Gene Disruption by Homologous Recombination in the Xylella fastidiosa Citrus Variegated Chlorosis Strain

Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for β-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to β-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant.     

Effects of DNA Size on Transformation and Recombination Efficiencies in Xylella fastidiosa

Horizontally transferred DNA acquired through transformation and recombination has the potential to contribute to the diversity and evolution of naturally competent bacteria. However, many different factors affect the efficiency with which DNA can be transformed and recombined. In this study, we determined how the size of both homologous and nonhomologous regions affects transformation and recombination efficiencies in Xylella fastidiosa, a naturally competent generalist pathogen responsible for many emerging plant diseases. Our experimental data indicate that 96 bp of flanking homology is sufficient to initiate recombination, with recombination efficiencies increasing exponentially with the size of the homologous flanking region up to 1 kb. Recombination efficiencies also decreased with the size of the nonhomologous insert, with no recombination detected when 6 kb of nonhomologous DNA was flanked on either side by 1 kb of homologous sequences. Upon analyzing sequenced X. fastidiosa subsp. fastidiosa genomes for evidence of allele conversion, we estimated the mean size of recombination events to be 1,906 bp, with each event modifying, on average, 1.79% of the nucleotides in the recombined region. There is increasing evidence that horizontally acquired genes significantly affect the genetic diversity of X. fastidiosa, and DNA acquired through natural transformation could be a prominent mode of this horizontal transfer.     

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.     

Calcium-Enhanced Twitching Motility in Xylella fastidiosa Is Linked to a Single PilY1 Homolog

The plant-pathogenic bacterium Xylella fastidiosa is restricted to the xylem vessel environment, where mineral nutrients are transported through the plant host; therefore, changes in the concentrations of these elements likely impact the growth and virulence of this bacterium. Twitching motility, dependent on type IV pili (TFP), is required for movement against the transpiration stream that results in basipetal colonization. We previously demonstrated that calcium (Ca) increases the motility of X. fastidiosa, although the mechanism was unknown. PilY1 is a TFP structural protein recently shown to bind Ca and to regulate twitching and adhesion in bacterial pathogens of humans. Sequence analysis identified three pilY1 homologs in X. fastidiosa (PD0023, PD0502, and PD1611), one of which (PD1611) contains a Ca-binding motif. Separate deletions of PD0023 and PD1611 resulted in mutants that still showed twitching motility and were not impaired in attachment or biofilm formation. However, the response of increased twitching at higher Ca concentrations was lost in the pilY1-1611 mutant. Ca does not modulate the expression of any of the X. fastidiosa PilY1 homologs, although it increases the expression of the retraction ATPase pilT during active movement. The evidence presented here suggests functional differences between the PilY1 homologs, which may provide X. fastidiosa with an adaptive advantage in environments with high Ca concentrations, such as xylem sap.     

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.     

Development and Application of Monoclonal Antibodies Against Xylella fastidiosa, the Causal Bacterium of Pear Leaf Scorch

Ten stable hybridoma cell lines, M As l -10, secreting monoclonal antibodies specific to the causal bacterium of pear leaf scorch (PLS), Xylella fastidiosa. were produced. The monoclonal antibodies can detect 3 × 10⁵ PLS-bacterium cells by indirect enzyme-linked immunosorbent assay (ELISA). In the antibody titer determination by indirect ELISA, hybridoma-culture supernatant from clone MA4 had the highest titer of 20480. In the antibody specificity tests, nine of the 10 monoclonal antibodies did not cross-react with 14 other bacterial strains belonging to nine genera. Only the antibody from hybridoma clone MAI cross-reacted with Xanthomonas campestris pv. cam-pestris and X. campestris pv. vesicatoria. In western blot analysis, all the monoclonal antibodies recognized the major 46.9-kDa polypeptide from all 12 X. fastidiosa strains and a distinct 21.5-kDa polypeptide only from PLS bacterium. In tissue-blotting detection, the PLS bacteria were specifically detected in blots of tissue sections from infected pear with the antibodies developed.     

Xylella fastidiosa in Europe: From the Introduction to the Current Status

Xylella fastidiosa is xylem-limited bacterium capable of infecting a wide range of host plants, resulting in Pierce’s disease in grapevine, citrus variegated chlorosis, olive quick decline syndrome, peach phony disease, plum leaf scald, alfalfa dwarf, margin necrosis and leaf scorch affecting oleander, coffee, almond, pecan, mulberry, red maple, oak, and other types of cultivated and ornamental plants and forest trees. In the European Union, X. fastidiosa is listed as a quarantine organism. Since its first outbreak in the Apulia region of southern Italy in 2013 where it caused devastating disease on Olea europaea (called olive leaf scorch and quick decline), X. fastidiosa continued to spread and successfully established in some European countries (Corsica and PACA in France, Balearic Islands, Madrid and Comunitat Valenciana in Spain, and Porto in Portugal). The most recent data for Europe indicates that X. fastidiosa is present on 174 hosts, 25 of which were newly identified in 2021 (with further five hosts discovered in other parts of the world in the same year). From the six reported subspecies of X. fastidiosa worldwide, four have been recorded in European countries (fastidiosa, multiplex, pauca, and sandyi). Currently confirmed X. fastidiosa vector species are Philaenus spumarius, Neophilaenus campestris, and Philaenus italosignus, whereby only P. spumarius (which has been identified as the key vector in Apulia, Italy) is also present in Americas. X. fastidiosa control is currently based on pathogen-free propagation plant material, eradication, territory demarcation, and vector control, as well as use of resistant plant cultivars and bactericidal treatments.     

Natural competence and recombination in the plant pathogen Xylella fastidiosa

Homologous recombination is one of many forces contributing to the diversity, adaptation, and emergence of pathogens. For naturally competent bacteria, transformation is one possible route for the acquisition of novel genetic material. This study demonstrates that Xylella fastidiosa, a generalist bacterial plant pathogen responsible for many emerging plant diseases, is naturally competent and able to homologously recombine exogenous DNA into its genome. Several factors that affect transformation and recombination efficiencies, such as nutrient availability, growth stage, and methylation of transforming DNA, were identified. Recombination was observed in at least one out of every 10(6) cells when exogenous plasmid DNA was supplied and one out of every 10(7) cells when different strains were grown together in vitro. Based on previous genomic studies and experimental data presented here, there is mounting evidence that recombination can occur at relatively high rates and could play a large role in shaping the genetic diversity of X. fastidiosa.     

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.     

Presence of Cicadomorpha in olive orchards of Greece with special reference to Xylella fastidiosa vectors

Insects of the infraorder Cicadomorpha cause serious economic impact on agriculture because of pathogens’ transmission. The vector-borne bacterium Xylella fastidiosa, which is associated with olive quick decline syndrome, is exclusively transmitted by insects of this infraorder and more specific by sharpshooters and spittlebugs. Efficient control practices of the insect vectors are necessary for the control of the disease in cases of outbreaks. Therefore, the knowledge of the vectors' species as well as the robust understanding of their biology and ecology is of crucial importance. In the present study, extensive sampling was conducted in 28 olive orchards of Greece during the years 2017 and 2018 aiming to investigate which species of this infraorder are present, with special interest to those which are considered as vectors of the bacterium. During the surveys, 4,350 Cicadomorpha insects were collected. Forty-two, five and one species of the families Cicadellidae, Aphrophoridae and Cercopidae, respectively, were identified. The results of the surveys revealed that the main potential vectors of X. fastidiosa in Greek olive orchards are the spittlebugs Philaenus spumarius and Neophilaenus campestris. Those species have wide distribution in Greece and are present for a long period during the year, and they inhabit both the ground vegetation and the olive trees' canopy. A representative portion of the captured spittlebugs was analysed with molecular methods, and the analysis confirmed the absence of X. fastidiosa in the tested individuals.     

Whole-genome analysis of transporters in the plant pathogen Xylella fastidiosa.

The transport systems of the first completely sequenced genome of a plant parasite, Xylella fastidiosa, were analyzed. In all, 209 proteins were classified here as constitutive members of transport families; thus, we have identified 69 new transporters in addition to the 140 previously annotated. The analysis lead to several hints on potential ways of controlling the disease it causes on citrus trees. An ADP:ATP translocator, previously found in intracellular parasites only, was found in X. fastidiosa. A P-type ATPase is missing-among the 24 completely sequenced eubacteria to date, only three (including X. fastidiosa) do not have a P-type ATPase, and they are all parasites transmitted by insect vectors. An incomplete phosphotransferase system (PTS) was found, without the permease subunits-we conjecture either that they are among the hypothetical proteins or that the PTS plays a solely metabolic regulatory role. We propose that the Ttg2 ABC system might be an import system eventually involved in glutamate import rather than a toluene exporter, as previously annotated. X. fastidiosa exhibits fewer proteins with > or =4 alpha-helical transmembrane spanners than any other completely sequenced prokaryote to date. X. fastidiosa has only 2.7% of all open reading frames identifiable as major transporters, which puts it as the eubacterium having the lowest percentage of open reading frames involved in transport, closer to two archaea, Methanococcus jannaschii (2.4%) and Methanobacterium thermoautotrophicum (2.4%).     

Heteroduplex DNA Formation Is Associated with Replication and Recombination in Poxvirus-Infected Cells

Poxviruses are large DNA viruses that replicate in the cytoplasm of infected cells and recombine at high frequencies. Calcium phosphate precipitates were used to cotransfect Shope fibroma virus-infected cells with different DNA substrates and the recombinant products assayed by genetic and biochemical methods. We have shown previously that bacteriophage lambda DNAs can be used as substrates in these experiments and recombinants assayed on Escherichia coli following DNA recovery and in vitro packaging. Using this assay it was observed that 2-3% of the phage recovered from crosses between point mutants retained heteroduplex at at least one of the mutant sites. The reliability of this genetic analysis was confirmed using DNA substrates that permitted the direct detection of heteroduplex molecules by denaturant gel electrophoresis and Southern blotting. It was further noted that heteroduplex formation coincided with the onset of both replication and recombination suggesting that poxviruses, like certain bacteriophage, make no clear biochemical distinction between these three processes. The fraction of heteroduplex molecules peaked about 12-hr postinfection then declined later in the infection. This decline was probably due to DNA replication rather than mismatch repair because, while high levels of induced DNA polymerase persisted beyond the time of maximal heteroduplex recovery, we were unable to detect any type of mismatch repair activity in cytoplasmic extracts. These results suggest that, although heteroduplex molecules are formed during the progress of poxviral infection, gene conversion through mismatch repair probably does not produce most of the recombinants. The significance of these observations are discussed considering some of the unique properties of poxviral biology.     

Twitching motility and biofilm formation are associated with tonB1 in Xylella fastidiosa

A mutation in the Xylella fastidiosa tonB1 gene resulted in loss of twitching motility and in significantly less biofilm formation as compared with a wild type. The altered motility and biofilm phenotypes were restored by complementation with a functional copy of the gene. The mutation affected virulence as measured by Pierce's disease symptoms on grapevines. The role of TonB1 in twitching and biofilm formation appears to be independent of the characteristic iron-uptake function of this protein. This is the first report demonstrating a functional role for a tonB homolog in X. fastidiosa .     

Fastidian gum: the Xylella fastidiosa exopolysaccharide possibly involved in bacterial pathogenicity

The Gram-negative bacterium Xylella fastidiosa was the first plant pathogen to be completely sequenced. This species causes several economically important plant diseases, including citrus variegated chlorosis (CVC). Analysis of the genomic sequence of X. fastidiosa revealed a 12 kb DNA fragment containing an operon closely related to the gum operon of Xanthomonas campestris. The presence of all genes involved in the synthesis of sugar precursors, existence of exopolysaccharide (EPS) production regulators in the genome, and the absence of three of the X. campestris gum genes suggested that X. fastidiosa is able to synthesize an EPS different from that of xanthan gum. This novel EPS probably consists of polymerized tetrasaccharide repeating units assembled by the sequential addition of glucose-1-phosphate, glucose, mannose and glucuronic acid on a polyprenol phosphate carrier.