Fig 1.
Natural transformation and twitching motility phenotypes of X. fastidiosa mutant strains used in this study.
Quantification of natural transformation was performed by enumerating total viable and recombinant culturable cells and results are expressed as the ratio of recipient cells transformed (recombination frequency; values shown in the x-axis of the chart). Twitching motility was determined by spotting cells on agar plates and measuring the movement fringe width after 4 days of growth at 28°C (values shown in the y-axis of the chart). Results of mutant strains for paralogous genes are shown using the same color for text and symbol. The non-motile and non-recombinant mutants are shown as a black dot. The WT is represented as a dark blue triangle and all other mutants are represented as black triangles. Data represent means and standard errors. Different letters in parenthesis indicate significant difference in fringe width as analyzed by ANOVA followed by Tukey’s HSD multiple comparisons of means (P<0.05; n = three to 14 independent replicates with eight to 48 internal replicates each). * and ** indicate significant difference (P<0.05 and P<0.005, respectively) of recombination frequency through natural competence in comparison to the WT as determined using Student’s t-test (n = three to 21 independent replicates with two internal replicates each). The detection limit for recombination frequency was 10−7 and for twitching motility was 10 μm. Mutants below the detection limit are shown in the non-motile and non-recombinant group.
Fig 2.
Deletion of pil genes negatively affects virulence of X. fastidiosa.
a Disease severity progression over time in inoculated tobacco plants. X. fastidiosa WT and mutant strains were inoculated into Nicotiana tabacum L. cv. Petite Havana SR1 plants (PBS mock inoculation used as control). Mutant strains chosen for inoculation presented different twitching motility phenotypes, including higher twitching motility (ΔpilA1 and ΔpilS), lower twitching motility (ΔpilA2) and non-motility (ΔpilA1pilA2, ΔpilQ and ΔpilR). Leaf scorch symptoms were recorded for measurements of disease incidence and severity once a week during nine weeks after appearance of the first disease symptoms. At the final time point of evaluation, disease incidence in all inoculated plants reached 100%, except for plants inoculated with ΔpilA1, which reached 88.88% disease incidence (±11.11%, standard error). On the other hand, disease severity reached 96% in the WT, 35% in ΔpilA1, 86% in ΔpilA2, 57% in ΔpilA1pilA2, 73% in ΔpilQ, 72% in ΔpilR and 56% in ΔpilS. Data represent means and standard errors from two independent experiments (n = seven to ten plants in each independent experiment). b Mean AUDPC per treatment group. AUDPC was calculated using data from disease severity over nine weeks after first disease symptom appearance. WT is highlighted in blue, and the dashed blue line indicates the mean value of AUDPC for WT-inoculated plants. AUDPC was significantly lower for plants inoculated with all mutant strains in comparison to WT-inoculated plants, except for ΔpilA2. Data represent means and standard errors. Different letters on top of bars indicate significant difference as analyzed by ANOVA followed by Tukey’s HSD multiple comparisons of means (P<0.05; n = two independent experiments with seven to ten plants each). c X. fastidiosa in planta population determined by qPCR at the last time point of evaluation. The population of WT and mutant strains throughout inoculated plants was calculated using petioles of basal and top leaves. ΔpilA1pilA2 cells showed a defect in colonizing the top leaf of plant hosts, while ΔpilQ and ΔpilR showed a significant higher population in the basal leaf of infected plants. This shows that mutant strains still move inside the xylem of plant hosts, but absence of deleted genes impairs full symptom development. Data represent means and standard errors. Different letters on top of bars indicate significant difference as analyzed by ANOVA followed by Tukey’s HSD multiple comparisons of means (P<0.05; n = two independent experiments with three leaf replicates each). d Figure panel with representative pictures of leaf scorch symptoms in WT- and mutant strains-inoculated plants (top and basal leaves), as well as control plants (PBS mock inoculation). Only WT-inoculated plants consistently presented severe leaf scorch symptoms. Similar events were captured in two independent experiments.
Fig 3.
a Transmission electron microscopy micrographs of pilus formation by X. fastidiosa WT and ΔfimT3 cells. Arrows are pointing to TFP. Similar events were captured in two independent experiments. Images were captured at 31,500× magnification. Scale bars, 500 nm. b Uptake of Cy-3-labeled pAX1-Cm plasmid into a DNase I resistant state by X. fastidiosa cells observed using a fluorescence microscope. The images correspond to the bright field (left), Cy-3 channel (center) and merged images (right). In merged images, arrows are pointing to fluorescent DNA foci at the cell poles. Similar events were captured in three independent experiments. Images were captured at 100× magnification. Scale bars, 1.5 μm. c DNA-binding ability of purified FimT1s, FimT2s and FimT3s assessed by agarose EMSA. Similar events were captured in three independent experiments. d Surface electrostatics representation of FimT3 merged with its ribbon representation. The arginine amino acid residues (R160 and R162) that were mutated for functional studies are highlighted in the figure. The electrostatic potential (eV) color scale is shown in the figure. e DNA-binding ability of purified FimT3s and FimT3s-R160AR162A assessed by agarose EMSA. Similar events were captured in three independent experiments. f Titration of the DNA binding activity of FimT3s and FimT3s-R160AR162A by native acrylamide EMSA. Similar events were captured in two independent experiments. g Densitometry analysis of the DNA binding activity of FimT3s and FimT3s-R160AR162A in acrylamide EMSA. The fluorescent intensity of the shifted DNA bands of Cy-3-labeled Km resistance cassette presented in f was measured using ImageJ. The fluorescent intensity of shifted bands when treated only with 1, 2, 5, and 10 μM of each protein was measured, since they presented higher shifts in electrophoretic mobility within this protein concentration range. h Area under the fluorescence curve. The area under the fluorescence curves in g was calculated to quantify the DNA-binding affinity of FimT3s and FimT3s-R160AR162A. Statistical significance was determined using Student’s t-test (* indicates P<0.05 in comparison to the wild-type protein; n = two independent replicates). Multiple bands of the pAX1-Cm plasmid observed in c and e are due to the multiple forms of the plasmid after purification, with each form displaying different sizes in agarose gels.
Fig 4.
Phylogenetic tree based on nucleotide sequences of the three fimT paralogs from Xanthomonadaceae bacterial strains with whole-genome sequences available.
The phylogenetic tree was built using the Maximum-likelihood method and visualized using FigTree. Branches with bootstrap values below 70% were collapsed, except where indicated. This was performed to keep conciseness of the figure while indicating the different clusters for fimT1, fimT2 and fimT3. X. fastidiosa is highlighted in blue within the tree. When known, clade I Xanthomonas spp. are highlighted in orange, and clade II Xanthomonas spp. are highlighted in purple. Bacterial species encoding all three fimT paralogs are indicated in bold, while those encoding two paralogs are shown in italics and those encoding only one paralog are underlined within the tree. The order of appearance of bacterial species in collapsed branches follows the original order of appearance within the tree. For representation purposes, branches are highlighted by color according to each fimT paralog. Brown: fimT1; Dark blue: fimT2; Red: fimT3. *** Indicates clusters within the fimT3 clade which contain few FimT3 sequences with no GRxR motif (see S5 Table and S14 Fig). Abbreviations are the following. Coralloluteibacterium: Cst–C. stylophorae. Luteimonas: Lab–L. abyssi; Lae–L. aestuarii; Laq–L. aquatica; Lar–L. arsenica; Lch–L. chenhongjianii; Lcu–L. cucumeris; Lde–L. deserti; Lfr–L. fraxinea; Lgi–L. gilva; Lgr–L. granuli; Lhu–L. huabeiensis; Llu–L. lumbrici; Lma–L. marina; Lme–L. mephitis; Lpd–L. padinae; Lpn–L. panaciterrae; Lsp–L. sp.; Lte–L. terrae; Lti–L. terricola; Lwe–L. wenzhouensis; Lyi–L. yindakuii. Lysobacter: Lys–L. sp. Pseudoxanthomonas: Pbe–P. beigongshangi; Pbr–P. broegbernensis; Pco–P. composti; Pda–P. daejeonensis; Pdo–P. dokdonensis; Pge–P. gei; Phe–P. helianthi; Pin–P. indica; Pja–P. japonensis; Pji–P. jiangjuensis; Pkl–P. kalamensis; Pko–P. kaohsiungensis; Pkr–P. koreensis; Pme–P. mexicana; Psc–P. sacheonensis; Psn–P. sangjuensis; Psp–P. sp.; Psx–P. spadix; Psu–P. suwonensis; Ptw–P. taiwanensis; Pwi–P. winnipegensis; Pwu–P. wuyuanensis; Pye–P. yeongjuensis. Rehaibacterium: Rte–R. terrae. Silanimonas: Sle–S. lenta. Stenotrophomonas: Sac–S. acidaminiphila; Sbe–S. bentonitica; Sch–S. chelatiphaga; Scy–S. cyclobalanopsidis; Sda–S. daejeonensis; Sge–S. geniculata; Sgi–S. ginsengisoli; Shu–S. humi; Sin–S. indicatrix; Skr–S. koreensis; Sla–S. lactitubi; Sma–S. maltophilia; Sne–S. nematodicola; Sni–S. nitritireducens; Spn–S. panacihumi; Spv–S. pavanii; Spi–S. pictorum; Srh–S. rhizophila; Sse–S. sepilia; Ssp–S. sp; Ste–S. terrae; Stu–S. tumulicola. Vulcaniibacterium: Vge–V. gelatinicum; Vte–V. tengchongense; Vth–V. thermophilum. Xanthomonas: Xab–X. albilineans; Xaf–X. alfalfae; Xar–X. arboricola; Xax–X. axonopodis; Xbr–X. bromi; Xcm–X. campestris; Xcn–X. cannabis; Xcs–X. cassavae; Xci–X. cissicola; Xct–X. citri; Xco–X. codiaei; Xcu–X. curcubitae; Xdy–X. dyei; Xer–X. euroxanthea; Xeu–X. euvesicatoria; Xfl–X. floridensis; Xfr–X. fragariae; Xho–X. hortorum; Xha–X. hyacinthi; Xhd–X. hydrangea; Xml–X. maliensis; Xms–X. massiliensis; Xme–X. melonis; Xna–X. nasturtii; Xor–X. oryzae; Xpe–X. perforans; Xpi–X. pisi; Xpo–X. populi; Xpr–X. prunicola; Xsa–X. sacchari; Xso–X. sontii; Xsp–X. sp.; Xth–X. theicola; Xtr–X. translucens; Xva–X. vasicola; Xve–X. vesicatoria. Xylella: Xtw–X. taiwanensis.
Fig 5.
Schematic representation of the functional role of TFP molecular components in natural competence and twitching motility of X. fastidiosa.
The figure shows the structure of a type IV pilus (PilA1, PilA2 and PilA3), the alignment subcomplex (PilM, PilN, PilO and PilP), the inner membrane motor subcomplex (PilB, PilC, PilD, PilT and PilU), the pore subcomplex (PilF and PilQ), minor pilins (PilE1, PilE2, PilV1, PilV2, PilW1, PilW2, PilX1, PilX2, FimT1, FimT2 and FimT3), TFP tip adhesins (PilY1-1, PilY1-2 and PilY1-3), as well as regulatory proteins (PilG, PilH, PilI, PilJ, PilL, PilR, PilS, PilZ, ChpB and ChpC). Proteins in the figure are color-coded according to their phenotype upon knockout mutation and thus functional role. More information about the role of each protein is described in S1 Table, throughout the manuscript and in SI Discussion in S1 Text. OM–outer membrane; P–periplasm; IM–inner membrane.