Virulence Comparison of a Comprehensive Panel of Xylella fastidiosa Pierce's Disease Isolates from California.

Xylella fastidiosa, the causal agent of Pierce's disease of grapevine, has been found in all major grape-growing regions in California, U.S.A. Large collections of X. fastidiosa isolates are available from these areas, which enable comparative studies of pathogen genetic traits and virulence. Owing to the significant resource requirements for experiments with X. fastidiosa in grapevine, however, most studies use only a single isolate to evaluate disease, and it is not clear how much variability between isolates impacts disease development in experimental or natural settings. In this study, a comprehensive panel of X. fastidiosa isolates from all California grape-growing regions was tested for virulence in susceptible grapevine and in the model host plant, tobacco. Seventy-one isolates were tested, 29 in both grapevine and tobacco. The results of this study highlight the inherent variability of inoculation experiments with X. fastidiosa, including variation in disease severity in plants inoculated with a single isolate, and variability between experimental replicates. There were limited differences in virulence between isolates that were consistent across experimental replicates, or across different host plants. This suggests that choice of isolate within the X. fastidiosa subsp. fastidiosa Pierce's disease group may not make any practical difference when testing in susceptible grape varieties, and that pathogen evolution has not significantly changed virulence of Pierce's disease isolates within California. The location of isolation also did not dictate relative disease severity. This information will inform experimental design for future studies of X. fastidiosa in grapevine and provide important context for genomic research.

Induction of Defense Responses and Protection of Almond Plants Against Xylella fastidiosa by Endotherapy with a Bifunctional Peptide.

Xylella fastidiosa is a plant pathogenic bacterium that has been introduced in the European Union (EU), causing significant yield losses in economically important Mediterranean crops. Almond leaf scorch (ALS) is currently one of the most relevant diseases observed in Spain, and no cure has been found to be effective for this disease. In previous reports, the peptide BP178 has shown a strong bactericidal activity in vitro against X. fastidiosa and to other plant pathogens, and to trigger defense responses in tomato plants. In the present work, BP178 was applied by endotherapy to almond plants of cultivar Avijor using preventive and curative strategies. The capacity of BP178 to reduce the population levels of X. fastidiosa and to decrease disease symptoms and its persistence over time were demonstrated under greenhouse conditions. The most effective treatment consisted of a combination of preventive and curative applications, and the peptide was detected in the stem up to 60 days posttreatment. Priming plants with BP178 induced defense responses mainly through the salicylic acid pathway, but also overexpressed some genes of the jasmonic acid and ethylene pathways. It is concluded that the bifunctional peptide is a promising candidate to be further developed to manage ALS caused by X. fastidiosa.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Xylella fastidiosa subsp. pauca Strains Fb7 and 9a5c from Citrus Display Differential Behavior, Secretome, and Plant Virulence.

Xylella fastidiosa colonizes the xylem of various cultivated and native plants worldwide. Citrus production in Brazil has been seriously affected, and major commercial varieties remain susceptible to Citrus Variegated Chlorosis (CVC). Collective cellular behaviors such as biofilm formation influence virulence and insect transmission of X. fastidiosa. The reference strain 9a5c produces a robust biofilm compared to Fb7 that remains mostly planktonic, and both were isolated from symptomatic citrus trees. This work deepens our understanding of these distinct behaviors at the molecular level, by comparing the cellular and secreted proteomes of these two CVC strains. Out of 1017 identified proteins, 128 showed differential abundance between the two strains. Different protein families were represented such as proteases, hemolysin-like proteins, and lipase/esterases, among others. Here we show that the lipase/esterase LesA is among the most abundant secreted proteins of CVC strains as well, and demonstrate its functionality by complementary activity assays. More severe symptoms were observed in Nicotiana tabacum inoculated with strain Fb7 compared to 9a5c. Our results support that systemic symptom development can be accelerated by strains that invest less in biofilm formation and more in plant colonization. This has potential application in modulating the bacterial-plant interaction and reducing disease severity.

Discriminating between viable and membrane-damaged cells of the plant pathogen Xylella fastidiosa.

Xylella fastidiosa is a plant pathogenic bacterium with devastating consequences to several crops of economic importance across the world. While this pathogen has been studied for over a century in the United States, several aspects of its biology remain to be investigated. Determining the physiological state of bacteria is essential to understand the effects of its interactions with different biotic and abiotic factors on cell viability. Although X. fastidiosa is culturable, its slow growing nature makes this technique cumbersome to assess the physiological state of cells present in a given environment. PMA-qPCR, i.e. the use of quantitative PCR combined with the pre-treatment of cells with the dye propidium monoazide, has been successfully used in a number of studies on human pathogens to calculate the proportion of viable cells, but has less frequently been tested on plant pathogens. We found that the use of a version of PMA, PMAxx, facilitated distinguishing between viable and non-viable cells based on cell membrane integrity in vitro and in planta. Additional experiments comparing the number of culturable, viable, and total cells in planta would help further confirm our initial results. Enhancers, intended to improve the efficacy of PMAxx, were not effective and appeared to be slightly toxic to X. fastidiosa.

Ectopic Expression of Xylella fastidiosa rpfF Conferring Production of Diffusible Signal Factor in Transgenic Tobacco and Citrus Alters Pathogen Behavior and Reduces Disease Severity.

The pathogenicity of Xylella fastidiosa is associated with its ability to colonize the xylem of host plants. Expression of genes contributing to xylem colonization are suppressed, while those necessary for insect vector acquisition are increased with increasing concentrations of diffusible signal factor (DSF), whose production is dependent on RpfF. We previously demonstrated that transgenic citrus plants ectopically expressing rpfF from a citrus strain of X. fastidiosa subsp. pauca exhibited less susceptibility to Xanthomonas citri subsp. citri, another pathogen whose virulence is modulated by DSF accumulation. Here, we demonstrate that ectopic expression of rpfF in both transgenic tobacco and sweet orange also confers a reduction in disease severity incited by X. fastidiosa and reduces its colonization of those plants. Decreased disease severity in the transgenic plants was generally associated with increased expression of genes conferring adhesiveness to the pathogen and decreased expression of genes necessary for active motility, accounting for the reduced population sizes achieved in the plants, apparently by limiting pathogen dispersal through the plant. Plant-derived DSF signal molecules in a host plant can, therefore, be exploited to interfere with more than one pathogen whose virulence is controlled by DSF signaling.

The Secreted Protease PrtA Controls Cell Growth, Biofilm Formation and Pathogenicity in Xylella fastidiosa.

Pierce's disease (PD) is a deadly disease of grapevines caused by the Gram-negative bacterium Xylella fastidiosa. Though disease symptoms were formerly attributed to bacteria blocking the plant xylem, this hypothesis is at best overly simplistic. Recently, we used a proteomic approach to characterize the secretome of X. fastidiosa, both in vitro and in planta, and identified LesA as one of the pathogenicity factors of X. fastidiosa in grapevines that leads to leaf scorching and chlorosis. Herein, we characterize another such factor encoded by PD0956, designated as an antivirulence secreted protease "PrtA" that displays a central role in controlling in vitro cell proliferation, length, motility, biofilm formation, and in planta virulence. The mutant in X. fastidiosa exhibited reduced cell length, hypermotility (and subsequent lack of biofilm formation) and hypervirulence in grapevines. These findings are supported by transcriptomic and proteomic analyses with corresponding plant infection data. Of particular interest, is the hypervirulent response in grapevines observed when X. fastidiosa is disrupted for production of PrtA, and that PD-model tobacco plants transformed to express PrtA exhibited decreased symptoms after infection by X. fastidiosa.

Three New Pierce's Disease Pathogenicity Effectors Identified Using Xylella fastidiosa Biocontrol Strain EB92-1.

Xylella fastidiosa (X. fastidiosa) infects a wide range of plant hosts and causes economically serious diseases, including Pierce's Disease (PD) of grapevines. X. fastidiosa biocontrol strain EB92-1 was isolated from elderberry and is infectious and persistent in grapevines but causes only very slight symptoms under ideal conditions. The draft genome of EB92-1 revealed that it appeared to be missing genes encoding 10 potential PD pathogenicity effectors found in Temecula1. Subsequent PCR and sequencing analyses confirmed that EB92-1 was missing the following predicted effectors found in Temecula1: two type II secreted enzymes, including a lipase (LipA; PD1703) and a serine protease (PD0956); two identical genes encoding proteins similar to Zonula occludens toxins (Zot; PD0915 and PD0928), and at least one relatively short, hemagglutinin-like protein (PD0986). Leaves of tobacco and citrus inoculated with cell-free, crude protein extracts of E. coli BL21(DE3) overexpressing PD1703 exhibited a hypersensitive response (HR) in less than 24 hours. When cloned into shuttle vector pBBR1MCS-5, PD1703 conferred strong secreted lipase activity to Xanthomonas citri, E. coli and X. fastidiosa EB92-1 in plate assays. EB92-1/PD1703 transformants also showed significantly increased disease symptoms on grapevines, characteristic of PD. Genes predicted to encode PD0928 (Zot) and a PD0986 (hemagglutinin) were also cloned into pBBR1MCS-5 and moved into EB92-1; both transformants also showed significantly increased symptoms on V. vinifera vines, characteristic of PD. Together, these results reveal that PD effectors include at least a lipase, two Zot-like toxins and a possibly redundant hemagglutinin, none of which are necessary for parasitic survival of X. fastidiosa populations in grapevines or elderberry.

Ionome changes in Xylella fastidiosa-infected Nicotiana tabacum correlate with virulence and discriminate between subspecies of bacterial isolates.

Characterization of ionomes has been used to uncover the basis of nutrient utilization and environmental adaptation of plants. Here, ionomic profiles were used to understand the phenotypic response of a plant to infection by genetically diverse isolates of Xylella fastidiosa, a gram-negative, xylem-limited bacterial plant pathogen. In this study, X. fastidiosa isolates were used to infect a common model host (Nicotiana tabacum 'SR1'), and leaf and sap concentrations of eleven elements together with plant colonization and symptoms were assessed. Multivariate statistical analysis revealed that changes in the ionome were significantly correlated with symptom severity and bacterial populations in host petioles. Moreover, plant ionome modification by infection could be used to differentiate the X. fastidiosa subspecies with which the plant was infected. This report establishes host ionome modification as a phenotypic response to infection.

Recent evolutionary radiation and host plant specialization in the Xylella fastidiosa subspecies native to the United States.

The bacterial pathogen, Xylella fastidiosa, infects many plant species in the Americas, making it a good model for investigating the genetics of host adaptation. We used multilocus sequence typing (MLST) to identify isolates of the native U.S. subsp. multiplex that were largely unaffected by intersubspecific homologous recombination (IHR) and to investigate how their evolutionary history influences plant host specialization. We identified 110 "non-IHR" isolates, 2 minimally recombinant "intermediate" ones (including the subspecific type), and 31 with extensive IHR. The non-IHR and intermediate isolates defined 23 sequence types (STs) which we used to identify 22 plant hosts (73% trees) characteristic of the subspecies. Except for almond, subsp. multiplex showed no host overlap with the introduced subspecies (subspecies fastidiosa and sandyi). MLST sequences revealed that subsp. multiplex underwent recent radiation (<25% of subspecies age) which included only limited intrasubspecific recombination (ρ/θ = 0.02); only one isolated lineage (ST50 from ash) was older. A total of 20 of the STs grouped into three loose phylogenetic clusters distinguished by nonoverlapping hosts (excepting purple leaf plum): "almond," "peach," and "oak" types. These host differences were not geographical, since all three types also occurred in California. ST designation was a good indicator of host specialization. ST09, widespread in the southeastern United States, only infected oak species, and all peach isolates were ST10 (from California, Florida, and Georgia). Only ST23 had a broad host range. Hosts of related genotypes were sometimes related, but often host groupings crossed plant family or even order, suggesting that phylogenetically plastic features of hosts affect bacterial pathogenicity.

Xylella fastidiosa: an in vivo system to study possible survival strategies within citrus xylem vessels based on global gene expression analysis

Xylella fastidiosa inhabits the plant xylem, a nutrient-poor environment, so that mechanisms to sense and respond to adverse environmental conditions are extremely important for bacterial survival in the plant host. Although the complete genome sequences of different Xylella strains have been determined, little is known about stress responses and gene regulation in these organisms. In this work, a DNA microarray was constructed containing 2,600 ORFs identified in the genome sequencing project of Xylella fastidiosa 9a5c strain, and used to check global gene expression differences in the bacteria when it is infecting a symptomatic and a tolerant citrus tree. Different patterns of expression were found in each variety, suggesting that bacteria are responding differentially according to each plant xylem environment. The global gene expression profile was determined and several genes related to bacterial survival in stressed conditions were found to be differentially expressed between varieties, suggesting the involvement of different strategies for adaptation to the environment. The expression pattern of some genes related to the heat shock response, toxin and detoxification processes, adaptation to atypical conditions, repair systems as well as some regulatory genes are discussed in this paper. DNA microarray proved to be a powerful technique for global transcriptome analyses. This is one of the first studies of Xylella fastidiosa gene expression in vivo which helped to increase insight into stress responses and possible bacterial survival mechanisms in the nutrient-poor environment of xylem vessels.

An engineered innate immune defense protects grapevines from Pierce disease.

We postulated that a synergistic combination of two innate immune functions, pathogen surface recognition and lysis, in a protein chimera would lead to a robust class of engineered antimicrobial therapeutics for protection against pathogens. In support of our hypothesis, we have engineered such a chimera to protect against the gram-negative Xylella fastidiosa (Xf), which causes diseases in multiple plants of economic importance. Here we report the design and delivery of this chimera to target the Xf subspecies fastidiosa (Xff), which causes Pierce disease in grapevines and poses a great threat to the wine-growing regions of California. One domain of this chimera is an elastase that recognizes and cleaves MopB, a conserved outer membrane protein of Xff. The second domain is a lytic peptide, cecropin B, which targets conserved lipid moieties and creates pores in the Xff outer membrane. A flexible linker joins the recognition and lysis domains, thereby ensuring correct folding of the individual domains and synergistic combination of their functions. The chimera transgene is fused with an amino-terminal signal sequence to facilitate delivery of the chimera to the plant xylem, the site of Xff colonization. We demonstrate that the protein chimera expressed in the xylem is able to directly target Xff, suppress its growth, and significantly decrease the leaf scorching and xylem clogging commonly associated with Pierce disease in grapevines. We believe that similar strategies involving protein chimeras can be developed to protect against many diseases caused by human and plant pathogens.

Real-time investigation of mannosyltransferase function of a Xylella fastidiosa recombinant GumH protein using QCM-D.

Xylella fastidiosa is a gram-negative bacterium that causes serious diseases in economically important crops, including grapevine, coffee, and citrus fruits. X. fastidiosa colonizes the xylem vessels of the infected plants, thereby blocking water and nutrient transport. The genome sequence of X. fastidiosa has revealed an operon containing nine genes possibly involved in the synthesis of an exopolisaccharide (EPS) named fastidian gum that can be related with the pathogenicity of this bacterium. The α-1,3-mannosyltransferase (GumH) enzyme from X. fastidiosa is involved in fastidian gum production. GumH is responsible for the transfer of mannose from guanosine diphosphate mannose (GDP-man) to the cellobiose-pyrophosphate-polyprenol carrier lipid (CPP-Lip) during the assembly and biosynthesis of EPS. In this work, a method for real-time detection of recombinant GumH enzymatic activity was successfully developed using a Quartz Crystal Microbalance with dissipation monitoring (QCM-D). The QCM-D transducer was strategically modified with CPP-Lip by using a solid-supported lipid bilayer that makes use of a self-assembled monolayer of 1-undecanethiol. Monitoring the real-time CPP-Lip QCM-D transducer in the presence of GDP-man and GumH enzyme shows a mass increase, indicating the transfer of mannose. The real-time QCM-D determination of mannosyltransferase function was validated by a High Performance Liquid Chromatography (LC) method developed for determination of GDP produced by enzymatic reaction. LC results confirmed the activity of recombinant GumH protein, which is the first enzyme involved in the biosynthesis of the EPS from X. fastidiosa enzymatically characterized.

Spatial genetic structure of a vector-borne generalist pathogen.

Vector-borne generalist pathogens colonize several reservoir species and are usually dependent on polyphagous arthropods for dispersal; however, their spatial genetic structure is generally poorly understood. Using fast-evolving genetic markers (20 simple sequence repeat loci, resulting in a total of 119 alleles), we studied the genetic structure of the vector-borne plant-pathogenic bacterium Xylella fastidiosa in Napa Valley, CA, where it causes Pierce's disease when it is transmitted to grapevines from reservoir plants in adjacent riparian vegetation. Eighty-three different X. fastidiosa multilocus microsatellite genotypes were found in 93 isolates obtained from five vineyards, resulting in an index of clonal fraction closer to 0 and a Simpson's genotypic diversity index (D) closer to a maximum value of 1. Moderate values of Nei's gene diversity (H(Nei); average H(Nei) = 0.41) were observed for most of the X. fastidiosa populations. The low Wright's index of genetic diversity among populations calculated by the FSTAT software (Wright's F(ST) index) among population pairs (0.0096 to 0.1080) indicated a weak or absent genetic structure among the five populations; a panmictic population was inferred by Bayesian analyses (with the STRUCTURE and BAPS programs). Furthermore, a Mantel test showed no significant genetic isolation by distance when both Nei (r = -0.3459, P = 0.268) and linearized (r = -0.3106, P = 0.269) indices were used. These results suggest that the riparian vegetation from which vectors acquire the pathogen prior to inoculation of grapevines supports a diverse population of X. fastidiosa.

Structural and biochemical characterization of peroxiredoxin Qbeta from Xylella fastidiosa: catalytic mechanism and high reactivity.

The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of various plant diseases. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including cysteine-based peroxidases named peroxiredoxins. This work is a comprehensive analysis of the catalysis performed by PrxQ from X. fastidiosa (XfPrxQ) that belongs to a peroxiredoxin class still poorly characterized and previously considered as moderately reactive toward hydroperoxides. Contrary to these assumptions, our competitive kinetics studies have shown that the second-order rate constants of the peroxidase reactions of XfPrxQ with hydrogen peroxide and peroxynitrite are in the order of 10(7) and 10(6) M(-1) S(-1), respectively, which are as fast as the most efficient peroxidases. The XfPrxQ disulfides were only slightly reducible by dithiothreitol; therefore, the identification of a thioredoxin system as the probable biological reductant of XfPrxQ was a relevant finding. We also showed by site-specific mutagenesis and mass spectrometry that an intramolecular disulfide bond between Cys-47 and Cys-83 is generated during the catalytic cycle. Furthermore, we elucidated the crystal structure of XfPrxQ C47S in which Ser-47 and Cys-83 lie approximately 12.3 A apart. Therefore, significant conformational changes are required for disulfide bond formation. In fact, circular dichroism data indicated that there was a significant redox-dependent unfolding of alpha-helices, which is probably triggered by the peroxidatic cysteine oxidation. Finally, we proposed a model that takes data from this work as well data as from the literature into account.

Nutritional deficiency in citrus with symptoms of citrus variegated chlorosis disease / Deficiência nutricional em citros com sintomas da doença da clorose variegada dos citros

It is well known that citrus plants that have been infected by Xylella fastidiosa display nutritional deficiencies, probably caused by production of extracellular polymers by the bacteria that block normal nutrient flow through the xylem. The aim of this work was to study the mineral composition of specific foliar areas in different stages of infection in citrus. Thus, the concentrations of macro and micronutrients in leaves of citrus infected by X. fastidiosa were measured. Samples from four infected citrus orchards in the State of São Paulo, Brazil, were respectively collected from Santa Rita do Passa Quatro, Neves Paulista, Gavião Peixoto and Paraíso counties. The presence of X. fastidiosa in leaves was confirmed by polymerase chain reaction (PCR) using specific PCR primers. To understand the variation in leaf-nutrient content in citrus plants, we used foliar nutrient values from control (non-symptomatic) plants as a reference. Chemometric analysis showed that the deficiency of P and K in symptomatic trees for all orchards and high concentrations of Fe, Mn and Zn were observed in chlorotic areas, although other studies revealed deficiency of zinc in leaves. This is the first report showing that a correlation between chlorotic citrus leaf and higher concentrations of Fe, Mn and Zn are observed when infected and healthy plants were compared.

Já é bem conhecido que cultivares cítricas que foram infectadas pela bactéria Xylella fastidiosa apresentam deficiências nutricionais devido à produção de polímero extracelular por esta bactéria, o qual bloqueia o fluxo normal de nutriente pelo xilema. O objetivo deste trabalho foi o de estudar a composição mineral em áreas foliares específicas em diferentes fases de infecção na planta. Assim, as concentrações de macro e micronutrientes em folhas de citros infectados por X. fastidiosa foram quantificadas. Foram coletadas amostras de quatro pomares cítricos infectados localizados em: Santa Rita do Passa Quatro, Neves Paulista, Gavião Peixoto e Paraíso, no Estado de São Paulo. A presença de X. fastidiosa em folhas foi confirmada através de reação da polimerase em cadeia (PCR) usando iniciadores específicos. Para entender a variação no conteúdo de nutriente foliar em plantas cítricas, utilizou-se de valores de nutrientes foliares de plantas não sintomáticas (controle) como referência. A análise quimiométrica mostrou que a deficiência de P e K em plantas sintomáticas e concentrações altas de Fe, Mn e Zn foram presentes em áreas foliares cloróticas, embora outros estudos mostrem a deficiência de zinco em folhas. Este é o primeiro relato indicando que uma correlação entre folhas cítricas cloróticas e elevadas concentrações de Fe, Mn e Zn foi observada quando plantas infectadas e saudáveis foram comparadas.

Intrinsic bent DNA sites in the chromosomal replication origin of Xylella fastidiosa 9a5c

The features of the nucleotide sequences in both replication and promoter regions have been investigated in many organisms. Intrinsically bent DNA sites associated with transcription have been described in several prokaryotic organisms. The aim of the present study was to investigate intrinsic bent DNA sites in the segment that holds the chromosomal replication origin, oriC, of Xylella fastidiosa 9a5c. Electrophoretic behavior analyses, as well as in silico analyses of both the 2-D projection and helical parameters, were performed. The chromosomal segment analyzed contains the initial sequence of the rpmH gene, an intergenic region, the dnaA gene, the oriC sequence, and the 5' partial sequence of the dnaN gene. The analysis revealed fragments with reduced electrophoretic mobility, which indicates the presence of curved DNA segments. The analysis of the helical parameter ENDS ratio revealed three bent DNA sites (b1, b2, and b3) located in the rpmH-dnaA intergenic region, the dnaA gene, and the oriC 5' end, respectively. The chromosomal segment of X. fastidiosa analyzed here is rich in phased AT tracts and in CAnT motifs. The 2-D projection indicated a segment whose structure was determined by the cumulative effect of all bent DNA sites. Further, the in silico analysis of the three different bacterial oriC sequences indicated similar negative roll and twist >34.00° values. The DnaA box sequences, and other motifs in them, may be associated with the intrinsic DNA curvature.

The iron stimulon of Xylella fastidiosa includes genes for type IV pilus and colicin V-like bacteriocins.

Xylella fastidiosa is the etiologic agent of a wide range of plant diseases, including citrus variegated chlorosis (CVC), a major threat to citrus industry. The genomes of several strains of this phytopathogen were completely sequenced, enabling large-scale functional studies. DNA microarrays representing 2,608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c were used to investigate transcript levels during growth with different iron availabilities. When treated with the iron chelator 2,2'-dipyridyl, 193 CDS were considered up-regulated and 216 were considered down-regulated. Upon incubation with 100 microM ferric pyrophosphate, 218 and 256 CDS were considered up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription-quantitative PCR. Several CDS involved with regulatory functions, pathogenicity, and cell structure were modulated under both conditions assayed, suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to functions of pili/fimbriae. We also investigated the contribution of the ferric uptake regulator Fur to the iron stimulon of X. fastidiosa. The promoter regions of the strain 9a5c genome were screened for putative Fur boxes, and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron stimulon of X. fastidiosa, and they present novel evidence for iron regulation of pathogenicity determinants.

Crystallization and preliminary X-ray analysis of BigR, a transcription repressor from Xylella fastidiosa involved in biofilm formation.

BigR (biofilm growth-associated repressor) is a novel repressor protein that regulates the transcription of an operon implicated in biofilm growth in both Xylella fastidiosa and Agrobacterium tumefaciens. This protein binds to a palindromic TA-rich element located in the promoter of the BigR operon and strongly represses transcription of the operon. BigR contains a helix-turn-helix (HTH) domain that is found in some members of the ArsR/SmtB family of metal sensors, which control metal resistance in bacteria. Although functional studies have suggested that BigR does not act as a metal sensor, the presence of two cysteines and a methionine in its primary structure raised the possibility of BigR being a metal-ligand protein. In order to gain new insights into the protein structure and its possible interaction with a metal ion or effector ligand, BigR from X. fastidiosa was crystallized in native and selenomethionine (SeMet) labelled forms using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from native and SeMet crystals to resolutions of 1.95 and 2.2 A, respectively. Both crystals belong to space group P321 and contain one molecule per asymmetric unit.

BigR, a transcriptional repressor from plant-associated bacteria, regulates an operon implicated in biofilm growth.

Xylella fastidiosa is a plant pathogen that colonizes the xylem vessels, causing vascular occlusion due to bacterial biofilm growth. However, little is known about the molecular mechanisms driving biofilm formation in Xylella-plant interactions. Here we show that BigR (for "biofilm growth-associated repressor") is a novel helix-turn-helix repressor that controls the transcription of an operon implicated in biofilm growth. This operon, which encodes BigR, membrane proteins, and an unusual beta-lactamase-like hydrolase (BLH), is restricted to a few plant-associated bacteria, and thus, we sought to understand its regulation and function in X. fastidiosa and Agrobacterium tumefaciens. BigR binds to a palindromic AT-rich element (the BigR box) in the Xylella and Agrobacterium blh promoters and strongly represses the transcription of the operon in these cells. The BigR box overlaps with two alternative -10 regions identified in the blh promoters, and mutations in this box significantly affected transcription, indicating that BigR competes with the RNA polymerase for the same promoter site. Although BigR is similar to members of the ArsR/SmtB family of regulators, our data suggest that, in contrast to the initial prediction, it does not act as a metal sensor. Increased activity of the BigR operon was observed in both Xylella and Agrobacterium biofilms. In addition, an A. tumefaciens bigR mutant showed constitutive expression of operon genes and increased biofilm formation on glass surfaces and tobacco roots, indicating that the operon may play a role in cell adherence or biofilm development.

Xylella fastidiosa infection and ethylene exposure result in xylem and water movement disruption in grapevine shoots.

It is conventionally thought that multiplication of the xylem-limited bacterium Xylella fastidiosa (Xf) within xylem vessels is the sole factor responsible for the blockage of water movement in grapevines (Vitis vinifera) affected by Pierce's disease. However, results from our studies have provided substantial support for the idea that vessel obstructions, and likely other aspects of the Pierce's disease syndrome, result from the grapevine's active responses to the presence of Xf, rather than to the direct action of the bacterium. The use of magnetic resonance imaging (MRI) to observe the distribution of water within the xylem has allowed us to follow nondestructively the development of vascular system obstructions subsequent to inoculation of grapevines with Xf. Because we have hypothesized a role for ethylene produced in vines following infection, the impact of vine ethylene exposure on obstruction development was also followed using MRI. In both infected and ethylene-exposed plants, MRI shows that an important proportion of the xylem vessels become progressively air embolized after the treatments. The loss of xylem water-transporting function, assessed by MRI, has been also correlated with a decrease in stem-specific hydraulic conductivity (K(S)) and the presence of tyloses in the lumens of obstructed water conduits. We have observed that the ethylene production of leaves from infected grapevines is greater than that from healthy vines and, therefore, propose that ethylene may be involved in a series of cellular events that coordinates the vine's response to the pathogen.