Differentiation of Xylella fastidiosa strains via multilocus sequence analysis of environmentally mediated genes (MLSA-E)

Isolates of the plant pathogen Xylella fastidiosa are genetically very similar, but studies on their biological traits have indicated differences in virulence and infection symptomatology. Taxonomic analyses have identified several subspecies, and phylogenetic analyses of housekeeping genes have shown broad host-based genetic differences; however, results are still inconclusive for genetic differentiation of isolates within subspecies. This study employs multilocus sequence analysis of environmentally mediated genes (MLSA-E; genes influenced by environmental factors) to investigate X. fastidiosa relationships and differentiate isolates with low genetic variability. Potential environmentally mediated genes, including host colonization and survival genes related to infection establishment, were identified a priori. The ratio of the rate of nonsynonymous substitutions to the rate of synonymous substitutions (dN/dS) was calculated to select genes that may be under increased positive selection compared to previously studied housekeeping genes. Nine genes were sequenced from 54 X. fastidiosa isolates infecting different host plants across the United States. Results of maximum likelihood (ML) and Bayesian phylogenetic (BP) analyses are in agreement with known X. fastidiosa subspecies clades but show novel within-subspecies differentiation, including geographic differentiation, and provide additional information regarding host-based isolate variation and specificity. dN/dS ratios of environmentally mediated genes, though <1 due to high sequence similarity, are significantly greater than housekeeping gene dN/dS ratios and correlate with increased sequence variability. MLSA-E can more precisely resolve relationships between closely related bacterial strains with low genetic variability, such as X. fastidiosa isolates. Discovering the genetic relationships between X. fastidiosa isolates will provide new insights into the epidemiology of populations of X. fastidiosa, allowing improved disease management in economically important crops.     

A genomic approach to the understanding of Xylella fastidiosa pathogenicity

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes several economically important plant diseases, including citrus variegated chlorosis (CVC). X. fastidiosa is the first plant pathogen to have its genome completely sequenced. In addition, it is probably the least previously studied of any organism for which the complete genome sequence is available. Several pathogenicity-related genes have been identified in the X. fastidiosa genome by similarity with other bacterial genes involved in pathogenesis in plants, as well as in animals. The X. fastidiosa genome encodes different classes of proteins directly or indirectly involved in cell-cell interactions, degradation of plant cell walls, iron homeostasis, anti-oxidant responses, synthesis of toxins, and regulation of pathogenicity. Neither genes encoding members of the type III protein secretion system nor avirulence-like genes have been identified in X. fastidiosa.     

Transposon mutagenesis of Xylella fastidiosa by electroporation of Tn5 synaptic complexes

Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects. Strains of X. fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. Although the complete genome sequence of X. fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X. fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions. We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X. fastidiosa. The four suicide vectors failed to produce any detectable transposition events. Electroporation of transposomes, however, yielded 6 x 10(3) and 4 x 10(3) Tn5 mutants per microg of DNA in two different grapevine strains of X. fastidiosa. Molecular analysis showed that the transposition insertions were single, independent, stable events. Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X. fastidiosa genome. Transposome-mediated mutagenesis should facilitate the identification of X. fastidiosa genes that mediate plant pathogenicity and insect transmission.     

Susceptibility and symptom development in Arabidopsis thaliana to Tobacco mosaic virus is influenced by virus cell-to-cell movement

To identify host factors that regulate susceptibility to Tobacco mosaic virus (TMV), 14 Arabidopsis thaliana ecotypes were screened for their ability to support TMV systemic movement. The susceptibility phenotypes observed included one ecotype that permitted rapid TMV movement accompanied by symptoms, nine ecotypes that allowed a slower intermediate rate of systemic movement without symptoms, and four ecotypes that allowed little or no systemic TMV movement. Molecular comparisons between ecotypes representing the rapid (Shahdara), intermediate (Col-1), and slow (Tsu-1) movement phenotypes revealed a positive correlation between the ability of TMV to move cell to cell and its speed of systemic movement. Additionally, protoplasts prepared from all three ecotypes supported similar levels of TMV replication, indicating that viral replication did not account for differences in systemic movement. Furthermore, induction of the pathogenesis-related genes PR-1 and PR-5 occurred only in the highly susceptible ecotype Shahdara, demonstrating that reduced local and systemic movement in Col-1 and Tsu-1 was not due to the activation of known host defense responses. Genetic analysis of F2 progeny derived from crosses made between Shahdara and Tsu-1 or Col-1 and Tsu-1 showed the faster cell-to-cell movement phenotypes of Shahdara and Col-1 segregated as single dominant genes. In addition, the Shahdara symptom phenotype segregated independently as a single recessive gene. Taken together, these findings suggest that, within Arabidopsis ecotypes, at least two genes modulate susceptibility to TMV.     

Natural variants of AtHKT1 enhance Na+ accumulation in two wild populations of Arabidopsis

Plants are sessile and therefore have developed mechanisms to adapt to their environment, including the soil mineral nutrient composition. Ionomics is a developing functional genomic strategy designed to rapidly identify the genes and gene networks involved in regulating how plants acquire and accumulate these mineral nutrients from the soil. Here, we report on the coupling of high-throughput elemental profiling of shoot tissue from various Arabidopsis accessions with DNA microarray-based bulk segregant analysis and reverse genetics, for the rapid identification of genes from wild populations of Arabidopsis that are involved in regulating how plants acquire and accumulate Na(+) from the soil. Elemental profiling of shoot tissue from 12 different Arabidopsis accessions revealed that two coastal populations of Arabidopsis collected from Tossa del Mar, Spain, and Tsu, Japan (Ts-1 and Tsu-1, respectively), accumulate higher shoot levels of Na(+) than do Col-0 and other accessions. We identify AtHKT1, known to encode a Na(+) transporter, as being the causal locus driving elevated shoot Na(+) in both Ts-1 and Tsu-1. Furthermore, we establish that a deletion in a tandem repeat sequence approximately 5 kb upstream of AtHKT1 is responsible for the reduced root expression of AtHKT1 observed in these accessions. Reciprocal grafting experiments establish that this loss of AtHKT1 expression in roots is responsible for elevated shoot Na(+). Interestingly, and in contrast to the hkt1-1 null mutant, under NaCl stress conditions, this novel AtHKT1 allele not only does not confer NaCl sensitivity but also cosegregates with elevated NaCl tolerance. We also present all our elemental profiling data in a new open access ionomics database, the Purdue Ionomics Information Management System (PiiMS; http://www.purdue.edu/dp/ionomics). Using DNA microarray-based genotyping has allowed us to rapidly identify AtHKT1 as the casual locus driving the natural variation in shoot Na(+) accumulation we observed in Ts-1 and Tsu-1. Such an approach overcomes the limitations imposed by a lack of established genetic markers in most Arabidopsis accessions and opens up a vast and tractable source of natural variation for the identification of gene function not only in ionomics but also in many other biological processes.     

Disruption of Xylella fastidiosa CVC gumB and gumF genes affects biofilm formation without a detectable influence on exopolysaccharide production

Xylella fastidiosa causes citrus variegated chlorosis (CVC), a destructive disease of citrus. Xylella fastidiosa forms a biofilm inside plants and insect vectors. Biofilms are complex structures involving X. fastidiosa cells and an extracellular matrix which blocks water and nutrient transport in diseased plants. It is hypothesized that the matrix might be composed of an extracellular polysaccharide (EPS), coded by a cluster of nine genes closely related to the xanthan gum operon of Xanthomonas campestris pv. campestris. To understand the role of X. fastidiosa gum genes on biofilm formation and EPS biosynthesis, we produced gumB and gumF mutants. Xylella fastidiosa mutants were obtained by insertional duplication mutagenesis and recovered after triply cloning the cells. Xylella fastidiosa gumB and gumF mutants exhibited normal cell characteristics; typical colony morphology and EPS biosynthesis were not altered. It was of note that X. fastidiosa mutants showed a reduced capacity to form biofilm when BCYE was used as the sustaining medium, a difference not observed with PW medium. Unlike X. campestris pv. campestris, the expression of the X. fastidiosa gumB or gumF genes was not regulated by glucose.     

Multilocus sequence type system for the plant pathogen Xylella fastidiosa and relative contributions of recombination and point mutation to clonal diversity

Multilocus sequence typing (MLST) identifies and groups bacterial strains based on DNA sequence data from (typically) seven housekeeping genes. MLST has also been employed to estimate the relative contributions of recombination and point mutation to clonal divergence. We applied MLST to the plant pathogen Xylella fastidiosa using an initial set of sequences for 10 loci (9.3 kb) of 25 strains from five different host plants, grapevine (PD strains), oleander (OLS strains), oak (OAK strains), almond (ALS strains), and peach (PP strains). An eBURST analysis identified six clonal complexes using the grouping criterion that each member must be identical to at least one other member at 7 or more of the 10 loci. These clonal complexes corresponded to previously identified phylogenetic clades; clonal complex 1 (CC1) (all PD strains plus two ALS strains) and CC2 (OLS strains) defined the X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. sandyi clades, while CC3 (ALS strains), CC4 (OAK strains), and CC5 (PP strains) were subclades of X. fastidiosa subsp. multiplex. CC6 (ALS strains) identified an X. fastidiosa subsp. multiplex-like group characterized by a high frequency of intersubspecific recombination. Compared to the recombination rate in other bacterial species, the recombination rate in X. fastidiosa is relatively low. Recombination between different alleles was estimated to give rise to 76% of the nucleotide changes and 31% of the allelic changes observed. The housekeeping loci holC, nuoL, leuA, gltT, cysG, petC, and lacF were chosen to form the basis of a public database for typing X. fastidiosa (www.mlst.net). These loci identified the same six clonal complexes using the strain grouping criterion of identity at five or more loci with at least one other member.     

Population genomic analysis of a bacterial plant pathogen: novel insight into the origin of Pierce's disease of grapevine in the U.S

Invasive diseases present an increasing problem worldwide; however, genomic techniques are now available to investigate the timing and geographical origin of such introductions. We employed genomic techniques to demonstrate that the bacterial pathogen causing Pierce's disease of grapevine (PD) is not native to the US as previously assumed, but descended from a single genotype introduced from Central America. PD has posed a serious threat to the US wine industry ever since its first outbreak in Anaheim, California in the 1880s and continues to inhibit grape cultivation in a large area of the country. It is caused by infection of xylem vessels by the bacterium Xylella fastidiosa subsp. fastidiosa, a genetically distinct subspecies at least 15,000 years old. We present five independent kinds of evidence that strongly support our invasion hypothesis: 1) a genome-wide lack of genetic variability in X. fastidiosa subsp. fastidiosa found in the US, consistent with a recent common ancestor; 2) evidence for historical allopatry of the North American subspecies X. fastidiosa subsp. multiplex and X. fastidiosa subsp. fastidiosa; 3) evidence that X. fastidiosa subsp. fastidiosa evolved in a more tropical climate than X. fastidiosa subsp. multiplex; 4) much greater genetic variability in the proposed source population in Central America, variation within which the US genotypes are phylogenetically nested; and 5) the circumstantial evidence of importation of known hosts (coffee plants) from Central America directly into southern California just prior to the first known outbreak of the disease. The lack of genetic variation in X. fastidiosa subsp. fastidiosa in the US suggests that preventing additional introductions is important since new genetic variation may undermine PD control measures, or may lead to infection of other crop plants through the creation of novel genotypes via inter-subspecific recombination. In general, geographically mixing of previously isolated subspecies should be avoided.     

A putative twin-arginine translocation system in the phytopathogenic bacterium Xylella fastidiosa

The twin-arginine translocation (Tat) pathway of the xylem-limited phytopathogenic bacterium Xylella fastidiosa strain 9a5c, responsible for citrus variegated chlorosis, was explored. The presence of tatA, tatB, and tatC in the X. fastidiosa genome together with a list of proteins harboring 2 consecutive arginines in their signal peptides suggested the presence of a Tat pathway. The functional Tat dependence of X. fastidiosa OpgD was examined. Native or mutated signal peptides were fused to the β-lactamase. Expression of fusion with intact signal peptides mediated high resistance to ampicillin in Escherichia coli tat+ but not in the E. coli tat null mutant. The replacement of the 2 arginines by 2 lysines prevented the export of β-lactamase in E. coli tat+, demonstrating that X. fastidiosa OpgD carries a signal peptide capable of engaging the E. coli Tat machinery. RT-PCR analysis revealed that the tat genes are transcribed as a single operon. tatA, tatB, and tatC genes were cloned. Complementation assays in E. coli devoid of all Tat or TatC components were unsuccessful, whereas X. fastidiosa Tat components led to a functional Tat translocase in E. coli TatB-deficient strain. Additional experiments implicated that X. fastidiosa TatB component could form a functional heterologous complex with the E. coli TatC component.     

Identification of an operon, Pil-Chp, that controls twitching motility and virulence in Xylella fastidiosa

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce's disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.     

Diverse RNA viruses elicit the expression of common sets of genes in susceptible Arabidopsis thaliana plants

Systemic infections of plants by viruses require that viruses modify host cells in order to facilitate infections. These modifications include induction of host factors required for replication, propagation and movement, and suppression of host defense responses, which are likely to be associated with changes in host gene expression. Past studies of the effects of viral infection on gene expression in susceptible hosts have been limited to only a handful of genes. To gain broader insight into the responses elicited by viruses in susceptible hosts, high-density oligonucleotide probe microarray technology was used. Arabidopsis leaves were either mock inoculated or inoculated with cucumber mosaic cucumovirus, oil seed rape tobamovirus, turnip vein clearing tobamovirus, potato virus X potexvirus, or turnip mosaic potyvirus. Inoculated leaves were collected at 1, 2, 4, and 5 days after inoculation, total RNA was isolated, and samples were hybridized to Arabidopsis GeneChip microarrays (Affymetrix). Microarray hybridization revealed co-ordinated changes in gene expression in response to infection by diverse viruses. These changes include virus-general and virus-specific alterations in the expression of genes associated with distinct defense or stress responses. Analyses of the promoters of these genes further suggest that diverse RNA viruses elicit common responses in susceptible plant hosts through signaling pathways that have not been previously characterized.     

Impact of duration versus frequency of probing by Homalodisca vitripennis (Hemiptera: Cicadellidae) on inoculation of Xylella fastidiosa

Successful infection of the plant pathogenic bacterium Xylella fastidiosa (Wells) from an infected plant to a new host involves three main steps: 1) acquisition of the bacterium by a vector; 2) inoculation of a noninfected host plant by the vector; and 3) establishment of sufficient titers of X. fastidiosa in the host plant to sustain a chronic infection. Understanding the basic biology of the transmission process is a key to limiting the spread of plant diseases induced by X. fasdidiosa and reducing agricultural losses, especially those experienced in California since the introduction of a new vector, Homalodisca vitripennis (Germar) (Hemiptera, Cicadellidae) (formerly H. coagulata Say), the glassy-winged sharpshooter. In this study, H. vitripennis adults that acquired X. fastidiosa were allowed access to chrysanthemum plant cuttings for 30, 60, 90, or 120 min. The numbers of X. fastidiosa acquired (i.e., cells present in the insect foregut) and the number inoculated to the plant cuttings were separately determined using quantitative real-time polymerase chain reaction (PCR). In addition, the number of times glassy-winged sharpshooter stylets probed plant cuttings and the amount of time glassy-winged sharpshooter spent actively ingesting were monitored using video surveillance. Linear regression did not indicate a relationship between the number of X. fastidiosa cells inoculated into the plant cutting and either the titer of pathogen present in the insect or amount of time spent ingesting per probe. However, the number of probes significantly influenced the number of X. fastidiosa cells inoculated. Due to the highly variable nature of transmission, our model could not account for all observed variation as indicated by low R2 values. However, our results suggest that the mechanism of transmission is dependent on probing behaviors more than ingestion duration.     

The endophyte Curtobacterium flaccumfaciens reduces symptoms caused by Xylella fastidiosa in Catharanthus roseus

Citrus variegated chlorosis (CVC) is a disease of the sweet orange [Citrus sinensis (L.)], which is caused by Xylella fastidiosa subsp. pauca, a phytopathogenic bacterium that has been shown to infect all sweet orange cultivars. Sweet orange trees have been occasionally observed to be infected by Xylella fastidiosa without evidencing severe disease symptoms, whereas other trees in the same grove may exhibit severe disease symptoms. The principal endophytic bacterial species isolated from such CVC-asymptomatic citrus plants is Curtobacterium flaccumfaciens. The Madagascar periwinkle [Citrus sinensis (L.)] is a model plant which has been used to study X. fastidiosa in greenhouse environments. In order to characterize the interactions of X. fastidiosa and C. flaccumfaciens, periwinkle plants were inoculated separately with C. flaccumfaciens, X. fastidiosa, and both bacteria together. The number of flowers produced by the plants, the heights of the plants, and the exhibited disease symptoms were evaluated. PCR-primers for C. flaccumfaciens were designed in order to verify the presence of this endophytic bacterium in plant tissue, and to complement an existing assay for X. fastidiosa. These primers were capable of detecting C. flaccumfaciens in the periwinkle in the presence of X. fastidiosa. X. fastidiosa induced stunting and reduced the number of flowers produced by the periwinkle. When C. flaccumfaciens was inoculated together with X. fastidiosa, no stunting was observed. The number of flowers produced by our doubly- inoculated plants was an intermediate between the number produced by the plants inoculated with either of the bacteria separately. Our data indicate that C. flaccumfaciens interacted with X. fastidiosa in C. roseus, and reduced the severity of the disease symptoms induced by X. fastidiosa. Periwinkle is considered to be an excellent experimental system by which the interaction of C. flaccumfaciens and other endophytic bacteria with X. fastidiosa can be studied.     

Genetics of drought adaptation in Arabidopsis thaliana II. QTL analysis of a new mapping population, KAS-1 x TSU-1

Despite compelling evidence that adaptation to local climate is common in plant populations, little is known about the evolutionary genetics of traits that contribute to climatic adaptation. A screen of natural accessions of Arabidopsis thaliana revealed Tsu-1 and Kas-1 to be opposite extremes for water-use efficiency and climate at collection sites for these accessions differs greatly. To provide a tool to understand the genetic basis of this putative adaptation, Kas-1 and Tsu-1 were reciprocally crossed to create a new mapping population. Analysis of F(3) families showed segregating variation in both delta(13)C and transpiration rate, and as expected these traits had a negative genetic correlation (r(g)=- 0.3). 346 RILs, 148 with Kas-1 cytoplasm and 198 with Tsu-1 cytoplasm, were advanced to the F(9) and genotyped using 48 microsatellites and 55 SNPs for a total of 103 markers. This mapping population was used for QTL analysis of delta(13)C using F(9) RIL means. Analysis of this reciprocal cross showed a large effect of cytoplasmic background, as well as two QTL for delta(13)C. The Kas-1 x Tsu-1 mapping population provides a powerful new resource for mapping QTL underlying natural variation and for dissecting the genetic basis of water-use efficiency differences.     

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.     

Interactive effects of water stress and xylem-limited bacterial infection on the water relations of a host vine

Xylella fastidiosa, a xylem-limited bacterial pathogen that causes bacterial leaf scorch in its hosts, has a diverse and extensive host range among plant species worldwide. Previous work has shown that water stress enhances leaf scorch symptom severity and progression along the stem in Parthenocissus quinquefolia infected by X. fastidiosa. The objective here was to investigate the mechanisms underlying the interaction of water stress and infection by X. fastidiosa. Using the eastern deciduous forest vine, P. quinquefolia, infection and water availability were manipulated while measuring leaf water potentials (psi(L)), stomatal conductance (g(s)), whole shoot hydraulic conductance (K(h)), per cent xylem embolism, and xylem vessel dimensions. No significant differences in any of the physiological measurements were found between control and infected plants prior to drought. Drought treatment significantly reduced psi(L) and g(s) at all leaf positions throughout the day in late summer in both years of the study. In addition, infection significantly reduced psi(L) and g(s) in the most basal leaf positions in late summer in both years. Whole shoot hydraulic conductance was reduced by both low water and infection treatments. However, per cent embolized vessels and mean vessel diameter were affected by drought treatment only. These results imply that the major effect of infection by X. fastidiosa occurs due to reduced hydraulic conductance caused by clogging of the vessels, and not increased cavitation and embolism of xylem elements. The reduced K(h) caused by X. fastidiosa infection acts additively with the water limitation imposed by Drought stress.