Isolation of Ralstonia solanacearum hrpB constitutive mutants and secretion analysis of hrpB-regulated gene products that share homology with known type III effectors and enzymes

Naoyuki Tamura, Yukio Murata and Takafumi Mukaihara

Research Institute for Biological Sciences, Okayama (RIBS), 7549-1 Yoshikawa, Kibichuo-cho, Okayama 716-1241, Japan

Correspondence
Takafumi Mukaihara
mukaihara{at}bio-ribs.com


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The Hrp type III secretion system (TTSS) is essential for the pathogenicity of the Gram-negative plant pathogen Ralstonia solanacearum. To examine the secretion of type III effector proteins via the Hrp TTSS, a screen was done of mutants constitutively expressing the hrpB gene, which encodes an AraC-type transcriptional activator for the hrp regulon. A mutant was isolated that in an hrp-inducing medium expresses several hrpB-regulated genes 4·9–83-fold higher than the wild-type. R. solanacearum Hrp-secreted outer proteins PopA and PopC were secreted at high levels into the culture supernatants of the hrpB constitutive (hrpBc) mutant. Using hrpBc mutants, the extracellular secretion of several hrpB-regulated (hpx) gene products that share homology with known type III effectors and enzymes was examined. Hpx23, Hpx24 and Hpx25, which are similar in sequence to Pseudomonas syringae pv. tomato effector proteins HopPtoA1, HolPtoR and HopPtoD1, are also secreted via the Hrp TTSS in R. solanacearum. The secretion of two hpx gene products that share homology with known enzymes, glyoxalase I (Hpx19) and Nudix hydrolase (Hpx26), was also examined. Hpx19 is accumulated inside the cell, but interestingly, Hpx26 is secreted outside the cell as an Hrp-secreted outer protein, suggesting that Hpx19 functions intracellularly but Hpx26 is a novel effector protein of R. solanacearum.


Abbreviations: PIP, plant-inducible promoter; TTSS, type III secretion system


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In many Gram-negative pathogenic bacteria, the type III secretion system (TTSS) plays essential roles in disease development in their respective hosts (Hueck, 1998; Galán & Collmer, 1999; Cornelis & Van Gijsegem, 2000). Components of the TTSS apparatus from animal- and plant-pathogenic bacteria are highly conserved at the amino acid level (Bogdanove et al., 1996). In certain animal pathogens, such as Salmonella, Shigella and Yersinia, the structure of the TTSS machinery complex, a membrane-embedded complex that is associated with a short needle-like structure, has been investigated by transmission electron microscopy (Kubori et al., 1998; Tamano et al., 2000; Blocker et al., 2001; Sekiya et al., 2001). In plant-pathogenic bacteria on the other hand, a long pilus-like filament, called the Hrp pilus, probably extends from the type III secretion apparatus, although direct observation of a membrane-bound basal-body complex has not been achieved (Roine et al., 1997; Van Gijsegem et al., 2000; Jin & He, 2001). At present, these secretion apparatuses are thought to act as a molecular needle or conduit for delivering bacterial virulence proteins, the so-called type III effector proteins, directly into host cells across the plasma membrane (Hueck, 1998).

In phytopathogenic bacteria, genes encoding the type III effector proteins have historically been identified as avirulence (avr) genes that are recognized by the corresponding resistance (R) gene product and induce a hypersensitive response (HR), a rapid and localized necrosis of plant tissues, on non-host plants (Hammond-Kosack & Jones, 1996). The HR-inducing activity of avr genes on non-host plants occurs in an Hrp-dependent manner, and the expression of avr genes in non-host plant cells leads to the HR, indicating that Avr proteins are translocated into plant cells (Alfano & Collmer, 1997). It is important to identify effector proteins without an HR-inducing activity because they may have pathogenicity functions in host plant cells. The exact number of effector proteins injected into plant cells from a certain plant pathogen and their functions in planta are the main issues to be resolved in this field. Recent research involving detection of the Hrp-dependent secretion of effector proteins into culture media or obtaining direct evidence for the translocation of effector proteins into plant cells using a Cya reporter fusion has unveiled the effector proteins of some plant-pathogenic bacteria (Casper-Lindley et al., 2002; Schechter et al., 2004; Cunnac et al., 2004; Roden et al., 2004).

Ralstonia solanacearum, the causal agent of bacterial wilt, is one of the most important plant-pathogenic bacteria because it causes diseases in various economically important crops (Hayward, 1991). In R. solanacearum, plant signals or poor nutritional conditions, which mimic conditions in the intracellular spaces of plants, induce the expression of hrp genes encoding the Hrp TTSS apparatus through the expression of an AraC-type transcriptional activator, HrpB (Genin et al., 1992). HrpB also regulates the expression of most of the effector genes in R. solanacearum (Cunnac et al., 2004). In R. solanacearum GMI1000 (race 1, biovar 3), in silico and expression analyses identified 48 novel hrpB-regulated (brg) genes, which have a plant-inducible promoter (PIP)-box motif in the putative promoter region (Salanoubat et al., 2002; Cunnac et al., 2004).

In a previous study, we screened for hrpB-regulated genes in Japanese strain RS1000 (race 1, biovar 4) using a transposon-based genetic screening system and identified 30 hpx (hrpB-dependent expression) genes (Mukaihara et al., 2004). Most of the hpx genes were considered to encode candidate effector proteins of R. solanacearum because they contained a PIP-box motif in the putative promoter region considered to be directly controlled by HrpB (Mukaihara et al., 2004). In fact, seven hpx genes encoded homologues of known type III effectors and type-III-related proteins found in other animal and plant pathogens (Mukaihara et al., 2004). In this study, we investigated the Hrp-dependent extracellular secretion of these effector homologues in R. solanacearum. Because the expression level of wild-type hrpB under the in vitro hrp-inducing conditions we used was insufficient to detect the secreted proteins in culture media, we constructed a strain constitutively expressing hrpB. Using hrpB constitutive (hrpBc) mutants, we demonstrated the secretion of several type III effector homologues of R. solanacearum. We also report on the secretion analysis of two hpx gene products, Hpx19 and Hpx26, which share homology with known enzymes, glyoxalase I and Nudix hydrolase, and discuss their possible roles in the infection of host plants by R. solanacearum.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, media and culture conditions.
The bacterial strains used in this study are listed in Table 1. All the R. solanacearum strains are derivatives of RS1002 (Mukaihara et al., 2004). Escherichia coli strains were grown in LB medium at 37 °C (Miller, 1992). R. solanacearum was grown at 28 °C in all experiments. For multiplication, R. solanacearum strains were grown in BG medium (Boucher et al., 1985). For the protein secretion assay or {beta}-galactosidase assay, a modified HDM medium (Huynh et al., 1989) including 20 mM potassium phosphate buffer (pH 6·0) containing 7·6 mM (NH4)2SO4, 1·7 mM MgCl2 and 1·7 mM NaCl supplemented with 10 mM L-glutamate and 10 mM sucrose was used as an hrp-inducing medium. In the secretion assay, Congo red was added to the hrp-inducing medium (100 µg ml–1). Antibiotics were used at the same concentrations as previously described (Mukaihara et al., 2004).


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Table 1. Bacterial strains

 
DNA handling and transformation.
Standard procedures were used for DNA handling (Sambrook et al., 1989). The transformation of R. solanacearum genomic loci was performed as previously described (Mukaihara et al., 2004).

Construction of a transposon vector for screening.
To clone the hrpB gene, pRS103 (Mukaihara et al., 2004), which contains an hrp gene cluster from RS1002, was digested with NspV and the 2·2 kb fragment containing the entire hrpB gene was cloned into the ClaI site of pMCL200 (Nakano et al., 1995) to yield pRS142. pRS142 was digested with NspI and the 0·6 kb fragment containing a 5' portion of hrpB was inserted into the SphI site of pHSG398 (TaKaRa) to yield pRS173. To produce a promoterless hrpB gene, pRS142 and pRS173 were digested with EcoRI and the 0·6 kb fragment of pRS173 was replaced with the 1·0 kb fragment of pRS142 to produce pRS766. Finally, pRS766 was linearized by BamHI digestion and inserted into pBSL202 (Alexeyev et al., 1995) to yield pRS767, in which a promoterless hrpB gene was associated with the O-end of a mini-Tn5 transposon (Fig. 1).



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Fig. 1. Structure of the mini-Tn5hrpB transposon. Filled boxes indicate the inner (I) and outer (O) ends of IS50, and the thick line between them indicates the transposon sequence. The promoterless hrpB gene is indicated by a filled arrow. Open arrows indicate antibiotic-resistance genes: Cmr, chloramphenicol resistance; Gmr, gentamicin resistance. The open circle represents the origin (ori) of ColE1.

 
Screening of hrpB constitutive mutants.
E. coli S17-1 harbouring pRS767 was mated with RS1207 as previously described (Mukaihara et al., 2004). Conjugates were spread on BG plates containing X-Gal (100 µg ml–1), gentamicin and nalidixic acid, and transconjugants were selected. After 3 days of incubation, blue colonies expressing the hpx24'–'lacZ fusion were chosen as candidates for hrpB constitutive mutants and used in further studies.

Construction of hrpB–lacZYA and hrpBclacZYA fusions.
A 6·3 kb BamHI–SalI promoterless lacZYA fragment from pUC-lacZYA (Mukaihara et al., 2004) was inserted into pARO191 (Parke, 1990) to yield pARO-lacZYA. A 1·7 kb PstI fragment containing a part of the coding sequence of the hrpB gene from pRS142 (Mukaihara et al., 2004) was inserted into pUC118 (TaKaRa) to yield pRS919. A 1·7 kb HindIII–BamHI hrpB fragment was inserted into pARO-lacZYA. The resulting pRS920 carrying the hrpB–lacZYA transcriptional fusion was integrated into the genome of strains RS1085 and RS1316, in which the wild-type hrpB locus was deleted by homologous recombination through conjugation with S17-1. The transconjugants contain the hrpB–lacZYA fusion and the hrpBclacZYA fusion, respectively.

{beta}-Galactosidase assay.
Cells of R. solanacearum strains were cultivated in BG medium for 16 h at 28 °C, collected by centrifugation, washed twice in distilled water and resuspended in 1 : 100 hrp-inducing medium or BG medium. After incubation for 16 h (hrp-inducing medium) or 8 h (BG medium), the {beta}-galactosidase activity was measured as described by Miller (1992).

Plasmid-based procedure for constructing R. solanacearum mutants that express FLAG-fused proteins.
To construct the FLAG fusion, a plasmid-based method was applied as follows. PCR-amplified fragments containing parts of the 3' coding sequences of the target genes were inserted into pNC123. pNC123 was constructed by inserting a 0·1 kb fragment including the FLAG-coding sequence into pARO181 (Parke, 1990). The resulting plasmid carrying the flag fusion was integrated into the genome of R. solanacearum EPS strain RS1085 and its {Delta}(hrpBhrcT) derivative, RS1204, through conjugation with S17-1. The transconjugants contained the plasmid insertion, expressing the FLAG fusion, in the chromosomal locus of the target gene. The primer sets used for the amplification of the hrpB-regulated genes and the resulting integration vectors are listed in Table 2.


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Table 2. Primers used to construct the flag fusions and plasmids used in this study

 
Preparation of protein samples.
R. solanacearum strains expressing the C-terminal FLAG fusion were grown overnight in BG medium at 28 °C. Cells were centrifuged, washed twice with distilled water and resuspended in hrp-inducing medium supplemented with Congo red (100 µg l–1) at OD600 0·5. After 16 h incubation, the culture (OD600 approx. 1·0) was centrifuged at 1500 g for 30 min, twice, and then the culture supernatant was filtered with 0·2 µm pore filter units (Millipore). To confirm the complete removal of bacteria, 50 µl of the filtered culture supernatant was plated on BG medium and no colony formation after 2 days of incubation at 28 °C was confirmed. The culture supernatant was concentrated 250-fold by ultrafiltration using a 30 000 Mr cutoff Amicon Ultra-15 filter (Millipore). In the experiment on Hpx19 (20 kDa), 10 000 Mr cutoff Amicon Ultra-15 filter was used. These samples were added to the same volume of 2x SDS sample buffer (Bio-Rad) and used as supernatant proteins for further analysis. To prepare total cell proteins, for comparison, an aliquot of the cultured medium was removed before centrifugation and mixed with the same volume of SDS sample buffer.

Visualization by silver staining or immunoblotting.
The protein samples were heated at 92 °C for 5 min, and 5 µl of cell and supernatant samples was separated using 8–16 % gradient SDS-polyacrylamide gels. To visualize proteins, gels were stained with Coomassie blue (Bio-Rad) or the Silver Stain Kit (Wako Chemicals). FLAG-fused proteins were transferred onto PVDF membranes using a semi-dry transblotter (Bio-Rad) and detected by immunoblotting. The procedures for detection were carried out according to the manual of the ECL plus Western Blotting Detection Kit, and exposure to Hyper Film ECL was carried out (Amersham Pharmacia Biotech). Anti-FLAG M2 monoclonal antibody (Sigma, 1 : 30 000 concentration) and anti-mouse IgG antibody (Amersham Pharmacia Biotech, 1 : 8000 concentration) were used as the primary and secondary antibodies, respectively. For the detection of neomycin phosphotransferase II (NptII), anti-NptII rabbit polyclonal IgG antibodies (US Biological, 1 : 10 000 concentration) and anti-rabbit IgG–HRP conjugate (Amersham Pharmacia Biotech, 1 : 30 000 concentration) were used as the primary and secondary antibodies, respectively.

Construction of {Delta}gloA mutants.
A 0·5 kb fragment upstream of gloA was PCR-amplified from RS1002 genomic DNA using the primers P1047 (5'-GCTCTAGACTCAGACAGCCTCCCCTCTT-3') and P1048 (5'-GCGGATCCTGGAGCATTCGC ATGGGATT-3') with XbaI (underlined) and BamHI (bold) restriction sites, respectively. Similarly, a 0·5 kb fragment downstream of gloA was PCR-amplified using the primers P1049 (5'-GCGGATCCCCGATGGCAACCGCCACTGA-3') and P1050 (5'-CGAATTCGCCGGATGCAGCATGCCATC-3') with BamHI (underlined) and EcoRI (bold) restriction sites, respectively. The two fragments were tandemly inserted into pK18mobsacB (Schäfer et al., 1994), and a 2·0 kb streptomycin/spectinomycin-resistant (Smr/Spr) gene cassette from pKRP13 (Reece & Phillips, 1995) was inserted between them to yield pRS926. Using pRS926, {Delta}gloA strains were constructed by the marker-exchange method (Schäfer et al., 1994).

Methylglyoxal sensitivity test.
R. solanacearum strains were grown overnight in BG medium at 28 °C. Cells were collected by centrifugation, washed twice with distilled water and resuspended in distilled water at OD600 0·5. Cell suspensions were spread on hrp-inducing minimal plates; a filter paper with a diameter of 1 cm was placed at the centre of the plate and then 10 µl 100 mM methylglyoxal was instilled into the filter paper. After 1 day incubation at 28 °C, the radius of the inhibition zone of bacterial growth that was formed around the filter paper was measured.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Isolation of R. solanacearum hrpB constitutive mutants
The expression of R. solanacearum hrpB-regulated genes is specifically activated under poor nutrient conditions (Arlat et al., 1992). Therefore, we attempted to detect the secretion of two well-known Hrp-secreted outer proteins of R. solanacearum, PopA (Arlat et al., 1994) and PopC (Guéneron et al., 2000), in several hrp-inducing minimal media. However, we failed to detect the secretion of either protein in the culture supernatants (data not shown). This seemed to be due to the low expression level of hrpB, because the expression of hrpB was markedly increased during co-cultivation with plant cells compared with an hrp-inducing medium (Marenda et al., 1998). We therefore attempted to construct a R. solanacearum mutant that constitutively expresses hrpB at a high level.

To isolate hrpB constitutive (hrpBc) mutants, we conjugatively transferred the plasmid pRS767 carrying a mini-Tn5hrpB transposon into R. solanacearum RS1207, an EPS {Delta}hrpB strain containing an in-frame 'lacZ fusion in an hrpB-regulated gene, hpx24. Mini-Tn5hrpB contains a promoterless hrpB gene and a gentamicin-resistance (Gmr) gene for the selection of transposition events (Fig. 1). Because the wild-type hrpB gene in RS1207 was entirely deleted, only an exoconjugant, in which mini-Tn5hrpB was transposed into its chromosome and inserted under a constitutive promoter, could express hrpB, simultaneously leading to the expression of the hpx24'–'lacZ fusion. The frequency of transposition was ~10–7 per recipient cell. We screened approximately 6000 Gmr colonies and isolated 29 mutants, B1–B29, which showed a blue colony on X-Gal-containing BG plates, on which the expression of the original hrpB gene was repressed (Arlat et al., 1992) (Fig. 2b). The {beta}-galactosidase activity of these mutants in liquid BG medium was measured and compared with that of wild-type RS1207 (part of the results are shown in Fig. 2b). One of the mutants, B9, exhibiting the highest {beta}-galactosidase activity –15-fold higher than that of the wild-type – was chosen and renamed RS1238 for further study (Fig. 2b). The chromosomal DNA of RS1238 was purified and electroporated into the EPS strain RS1085, and the Gmr transformant RS1239, which contained the mini-Tn5hrpB-B9 insertion but not the hpx24'–'lacZ fusion, was isolated. Using the chromosomal DNA purified from RS1239, the mini-Tn5hrpB-B9 was reintroduced into RS1207 and its ability to induce the constitutive expression of hpx24'–'lacZ was confirmed (Table 3). Enhanced expression of hpx24'–'lacZ was also observed in an hrp-inducing minimal medium (Table 3). This suggests that the hrpB on mini-Tn5hrpB-B9 is expressed at a level higher than that of the wild-type.



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Fig. 2. Screening and characterization of hrpB constitutive (hrpBc) mutants. (a) Primary screening of hrpBc mutants on an hrp-repressing BG plate. Transconjugants with a mini-Tn5hrpB insertion form colonies on the selective plate. Blue colonies expressing the hpx24'–'lacZ fusion are arrowed. (b) Expression of hpx24'–'lacZ in strain RS1207 (WT) and its hrpBc derivatives (B8, B9, B13, B24, B25 and B28) in hrp-repressing BG medium. (c) Expression of a transcriptional lacZYA fusion in the wild-type hrpB gene (hrpB–lacZYA, white columns) and in the hrpB gene on the mini-Tn5hrpB-B9 transposon (hrpBclacZYA, black columns) in BG medium (BG) and hrp-inducing minimal medium (MM). {beta}-Galactosidase activity was measured as described in Methods; means±SD of four experiments are shown.

 

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Table 3. Expression of hrpB-regulated genes in two hrpB backgrounds

The table shows {beta}-galactosidase activity in Miller units produced by a translational fusion for each gene in rich (BG) or minimal (MM) medium. The values are means±SD for four replicates. Fold induction indicates the ratio of activity of the fusion in the hrpB constitutive (hrpBc) background to that in the wild-type (hrpB+) background.

 
The chromosomal locus including mini-Tn5hrpB-B9 was rescued from RS1239 and the sequences flanking the transposon were determined. A comparison of those sequences and the genome sequence of GMI1000 (Salanoubat et al., 2002) revealed that mini-Tn5hrpB was inserted into a hypothetical gene, RSc2836, in the same transcriptional direction (data not shown). We constructed strains carrying a transcriptional lacZYA fusion in the hrpB gene on mini-Tn5hrpB-B9 and in the wild-type locus and measured their {beta}-galactosidase activities in the BG and hrp-inducing minimal medium. The expression of hrpB on mini-Tn5hrpB-B9 increased by 15·3-fold and 2·8-fold compared with that of the wild-type hrpB in the BG and hrp-inducing minimal medium, respectively (Fig. 2c). This indicates that the promoterless hrpB gene on the transposon is expressed from an as yet unknown constitutive promoter upstream of RSc2836, although the transcriptional regulation of this gene remains unclear.

Expression of other hrpB-regulated genes is enhanced in the hrpBc background
To confirm that the expression of other hrpB-regulated genes is also enhanced in the hrpBc background, we introduced RSc2836 : : mini-Tn5hrpB into several R. solanacearum strains containing an in-frame 'lacZ fusion in hrpB-regulated genes. We monitored the expression of an Hrp component gene, hrcQ (Bogdanove et al., 1996), known Hrp-secreted protein genes, popA (Arlat et al., 1994) and popC (Guéneron et al., 2000), and some of the recently identified hpx genes, hpx19, hpx24, hpx25 and hpx26 (Mukaihara et al., 2004). The {beta}-galactosidase activity of the fusion in the hrpBc mutants was 10–90·6-fold higher in the BG medium and 4·9–84-fold higher in the hrp-inducing minimal medium compared with that of the wild-type (Table 3). These results suggest that the hrpB-regulated genes are constitutively expressed in the hrpBc background. In the hrpBc background, interestingly, the expression of hrpB-regulated genes was 2·0–13·8-fold higher in the hrp-inducing minimal medium than in the BG medium, whereas the expression of hrpB was 15-fold higher in the BG medium (Table 3).

Detection of known R. solanacearum Hrp-secreted proteins, PopA and PopC, in the culture supernatant of hrpBc mutants
To investigate whether the Hrp secretion apparatus and its substrate proteins are highly expressed in the hrpBc background, we analysed the secretion of PopA by constructing strain RS1338, which expresses the C-terminal FLAG-tagged PopA protein, using a plasmid-based recombination procedure (see Methods). An hrpBc derivative, RS1340, and its Hrp-defective mutant, RS1341, expressing FLAG-tagged PopA were also constructed. These strains were grown in an hrp-inducing medium and their cellular and extracellular proteins were analysed by immunoblot analysis using the anti-FLAG antibody. For the assay, several hrp-inducing media, including one-fourth-strength M63 glutamate medium (Arlat et al., 1994), XOM2 medium (Tsuge et al., 2002) and hrp-derepressing medium (HDM) (Huynh et al., 1989), were tested. We found modified HDM medium supplemented with 10 mM L-glutamate and 10 mM sucrose to be the most effective medium for the secretion of Hrp-secreted proteins into culture supernatants (data not shown). We also confirmed that, as reported by Guéneron et al. (2000), the presence of Congo red in the culture medium enables the detection of Hrp-secreted proteins (data not shown). As shown in Fig. 3(a), 38 kDa and 28 kDa proteins, which represented PopA1 and PopA3, a full-size and N-terminal-truncated form of PopA, respectively (Arlat et al., 1994), were observed in the culture supernatant of the wild-type strain. However, much higher amounts of PopA1 and PopA3 were detected in the culture supernatant from the hrpBc mutant. Under the conditions we used, the signal of FLAG-tagged PopA was hard to detect in cell samples of the wild-type strain but became visible in the hrpBc background (Fig. 3a). The secretion of FLAG-tagged PopA was completely abolished in the Hrp-defective mutant (Fig. 3a), indicating that the secretion occurred in an Hrp-dependent manner.



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Fig. 3. Detection of Hrp-secreted outer proteins PopA and PopC. (a) Secretion of the FLAG-tagged PopA protein and FLAG-tagged PopC protein in the wild-type (hrpB+ Hrp TTSS+), hrpB constitutive (hrpBc Hrp TTSS+) and Hrp-defective hrpB constitutive (hrpBc Hrp TTSS) backgrounds. Each strain expressing the FLAG fusion was grown in secretion medium, and equal volumes of samples of total cell extracts (Cell) and culture supernatants (Sup.) were analysed by SDS-PAGE and immunoblotting using the anti-FLAG antibody. The neomycin phosphotransferase II (NptII) protein was detected by immunoblotting with NptII antibody and used as a cytoplasmic protein marker. (b) Visualization of secreted proteins by silver staining. Culture supernatant samples were separated by SDS-PAGE and silver-stained. Arrows indicate protein bands appearing in an Hrp-dependent manner. Filled circles indicate hrpB-dependent but Hrp-independent outer proteins. The bracket indicates the range covered by Congo red.

 
We also examined the secretion of PopC, another known Hrp-secreted outer protein of R. solanacearum (Guéneron et al., 2000). Although the signal of cellular FLAG-tagged PopC was hard to detect even in the hrpBc background, the Hrp-dependent secretion of FLAG-tagged PopC in the culture supernatant was clearly observed in the hrpBc mutant (Fig. 3a).

To exclude the possibility that FLAG-tagged proteins in the culture supernatant resulted from cell autolysis during cultivation, we screened for the presence of neomycin phosphotransferase II (NptII), a well-known cytoplasmic protein, in each protein sample. The NptII signal was detected only in the cell samples, not in the supernatant samples (Fig. 3a), demonstrating that no cell autolysis occurred during cultivation.

SDS-PAGE analysis of proteins secreted into the culture supernatants of hrpBc mutants
To compare the secretion of extracellular proteins among mutants, we silver-stained an SDS-PAGE gel loaded with the respective supernatant proteins. Compared with the wild-type, large protein bands with a molecular mass of 80–240 kDa were observed in the culture supernatant of the hrpBc mutant (Fig. 3b). These proteins were presumably Hrp-secreted proteins because they were not observed in the culture supernatant of the Hrp-defective hrpBc mutant. This result and the results outlined above indicate that the hrpBc mutant secretes a high amount of Hrp-secreted proteins into the culture supernatants and that this characteristic is useful for analysing the secretion of candidate effector proteins in R. solanacearum.

Secretion analysis of Hpx proteins that are homologues of known type III effector proteins
In a previous study, we isolated several hpx genes encoding homologues of known type III effector proteins found in other animal and plant pathogens (Mukaihara et al., 2004). Therefore, we next determined whether these gene products are also secreted as Hrp-secreted outer proteins in R. solanacearum as above. We tested the hpx23, hpx24 and hpx25 gene products, which share homology with HopPtoA1, HolPtoR and HopPtoD1 from P. syringae pv. tomato, respectively (Badel et al., 2002; Buell et al., 2003; Petnicki-Ocwieja et al., 2002). Of the three proteins tested, FLAG-tagged proteins were secreted into the culture supernatant in the hrpBc mutant but not in its Hrp-defective mutant (Fig. 4a). The Hpx23-FLAG and Hpx25-FLAG signals were hard to detect in the cell fraction, probably due to their low expression levels (Mukaihara et al., 2004). We also performed immunodetection of NptII in each protein sample and confirmed that no cell autolysis occurred during cultivation (data not shown). These results clearly indicate that these effector homologues, Hpx23, Hpx24 and Hpx25, are also Hrp-secreted outer proteins in R. solanacearum.



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Fig. 4. Detection of FLAG-tagged Hpx proteins in the hrpB constitutive background. (a) Secretion of Hpx proteins with homology to known type III effector proteins in the wild-type (Hrp TTSS+) and Hrp-defective (Hrp TTSS) backgrounds. (b) Secretion of Hpx proteins with homology to known enzymes in the wild-type (Hrp TTSS+) and Hrp-defective (Hrp TTSS) backgrounds. The procedures for the preparation and analysis of protein samples were as outlined in Fig. 3.

 
Secretion analysis of the Hpx19 and Hpx26 proteins, which share homology with known enzymes
We have identified four hpx genes encoding protein products that share homology with known enzymes: glyoxylase I (hpx19), Nudix hydrolase (hpx26), spermidine synthase (hpx7) and transposase (hpx22) (Mukaihara et al., 2004). The hpx19 and hpx26 genes seem to be directly regulated by HrpB because these two genes contain a PIP-box-like motif in their putative promoter regions (Mukaihara et al., 2004). To determine whether these gene products are secreted via the Hrp TTSS, we performed immunoblot analysis as above. FLAG-tagged Hpx19 (20 kDa) was not secreted outside the cell but accumulated to high levels in both the hrpBc mutant and its Hrp-defective mutant (Fig. 4b). On the other hand, FLAG-tagged Hpx26 (51 kDa) was secreted outside the cell in the hrpBc mutant but not in its Hrp-defective mutant (Fig. 4b). This shows that Hpx19 is an intracellular protein but Hpx26 is an Hrp-secreted outer protein.

Hpx19 is partly involved in the detoxification of methylglyoxal
Glyoxalase I is known to detoxify methylglyoxal, a cytotoxic metabolic intermediate (Iyengar & Rose, 1981; Rahman et al., 1990; Lo et al., 1994; Papoulis et al., 1995). Hpx19 shares homology with E. coli glyoxalase I (27·4 % identity and 54·8 % similarity). The genome of R. solanacearum GMI1000 contains another gene, gloA (RSc0520), whose product shares a higher homology with E. coli glyoxalase I (77·8 % identity and 92·6 % similarity) than Hpx19. To explore whether Hpx19 and GloA can detoxify exogenous methylglyoxal, a sensitivity test was performed on several mutant derivatives of the hrpBc strain. When an hpx19 Tn-insertion mutant, RS1243, was compared with the wild-type RS1239, no difference was observed in the size of the growth-inhibition zone by spotted methylglyoxal (Table 4). In contrast, a {Delta}gloA derivative, RS1384, showed an apparent increased sensitivity to methylglyoxal, indicating that GloA mainly functions in the detoxification of methylglyoxal under this experimental condition. However, a {Delta}gloA hpx19 derivative, RS1385, showed a slightly increased sensitivity to methylglyoxal compared with a single gloA mutant (Table 4). This suggests that Hpx19 has some role in the detoxification of methylglyoxal.


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Table 4. Methylglyoxal sensitivity test

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we examined the secretion of several candidate effector proteins encoded by hrpB-regulated genes of R. solanacearum, previously isolated by genetic screening (Mukaihara et al., 2004). The expression level of wild-type hrpB in an hrp-inducing medium was insufficient to detect the secretion of known R. solanacearum Hrp-secreted outer proteins, PopA and PopC, into culture supernatants. Therefore, we isolated a mutant constitutively expressing hrpB (Fig. 2). In hrpBc mutants, all the hrpB-regulated genes we tested were highly expressed (Table 3) and, as expected, high-level secretion of PopA and PopC into culture supernatants was observed (Fig. 3). When hrpBc mutants were cultivated in an hrp-inducing medium, as compared with the wild-type, numerous Hrp pili were observed on the cell surface by transmission electron microscopy (data not shown). These results indicated that the Hrp TTSS machinery complex and its substrate proteins were strongly expressed in the hrpBc background, resulting in the high-level secretion of effector proteins. In Xanthomonas campestris pv. vesicatoria, which has an hrp regulatory system similar to that of R. solanacearum (Wengelnik & Bonas, 1996), a constitutive active form of an hrp-regulatory protein, HrpG*, was constructed by amino acid substitutions and used to express Hrp TTSS at high levels (Wengelnik et al., 1999). Our system, using the high-level expression of an hrp-regulatory protein, provides a new strategy for the high-level production of the Hrp TTSS.

It is noteworthy that, in the hrpBc background, the expression of the hrpB-regulated genes was higher in the hrp-inducing minimal medium than in the BG medium, whereas the expression of hrpB was high in the BG medium (Table 3). This suggests that HrpB activity is regulated at the post-transcriptional level by an unknown regulatory mechanism in addition to the previously reported transcriptional regulation of the hrpB gene (Genin et al., 1992; Arlat et al., 1994; Marenda et al., 1998). Some component of the hrp-inducing minimal medium or BG medium might directly bind HrpB and activate or repress its activity. Although the mechanism is currently unclear, hrpBc mutants may be a powerful tool to solve the problem.

We found several Hrp-dependent outer proteins in the culture supernatants of hrpBc mutants (Fig. 3). These proteins may include protein products from the hpx genes and other hrpB-dependent or -independent genes that were missed in our screening. Analysing these proteins will help us identify novel type III effector proteins of R. solanacearum. Using hrpBc mutants, we examined the secretion of Hpx23, Hpx24 and Hpx25, which are homologues of Hrp-secreted outer proteins, HopPtoA1, HolPtoR and HopPtoD1, of P. syringae pv. tomato, respectively (Badel et al., 2002; Buell et al., 2003; Petnicki-Ocwieja et al., 2002). In P. syringae pv. tomato, these genes are controlled by HrpL, an alternative sigma factor regulating the expression of hrp genes and most of the effector genes (Xiao et al., 1994). In contrast, in R. solanacearum, these genes are controlled by HrpB, an AraC-type transcriptional activator (Mukaihara et al., 2004). We demonstrated that these proteins are also Hrp-secreted outer proteins of R. solanacearum (Fig. 4). This suggests that the above-mentioned Hpx proteins are also effector proteins of R. solanacearum and that they are injected into plant cells and exercise their pathogenicity functions.

We also examined the secretion of two hpx gene products that share homology with known enzymes, glyoxalase I (Hpx19) and Nudix hydrolase (Hpx26). The Hpx19 protein is accumulated to high levels in bacterial cells but not secreted into the culture medium (Fig. 4). Glyoxalase I is known to detoxify methylglyoxal, a cytotoxic metabolic intermediate mainly formed in glycolysis as a by-product (Iyengar & Rose, 1981; Rahman et al., 1990; Lo et al., 1994; Papoulis et al., 1995). In yeast and plants, increasing the protein level of glyoxalase I results not only in elevated tolerance to high-level methylglyoxal but also in improved tolerance to various environmental stresses (Espartero et al., 1995). It has also been reported that the glyoxalase I activity is increased in rapidly dividing cells (Seraj et al., 1992; Paulus et al., 1993). The expression of hpx19 was controlled by HrpB and seemed to be induced at high levels in host plants (Mukaihara et al., 2004). Although the involvement of Hpx19 in the detoxification of methylglyoxal was apparently slight (Table 4), it may function as glyoxalase I. The low activity might be due to its low expression in our experimental system, even in the hrpBc background. The expression of hrpB-regulated genes is activated more than 20-fold higher in co-culture with plant cells compared with that in an hrp-inducing minimal medium (Marenda et al., 1998). Under such an in planta condition, Hpx19 might make a greater contribution to the detoxification of methylglyoxal. Alternatively, Hpx19 may mainly detoxify toxic compounds other than methylglyoxal, formed during bacterial metabolism or derived from plants. Although the biochemical activities of Hpx19 are still unclear, it may function to promote the rapid cell division of the pathogen or confer tolerance to stresses in host plants.

It is interesting to note that Hpx26 is secreted outside the cell as an Hrp-secreted outer protein in R. solanacearum (Fig. 4). Sequence analysis of the N-terminal 50 amino acids of Hpx26 revealed that the contents of serine and proline residues, 10·0 % and 18·0 %, are higher than those of the 3'-terminus, 4·7 % and 5·9 % (data not shown). This is one of the characteristics of type III effector proteins of phytopathogenic bacteria (Guttman et al., 2002; Petnicki-Ocwieja et al., 2002; Cunnac et al., 2004). Several Nudix motif proteins related to pathogenicity have been identified in several animal pathogens. The NudA protein of the gastric pathogen Helicobacter pylori is involved in oxidative stress response (Lundin et al., 2003). The IalA protein of Bartonella bacilliformis is involved in the invasiveness of the pathogen into human erythrocytes, in cooperation with IalB (Mitchell & Minnick, 1995). The above two Nudix motif proteins are intracellular proteins (Lundin et al., 2003; Mitchell & Minnick, 1995). As far as we know, Hpx26 seems to be the first Nudix motif protein secreted extracellularly. The Nudix motif, GX5EX7REUXEEXGU (where U is usually Ile, Leu or Val) (Bessman et al., 1996), was first identified as an active site of the MutT protein, an 8-oxo-dGTPase, of E. coli (Mejean et al., 1994) (Fig. 5). The Nudix motif protein family consists of more than 1100 proteins in more than 250 species from all kingdoms. The major substrates of these enzymes are nucleoside diphosphates linked to some other moiety X, hence the acronym ‘Nudix’: ADP-ribose, dinucleoside polyphosphate, NADH, nucleoside sugars, or ribo- and deoxyribonucleoside triphosphates (Dunn et al., 1999) (Fig. 5). These substrates are potential signalling molecules and metabolic intermediates of which the concentrations change due to the modulation of cellular functions (Kisselev et al., 1998). Therefore, it is possible that Hpx26 changes the concentration of some of the above substrates to modulate plant cellular functions. The identification of the substrate will help us clarify the pathogenic role of Hpx26 in host plant cells.



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Fig. 5. Comparison of the Nudix motif of Hpx26 and Nudix hydrolases from several organisms. The positions of the N-terminal and C-terminal residues relative to mature proteins are indicated at the ends of each amino acid sequence. The consensus amino acid residues of the Nudix motif are shown in bold. Filled circles indicate the previously described conserved amino acid residues that exhibit substrate specificity (Dunn et al., 1999). Sequence accession numbers are P08337 for MutT of E. coli (Akiyama et al., 1987), AY250081 for NudA of Helicobacter pylori (Lundin et al., 2003), L25276 for IalA of Bartonella bacilliformis (Mitchell & Minnick, 1995), Z35980 for Ysa1 of Saccharomyces cerevisiae (Dunn et al., 1999), U89841 for Ap4Ase of Lupinus angustifolius (Maksel et al., 2001) and P32664 for ORF257 of E. coli (Frick & Bessman, 1995).

 


   ACKNOWLEDGEMENTS
 
We thank Miss Nanba for her excellent technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (A) (no. 15028218) to T. M. from the Ministry of Education, Culture, Sports, Science and Technology, Japan.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 29 April 2005; revised 16 June 2005; accepted 22 June 2005.



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