Received date: August 14, 2015; Accepted date: August 21, 2015; Published date: August 25, 2015
Citation: Shi X, Lin H (2015) Characterization of Xylella fastidiosa popP Gene Required for Pathogenicity. J Plant Pathol Microb 6:295. doi:10.4172/2157-7471.1000295
Copyright: © 2015 Shi X, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Xylella fastidiosa (Xf) possess a two component regulatory system (TCS) PopP-PopQ which differentially regulates genes in response to environmental stimuli. To elucidate the role of popP in the pathogenicity of X. fastidiosa causing Pierce’s disease (PD) of grapevine, a site-directed deletion method and chromosome-based genetic complementation strategy were employed to create popP deletion mutant XfΔpopP and its complementary strain XfΔpopP-C. In vitro studies showed that while all strains had similar growth curves, XfΔpopP showed significant reduction in cell-cell aggregation and cell-matrix adherence. Biofilm production of XfΔpopP was about 42% less than that of wild type X. fastidiosa and XfΔpopP-C. No symptoms were observed in grapevines inoculated with XfΔpopP, whereas grapevines inoculated with wild type X. fastidiosa and XfΔpopP-C showed typical PD symptoms. Several biofilm-related genes and genes involving protein secretary systems were down regulated in XfΔpopP in compared with wild type X. fastidiosa and XfΔpopP-C. These in vitro and in planta assay results provide strong evidence that the role of PopP is required for pathogenicity of X. fastidiosa on grapevine.
Xylella fastidiosa (Xf) is a xylem-limited gram-negative plant pathogenic bacterium, which causes diseases in many plants, including Pierce’s disease (PD) in grapevine . In diseased plants, X. fastidiosa cells are embedded in the plant vessel matrix in clumps (biofilm) and result in the blockage of the water flow within the xylem vessels. The formation of biofilms allows the pathogenic bacteria to adapt in low nutrition and sub-optimal osmolarity conditions, potentially protecting them from a hostile environment . The bacteria to sense changes in the environment and to differentially regulate genes in response to environmental stimuli employ the regulatory systems. These regulatory circuits are generally involved in the two-component systems (TCS) in pathogenic bacteria . One of the TCS used by bacteria to sense the environment is TCS PhoP-PhoQ, which senses specific nutrients and regulates responses in the bacteria .
The PhoP-PhoQ is a well-studied and highly conserved TCS responsible for the regulation of genes involved in the adaptation to new environments. PhoQ is a transmembrane histidine kinase protein with a long C-terminal tail residing in the cytoplasm, involved in sensing for extracellular signal . Upon activation via environmental stimuli, PhoQ phosphorylates the corresponding response regulator PhoP, which regulates gene expression in response to environment stress . In phytopathogenic Xanthomonas oryzae pv. oryzae, TCS PhoP-PhoQ was recently shown to be a requirement for the induction of density-dependent gene expression, including genes related with biofilm formation [4-7]. PhoP-PhoQ controls virulence functions in the plant pathogen Erwinia carotovora and E. chrysanthemi [8-11]. A cell wall degrading enzyme endopolygalacturonase (designated PehA) secreted by Erwinia is transcriptionally regulated by the PhoP-PhoQ homologue PehR-PehS, and the pehR or pehS deficient of Erwinia are attenuated for virulence in tobacco seedlings .
Evidently, the PhoP-PhoQ system is required for virulence in several animal pathogens. Salmonella species are facultative intracellular pathogens that can infect a wide variety of animals causing different diseases . A phoP mutant of the etiologic agent of bubonic plagues Yersinia pestis [13,14], reduced the abilities to survival within macrophages, and increased sensitivity to low pH, oxidative stress, and high osmolarity . A phoP deficient of Shigella is hypersensitive to killing by neutrophils . The phoP or phoQ deletion mutant of Salmonella is highly attenuated for virulence [17-19]. More than forty genes regulated by phosphorylated PhoP have been shown to play important roles in the survival and the pathogenesis of Salmonella within macrophages [19,20]. While Salmonella, Shigella, and Yersinia cause different diseases in animal, inactivation of the phoP gene results in the defective for survival and virulence in their hosts.
The homologue of PopP-PopQ was identified in the X. fastidiosa genome . To elucidate the role of TCS regulator popP in X. fastidiosa, here, we characterize X. fastidiosa popP gene by comparing the phenotypes of a deletion mutant XfΔpopP, complementary strain XfΔpopP-C and its wild-type X. fastidiosa in vitro and in planta studies. These results confirm the role of popP pathogenicity in X. fastidiosa on grapevine.
Construction of XfΔpopP mutant and complementation strain XfΔpopP-C
A crossover PCR-based strategy for a site-directed deletion was used to construct an XfΔpopP mutant of X. fastidiosa . The replacement of the popP ORF with a Gentamicin cassette in the genome of XfΔpopP was confirmed by sequencing. The complementation strain XfΔpopP-C was generated by the chromosome-based genetic complementation strategy . The location of popP gene incorporating into the chromosome of XfΔpopP-C was confirmed by PCR. All amplified DNA fragments were confirmed by resequencing. The bacterial strains, plasmids, and primers used in this work are listed in Tables 1 and 2.
|Strain or plasmid||Characteristic(s)||Source or reference|
|Escherichia coli DH5||DH1 F- Φ80ΔlacZΔM15Δ(lacZYA-argF)U169||Promega|
|X. fastidiosa (Xf)|
|Temecula1||X. fastidiosawild type|
|XfΔpopP||Gentamicin (Gm) cassette replacing entire popP ORF (ΔpopP::Gm) of X. fastidiosa wild type||This work|
|XfΔpopP-C||GmrCmr; a fragment including chloramphenicol (Cm) cassette and the popP promoter and ORF of X. fastidiosa insert the chromosome of XfΔpopP||This work|
|pGEM-T Easy||Apr; cloning vector||Promega|
|pBBR1MCS-5||Gmr; broad-range plasmid||Kovach et al., |
|pGEM-T-GM||Apr Gmr; Gm cassette from pBBR1MCS-5 cloned into pGEM-T||This work|
|pUC129||Apr; cloning vector||New England Biolabs|
|pUC1679||Apr; mutagenized PCR fragment of the flanking regions of popP ORF of X. fastidiosa cloned into pUC129||This work|
|pUC16791||Apr Gmr; Gm cassette from pGEM-T-GM cloned into the AscI site of pUC1679||This work|
|pBBR1MCS||Cmr; broad-range plasmid||Kovach et al., |
|pUC129Cm||Cmr; Cm cassette from pBBR1MCS-3 cloned into pUC129||This work|
|pUCpopPXf-Exp||Apr Cmr; a fragment including the popP promoter and ORF of X. fastidiosa cloned into pUC129Cm||This work|
Table 1: Bacterial strains and plasmids used in this study.
|popPA||5’- AGTAATAGTACGATGCCAGCA-3'||This work|
|popPB||5’- CGGCGCGCCGGATCTGCTGTGCACCATGTT-3'||This work|
|popPC||5’- CGGCGCGCCGTATCTAAAGGTTATCGGCAC-3'||This work|
|popPD||5’- ATTAGAGCTTCTCCTCCAAT-3'||This work|
|popPORF For||5’- GCGTCAAGCGCATAACCAGC-3'||This work|
|popPORF Rev||5’- CTCTTCACGCATGGACGTTG-3'||This work|
|popPXFExpFor||5’- ACATGGTGCACAGCAGATCT-3'||This work|
|popPXFExpRev||5’- ATACGATAGATTTTGTGGCT-3'||This work|
|Rst31||5’-GCGTTAATTTTCGAAGTGATTCGATTGC-3'||Minsavage et al., |
|Rst33||5’-CACCATTCGTATCCCGGTG-3'||Minsavage et al., |
Table 2: Primers used in this study.
The detection of expression of popP
A modified hot-phenol RNA preparation procedure was used to extract the total RNA from wild type X. fastidiosa, XfΔpopP, and XfΔpopP-C . Bacterial cultures were incubated in 50 ml of PD2 broth at 28°C for 5 days under constant agitation at 200 rpm. After the hot-phenol extraction, RNA was dissolved in RNase-free distilled H2O and treated by Turbo DNA-free DNase (2U/μl) (Ambion, TX). The quality of isolated RNAs was determined by denaturing RNA formaldehyde gel electrophoresis . The expression of popP was analyzed by reverse transcription polymerase chain reaction (RT-PCR) with primers popPmRNAXf-F/R (Table 2), using the AccessQuick RTPCR System according to the manufacturer’s instructions (Promega, WI). The expression of DNA polymerase III related gene dnaQ served as a positive control (Table 1). RT-PCR was conducted in three independent experiments.
Wild type X. fastidiosa, XfΔpopP, and XfΔpopP-C were grown on PD2 agar medium for 5 days at 28°C, suspended in sterile deionized water, and adjusted to an OD600 of 0.10. A 20-μl of cell suspension was used to inoculate five plants of Cabernet Sauvignon by a needle inoculation procedure described previously . A water inoculation served as a negative control. The inoculated grapevines were kept on benches in a greenhouse at 24 to 32°C with 18 hr of exposure supplemented with High-Pressure Sodium lamp (20 watts per sq. ft.) . Pierce’s disease symptoms were observed two months post inoculation. The symptoms were rated on a scale from 0 to 5 as described previously  with 0 representing healthy grapevines without any scorched leaves (water control) and 5 representing plants with severely scorching symptoms. The final disease index was an average from 5 independent replications in each X. fastidiosa strain. The pathogenicity assays on grapevine were conducted in three independent inoculation experiments in greenhouse from May 2012 to July 2014.
Bacterial titer assessment
To confirm and detect the bacterial population in inoculated grapevines, 10-week post inoculation petioles (2 to 3 cm) from each grapevine inoculated with wild type X. fastidiosa, XfΔpopP, and XfΔpopP-C were harvested at 50cm above the inoculation points. Total DNAs (plant and bacteria) were prepared from the petioles according to standard DNA extraction procedure . The DNA samples were amplified by PCR using specific Rst31/33 primers  to confirm X. fastidiosa in the samples. The concentrations of DNA extracted from the leaf petiole of healthy and inoculated grapevine were quantified by PicoGreen Dye using DNA Auant Kit (Invitrogen, CA), and diluted to 5 ng/μl. The copy numbers of X. fastidiosa genomic DNA in the samples were estimated using quantitative PCR (qPCR) according to the method described earlier . The assessment of bacterial titer was evaluated from three independent experiments.
In vitro growth curves of wild type X. fastidiosa, XfΔpopP, and XfΔpopP-C were determined after 3 to 21 days of growth at 28°C as previously described . Cell concentration was determined by measuring turbidity at OD600. Cell aggregations were analyzed as described previously [31,32]. Due to the aggregation of the cells in broth, cells of all tested strains cultured in PD2 broth were dispersed by repeated pipetting or vortexing, and processed to measure the turbidity of bacterial cells at the OD540nm according to the previously described methods [31,32]. Biofilm formation analyses were done by culturing all tested stains in 96-well plates as described previously . The data were averaged from three replications.
The differentially expressed genes between wild type, XfΔpopP, and XfΔpopP-C in vitro
Previously, studies showed that PhoP differentially regulated genes involving pathogenesis on the host . Based on the genome sequences of X. fastidiosa 9a5c (a CVC strain)  and X. fastidiosa Temecula1 (a PD strain) , genes associated with putative roles in X. fastidiosa virulence, the metabolism of nucleic acids and proteins, and cellular transport and stress tolerance were selected for differential expression analysis (Table S1). Total RNA was extracted from wild type X. fastidiosa, XfΔpopP, and XfΔpopP-C strains grown in PD2 at an initial OD600nm of 0.05 of in final volumes of 2 ml in glass tubes, and were agitated at 200 rpm at 28ºC for 7 days [34,35]. Total RNA was extracted, DNase-treated, and purified to assure no contamination of DNA as described previously [26,34,35]. RT-PCR was conducted with primers specifically designed to amplify internal regions of the ORFs of the selected genes (S1). Ten microliters of amplified product was run on agarose gels and visualized under UV light. RT-PCR was conducted for three biological replicates from three independent experiments.
Physiological properties of XfΔpopP in vitro
Sequence analysis confirmed that gentamicin cassette physically replaced the entire popP ORF from start codon ATG to terminal codon TAA in the wild type X. fastidiosa genome. The expression of popP in XfΔpopP was not detectable with RT-PCR. Successfully complemented cells from chloramphenicol-resistance clones were confirmed by PCR. Sequence analysis further confirmed that popP gene (promoter and ORF) was incorporated into the site between PD0702 and PD0703 of chromosome of XfΔpopP as expected. Stable XfΔpopP and XfΔpopP-C colonies were obtained after five to eight streaks on PD2 agar medium supplemented with 10μg/ml Gm or 10μg/ml Cm, respectively.
In vitro growth curves showed that XfΔpopP reached the exponential and stationary phase in a manner similar to wild type X. fastidiosa after twelve days in culture (data not shown). Whereas there was no obvious difference in colony morphology between wild type X. fastidiosa and XfΔpopP-C, XfΔpopP had less sticky colonies when touched with a bacteriological loop (data not shown). The quantitative assessment of cell aggregation showed that XfΔpopP had about a 36% reduction in cell-to-cell aggregation comparing to that of wild type X. fastidiosa (Figure 1A). However, XfΔpopP-C had about a 19% reduction in cellto- cell aggregation in compared with the wild type X. fastidiosa. Biofilm formation by XfΔpopP was found to have about 42% less than that of wild type X. fastidiosa after ten days of static incubation determined by the crystal violet assay while complement strain XfΔpopP-C had about only 16.6% of reduction in biofilm formation in comparison with that of wild type X. fastidiosa (Figure 1B).
Figure 1: Quantitative assessment of cell-to-cell aggregation and biofilm formation of wild type X. fastidiosa, XfΔpopP and complement XfΔpopP-C. A) The percentages of aggregated cells of X. fastidiosa strains in PD2 broth . B) Quantitative assessment of biofilm formation of all tested X. fastidiosa strains. Averages and standard deviations of three independent experiments are shown for all assessment experiments. Error bars indicate standard deviation. Letters indicate groups assigned by significant difference test (t test, P<0.05).
Pathogenicity of XfΔpopP and XfΔpopP-C on grapevine
In contrast to the grapevines inoculated with wild type X. fastidiosa and XfΔpopP-C, grapevines infected by XfΔpopP showed no symptom 12 weeks post inoculation (Figures 2A and 2B). Mock-inoculated control grapevines did not show any PD symptoms (Figures 2A and 2B). All diseased grapevines were detected positive for X. fastidiosa by PCR with X. fastidiosa specific primers Rst31/33 (data not shown), and grapevines in mock-inoculated group were detected negative. To further evaluate of the reduced virulence of XfΔpopP, the bacterial titers in infected grapevines with wild type X fastidiosa, XfΔpopP, and XfΔpopP-C were estimated by qPCR. The bacterial populations in grapevines infected with XfΔpopP showed about 66% less than those of grapevines inoculated with wild type X. fastidiosa (Figure 2C).
Figure 2: PD progression of Cabernet sauvignon grapevines inoculated with wild type X. fastidiosa, XfΔpopP, and XfΔpopP-C. A) Cabernet sauvignon grapevines infected with wild type X. fastidiosa and XfΔpopP-C, showing typical PD symptom of the scorched leaves, whereas no PD symptom was shown on grapevines infected with XfΔpopP. Controls are grapevines inoculated with water, which all shown no PD symptom. B) Disease severity of grapevine inoculated by all tested X. fastidiosa strains. Disease severity was based on a visual disease scale of 0 to 5 and was assessed 20 weeks after inoculation . C) The genomic DNA copy number of wild type X. fastidiosa, XfΔpopP, and XfΔpopP-C in infected grapevines was estimated by qPCR. Averages and standard deviations of 5 independent replicates. Error bars indicate standard deviation. Letters indicate groups assigned by significant difference test (t test, P<0.05).
popP regulated gene expression
Most of selected genes were not differentially expressed among tested strains. However, TCS regulator popP, surface-exposed outer membrane gene uspA1, gum synthesized gene gumB, two-component system regulatory gene algR, type II secretory pathway protein-export membrane gene secG, and type V secretory pathway gene mttC were down regulated in XfΔpopP in comparison to wild type X. fastidiosa and XfΔpopP-C (Figure 3). Both housekeeping genes, tapB (temperature acclimation) and dnaQ (DNA polymerase III) were expressed almost equally in wild type X. fastidiosa, XfΔpopP, and XfΔpopP-C.
Figure 3: The detection of differential expression of virulence-related genes in wild type X. fastidiosa, XfΔpopP, and XfΔpopP-C by RT-PCR. Controls included positive control (the detectable expression of dnaQ) and negative controls [RT(-) and NTC], where RT(-) was a reaction with all components except RT reverse enzyme and NTC was a reaction with all components except RNA template. RT-PCR was conducted in three independent experiments.
TCS PhoP-PhoQ system of Salmonella and Yersinia pestis is responsible for the virulence within animal host cells [13-15]. PhoPPhoQ of X. oryzae pv. oryzae (Xoo), E. carotovora, and E. chrysanthemi appears to be cell density-dependent in plant host [4-10]. In this study, while both wild type and popP mutant X. fastidiosa strains showed similar growth curves in vitro, grapevines infected with popP deletion strain have significantly decreased bacterial populations in comparison to the bacterial populations of grapevines inoculated with wild type. The severity of the disease has been known to be positively correlated with the titer of X. fastidiosa. Fritschi, et al.  investigated 18 grape genotypes in response to X. fastidiosa infection under greenhouse conditions and concluded that an inverse relationship was found between the level of PD resistance and bacterial populations in grapevines. No PD symptom was observed in the grapevines inoculated with popP deletion strain in compared with severe PD symptom developed by grapevines infected with wild type X. fastidiosa and complementary strain XfΔpopP-C consistently observed in repeated greenhouse inoculation tests. Similarity results were recently reported on the pathogenicity of Xf popP . Our results demonstrate that popP is an important virulence factors and plays critical role for the survivals and the pathogenicity of X. fastidiosa.
Colonization, proliferation and biofilm formation of X. fastidiosa in water-conducting xylem vessels of host plants result in the blockage of water and nutrient in diseased plants [38,39]. In this study, the abilities of cell aggregation and biofilm formation of popP deficient strain were decreased while complementary strain XfΔpopP-C significantly restored biofilm formation. popP is required for the regulation of density-dependent gene expression in X. fastidiosa, including genes responsible for biofilm formation. Biofilms are complex structures involving X. fastidiosa cells and an extracellular matrix. X. fastidiosa produces abundant multiple putative afimbrial adhesins, such as exopolysaccharide (EPS), lipopolysaccharide (LPS), and surface proteins, which contribute to the virulence of X. fastidiosa by attaching to the xylem wall in the plant and enhancing biofilm formation [33,40-43]. Previously study showed that X. fastidiosa EPS was coded by a cluster of nine gum genes closely related to the xanthan gum operon of Xanthomonas campestris pv. campestris . The disruption of gumB in X. fastidiosa did not affect exopolysaccharide production, the gumB mutant, however, does show a reduced capacity to form biofilm . The two-component system regulatory algR in X. fastidiosa was previously reported to regulate the synthesis of the LPS, which could play a role in biofilm formation and cell attachment as well [2,45-47]. Surface-exposed outer membrane gene uspA1 was shown to be related to the virulence of X. fastidiosa . Interestingly, in this study the expressions of gumB, uspA1, and algR were repressed in popP mutant strain in contrast to the wild type X. fastidiosa, the mutation of popP might reduce the production of LPS, resulting in the reduced capacity to form biofilm and cell-cell aggregation, both are important events of X. fastidiosa in successful colonization of the grapevine xylem. This might account for the significantly reduced disease severity of grapevines inoculated with XfΔpopP.
The ability of X. fastidiosa to colonize grapevines and to incite disease development is also dependent upon the capacity of this bacterium to produce a diverse set of virulence factors [33,49], which must be secreted to the bacterial cell surface or released them into the external environment. In X. fastidiosa, the important virulence determinants are delivered to the bacterial cell surface through type I, type II, and type V secretion systems [21,33,50,51]. It has been demonstrated that these actively secreted proteins are associated with bacterial pathogenicity or suppressing host defenses [52-54]. Matsumoto et al.  showed that XatA was one of six members of the AT-1 autotransporter family secreted by type V secretion system in X. fastidiosa . xatA mutant of X. fastidiosa shows a significant decrease in cells autoaggregation, biofilm formation, and disease symptoms on grapevines . In this study type II sec-dependent secretion system secG and type V secretory pathway mttC were positively regulated by popP. The loss of popP in X. fastidiosa might affect the synthesis of type II and type V secretory pathway, resulting in the blocking of the secretions of virulence factors, and the lost the pathogenicity on grapevine.
Although TCS PhoPQ is believed to be primarily involved in phosphate metabolism as it regulates the expression of a nonspecific acid phosphatase , it has been experimentally verified that the PhoPQ regulates a range of genes responsible for virulence via the sensing of extracellular signal, such as nutrient and antimicrobial peptides [3,57]. Previous studies indicated the PhoPQ was necessary for X. oryzae pv. oryzae to be able to tolerate a change in acidic conditions (pH 4.5 to 6.5) after invasion of its host rice . Those results imply that PhoPQ signal transduction in phytopathogenic bacteria might be induced by fluctuations pH in host and the presence of extracellular molecule(s). Since grape xylem is a nutritionally limited environment, subject to a fluctuating pH and differentially available minerals [34,59-61], X. fastidiosa deficient in popP may be incapable of sensing the extracellular molecule(s) or fluctuations of pH in host or producing the biofilm formation-related factors, secrete virulence factors, resulting in complete loss of pathogenicity on grapevine. The preliminary studies identified several heat shock proteins, translation factors, and virulence regulator likely involving interactions with PopP, implicating that PopP in X. fastidiosa might regulate a wide variety of biological processes in sensing host xylem fluctuation environmental stimuli to regulate the virulence factor of X. fastidiosa in hosts [64,65].
While the exact molecular mechanisms by which PopP regulates in X. fastidiosa require further investigation, results from this study demonstrate that PopP plays a key role in controlling a variety of genes affecting biofilm formation and secreted virulence factors. These findings suggest that PopP is a critical component of the regulatory hierarchy governing the pathogenicity of X. fastidiosa in response to environmental fluctuations. These results constitute the first attempt in characterizing the pathogenicity role of popP to PD of grapevines, which provides new information for understanding the pathogenicity in X. fastidiosa.
Thanks to Guimei Qi for technical support. Our appreciation is also extended to Dr. Joseph Morse for comments on the manuscript. Funding for the project was supported by the United States Department of Agriculture, Agricultural Research Service. Trade names or commercial products in this publication are mentioned solely for the purpose of providing specific information and does not imply recommendation or endorsement by the United States Department of Agriculture. USDA is equal opportunity provider.
|Table 1||Table 2|
|Figure 1||Figure 2||Figure 3|