the phosphate to response regulators (RRs), which direct an output response, usually by acting as transcriptional regulators. In the X. fastidiosa genome, 19 RRs were found. A site-directed knockout mutant in one unusual RR, designated XhpT, composed of a receiver domain and a histidine phosphotransferase output domain, was constructed. The resulting mutant strain was analysed for changes in phenotypic traits related to biofilm formation and gene expression using microarray analysis. We found that the xhpT mutant was altered in surface attachment, cell-cell aggregation, exopolysaccharide (EPS) production and virulence in grapevine. In addition, this mutant had an altered transcriptional profile when compared with wild-type X. fastidiosa in genes for several biofilm-related traits, such as EPS production and haemagglutinin adhesins.

Exogenous Applications of Abscisic Acid Increase Curing of Pierce's Disease-Affected Grapevines Growing in Pots.

Foliar and drench applications of the plant growth regulator abscisic acid (ABA) were applied to 1-year-old potted Vitis vinifera 'Pinot Noir' and 'Cabernet Sauvignon' vines infected with Xylella fastidiosa, the bacterial pathogen that causes Pierce's disease (PD). A naturally occurring ABA and a synthetic ABA were applied, and both materials showed some effectiveness at increasing curing rates of PD-affected grapevines. Pinot Noir grapevines treated with the drench ABA treatments had significantly greater disease curing effects than the unsprayed control plants. It has been shown that plant phenolics have antimicrobial properties, and we found a positive correlation between effective ABA treatments and the total phenolic compound content of xylem sap extracted from Pinot Noir vines.

Influence of Inoculation Date on the Colonization of Xylella fastidiosa and the Persistence of Almond Leaf Scorch Disease Among Almond Cultivars.

Field grown 2-year-old almond trees (Prunus dulcis cvs. Butte, Carmel, Mission, Ne Plus Ultra, Padre, Peerless, Price, Solano, Sonora, and Thompson) were mechanically inoculated with Xylella fastidiosa in the growing seasons of 2002 and 2003 to study the effect of inoculation date on the movement and colonization of X. fastidiosa and the overwintering persistence of almond leaf scorch disease (ALS) in these cultivars. X. fastidiosa was inoculated into the base of current-season growing shoots in April, May, June, July, August, September, and October. Almond trees inoculated in spring months developed more ALS-symptomatic leaves and more extensive within-plant spread of X. fastidiosa by the end of the current growing season compared with trees inoculated in July, August, September, and October. Trees inoculated in June developed the most severe ALS symptoms during the season in which they were inoculated. Trees inoculated in June and July 2002 had significantly higher disease ratings in 2003 than inoculations made in August and October 2002. Based on disease ratings observed in 2003, 1 year after inoculation, Sonora and Solano were the most ALS susceptible, Mission and Price intermediate, and Carmel, Padre, Ne Plus Ultra, Butte, Peerless, and Thompson were the least susceptible cultivars for allowing X. fastidiosa to overwinter and cause disease the following year. Assessment of all trees in August 2004 indicated that trees inoculated in June and July 2002 had a significantly higher amount of ALS-infected branches than trees inoculated in other months. Butte, Carmel, Padre, and Thompson cultivars had no symptomatic branches, while X. fastidiosa infections persisted or colonized new branches in Sonora, Solano, Peerless, Price, Mission, and Ne Plus Ultra. Based on the 2004 assessment, Sonora was the most susceptible cultivar. Surveys of a diseased orchard in Chico, CA showed large differences in ALS incidence in four almond cultivars. Nonpareil and Peerless had significantly greater incidence of disease than Butte and Carmel over the 2 years surveyed. These data suggest that cultivar susceptibility and the time of X. fastidiosa infection are important factors in determining the persistence of ALS in almond trees.

Analysis of Xylem Fluid Components in Almond Cultivars Differing in Resistance to Almond Leaf Scorch Disease.

Almond leaf scorch (ALS) is caused by the pathogenic bacterium Xylella fastidiosa and poses a threat to the California almond industry. Almond cultivars are differentially resistant or susceptible to ALS. X. fastidiosa can infect but does not overwinter in resistant cultivars in sufficient numbers to cause symptoms or be detected by polymerase chain reaction. To better understand the biochemical or morphological factors mediating resistance, we extracted and analyzed almond xylem fluid from four almond cultivars differing in ALS susceptibility, including Butte and Carmel cultivars that are field resistant and Peerless and Sonora that are ALS susceptible. Xylem fluid was collected over winter months in 2007 to 2009, as well as July 2008 and April 2009, and analyzed for the following: pH, osmolarity, concentrations of sugars, calcium, magnesium, organic acids, and total phenolics. For most of these analyses, we found no clear differences in xylem fluid from resistant and susceptible almond cultivars. However, during the winter months, resistant cultivars tended to have higher concentrations of total phenolic compounds compared with susceptible cultivars (P = 0.05). In February 2009, Carmel had the highest total phenolic concentration measured, 233 µg/ml of gallic acid equivalents. The lowest phenolic concentrations occurred in April 2009. The cross-sectional areas of xylem vessels in Butte (resistant) and Peerless (susceptible) trees were not significantly different between cultivars.

Localization and characterization of Xylella fastidiosa haemagglutinin adhesins.

Xylella fastidiosa is a gram-negative, xylem-inhabiting, plant-pathogenic bacterium responsible for several important diseases including Pierce's disease (PD) of grapevines. The bacteria form biofilms in grapevine xylem that contribute to the occlusion of the xylem vessels. X. fastidiosa haemagglutinin (HA) proteins are large afimbrial adhesins that have been shown to be crucial for biofilm formation. Little is known about the mechanism of X. fastidiosa HA-mediated cell-cell aggregation or the localization of the adhesins on the cell. We generated anti-HA antibodies and show that X. fastidiosa HAs are present in the outer membrane and secreted both as soluble proteins and in membrane vesicles. Furthermore, the HA pre-proteins are processed from the predicted molecular mass of 360 kDa to a mature 220 kDa protein. Based on this information, we are evaluating a novel form of potential resistance against PD by generating HA-expressing transgenic grapevines.

Detection and visualization of an exopolysaccharide produced by Xylella fastidiosa in vitro and in planta.

Many phytopathogenic bacteria, such as Ralstonia solanacearum, Pantoea stewartii, and Xanthomonas campestris, produce exopolysaccharides (EPSs) that aid in virulence, colonization, and survival. EPS can also contribute to host xylem vessel blockage. The genome of Xylella fastidiosa, the causal agent of Pierce's disease (PD) of grapevine, contains an operon that is strikingly similar to the X. campestris gum operon, which is responsible for the production of xanthan gum. Based on this information, it has been hypothesized that X. fastidiosa is capable of producing an EPS similar in structure and composition to xanthan gum but lacking the terminal mannose residue. In this study, we raised polyclonal antibodies against a modified xanthan gum polymer similar to the predicted X. fastidiosa EPS polymer. We used enzyme-linked immunosorbent assay to quantify production of EPS from X. fastidiosa cells grown in vitro and immunolocalization microscopy to examine the distribution of X. fastidiosa EPS in biofilms formed in vitro and in planta and assessed the contribution of X. fastidiosa EPS to the vascular occlusions seen in PD-infected grapevines.

Xylella fastidiosa requires polygalacturonase for colonization and pathogenicity in Vitis vinifera grapevines.

Xylella fastidiosa is the causal agent of Pierce's disease of grape, an economically significant disease for the grape industry. X. fastidiosa systemically colonizes the xylem elements of grapevines and is able to breach the pit pore membranes separating xylem vessels by unknown mechanisms. We hypothesized that X. fastidiosa utilizes cell wall degrading enzymes to break down pit membranes, based on the presence of genes involved in plant cell wall degradation in the X. fastidiosa genome. These genes include several beta-1,4 endoglucanases, several xylanases, several xylosidases, and one polygalacturonase (PG). In this study, we demonstrated that the pglA gene encodes a functional PG. A mutant in pglA lost pathogenicity and was compromised in its ability to systemically colonize Vitis vinifera grapevines. The results indicate that PG is required for X. fastidiosa to successfully infect grapevines and is a critical virulence factor for X. fastidiosa pathogenesis in grapevine.

Characterization of putative rolling-circle plasmids from the Gram-negative bacterium Xylella fastidiosa and their use as shuttle vectors.

This study was initiated to characterize a small Xylella fastidiosa (X. fastidiosa) plasmid and attempt to create a X. fastidosa/Escherichia coli shuttle vector that was stable in planta. Restriction enzyme analysis of a 1.3kb plasmid DNA from a grape-infecting strain of X. fastidiosa (UCLA) revealed the presence of three similar, but genetically distinct, plasmids, pUCLAs. Evidence that suggests the pUCLA plasmids replicate via a rolling-circle (RC) mechanism include: (i) the presence of ssDNA in X. fastidiosa cells; (ii) the presence of conserved motifs in the predicted ORF1 that are typical of initiator (Rep) proteins associated with RC replication; (iii) high amino acid identity between the putative Rep proteins of pUCLAs and Pf3, a filamentous bacteriophage of Pseudomonas aeruginosa that replicates by a RC mechanism; and (iv) the presence of a putative origin of replication upstream of ORF1 that has the potential to form secondary hairpin structures. One DNA motif present in pUCLA shared sequence similarity to known nicking sites in the origins of replication of other RC plasmids and phages. The shuttle vector, pXF001, successfully transformed grape X. fastidiosa strains and was found to be present as autonomous, structurally unchanged DNA molecules in X. fastidiosa. However, pXF001 was not stably maintained in X. fastidiosa without antibiotic selection.

Influence of Ring Nematode Infestation and Calcium, Nitrogen, and Indoleacetic Acid Applications on Peach Susceptibility to Pseudomonas syringae pv. syringae.

ABSTRACT Two field experiments were conducted to study the effects of added nitrogen, calcium, and indoleacetic acid, in the presence or absence of ring nematodes (Mesocriconema xenoplax), on susceptibility of peach to bacterial canker. When noninfested soil was inoculated with ring nematodes, peach tree susceptibility to bacterial canker infection caused by Pseudomonas syringae pv. syringae was dramatically increased after a period of 2 years. However, no evidence was found that ring nematode infestation increased tree water stress or, in turn, altered plant calcium uptake. Soil fumigation with methyl bromide prior to planting in a commercial orchard significantly reduced both nematode populations and peach tree susceptibility to bacterial canker infection when compared with nonfumigated treatments. In both experiments, tree susceptibility, as measured by canker length following inoculation of stems with P. syringae pv. syringae, was negatively correlated with plant tissue nitrogen content and positively correlated with tissue calcium content. A principal components analysis showed that tissue nitrogen and calcium levels were negatively correlated, and that high-nitrogen, low-calcium tissues were less susceptible to bacterial canker than low-nitrogen, high-calcium tissues. These results indicate that the increased susceptibility of peach to P. syringae pv. syringae under nematode infestation conditions is mediated by both nutritional effects (primarily nitrogen) and nutritional-independent effects, but do not support previous reports of beneficial effects of calcium for reducing bacterial canker.

Identification of Xylella fastidiosa antivirulence genes: hemagglutinin adhesins contribute a biofilm maturation to X. fastidios and colonization and attenuate virulence.

Xylella fastidosa, a gram-negative, xylem-limited bacterium, is the causal agent of several economically important plant diseases, including Pierce's disease (PD) and citrus variegated chlorosis (CVC). Until recently, the inability to transform or produce transposon mutants of X. fastidosa had been a major impediment to identifying X. fastidosa genes that mediate pathogen and plant interactions. A random transposon (Tn5) library of X. fastidosa was constructed and screened for mutants showing more severe symptoms and earlier grapevine death (hypervirulence) than did vines infected with the wild type. Seven hypervirulent mutants identified in this screen moved faster and reached higher populations than the wild type in grapevines. These results suggest that X. fastidosa attenuates its virulence in planta and that movement is important in X. fastidosa virulence. The mutated genes were sequenced and none had been described previously as antivirulence genes, although six of them showed similarity with genes of known functions in other organisms. One transposon insertion inactivated a hemagglutinin adhesin gene (PD2118), which we named HxfA. Another mutant in a second putative X. fastidosa hemagglutinin gene, PD1792 (HxfB), was constructed, and further characterization of these hxf mutants suggests that X. fastidosa hemagglutinins mediate contact between X. fastidosa cells, which results in colony formation and biofilm maturation within the xylem vessels.

Interaction Between Nitrogen-Fertilized Peach Trees and Expression of syrB, a Gene Involved in Syringomycin Production in Pseudomonas syringae pv. syringae.

ABSTRACT The in vitro expression of the syrB gene that controls the synthesis of syringomycin, a non-host-specific phytotoxin produced by Pseudomonas syringae pv. syringae van Hall, was studied using aqueous extracts derived from bark tissues collected from nitrogen-fertilized and nonfertilized peach trees. Expression of the syrB gene was quantified as beta- galactosidase activity expressed by P. syringae pv. syringae B3AR-132 containing a syrB::lacZ fusion. Gene expression was significantly less in three of four paired comparisons using extracts derived from fertilized versus nonfertilized trees; however, canker lengths were significantly different in only one of four comparisons. Expression was negatively correlated with plant tissue nitrogen content and positively correlated with a plant carbon/nitrogen ratio. Bark tissue from ring nematodeinfested trees had significantly higher concentrations of total soluble phenolic compounds and carbon/nitrogen ratios than bark samples from trees without nematodes, and canker size was significantly greater in trees growing in ring nematode-infested soil compared with noninfested soil. Nitrogen fertilization significantly decreased the plant carbon/nitrogen ratio, which was positively correlated with the concentration of total soluble phenolic compounds. Canker size developing after bacterial inoculation was positively correlated with higher plant carbon/nitrogen ratios and total soluble phenolic compounds. These results support the hypothesis that one reason why nitrogen fertilization decreases host susceptibility to bacterial canker is by either reducing the amount of plant metabolites that can induce syrB gene expression, or producing or increasing the concentration of compounds that antagonize syrB inducing compounds.

Sequence analysis of two plasmids from the phytoplasma beet leafhopper-transmitted virescence agent.

The complete nucleotide sequences of the two plasmids from the phytoplasma beet leafhopper-transmitted virescence agent (BLTVA) have been determined. The larger plasmid, pBLTVA-1, was 10 785 nt in length and contained 11 putative ORFs, almost all of which were duplicated or triplicated on the plasmid due to the presence of large repeated regions. The sequence contained a series of tandem repeats, the largest of which was 338 nt long. The sequences of ORFs 4 and 11 showed homology with the replication genes of plasmids from other phytoplasmas and from geminiviruses. ORF9, the only ORF present as a single copy, showed homology with DNA primase genes from bacterial chromosomes and contained the conserved zinc finger and topoisomerase/primase domains. None of the other eight ORFs showed homology with known sequences in the GenBank database. pBLTVA-2 was 2587 nt in length, and all of its sequence was nearly identical to sequences from pBLTVA-1, most of which spanned ORFs 10 and 11, including the 338 nt tandem repeat. Analysis of 30 strains of BLTVA showed that most of the 11 putative ORFs were present, but the size of the plasmids varied in these strains.

Cosmid cloning and sample sequencing of the genome of the uncultivable mollicute, Western X-disease phytoplasma, using DNA purified by pulsed-field gel electrophoresis.

Western X-disease (WX) phytoplasma is an uncultivable, intracellular pathogen of plants and insects with an AT-rich genome of 670 kb. As part of the genome sequencing project of WX phytoplasma, we have cloned approximately 50% of its genome into the pcosRW2 cosmid vector using DNA purified from pulsed-field gels. One of the cosmid clones with an insert of 24.6 kb was sequenced, which along with the cosmid end sequences, represents 60 kb of unique sequence from the WX phytoplasma genome. The putative genes identified in this sequence represent a wide variety of functions and many had not been previously identified in a phytoplasma.

Transformation of Xylella fastidiosa with broad host range RSF1010 derivative plasmids.

SUMMARY The Gram-negative, fastidious bacterium Xylella fastidiosa was successfully transformed with two RSF1010 derivative plasmids belonging to the incompatibility group IncQ, using electroporation. These two derivative plasmids, pXF004 and pXF005, were found to be present as autonomous, structurally unchanged DNA molecules when propagated in X. fastidiosa. However, neither pXF004 nor pXF005 were stably maintained in X. fastidiosa without antibiotic selection. When plasmid DNAs were isolated from X. fastidiosa, or plasmid DNAs isolated from E. coli were supplemented with a TypeOne inhibitor, TRI, the frequency of transformation was increased by 13- or 5-fold, respectively. Plasmid pXF005 was also used to transform one additional grapevine strain of X. fastidiosa.

An immunodominant membrane protein gene from the Western X-disease phytoplasma is distinct from those of other phytoplasmas.

Membrane proteins mediate several important processes, including attachment, in several Mollicute species. Phytoplasmas are non-culturable plant pathogenic mollicutes that are transmitted in a specific manner by certain phloem-feeding insect vectors. Because it is likely that phytoplasma membrane proteins are involved with some aspect of the transmission process, their identification, isolation and characterization are important first steps in understanding phytoplasma transmission. A 32 kDa immunodominant protein (IDP) from the Western X-disease (WX) phytoplasma was purified from infected plants by immunoprecipitation using monoclonal antibodies, and two peptides from a tryptic digest were sequenced. PCR primers designed from these sequences amplified a 145 bp product which hybridized with WX-related phytoplasmas in Southern blots. This PCR product was used to identify a 2.5 kbp ECO:RI-HIN:dIII fragment that was cloned and sequenced. A complete 864 bp ORF (idpA) was identified for which the putative translation product contained both of the tryptic digest peptide sequences that were used to design the PCR primers. Analysis of the predicted IdpA sequence indicated two transmembrane domains but no cleavage point. The amino acid sequence had no significant homology with other known phytoplasma IDP genes. The idpA ORF was cloned into an Escherichia coli expression vector and a fusion protein of the predicted size was identified in Western blots using a WX-specific antiserum. A rabbit polyclonal antiserum was prepared to the purified expression protein and this reacted with both the E. coli-expressed and native WX phytoplasma proteins. This newly identified WX IDP (IdpA) is distinct from other known mollicute membrane proteins.