Departments of Plant Pathology1 and Microbiology2, University of Georgia, Athens, GA 30602, USA
Laboratoire de Biologie Moléculaire des Relations Plantes-Micro-organismes, INRA-CNRS, Toulouse, France3
Author for correspondence: Timothy P. Denny. Tel: +1 706 542 1282. Fax: +1 706 542 1262. e-mail: tdenny{at}arches.uga.edu
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ABSTRACT |
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Keywords: pili, fimbriae, bacterial wilt, bacterial cell movement
Abbreviations: EPS, extracellular polysaccharide; HR, hypersensitive reaction; Tfp, type IV pili
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INTRODUCTION |
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Efficient colonization of a host plant requires both EPS1 and multiple extracellular proteins (Araud-Razou et al., 1998 ; Kang et al., 1994
; Saile et al., 1997
; Schell, 2000
). Some extracellular proteins made by R. solanacearum are delivered to host cells via a type III secretion system encoded by a cluster of >20 hrp genes (Cornelis & van Gijsegem, 2000
; van Gijsegem et al., 1995
). These effector proteins promote bacterial multiplication in hosts and trigger defence responses in nonhosts. Other extracellular proteins, like the plant-cell-wall-degrading enzymes, transit the general secretory pathway (type II secretion) (Hueck, 1998
; Pugsley, 1993
). Individual enzymes enhance virulence of R. solanacearum (e.g. the rate and severity of wilting) (Denny et al., 1990
; Huang & Allen, 1997
; Tans-Kersten et al., 1998
), but together they may be essential for disease (Kang et al., 1994
). Expression of virulence factors, plus other genes not obviously required for virulence, is controlled by a complex, environmentally responsive regulatory network (Schell, 2000
). Central to this network is the Phc confinement-sensing system, which employs the PhcA global transcriptional regulator to control multiple genes directly and indirectly. Some PhcA-regulated genes, like the egl endoglucanase and the eps gene cluster, are expressed strongly only at high cell density, whereas others, such as the pglA endo-polygalacturonase, are more highly expressed at low cell density.
To understand better the process of systemic colonization by R. solanacearum, we are investigating what role the cell surface components of the pathogen may play during interactions with plant tissues. We are particularly interested in surface molecules that would be exposed when EPS1 is not produced, a condition that occurs when cells are at low density (e.g. early in colonization) (Kang et al., 1999 ). The obvious candidates are the lipopolysaccharide and pili (fimbriae). However, biosynthesis of lipopolysaccharides is complex, requiring multiple enzymic steps, and the R. solanacearum LPS-deficient mutants described to date also have defects in EPS1 synthesis or membrane function (Cook & Sequeira, 1991
; Titarenko et al., 1997
). In contrast, pili consist primarily of a single structural protein (pilin), so they are more amenable to reverse genetics to evaluate their role during pathogenesis.
Numerous Gram-negative bacteria, including some animal and plant pathogens, produce type IV pili (Tfp) (Ojanen-Reuhs et al., 1997 ; Roine et al., 1998
; Strom & Lory, 1993
; Su et al., 1999
; van Doorn et al., 1994
; Wall & Kaiser, 1999
). Depending on the organism, Tfp can be important for adhesion, aggregation, biofilm formation, horizontal gene transfer, multicellular development, pathogenesis and twitching motility (Fussenegger et al., 1997
; Merz et al., 2000
; Strom & Lory, 1993
; Wall & Kaiser, 1999
). Twitching motility, which is a form of flagella-independent translocation of bacteria over solid surfaces, now appears to be somewhat misnamed, because cell movements can be intermittent and jerky or smooth and prolonged (Henrichsen, 1983
; Semmler et al., 1999
; Wall & Kaiser, 1999
). The amount of movement necessary to qualify as twitching also varies from almost nonobservable to quite large (e.g.
0·6 mm h-1 for Pseudomonas aeruginosa) (Alm & Mattick, 1997
; Henrichsen, 1972
, 1983
; Semmler et al., 1999
; Strom & Lory, 1993
). Retraction of Tfp, probably by filament disassembly, has long been considered the likely mechanism for twitching motility (Bradley, 1980
; Wall & Kaiser, 1999
; Wolfgang et al., 2000
). Very recently, sophisticated microscopic techniques have provided direct evidence that Tfp are the actuators of cell movement in P. aeruginosa and Myxococcus xanthus (Skerker & Berg, 2001
; Sun et al., 2000
) and shown that Neisseria gonorrhoeae Tfp exert sufficient retractile force to move bacterial cells (Merz et al., 2000
). Tfp and twitching motility have been studied most in P. aeruginosa, where at least 35 genes are required for synthesis, display and function of polar, retractable Tfp (Alm & Mattick, 1997
; Wall & Kaiser, 1999
). Many genes for production of Tfp, including that for the pilin subunit protein, are well conserved in a variety of bacteria.
We became suspicious that R. solanacearum exhibits twitching motility when we noted that young, microscopic colonies growing on the surface of agar media had margins that were serrated and layered in appearance, resembling colonies of twitching P. aeruginosa pictured in the literature (Bradley, 1980 ; Darzins, 1993
; OToole & Kolter, 1998
; Semmler et al., 1999
). Our suspicion was reinforced when a subsequent search of the preliminary genomic DNA sequence of R. solanacearum strain GMI1000 revealed ORFs that were predicted to encode proteins very similar to many of those that are essential for biosynthesis of Tfp, and hence twitching, in P. aeruginosa and other bacteria. In this paper we present strong evidence that R. solanacearum exhibits twitching motility using a Tfp system, and that Tfp contribute to pathogenesis on tomato plants.
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METHODS |
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To test the virulence of R. solanacearum on tomato plants, seeds (Lycopersicon esculentum Mill., cultivar Marion) were germinated in vermiculite and 2-week old seedlings were transplanted into 4 inch (10 cm) plastic pots containing a mixture of composted pine bark and vermiculite (3:1) amended with lime and fertilizer. Plants were incubated in a growth chamber (constant 30 °C, 13 h photoperiod) for 45 weeks before inoculation. Unwounded roots were inoculated by drenching the soil with 40 ml of a bacterial suspension in water, containing 2x108 c.f.u. ml-1, prepared as described by Saile et al. (1997)
. Leaf petioles were inoculated by excising the first leaves above the cotyledon 0·5 to 1 cm from their base, and immediately applying to the cut surfaces a 2 µl droplet of a water suspension containing 8x104 c.f.u. of R. solanacearum cells taken from a 2-d-old BG agar plate. To test for elicitation of the defensive hypersensitive reaction (HR), water suspensions with 1x107 c.f.u. ml-1 of R. solanacearum taken from BG agar plates were infiltrated into leaves of tobacco (a nonhost plant) by making a hole in the leaf with a 25-gauge hypodermic needle, pressing the blunt end of a filled tuberculin syringe against the leaf surface while supporting the other side of the leaf with a finger, and gently injecting a small volume of inoculum (Carney & Denny, 1990
).
Microscopy.
Colonies were examined for twitching motility by placing a Petri dish without its lid on the stage of an upright light microscope (Nikon Labophot) equipped with 4x and 10x objectives. Digital images were acquired using a colour CoolSnap CCD camera (Roper Scientific) and saved as uncompressed TIFF files. Image processing, which was limited to cropping, conversion to greyscale, and adjustment of brightness and/or contrast, was done with CoolSnap or Photoshop (Adobe) software.
Isolation of an R. solanacearum cosmid clone with PilD-like activity.
A pre-existing, en masse cosmid library of total DNA from strain AW1 (Carney & Denny, 1990 ) was introduced into P. aeruginosa strain PAK-D
(pilD::
) by triparental mating, and Sp- and Tc-resistant transconjugants were selected on lipase activity agar plates. Colonies that acidified the surrounding medium were identified and restreaked on the same medium to confirm restoration of lipase activity and to check for twitching motility.
Plasmid construction.
To create pKS-Gm, the 0·85 kb aacC1 gentamicin-resistance gene cartridge was released from pUCGM (Schweizer, 1993 ) by digestion with SmaI and was cloned into pBluescript KS II+ (Stratagene) that had been digested with SspI (which releases a 131 bp fragment outside of the multiple cloning site). pKSL was created by deleting the SmaIHincII fragment from within the multiple cloning site of pKS-Gm.
Cloning of R. solanacearum pil genes and creation of pil mutants.
R. solanacearum genes involved in Tfp production were assigned names based on homology and the naming scheme used for P. aeruginosa (Alm & Mattick, 1997 ). The putative pilQ and pilT genes in R. solanacearum were identified in the genome sequence of strain GMI1000 using tBLASTn (Altschul et al., 1997
) with the P. aeruginosa PilQ and PilT protein sequences as probes. PCR primers were designed to amplify the complete pilQ gene (2178 bp fragment: forward 5' - TACCTCTAGAGACCCTGAAAGTTCAGGAGGGCGG-3', reverse 5'-TACCTCTAGACTTCAGCGACAGCTGGTCGGACAG-3'), an internal portion of pilQ (1336 bp fragment: forward 5'-TACCTCTAGAGCACCCGCGTCGTGCTGGATCTGG, reverse 5'-TACCTCTAGATCGGTACCCTGCTCGATCAGCGCC-3') and the complete pilT gene (1190 bp fragment: forward 5'-GGTACCTCTAGACATCGTGGCACTCCGGAGC-3', reverse 5'-GGTACCTCTAGACAAGTCCGAGCCACGGCTG-3'). Each primer had an XbaI site incorporated into it (nucleotides underlined above) for use in subsequent cloning steps. PCR was performed using standard conditions (94 °C for 4 min; 30 cycles of 94 °C for 1 min, 60 °C for 1·5 min, 72 °C for 2 min; 72 °C for 10 min) with AW1 genomic DNA as the template and the reaction products were resolved by agarose gel electrophoresis. Amplified fragments were purified from the gel using the QIAquick Gel Extraction Kit (Qiagen), cloned onto pGEM-T easy, and sequenced using vector primer sites. The XbaI fragments with the 1·3 kb pilQ internal fragment and the pilT gene were subcloned separately onto the XbaI site in pKSL to create pKSLQ1and pKSLT, respectively. Inactivated alleles of each gene were made by inserting the 0·9 kb nonpolar nptI cartridge (encoding Km resistance) (Galan et al., 1992
) to create pKSLQ1::npt1 and pKSLT::npt1. The pilQ::nptI and pilT::nptI constructs were released by digestion with NotI or XbaI, respectively, and cloned into similarly digested pTOK2 to create pTOQ1::npt1 and pTOT::npt1. The inactivated alleles were introduced into AW1 and K60 wild-type strains by electroporation of purified plasmid DNA. pTOK2 cannot replicate in R. solanacearum, so selection for Km-resistance followed by screening for Tc-sensitivity returned colonies where the wild-type gene on the chromosome had been site-specifically replaced with the inactivated allele. Fidelity of replacement was checked by: (1) determining whether genomic DNA from a mutant simultaneously transformed wild-type strains to Km resistance and the mutants phenotype; and (2) PCR amplification of the wild-type or inactivated allele using primers flanking the gene and analysis of the fragment sizes by agarose-gel electrophoresis. The complete pilQ and pilT genes also were subcloned in both orientations in pRK415 (Keen et al., 1988
) to create plasmids pRQ2+, pRQ2-, pRT+ and pRT-, respectively.
Recombinant DNA techniques and DNA sequence analysis.
Standard protocols were used for cloning, conjugation, electroporation, preparation of competent cells and Southern blotting (Ausubel et al., 1989 ; Carney & Denny, 1990
). Hybridization to Southern blots used the DIG High Prime labelling and detection kit (Roche Molecular Biochemicals) and followed the manufacturers protocols. Plasmid DNA was isolated using a Qiagen mini kit, and genomic DNA by the method of Chen & Kuo (1993)
. Natural transformation of R. solanacearum with genomic DNA was similar to that described by Bertolla et al. (1997)
, except that BG agar plates were used and the mixture of bacteria and DNA was incubated directly on the agar plate for 1824 h. Restriction enzymes were purchased from New England Biolabs and the MasterAmp Tfl DNA polymerase PCR kit was from Epicentre Technologies. DNA primer synthesis and DNA sequencing were performed at the University of Georgia Molecular Genetics Instrumentation Facility.
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RESULTS |
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Many of the proteins essential for type IV piliation are similar to those required for type II protein secretion across the outer membrane (Nunn, 1999 ). In P. aeruginosa these similarities are so strong that some proteins in both systems are processed by PilD, a specialized leader peptidase (Strom & Lory, 1993
). However, R. solanacearum strain AD4, which has its type II protein secretion system inactivated (Kang et al., 1994
), exhibited wild-type twitching activity.
R. solanacearum has at least three orthologues of genes essential for production of functional Tfp in P. aeruginosa
Movement of R. solanacearum cells over the surface of agar plates strongly suggests twitching motility, but proof requires documenting that cells become nonmotile when production of functional Tfp is inactivated. Careful examination of the preliminary GMI1000 genomic sequence revealed six ORFs that strongly resemble genes encoding pilin proteins in P. aeruginosa and other bacteria, so it will require substantial effort to determine which ORF encodes the pilin protein, as opposed to a pilin-like protein that is involved in biogenesis of Tfp. Therefore, to test more immediately the connection between twitching motility and Tfp, we targeted conserved genes likely to be essential for Tfp production and function.
One of the more conserved R. solanacearum GMI1000 orthologues of interest encodes the putative PilD, which is 4152% identical to PilD from P. aeruginosa (GenBank accession no. M32066), N. gonorrhoeae (U32588) and M. xanthus (AF003632). In P. aeruginosa, pilD encodes a leader peptidase that processes the Tfp prepilin protein and N-methylates the pilin N-terminal amino acid (Lory & Strom, 1997 ; Strom & Lory, 1993
). PilD peptidase also processes at least six other proteins involved in pilus biogenesis (FimTU, PilE, PilVWX), and five pseudopilins (XcpTUVWX) in the type II secretion system (Alm & Mattick, 1997
; Nunn, 1999
). Therefore, P. aeruginosa pilD mutants lack twitching motility and do not secrete a lipase and several other extracellular enzymes. We found the R. solanacearum pilD orthologue in a cosmid library of AW1 genomic DNA by its ability to restore lipase activity and twitching motility to a P. aeruginosa pilD mutant. After an en masse mating, two transconjugant colonies were positive for lipase activity on plates with olive oil as the sole carbon source. Cosmid DNA (pLAD1 and pLAD2) was extracted from each transconjugant, transformed into E. coli DH5
, and reconjugated into the P. aeruginosa pilD mutant. Both cosmids, which contained overlapping regions of the genome, again restored lipase activity and also restored twitching motility, indicating that R. solanacearum AW1 has a functional PilD orthologue. Unfortunately, because pilD mutants are pleiotropic, this gene was not a good target to test genetically the connection between Tfp and twitching motility.
A second orthologue of interest was that for pilQ. In M. xanthus, N. gonorrhoeae and P. aeruginosa PilQ is a secretin located in the outer membrane, where it is thought to form a gated channel through which the pilin subunits travel (Alm & Mattick, 1997 ; Fernandez & Berenguer, 2000
; Wall et al., 1999
; Wolfgang et al., 2000
). Inactivation of pilQ prevents biogenesis of Tfp, but should have little effect on other secretion systems. In GMI1000, the putative pilQ orthologue is the last gene in what appears to be the pilMNOPQ operon, an organization like that in P. aeruginosa and M. xanthus (Alm & Mattick, 1997
; Wall & Kaiser, 1999
). We PCR-amplified pilQ from the genome of AW1 on a 2·2 kb fragment, and DNA sequencing showed it to be 89·9% identical to pilQ in GMI1000. The predicted amino acid sequence of AW1 PilQ has 714 residues and is 91% identical to the predicted GMI1000 PilQ, and 3038% identical to PilQ from M. xanthus, N. gonorrhoeae and P. aeruginosa over its entire length (Fig. 5
). Like other members of the secretin superfamily (Genin & Boucher, 1994
; Wall et al., 1999
), the AW1 PilQ sequence is better conserved in its C-terminal half, with three of the four conserved sequence motifs being present (PRINTS accession PR00811).
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Inactivation of pilQ or pilT eliminates twitching motility and reduces virulence of R. solanacearum
To inactivate pilQ in R. solanacearum, we inserted an nptI (Km resistance) cartridge into an internal 1·3 kb pilQ gene fragment and introduced this into the genome of AW1 and K60 by allelic replacement to create strains AW1-Q and K60-Q, respectively. As expected, the R. solanacearum pilQ mutants did not exhibit twitching motility on BG agar plates, since colonies less than 24 h old had entire margins, and were smaller and more domed than those of the wild-type parents (Fig. 4e). Nevertheless, 2-d-old colonies of the pilQ mutants on BG agar plates were very similar to those of the wild-types. Introduction of wild-type pilQ on pRQ2+, which has the gene on a 2·2 kb fragment aligned with the vectors lac promoter, into the pilQ mutants restored twitching motility (not shown), so the nptI insertion in the genome did not have any obvious effects on downstream genes. pRQ2-, in which pilQ is aligned opposite to the vectors lac promoter, did not restore motility, so the cloned fragment lacks its native promoter as one would expect for a gene located at the end of a large operon.
pilT was inactivated in the genome of AW1 and K60 by allelic replacement, creating strains AW1-T and K60-T. Compared to their wild-type parents, PilT mutants formed a more substantial pellicle at the surface of unshaken cultures or developed as macroscopic clumps of cells that settled to the bottom of the culture tube, suggesting that they are hyperpiliated. Neither AW1-T nor K60-T exhibited twitching motility on BG agar plates, since 24-h-old colonies looked the same as pilQ mutants (not shown). However, 2-d-old cultures of the pilT mutants on BG agar plates had smaller colonies than wild-type, less EPS1 slime, and the slime produced was abnormally transparent due to there being fewer suspended cells.
pilQ and pilT mutants of AW1 and K60 multiplied normally in BG broth cultures, and AW1-Q survived as well as AW1 during the first 7 d after application to the potting mixture used for growing tomato plants (results not shown). To test whether inactivation of pilQ or pilT affected important extracellular products that contribute to virulence of R. solanacearum, we quantified EPS1 production, and the activities of two secreted enzymes (endoglucanase and endo-polygalacturonase). The mutants were comparable to the wild-type in all three traits, so the membrane-localized functions for EPS1 biosynthesis and type II secretion were not affected. Like AW1, both AW1-Q and AW1-T elicited a normal HR defence response when infiltrated into nonhost tobacco leaves, so Hrp pili and the rest of the type III protein secretion system also were not affected. However, AW1-Q induced wilt symptoms less quickly than did AW1 when unwounded tomato roots were inoculated by a soil drench (Fig. 6). Although tested less extensively, K60-Q was comparable to AW1-Q in virulence (results not shown). The difference between AW1 and AW1-Q was clearly affected by the inoculum concentration, with higher concentrations of bacteria (e.g. 2x108 c.f.u. ml-1) markedly reducing the differential between the wild-type and the mutant in some experiments. In three experiments, K60-T wilted plants less quickly than K60 when inoculated via wounded petioles (results not shown). These results suggest that R. solanacearum Tfp, or functions that require Tfp, are important during both invasion and colonization of young tomato plants.
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DISCUSSION |
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We were surprised to find, when researching the older literature on twitching motility, that Henrichsen (1975a , 1983
) reported many years ago that five strains of R. solanacearum exhibited twitching motility. These results were largely forgotten by the scientific community studying this plant pathogen for several reasons. First, until recently twitching motility was not a commonly studied phenomenon so most plant pathologists were (and are) unfamiliar with it. Second, because Henrichsen surveyed many bacteria in addition to R. solanacearum, it was not mentioned by name in the abstract, so only the most thorough or directed literature search would have found these reports.
In our study, motility by R. solanacearum clearly was much better on a normal agar surface than in the interstitial space between the agar and the plastic Petri dish, in contrast with P. aeruginosa, which migrated quickly in both locations. Unlike Semmler et al. (1999) , we observed that P. aeruginosa (i) readily developed rafts when growing on the agarair interface of BG plates and (ii) did not twitch well on the basal agar surface that was exposed by inverting the agar layer. In addition, in contrast to the results of Henrichsen (1975b
) with Acinetobacter calcoaceticus, motility of R. solanacearum was observed on media solidified with agarose. Therefore, neither BG agarose nor a BG agar surface that solidified while exposed to air was inherently suboptimal for observing twitching motility in vitro. At least for R. solanacearum, we found that rather than special conditions being required to observe twitching motility, it is more important that one observe the appropriate strains at the correct time.
Twitching motility has not been reported for other plant-pathogenic bacteria, but several xanthomonads and one pseudomonad make typical Tfp. Pili of Xanthomonas campestris pv. hyacinthi were purified, and the N-terminal amino acid sequence strongly resembled the conserved portion of Tfp (van Doorn et al., 1994 ). Cells and pili bound to the stomata of hyacinth leaves, but a nonpiliated mutant was not tested so the role of Tfp in pathogenesis on hyacinth remains unclear. For Xanthomonas campestris pv. vesicatoria, the pilin structural gene was found by reverse genetics and subsequently inactivated. The nonpiliated mutant retained full virulence on tomato plants when tested under controlled conditions, but because Tfp promoted aggregation on leaf surfaces and increased resistance to killing by UV light the authors speculated that piliation might increase survival on leaf surfaces (Ojanen-Reuhs et al., 1997
). Similarly, the Tfp of Xanthomonas campestris pv. citri were reportedly not important for virulence when a nonpiliated mutant was sprayed on citrus leaves in a humid environment (Su et al., 1999
). It would have been more interesting and informative if these pathogens had been tested on plants in environmental conditions that resemble natural settings. One example where a mutant strain lacking Tfp was tested in the field was for Pseudomonas syringae pv. tomato (Roine et al., 1998
). Even here, however, Tfp did not contribute measurably to virulence, although piliation was again associated with UV tolerance and survival on the leaf surface. Xylella fastidiosa probably makes Tfp, because 26 orthologous genes for pili synthesis and function were found in its genomic sequence (Simpson et al., 2000
), but this remains to be proven.
We did not demonstrate the physical presence of Tfp on R. solanacearum, but the literature suggests that this organism can make two or more types of pili. In the only contemporary research, van Gijsegem et al. (2000) observed two types of polar pili on GMI1000 during electron microscopy of negatively stained cells. One type of pilus was 35 nm wide and was produced preferentially when cells were cultured on rich medium, but was not further characterized. The second type of pilus had a constant diameter of 6·6±1 nm and it is composed of 7 kDa pilin monomers encoded by hrpY. The HrpY pilin has a much lower molecular mass than a typical type IV pilin (1617 kDa), and its amino acid sequence is completely unrelated. It was clear from our results that neither Hrp pili nor any of the Hrp regulatory system tested were necessary for twitching motility.
In the older literature on R. solanacearum, it is not clear what type of pili were detected. The first report was by Fuerst & Hayward (1969) , who observed sparse, polar pili on wild-type biovar 2, 3 and 4 strains. Interestingly, the biovar 4 strain 003A they reported using is probably similar to UW141 (originally designated as 007A), which we found to exhibit active twitching motility, because both were isolated by Hayward from ginger plants in Australia during April, 1965. Henrichsen & Blom (1975)
observed polar pili on what appears to be the same biovar 2 strain as used by Fuerst & Hayward (1969)
. More recently, a pilin protein was purified two separate times from K60 (Stemmer & Sequeira, 1987
; Young et al., 1985
), and neither the molecular mass (9·5 kDa) nor the amino acid compositions matched either R. solanacearum HrpY pilin or typical Tfp. Because these authors used very different culture conditions and methods to observe or isolate pili, the discrepancies in their results do not necessarily indicate production of different pili.
In contrast to the results with other Tfp- phytopathogenic bacteria, eliminating the PilQ secretin in R. solanacearum (and presumably Tfp, since the mutants were nonmotile), markedly reduced virulence on tomato plants, indicating that motility and/or Tfp play a significant role during pathogenesis. Although there was no change in type II or type III protein secretion or in EPS1 production when pilQ was inactivated, we cannot exclude the possibility that loss of this outer-membrane protein has unknown secondary effects. Addressing this concern will require additional work to make a mutant that is defective only in synthesis of the pilin subunit. Nevertheless, that twitching motility appears to be negatively regulated by PhcA, the global virulence regulator, supports the likelihood that motility and/or Tfp are important factors in pathogenesis. Regulation of twitching appears to be on a separate circuit from most other known virulence and pathogenicity factors, since none of the other 12 regulatory genes or two-component systems tested seemed to have any effect. However, we did not test the R. solanacearum pehSR two-component system, which positively regulates both transcription of pehA, the endo-polygalacturonase structural gene, and flagellar motility (Allen et al., 1997 ; Tans-Kersten et al., 2001
). Based on the similarity of PehS and PehR to the two-component regulatory system that regulates pilin synthesis in P. aeruginosa, Allen et al. (1997)
speculated that this system might also positively regulate Tfp production in R. solanacearum. Because expression of pehSR is negatively controlled by PhcA (Allen et al., 1997
), our observation of prolonged twitching motility by phcA mutants supports this proposed regulatory pathway.
It is interesting to consider why type IV piliation is more important for virulence of R. solanacearum than it appears to be for X. campestris or P. syringae. One explanation might be that, because R. solanacearum must systemically colonize tomato plants to cause disease symptoms (Saile et al., 1997 ; T. P. Denny, unpublished observations), it has different requirements for Tfp in planta than do the other pathogens tested, which cause only localized lesions. It would be premature to suggest what these requirements might be, because the process of systemic colonization of a host by R. solanacearum is not well understood. Another possibility is that contact with and invasion of unwounded tomato roots by R. solanacearum when poured into the soil is a more demanding process than that when the foliar pathogens are sprayed directly onto leaf surfaces. It also is fair to wonder what role twitching motility might have for R. solanacearum in natural settings. Here, the best possibility is that, as proposed for M. xanthus and P. aeruginosa (Wall & Kaiser, 1999
), which are common soil microbes, Tfp-mediated migration over surfaces could promote acquisition of nutrients. Regardless of whether these explanations are correct, our results with R. solanacearum reinforce the conclusion by Semmler et al. (1999)
that twitching motility is a highly organized process requiring cell-cell interactions similar to the social gliding motility in M. xanthus, rather than the slow, disorganized mode of movement by individual cells originally proposed by Henrichsen (1972)
. Consequently, the role of twitching motility for pathogenesis and survival of R. solanacearum will be all the more interesting and important to understand.
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ACKNOWLEDGEMENTS |
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Received 10 July 2001;
revised 27 September 2001;
accepted 3 October 2001.