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Appl Environ Microbiol. Mar 2007; 73(5): 1612–1621.
Published online Jan 12, 2007. doi:  10.1128/AEM.00261-06
PMCID: PMC1828793

In Planta Horizontal Transfer of a Major Pathogenicity Effector Gene[down-pointing small open triangle]

Abstract

Xanthomonas citri pv. citri is a clonal group of strains that causes citrus canker disease and appears to have originated in Asia. A phylogenetically distinct clonal group that causes identical disease symptoms on susceptible citrus, X. citri pv. aurantifolii, arose more recently in South America. Genomes of X. citri pv. aurantifolii strains carry two DNA fragments that hybridize to pthA, an X. citri pv. citri gene which encodes a major type III pathogenicity effector protein that is absolutely required to cause citrus canker. Marker interruption mutagenesis and complementation revealed that X. citri pv. aurantifolii strain B69 carried one functional pthA homolog, designated pthB, that was required to cause cankers on citrus. Gene pthB was found among 38 open reading frames on a 37,106-bp plasmid, designated pXcB, which was sequenced and annotated. No additional pathogenicity effectors were found on pXcB, but 11 out of 38 open reading frames appeared to encode a type IV transfer system. pXcB transferred horizontally in planta, without added selection, from B69 to a nonpathogenic X. citri pv. citri (pthA::Tn5) mutant strain, fully restoring canker. In planta transfer efficiencies were very high (>0.1%/recipient) and equivalent to those observed for agar medium with antibiotic selection, indicating that pthB conferred a strong selective advantage to the recipient strain. A single pathogenicity effector that can confer a distinct selective advantage in planta may both facilitate plasmid survival following horizontal gene transfer and account for the origination of phylogenetically distinct groups of strains causing identical disease symptoms.

The genus Xanthomonas is comprised of strains that exhibit a high level of host specificity; over 125 different pathogenic variants (pathovars) of X. campestris that differ primarily in host range have been described (29). Host specificity in Xanthomonas can be due to gene-for-gene interactions involving avirulence genes that act in a negative fashion to limit host range (18, 29, 38) but also can be due to positive acting pathogenicity factors that condition host range in a host-specific manner, e.g., pthN and avrb6 of X. campestris pv. malvacearum (6, 70), opsX of X. campestris pv. citrumelo (32), and pthA of X. citri (60, 61).

A highly clonal population structure is typical of many Xanthomonas pathovars that cause serious diseases (22). Surprisingly, some pathovars are comprised of clonal groups that are phylogenetically distinct but have similar or identical host ranges and cause identical disease symptoms. Examples of this phenomenon are observed for (i) common bean blight, caused by two major groups of strains within X. campestris pv. phaseoli that are only 20% related by DNA-DNA hybridization (28); (ii) bacterial spot of tomato and pepper, caused by two major groups of strains within X. campestris pv. vesicatoria (30) that are less than 50% related by DNA-DNA hybridization (53); and (iii) citrus canker disease, caused by two major groups of strains within X. citri (5) that are only 62 to 63% related by DNA-DNA hybridization (15). Strains with less than 70% DNA-DNA relatedness can be considered as different species (67). The purpose of this study was to attempt to determine how phylogenetically distinct X. citri strains can cause identical disease symptoms.

The most widespread and historically the oldest described group of strains that causes citrus canker disease is Xanthomonas citri pv. citri ex Hasse (synonymous with X. campestris pv. citri Dye pathotype A and X. axonopodis pv. citri Vauterin). The second phylogenetically distinct group is X. citri pv. aurantifolii Gabriel (synonymous with X. campestris pv. citri Dye and X. axonopodis pv. aurantifolii Vauterin). X. citri pv. citri appears to have originated in Asia, has a very wide range of citrus hosts, and is found worldwide, including in South America. X. citri pv. aurantifolii has a narrower range of citrus hosts and appears to have originated, and is found, only in South America. Both X. citri groups cause identical disease symptoms—hyperplastic, raised lesions that become dark and thick as canker progresses (5, 24, 25, 54).

The hyperplastic symptoms elicited by X. citri pv. citri are thought to be caused by type III secretion/injection of the pathogenicity effector protein PthA into citrus host cells (for a review, refer to reference 5). DNA fragments that hybridize with pthA are found in all strains known to cause citrus canker disease, regardless of origin (7). This raised the possibility that horizontal transfer of a single pthA homolog into a citrus-compatible xanthomonad (e.g., X. axonopodis pv. citrumelo [21] or X. axonopodis pv. alii [23]) might have created the new South American group (5).

Horizontal gene transfer is thought to be a major force in bacterial evolution (4, 13, 16, 36, 45, 62), and comparative genomic studies have provided numerous examples of likely horizontal transfer events (45, 47). Some selective advantage, such as antibiotic or heavy metal resistance, is thought to be important in the establishment of populations carrying the transferred genes (2, 35, 40, 41, 50, 51, 55, 70). Similarly, DNA “islands” containing large blocks of pathogenicity genes are thought to have been acquired by horizontal transfer at some time in the distant past by a variety of pathogens, including Salmonella enterica serovar Typhimurium, Yersinia pestis, Dichelobacter nodosus, Helicobacter pylori, and variants of Escherichia coli (26, 46). These large gene islands are also thought to provide a selective advantage (27, 33, 42, 56-59). Plasmid conjugation is thought to be a major player in horizontal transfer of DNA between bacteria (52, 65).

Environments associated with plants have been shown to be particularly conducive to horizontal gene transfer, and it has been shown that conjugation-mediated gene exchange can occur at relatively high frequency in specific zones or “hot spots” in the rhizosphere and the phyllosphere, including within plant tissues (2, 31, 40, 43, 51, 52, 63, 64, 66). In such plant-associated hot spots, it is thought that optimal conditions for conjugation are reached, including high bacterial concentrations of both donor and recipient.

There are many bioinformatics studies indicating that horizontal gene transfer occurred at some point in a bacterial species history (12, 34, 45), and there is growing evidence that conjugative plasmids carry genes of unknown function that confer selective advantages to plant-associated bacterial communities (63). To our knowledge, this study describes the first direct evidence of high-efficiency, in planta conjugative transfer of a native plasmid that carries a pathogenicity effector gene of known function and high selective value to the recipient microbe on its host.

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture media.

Bacterial strains and plasmids used in this study are listed in Table Table1.1. Xanthomonas spp. were cultured in PYGM medium (10) at 30°C. Escherichia coli spp. were grown in Luria-Bertani (LB) medium (48). The following antibiotics were used at the concentrations indicated (in μg/ml): ampicillin (Ap), 100; chloramphenicol (Cm), 35; rifamycin (Rif), 60; streptomycin (Sm), 100; tetracycline, 15; kanamycin (Kn), 12.5 or 25 (when used to grow Xanthomonas or E. coli, respectively); and spectinomycin (Sp), 35. Spontaneous Rif- or Sp-resistant mutants of X. citri pv. aurantifolii strain B69 were generated by isolating colonies that were resistant to increasing levels of Rif (25, 50, and then 75 μg/ml) or Sp (10, 20, and then 35 μg/ml) on solid PYGM medium.

TABLE 1.
List of strains and plasmids used in this study

Plasmid conjugal transfer on agar media.

Plasmid transfer by biparental or triparental mating from E. coli strains HB101 or DH5α to various Xanthomonas strains, using either helper strain ED8767/pRK2073 or pRK2013 as needed, was performed essentially as described previously (9). For plasmid transmission experiments on artificial media, overnight cultures of E. coli grown without antibiotics were mixed with 50×-concentrated mid-log-phase cultures of Xanthomonas, also grown without antibiotics. Sequentially, 10-μl droplets of recipient, donor and, when needed, helper cells were placed on PYGM agar medium without antibiotics. In each case, excess liquid was allowed to absorb into the plate before the addition of the next droplet of cells. The mating plates were incubated at 30°C overnight. For E. coli-to-Xanthomonas matings, the spotted mixtures were then streaked on PYGM agar supplemented with the appropriate antibiotics. For Xanthomonas-to-E. coli matings, the mixtures were streaked on MacConkey agar (DIFCO Laboratories, Detroit, MI) with 35 μg/ml chloramphenicol. For E. coli-to-E. coli matings, agar plates were incubated at 37°C overnight and the spotted mixtures were streaked on LB supplemented with the appropriate antibiotics. Exconjugants were screened for the presence of autonomously replicating plasmids by DNA mini-prep analysis.

For assays of frequency of transfer from one E. coli strain to another, donor and recipient strains were grown overnight at 37°C to an optical density at 600 nm of 0.5. Twenty-microliter portions of each culture were combined in a 1.5-ml reaction tube containing 160 μl of LB and grown overnight at 37°C. Cells were then resuspended in 1 ml of LB, pelleted, and then serially diluted on medium containing Cm and Sm to select for E. coli HB101 transconjugants. All frequency-of-transfer experiments were performed at least twice with duplicate samples in each experiment, and the numbers were averaged.

Marker integration.

Gene-specific knockout mutations of pthB and virB4 were created by marker interruptions following triparental matings. An E. coli DH5α strain carrying a truncated fragment of the target gene internal to the open reading frame (ORF) and cloned in suicide vector pUFR004 was used as the donor. A DH5α strain carrying pRK2013 was used as the helper. A single crossover in the recipient X. citri transconjugant cell results in duplication of the internal fragment at the integration site and in interruption of the target gene with the vector.

To disrupt pthB, pYY40.10 was constructed by cloning a 2.0-kb internal StuI-HincII fragment of pthA (all members of the pthA gene family are highly similar [19, 38]) in pUFR004. Similarly, to disrupt virB4, a PCR-generated 270-bp internal fragment of virB4 (virB4270) was cloned in pGEM-T Easy (Promega Corporation, Madison WI) and recloned in pUFR004, resulting in pBY13 (pUFR004::virB4270). To mark pXcB with Cm resistance, a similar homologous integration strategy was used, but without disrupting any likely ORF. Plasmid pQY92.1 was constructed by cloning a 6.2-kb DNA fragment immediately upstream of gene pthB in pUFR004, creating pQY92.1, which was then used to integrate pUFR004 into pXcB, creating pBIM6. In all cases, exconjugant integration mutants were selected in PYGM agar medium containing Cm to select for the integrated plasmid and Sp to counterselect E. coli. The localization of inserts was determined by plasmid DNA extractions, Southern blot analyses, and DNA sequencing.

Recombinant DNA techniques.

Plasmid and total DNAs were prepared from Xanthomonas as described previously (20). E. coli plasmid preparation, restriction enzyme digestion, alkaline phosphatase treatment, DNA ligation, and random primer-labeling reactions were performed using standard techniques (48). Southern hybridization was performed using nylon membranes as described previously (37). Colony hybridizations were used to identify cloned fragments carrying pthB from plasmid DNA of B69 by probing with a 32P-labeled, gel-purified internal BamHI fragment of pthA.

Plant inoculations and plasmid conjugal transfer in planta.

All citrus plants were grown under greenhouse conditions. Plant inoculations involving citrus canker strains were carried out under quarantine at the Division of Plant Industry, Florida Department of Agriculture, Gainesville, FL. Bacterial cells were harvested from log-phase cultures by centrifugation (5,000 × g, 10 min), washed (1×), and resuspended in sterile tap water or distilled water saturated with calcium carbonate to 108 CFU/ml. Inoculations were performed by pressure infiltration into the intercellular space of the spongy mesophyll of citrus leaves through the stomata of the abaxial leaf surface by use of tuberculin syringes without needles as described previously (21). Experimental inoculations were repeated at least three times.

For assays of frequency of transfer in planta of pBIM6 from X. citri BIM6 to B21.2, both BIM6 and B21.2 were inoculated by pressure infiltration as described above. Strains were inoculated at equal concentrations (107/ml) and so that the inoculated areas partially overlapped. For reisolation, three leaf discs of 0.28 mm in diameter were cut with a cork borer from entirely within the overlapping zones on different leaves and ground in 1 ml of sterile tap water. After serial dilution, frequencies of conjugation in planta were measured by screening on PYGM agar containing Sp and Kn for B21.2, Cm for BIM6 and Sp, and Kn and Cm for B21.2/pBIM6 4 days after inoculation. Bacterial cell count determinations represent the averages of at least three replicate experiments. For controls, the same procedure was followed but leaf disks were taken entirely from within nonoverlapping inoculation areas.

DNA sequencing and bioinformatics.

DNA sequencing was performed by the ICBR sequencing core (University of Florida), using purified pBIM2, pBIM6, and pQY96 plasmid DNAs extracted from E. coli DH5α. The GCG sequence analysis software package (Wisconsin Package version 10.3; Accelrys, Inc., San Diego, CA) was used to assemble the sequence data. Putative ORFs encoding predicted proteins of at least 50 amino acids were identified using ORF Finder from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/projects/gorf) and the Open Reading Frame Identification feature by the Bayer College of Medicine (BCM) gene feature searches (http://searchlauncher.bcm.tmc.edu/seq-search/gene-search.html). The program Codonpreference (Wisconsin Package version 10.3; Accelrys, Inc., San Diego, CA) was used for further analysis of ORFs. BlastX was used to identify ORFs for which homologs exist in the GenBank nonredundant database. For lipoprotein identification, the DOLOP (Database of Bacterial Lipoproteins) program was used (http://www.mrc-lmb.cam.ac.uk/genomes/dolop/analysis.htm).

RESULTS

Identification of the active pthA homolog in B69.

In order to determine whether the South American strain (X. citri pv. aurantifolii) B69 genome contained pthA homolog(s), a 32P-labeled internal BamHI fragment from pthA was used to probe both total and plasmid DNAs isolated from B69. EcoRI-digested total and plasmid DNAs showed identical hybridization profiles—one large fragment of 23 kb and one smaller fragment of 4.4 kb hybridized (Fig. (Fig.1A1A).

FIG. 1.
(A) Southern hybridization profiles of total and plasmid DNAs isolated from Xanthomonas citri pv. aurantifolii strain B69 and its Rif-cured derivative, B69.4. (B) Southern hybridization profiles of total DNA from two integrative B69 mutants, BIM1 and ...

A 14-kb HindIII fragment within the larger hybridizing fragment was cloned into pUFR053, yielding pQY96. The smaller (EcoRI-digested) hybridizing fragment was cloned into pUFR053, forming pQY222. Transformation of X. citri pv. citri B21.2 (pthA::Tn5) with pQY96 (carrying 14 kb from the large EcoRI fragment) complemented the PthA phenotype, restoring canker on citrus. pQY222, however, did not complement B21.2. These results indicated that one functional pthA homolog, named pthB, was present in the larger EcoRI fragment, and one putative nonfunctional homolog, named pthB0, was present in the smaller EcoRI fragment.

To disrupt pthB, pYY40.10 was used to generate B69 exconjugants by homologous recombination, resulting in the integration of pYY40.10 into two different sites at equal frequencies (Fig. (Fig.1B).1B). Class I exconjugants, represented by BIM1, appeared to carry disruptions of pthB0, residing on the 4.4-kb hybridizing fragment (Fig. (Fig.1B).1B). Class II exconjugants, represented by BIM2, appeared to carry disruptions of pthB residing on the 23-kb hybridizing fragment (Fig. (Fig.1B1B).

Pathogenicity tests of at least four strains from both classes of exconjugants were conducted by inoculation of Duncan grapefruit and Mexican lime. Class I integrative mutants (mutations in pthB0) were fully pathogenic, while class II integrative mutants were completely nonpathogenic on citrus. pQY99, carrying pthB, complemented the nonpathogenic phenotype of two class II exconjugants tested (BIM2 and BIM4), restoring canker (Fig. (Fig.2).2). By contrast, transformation of BIM2 and BIM4 with pQY222, carrying pthB0, failed to restore pathogenicity. These results confirmed that pthB was a functional homolog of pthA present in X. citri pv. aurantifolii, while pthB0 was a nonfunctional homolog.

FIG. 2.
Complementation of X. citri. pv. citri strain B21.2 (PthA) with pthB from X. citri pv. aurantifolii. B21.2/pQY99 (pthB+) was pressure infiltrated in zones labeled a, b, c, and d and developed the green canker symptomatic of X. citri pv. ...

Interchangeability of pthA and pthB.

To test whether pthA from X. citri pv. citri and pthB from X. citri pv. aurantifolii were interchangeable, the pthB knockout mutants BIM2 and BIM4 were transformed with plasmid pZit45, carrying pthA. Both strains were fully restored in their ability to cause canker symptoms typical of those caused by the wild-type B69 (not shown). Similarly, the pthA knockout mutant B21.2 was complemented with pQY99, carrying pthB and fully restoring the ability to cause canker symptoms typical of those caused by the wild type (Fig. (Fig.2),2), indicating that both pathogenicity genes were interchangeable.

pthB and pthB0 reside on separate plasmids.

In order to provide B69 with a selectable marker as a conjugation recipient and to counterselect E. coli strains to be used as donors and helpers, 18 spontaneous Rif-resistant derivates of B69 were selected and subsequently tested for pathogenicity. Surprisingly, all 18 Rif-resistant strains had lost pathogenicity on citrus. Southern blot hybridizations using EcoRI-digested DNA from wild-type B69 and several nonpathogenic Rif-resistant strains showed that the Rif-resistant strains all lacked pthB while retaining the putative homolog, pthB0 (see results for B69.4 in Fig. Fig.1A).1A). This indicated that pthB and pthB0 were carried on separate plasmids and that the plasmid carrying pthB, designated as pXcB, was cured by Rif. The other plasmid, carrying pthB0, was designated pXcB0.

Sequencing of pXcB and identification of a type IV system.

In order to define the genetic context of pathogenicity gene pthB, the entire plasmid was sequenced. Since both BIM2 and BIM4 carried pYY40.10 integrated in pthB on pXcB, the pXcB (pthB::pYY40.10) fusion plasmid was expected to replicate in E. coli due to the ColE1 replicon on pYY40.10. The fusion plasmid in BIM2, called pBIM2, was therefore transferred by conjugation from BIM2 to E. coli and appeared to replicate normally and without rearrangement, based on restriction digestion analyses (not shown). pBIM2 was fully sequenced. The complete sequence of pXcB (37,106 bp) (Fig. (Fig.3)3) was deduced primarily from pBIM2, with some sequence information derived from pBIM6 and pQY96.

FIG. 3.
Plasmid pXcB (gi:32347275) from X. citri pv. aurantifolii strain B69, with called ORFs indicated.

pXcB (NC_005240, gi:32347275) was found to contain 38 putative ORFs with an average gene density of 1.02 ORFs/kb. The closest BlastX hits in GenBank are provided in Table Table2.2. The average percent G+C content was 60.9%, with a minimum of 46.25% and a maximum of 71.75%. Two regions were found to have a percentage of G+C markedly different from that seen for the rest of pXcB. The G+C content between positions 7200 and 7800, corresponding to ORFs 105 and 106, was relatively low (56%). The G+C content from position 32560 to 34475, corresponding to the 102-nucleotide direct tandem repeat region of pthB, was high (69%). Twenty-three of 38 genes in pXcB, including pthB, showed high similarity to pXAC64 (NC_003921, gi:21264228), a 64-kb plasmid that carries pthA in X. citri pv. citri strain 306 (5, 8). PthB was found to be 87% identical to PthA of X. citri; both gene products carried sequences corresponding to 17.5 nearly identical direct tandem repeats that were very similar to each other (Fig. (Fig.44).

FIG. 4.
Alignment of PthA and PthB. The predicted proteins are 87% similar over their entire lengths. Underlined are the first and the last repeats.
TABLE 2.
Translated (BlastX) results for pXcB ORFsa

A cluster of 12 pXcB genes appeared to correspond to a type IV transfer system or virB operon (Fig. (Fig.5).5). Sequence similarity analyses revealed that the pXcB virB cluster appeared to encode a complete type IV transfer system. Of the 12 ORFs found in the cluster, 10 contained high sequence similarities to genes of previously described virB operons (98% similarity to the virB cluster of plasmid pXAC64 of X. citri pv. citri) as well as similar relative positions within the cluster. Because orf105 occupied a position within the cluster (7229 to 7295) similar to those for other virB7 members, a DOLOP algorithm analysis was conducted. This revealed that orf105 encoded a putative lipoprotein and was a virB7 homolog, encoding a lipobox sequence (LAGC) and with both virB7-conserved cysteines present at residues 36 and 48, respectively.

FIG. 5.
Organization of the virB operon found on pXcB (gi:32347275) in comparison to those of other described type IV transfer systems. ORFs lacking homology to Agrobacterium tumefaciens virB genes are shown as insertions. Abbreviations: X.c.c, Xanthomonas citri ...

pXcB is self-mobilizing, but its host range is limited.

Since pXcB appeared to carry a complete type IV transfer system, the transmissibility of the plasmid was investigated. An additional pXcB insertional derivative, pBIM6, was generated to place an antibiotic marker on pXcB but without mutating any pXcB genes. This was accomplished by again integrating suicide vector pUFR004 in pXcB, but this time outside of any coding region. pQY96.1 was used to generate B69 exconjugants as before by homologous recombination in a region upstream of pthB. One of the resulting exconjugants, BIM6, was inoculated on grapefruit and Mexican lime and found to remain fully pathogenic to citrus.

BIM2, BIM4, and BIM6, harboring plasmids pBIM2, pBIM4 and pBIM6, respectively, were used as donor strains in biparental mating experiments without helper plasmids and using X. citri B21.2, E. coli HB101, and E. coli DH5α as recipients. pBIM2, pBIM4, and pBIM6 were all found to transfer efficiently (>0.1% per recipient) into strains B21.2, HB101, and DH5α. Since the integrated pUFR004 suicide vector carried some Mob genes but no transfer genes (19), this result suggested that pXcB was a self-mobilizing plasmid.

To investigate the host range of pXcB, plasmid transfer from E. coli HB101/pBIM6 and DH5α/pBIM6 to X. axonopodis pv. citrumelo (3048Sp, 4600Sp), X. axonopodis pv. alfalfae (KX-1Sp), and X. axonopodis pv. malvacearum (XcmH1047), as well as to the Asiatic and South American strains of X. citri, was attempted by conjugation. The plasmid transferred to both of the X. citri strains but not to the X. citrumelo, X. alfalfae, or X. malvacearum strains, suggesting that the native pXcB replicon is not a broad-host-range replicon.

Role of the virB operon of pXcB in plasmid transfer.

To define the role and show the integrity and functionality of the virB operon of pXcB, a gene knockout strategy was undertaken. A 270-bp internal fragment of virB4 (virB4270) was cloned in pUFR004, forming pBY13, and used in triparental matings to generate virB4 insertion mutants carrying the same integrated plasmid (pUFR004) found in the BIM mutants. Southern blots revealed the existence of two DNA fragments homologous to virB4 in the B69 strain (using virB4270 as probe; Fig. Fig.6,6, second lane). One was virB4 on pXcB, which was absent in the pXcB-cured strain B69.4 (Fig. (Fig.6,6, third lane). A second homologous region was found on pXcB0, which remained in B69.4. Marker insertion resulted in two categories of exconjugants. Exconjugant strain B13.1 appeared to carry an interruption of the homologous region on pXcB0 (Fig. (Fig.6,6, seventh lane), while exconjugants B13.2, B13.4, and B13.5 appeared to carry interruptions of virB4 on pXcB (Fig. (Fig.6,6, fourth, fifth, and sixth lanes). All tested strains from both insertion mutant classes were fully pathogenic.

FIG. 6.
Southern hybridizations of virB4 knockout mutations. Total DNA was digested with HindIII and probed with a 32P-labeled 270-bp internal fragment of virB4. B13.2, B13.4, and B13.5 were integrated with pUFR004 in virB4 of pXcB, and B13.1 was integrated with ...

Plasmids pB13.1 and pB13.2 of strains B13.1 and B13.2 (marker interruptions in the virB4 homologous regions found on pXcB0 and pXcB, respectively) were further analyzed for their abilities to transfer to E. coli. Matings with and without a transfer helper plasmid resulted in DH5α exconjugants carrying plasmids that were Cm resistant, indicating that both plasmids were still mobilizable. Restriction enzyme digests of plasmid DNA extracted from the B13.2-to-E. coli exconjugant (DH5α/pB13.2) corresponded to the expected profile of the original plasmid with pBY13 integrated in virB4 (data not shown).

The abilities of pB13.1 and pB13.2 to self-mobilize were then analyzed by performing matings from DH5α to E. coli HB101 with DH5α/pBIM2 used as a positive control. pBIM2 was self-transferable from E. coli DH5α to E. coli HB101 at a frequency of 7.1 × 10−3 per donor. By contrast, E. coli-to-E. coli transconjugants harboring pB13.1 or pB13.2 were recovered only when matings were performed using a transfer helper strain, demonstrating that the self-mobilizing capacity of pXcB depended on the presence of an intact virB cluster and that X. citri pv. aurantifolii carries at least some plasmid transfer functions redundantly.

Evaluation of in planta horizontal transfer capacity of pXcB.

To evaluate the in planta horizontal transfer capacity of pXcB, BIM6 was inoculated onto citrus leaves so as to overlap an area simultaneously inoculated with B21.2 (PthA). Equal concentrations of inoculum (107/ml) were used. BIM6, when inoculated alone, elicited on grapefruit a whitish canker phenotype characteristic of X. citri pv. aurantifolii strains (Fig. (Fig.7).7). Due to the absence of a functional pthA gene, B21.2 showed no canker symptoms. At the convergence zone between B21.2 and BIM6 inoculations, a strong green canker characteristic of X. citri pv. citri was observed, indicating that pBIM6 had transferred to B21.2 in planta (Fig. (Fig.7).7). The frequency of transconjugants in planta was 0.62% per recipient, as measured by reisolating bacteria from inoculated leaves and screening on PYGM agar containing Sp and Kn for B21.2 or Cm for pBIM6 4 days after inoculation.

FIG. 7.
In planta mobilization of pXcB (gi:32347275) from X. citri pv. aurantifolii strain B69 to X. citri pv. citri strain B21.1 (pthA null), restoring its ability to cause canker. (Left) BIM6 (a), B21.1/pBIM6 (pthA+) (b), and B21.1 (PthA) (c). ...

The high efficiency of transfer in planta was similar to that observed for BIM2, BIM4, and BIM6 on agar medium with selection (i.e., >0.1% per recipient), indicating strong selection of B21.2/pBIM6 in planta. Several B21.2/pBIM6 strains isolated on plates using Cm selection or on plants using green canker selection were colony purified and then reinoculated on grapefruit leaves and again found to elicit green canker lesions, confirming stable in planta transfer of pXcB between the two Xanthomonas strains. Plasmid extracts from these colonies, separately digested with EcoR1 and HindIII, revealed restriction fragment lengths identical to that of the original plasmid, indicating no rearrangements (data not shown).

DISCUSSION

There are two phylogenetically distinct clonal groups of Xanthomonas citri that cause identical disease symptoms on susceptible citrus. Xanthomonas citri pv. citri appears to have originated in Asia and is now present in over 30 countries (24). X. citri pv. aurantifolii arose more recently in South America and causes economic losses in Brazil and Argentina mainly (24). In order to investigate how these two phylogenetically distinct groups cause identical symptoms on susceptible citrus, the role of pthA homologs was investigated with X. citri pv. aurantifolii. Both elicitation of canker symptoms and growth in planta of X. citri pv. citri requires pthA, which encodes a citrus-specific effector protein (14, 60, 61, 69).

In this study, X. citri pv. aurantifolii strain B69 was shown to carry two DNA fragments that hybridize to pthA, each residing on separate plasmids. Integration mutagenesis revealed that one fragment, encoding pthB and carried on pXcB, was required to cause canker on citrus by B69. Complementation experiments were used to demonstrate that pthA from the Asiatic strain 3213 and pthB from the South American strain B69 were functionally interchangeable in eliciting hyperplastic cankers on citrus. Sequence analysis revealed that the predicted PthA and PthB proteins were 87% identical. Comparison of the PthA and PthB repeat regions, which are known to carry host specificity (69), revealed an imperfect conservation in the arrangement of the repeats (Fig. (Fig.44).

The complete DNA sequence of pXcB revealed that almost one-third of the plasmid was occupied by a type IV transfer system. Type IV systems are defined on the basis of homologies between Agrobacterium tumefaciens tDNA transfer system, the conjugal transfer system Tra, and the Bordetella pertussis toxin exporter Ptl (68). Most type IV systems function primarily in conjugational transfer. All components of a type IV transfer system were found in the virB cluster of pXcB, including a putative virB7 homolog (5). Interestingly, the highest overall homology was found with the virB cluster of plasmid pXAC64 of the Brazilian X. citri pv. citri strain 306 (8).

pBIM6, a marked pXcB derivative with an added ColE1 replicon, was experimentally determined to be self-transferable by conjugation, without transfer helper strains, from X. citri to X. citri, from X. citri to E. coli, from E. coli to E. coli, and from E. coli to X. citri. However, pXcB did not transfer to strains from different X. axonopodis pathovars tested, suggesting that pXcB is not likely to be a broad-host-range plasmid, as was shown to be the case for other self-mobilizing plasmids isolated from plant-associated bacteria, for example pIPO2 and pSB102 (49, 63, 66).

Marker interruption of virB4 of the type IV transfer system on pXcB resulted in loss of self-transferability of pBIM6 from E. coli but not from X. citri B69, demonstrating redundancy of plasmid transfer functions in B69, most likely from pXcB0. Importantly, pBIM6 transferred by conjugation at high efficiencies between two phylogenetically distinct Xanthomonas strains in planta. pBIM6 was found to transfer in planta, without added selection, from X. citri pv. aurantifolii to X. citri pv. citri at frequencies equivalent to those observed using antibiotic selection on agar plates.

These experiments showed for the first time that a major pathogenicity effector gene could efficiently transfer in planta from one xanthomonad to another, dramatically changing the recipient's pathogenicity phenotype and conferring a significant selective advantage. The type IV transfer system, together with the pthB effector of pXcB, can therefore be considered to comprise a self-mobilizing pathogenicity enhancer plasmid capable of spreading among compatible bacteria by horizontal gene transfer (27). The presence of a pthA homolog on a self-mobilizing plasmid suggests that X. citri pv. aurantifolii may have arisen following a single horizontal transfer event to a citrus-compatible xanthomonad. The pthA homolog might have originated either fully adapted to citrus from an X. citri pv. citri donor or else unadapted to citrus from some other source, with subsequent adaptive evolution to citrus.

Residence of any host-specific effector gene, such as pthB, on a self-mobilizing plasmid virtually guarantees its transfer into a strain that is pathogenic on hosts to which the encoded effector is not adapted or even maladapted (18). If the effector is maladapted and injected by type III secretion, it may be recognized by the plant host as an avirulence effector, resulting in a defense response. However, if the secreted effector is simply unadapted and not recognized as an avirulence effector, it would allow time for the effector gene to evolve. Members of the pthA gene family have multiple internal tandem repeat and flanking inverted repeat structures that allow very rapid evolution by intragenic and intergenic recombination (71), which can readily result in new and major pathogenic effects on plants (69). Because of these unique structural characteristics, rapid adaptation and thus convergent and independent evolution of pthA in Asiatic strains and pthB in South American strains for specificity to citrus is quite possible. If so, the pthA effector gene family may be a more pathogenically significant and generally capable group than previously realized.

Members of the pthA gene family are noted for having major effects on pathogenicity, while most type III effectors have only minor effects. The combination of a major host-adaptable pathogenicity gene on a self-mobilizing plasmid such as pXcB may represent the minimal epidemiologically significant pathogenicity-enhancing machine. pXcB carried no identified type III effector genes other than pthB. Indeed, pXcB carried few other genes in addition to those needed for plasmid replication and self-transmissibility and the one host-specific pathogenicity gene, pthB. It may be of interest to determine how often pthA family members are found on self-transmissible plasmids.

Footnotes

[down-pointing small open triangle]Published ahead of print on 12 January 2007.

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