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Journal of Bacteriology, March 2004, p. 1374-1380, Vol. 186, No. 5
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.5.1374-1380.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Evidence for HrpXo-Dependent Expression of Type II Secretory Proteins in Xanthomonas oryzae pv. oryzae

Ayako Furutani,¹ Seiji Tsuge,^1* Kouhei Ohnishi,² Yasufumi Hikichi,³ Takashi Oku,⁴ Kazunori Tsuno,⁵ Yasuhiro Inoue,⁶ Hirokazu Ochiai,⁶ Hisatoshi Kaku,⁶ and Yasuyuki Kubo¹

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto Prefectural University, Kyoto 606-8522,¹ Research Institute of Molecular Genetics,² Laboratory of Plant Pathology and Biotechnology, Kochi University, Nankoku, Kochi 783-8502,³ Laboratory of Molecular Plant Pathology, School of Bioresources, Hiroshima Prefectural University, Shobara 727-0023,⁴ Faculty of Agriculture, Miyazaki University, Miyazaki 889-2155,⁵ National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan⁶

Received 17 June 2003/ Accepted 10 November 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
Xanthomonas oryzae pv. oryzae is a causal agent of bacterial leaf blight of rice. Recently, an efficient hrp-inducing medium, XOM2, was established for this bacterium. In this medium, more than 10 proteins were secreted from the wild-type strain of X. oryzae pv. oryzae. Many of these proteins disappeared or decreased in amount in culture on XOM2 when incubated with the strain that has a mutation in the hrp regulatory gene. Interestingly, the secretory protein profile of a mutant lacking a type III secretion system (TTSS), components of which are encoded by hrp genes, was similar to that of the wild-type strain except that a few proteins had disappeared. This finding suggests that many HrpXo-dependent secretory proteins are secreted via systems other than the TTSS. By isolating mutant strains lacking a type II secretion system, we examined this hypothesis. As expected, many of the HrpXo-dependent secretory proteins disappeared or decreased when the mutant was cultured in XOM2. By determining the N-terminal amino acid sequence, we identified one of the type II secretory proteins as a cysteine protease homolog, CysP2. Nucleotide sequence analysis revealed that cysP2 has an imperfect plant-inducible-promoter box, a consensus sequence which HrpXo regulons possess in the promoter region, and a deduced signal peptide sequence at the N terminus. By reverse transcription-PCR analysis and examination of the expression of CysP2 by using a plasmid harboring a cysP2::gus fusion gene, HrpXo-dependent expression of CysP2 was confirmed. Here, we reveal that the hrp regulatory gene hrpXo is also involved in the expression of not only hrp genes and type III secretory proteins but also some type II secretory proteins.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In general, plant-pathogenic bacteria possess hypersensitive response and pathogenicity (hrp) genes, which are clustered in their chromosomes. hrp genes encode a type III secretion system (TTSS) that delivers virulence and avirulence factors from the bacteria to plant cells and are required for pathogenesis in host plants and for triggering a hypersensitive response in nonhost plants (1, 38). Transcriptional regulation of hrp genes depends on environmental conditions. The expression of hrp genes is generally suppressed in complex media and induced in planta and under certain in vitro conditions (6, 24, 35, 39).

In xanthomonads, the hrp cluster comprises six hrp loci, hrpA to hrpF, which are all required for full pathogenicity, and their expression is regulated by two genes, hrpX and hrpG, which are located outside the hrp gene cluster region (7, 36). The HrpG protein belongs to the OmpR family of two-component regulatory systems and activates the expression of hrpA and hrpX (37). HrpX, an AraC-type transcriptional activator, has been reported to control the expression of the operons hrpB to hrpF, which contain the hrp genes encoding a component of TTSS (36). It has also been suggested that HrpX controls some effector proteins (5). Several genes that are regulated in a HrpX-dependent manner possess the consensus nucleotide sequence TTCGC(N₁₅)TTCGC, which has been termed the plant-inducible-promoter (PIP) box (12).

Xanthomonas oryzae pv. oryzae is a causal agent of bacterial leaf blight of rice (28). Recently, an efficient hrp-inducing medium, XOM2, was established for the bacterium (31). Using this medium, we have identified Hpa1 as one of the HrpXo-regulated type III secretory proteins in X. oryzae pv. oryzae (14). Hpa1 is encoded by an hrp cluster with a PIP box, and its requirement for disease development in rice plants has been reported (14, 41). We also detected HrpXo-regulated secretory proteins other than HpaI, none of which have been identified (14). Some of these proteins might be involved in pathogenicity.

Other than hrp gene products, extracellular polysaccharide, extracellular enzymes, and toxins have been proposed as possible virulence factors in X. oryzae pv. oryzae (3, 4, 22, 40). Suvendra et al. (27) reported that mutants of X. oryzae pv. oryzae deficient in a type II secretion system also lack virulence.

In this study, we detected not only HrpXo-regulated type III secretory proteins but also some HrpXo-regulated type II secretory proteins in culture supernatant from the hrp-inducing medium XOM2. We identified HrpE1 and HrpF as HrpXo-regulated type III proteins and a protein homologous with cysteine protease as one of the HrpXo-regulated type II secretory proteins.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli DH5MCR (Stratagene) was grown at 37°C in Luria-Bertani medium (25). X. oryzae pv. oryzae strains were usually grown at 28°C in nutrient broth-yeast extract (NBY) medium (32) or in an hrp-inducing medium, XOM2 (31). Xanthomonas axonopodis pv. citri was grown at 28°C in NBY medium. All media were supplemented with the following antibiotics at the indicated concentrations: rifampin, 20 µg/ml; ampicillin, 50 µg/ml; cycloheximide, 50 µg/ml; kanamycin, 25 µg/ml for X. oryzae pv. oryzae and 50 µg/ml for E. coli; spectinomycin, 25 µg/ml for X. oryzae pv. oryzae and 50 µg/ml for E. coli.

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TABLE 1. Bacterial strains and plasmids used in this study

Recombinant DNA techniques. DNA manipulations were performed by standard procedures (25).

Sequence analysis. A dye terminator cycle sequencing reaction was performed with a DNA sequencing kit (Applied Biosystems, Piscataway, N.J.) according to the manufacturer's instructions followed by electrophoresis and analysis with an autosequencer (model 373A; Applied Biosystems). Similarity searches were made by using the BLAST program (2). A potential signal peptide at the N terminus was predicted by PSORT (21).

Isolation of mutants lacking a type II secretion system of X. oryzae pv. oryzae. An EZ::TN <KAN-2> transposome, a mixture of the transposon EZ::TN <KAN-2> and EZ::TN transposase (Epicentre, Madison, Wis.), was introduced directly into X. oryzae pv. oryzae strain T7174R by electroporation. Three strains out of 1,000 kanamycin-resistant clones were then selected for deficiencies in extracellular cellulase and xylanase activities, which are known to be secreted by the type II secretion system (10, 14, 15). Assays for these enzymatic activities were done according to the procedures described by Tsuchiya et al. (29) and Keen et al. (18), respectively. Sequence analysis of the regions flanking the transposon revealed that the transposon was inserted into homologs of xpsE, xpsL, and xpsN of Xanthomonas campestris pv. campestris (DDBJ accession no. AE012165), which are deduced to be genes encoding a component of the type II secretion system (26), and the mutant strains were named 74XpsE, 74XpsL, and 74XpsN, respectively.

Detection of secretory proteins of X. oryzae pv. oryzae in XOM2. X. oryzae pv. oryzae strains were preincubated on NBY agar medium for 1 day and adjusted to an optical density at 600 nm of 2 with sterilized water. Forty microliters of the bacterial suspension was inoculated into 1 ml of XOM2 (pH 6.0). After 2 days of incubation (28°C, 180 rpm), bacteria were removed by centrifugation at 10,000 x g for 5 min and filtration, and the supernatant was precipitated on ice with 10% (vol/vol) trichloroacetic acid. After centrifugation at 16,000 x g for 30 min at 4°C, protein precipitates were washed twice with acetone and resuspended in 150 µl of Laemmli buffer (20). Protein samples were boiled for 3 min and separated by sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis (SDS-15% PAGE). Proteins were detected by silver staining with a Wako (Osaka, Japan) silver stain kit.

Amino acid sequence analysis. For the analysis of N-terminal amino acid sequences of secretory proteins from 14 ml of XOM2 culture medium, proteins were separated on a large preparative SDS-15% PAGE gel or Tricine-SDS-17.5% PAGE gel and transferred to a polyvinylidene difluoride membrane (Immobilon-P [Millipore, Bedford, Mass.] or a 0.2 µm-pore-size Immun-Blot polyvinylidene difluoride membrane [Bio-Rad, Richmond, Va.], respectively). The membranes were stained with 0.025% Coomassie brilliant blue R-250, and the protein bands were excised. The N-terminal amino acid sequences of the proteins were determined with an Applied Biosystems model 492 Procise protein sequencing system. Homology searches were done with the NCBI BLAST server (http://www.ncbi.nlm.nih.gov/BLAST/).

Reverse transcription (RT)-PCR. Total RNA from bacteria cultured in XOM2 for 1 day was extracted with an RNeasy kit (QIAGEN, Valencia, Calif.). cDNA synthesis and PCR were conducted with RiverTra-Ace (Toyobo, Osaka, Japan) and KOD Dash (Toyobo), respectively.

Cloning of a DNA fragment containing cysP2. pGLCysP, a cosmid clone from a genomic library of X. oryzae pv. oryzae T7174R containing cysP2, was selected by colony hybridization with the internal fragment of the X. axonopodis pv. citri cysteine protease gene (XAC2853; refer to GenBank accession no. AE011926) as a probe. The internal fragment of XAC2853 was amplified by PCR with genomic DNA of X. axonopodis pv. citri MAFF302104 used as a template and the primers 5'-ATGGGCCTGAAGCCTTCGTC-3' and 5'-TCGGCGCCGATCACATCCTT-3'. An approximately 5-kb EcoRI-HindIII fragment from pGLCysP containing cysP2 was subcloned into pUC119 (33) to give pUCCysP2.

Construction of a plasmid harboring a cysP2::gus fusion gene. A 459-bp fragment containing the 91-bp 5' coding region and 368-bp noncoding region of cysP2 was amplified by PCR using pUCCysP2 as a template and the primers 5'-GAGGCGAATTCGAAAACGAATGTGACG-3' and 5'-AGGCCCTTTCCGAGCTCTTCCGCCTGT-3'. The PCR product which was digested with EcoRI and SacI was cloned into a broad-host-range vector, pHM1 (17), to obtain pHMCysP2PIP. An approximately 1.8-kb SacI-KpnI fragment containing the gus gene from pBSGUS (31) was then inserted into pHMCysP2PIP, and the plasmid obtained was named pHMCysP2GUS. Plasmid pHMCysp2GUS was then introduced into X. oryzae pv. oryzae.

Assay of GUS activity. ß-Glucuronidase (GUS) activity was assayed as described previously (31).

Isolation of a cysP2 mutant of X. oryzae pv. oryzae T7174R. The transposon EZ::TN <KAN-2> was then introduced into pUCCysP2 according to the manufacturer's instructions. Plasmid pUCCysP2, which has the transposon inserted in cysP2 at the +198-bp position (+1 represents A of the start codon ATG) was selected by Southern blot analysis and sequence analysis. The plasmid was introduced into strain T7174R by electroporation, and kanamycin-resistant clones were selected. Marker exchange mutagenesis was confirmed by genomic Southern blot analysis (data not shown), and one of the clones was named 74CysP2.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Detection of secretory proteins from a mutant strain lacking a type II secretion system or a TTSS. Secretory proteins from the wild-type strain X. oryzae pv. oryzae T7174R in the hrp-inducing medium XOM2 were compared with those from strain 74HrpXo, in which an hrp-regulatory gene, hrpXo, is disrupted. Many of the proteins which were detected in the culture of T7174R disappeared or decreased in that of 74HrpXo (Fig. 1). To clarify whether those proteins were secreted via a TTSS, secretory proteins in 74HrcV, which has a transposon insertion in a conserved TTSS component gene and cannot secrete Hpa1, a type III secretory protein (13), were investigated. Interestingly, most of the proteins detected in the culture of T7174R were also detected in that of 74HrcV, although a few proteins containing Hpa1 were not detected (Fig. 1). These results suggest that many of the secretory proteins from the wild-type strain detected in XOM2 are secreted not via a TTSS but via other systems. To examine the involvement of the type II system in the secretion of these proteins, mutants lacking this system were isolated (74XpsE, 74XpsL, and 74XpsN; see Materials and Methods). A deficiency in secretion in these mutants was confirmed by a decrease of extracellular cellulase and xylanase activities (Fig. 2). The secretory proteins of 74XpsL were compared with those of 74HrcV, wild-type strain T7174R, and 74HrpXo (Fig. 1). Several signals detected in strains T7174R and 74HrcV had disappeared or weakened in 74XpsL along with 74HrpXo. Protein profiles from another two mutants lacking a type II secretion system (74XpsE and 74XpsN) were similar to the profile for 74XpsL (data not shown). To test whether a mutation in hrpXo influences the construction of the type II secretion system, extracellular cellulase and xylanase activities of an hrpXo mutant were investigated (Fig. 2). While 74XpsL showed low levels of these enzymatic activities, the hrpXo mutant 74HrpXo showed both activities to the same extent as the wild-type strain did. These results suggest that HrpXo regulates not only the expression of type III secretory proteins but also that of some type II secretory proteins.

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FIG. 1. Comparison of secretory proteins from the wild type (T7174R), the hrpXo mutant (74HrpXo), and mutants lacking a type II and type III secretion system (74XpsL and 74HrcV, respectively) in an hrp-inducing medium, XOM2. The proteins in the supernatants were separated by SDS-PAGE and detected by silver staining. An arrow indicates Hpa1.

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FIG. 2. Extracellular cellulase and xylanase activities in strains of X. oryzae pv. oryzae. Strains T7174R (WT), 74HrpXo (HrpXo), 74XpsL (XpsL), and 74HrcV (HrcV) were cultured on XOM2 agar plates containing carboxymethyl cellulose (upper panel) or RBB-xylan (4-O-methyl-D-glucurono-D-xylan-remazol brilliant blue R) (lower panel). The presence of a halo around a colony in T7174R, 74HrpXo, or 74HrcV indicates cellulase (CEL) and xylanase (XYL) proficiency. The halo was highly reduced when 74XpsL was cultured with XOM2 agar containing carboxymethyl cellulose, and no halo was observed on XOM2 agar containing RBB-xylan. The same results as those for 74XpsL were obtained for 74XpsE and 74XpsN.

Identification of an HrpXo-regulated secretory protein. We identified one of the type II secretory proteins, whose molecular mass was about 30 kDa, which was not detected in 74HrpXo nor in a type II secretion system-deficient mutant (Fig. 3A). The N-terminal sequence (EVHGKGLKPS) of the 30-kDa protein was almost identical to the internal sequence of X. axonopodis pv. citri cysteine protease (AVHGMGLKPS, amino acids [aa] 25 to 34; XAC2853 [refer to GenBank accession no. AE011926]) and that of X. campestris pv. campestris (AMHGMGLKPS, aa 25 to 34; XCC2693 [refer to GenBank accession no. AE012381]). The PSORT program predicted the presence of a signal peptide sequence at the N terminus (aa 1 to 24) of these proteins. These cysteine proteases are predicted to be secreted via the type II secretion system, and the N terminus of the mature form must start from aa 25.

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FIG. 3. HrpXo-regulated type II and type III secretory proteins identified in this study. Proteins in 14 ml of culture supernatant incubated with T7174R, 74HrpXo, 74XpsL, and 74HrcV were separated on a Tricine-SDS-17.5% PAGE gel (A) and an SDS-7.5% PAGE gel (B) and transferred to polyvinylidene difluoride membranes. The membranes were stained with 0.025% Coomassie brilliant blue R-250. Triangles indicate the protein bands whose N-terminal amino acid sequences were determined.

We also identified three type III secretory proteins whose molecular masses were approximately 50, 7, and 6 kDa (Fig. 3). The N-terminal sequence (NDEFNPKDIKGS) of the 50-kDa protein was perfectly consistent with the internal sequence of HrpF of X. oryzae pv. oryzae. The predicted size of HrpF is 84.9 kDa, and the consistency of the sequence determined with HrpF started from aa 223. Therefore, the 50-kDa protein is likely to be a processed or degraded product of HrpF. The N-terminal sequences of the 7- and 6-kDa proteins were MEILPQISSL and SLNSRFQQGM, respectively, perfectly consistent with the start and internal (aa 9 to 18) sequences of HrpE1 of X. oryzae pv. oryzae, whose molecular mass is predicted to be 9.7 kDa. The smaller protein might be a degraded product.

Identification of the gene encoding the 30-kDa secretory protein. Sequence analysis of pGLCysP, which is a cosmid clone from the genomic library for X. oryzae pv. oryzae T7174R and contains the region hybridized with the cysteine protease gene from X. axonopodis pv. citri, revealed that three homologs are tandemly located in an approximately 10-kb genomic region (Fig. 4). The homology among the homologs (cysP1, cysP2, and cysP3) was 95 to 97%, and all encoded 271 amino acid residues with a predicted molecular mass of 29.1 to 29.5 kDa. These homologs possessed a deduced signal sequence at the N terminus. The amino acids of the homologs at positions 25 to 34 were EVHAKGLKPS for CysP1 and EVHGKGLKPS for CysP2 and CysP3, indicating that the 30-kDa protein was CysP2 or CysP3. To determine which of these was the 30-kDa secretory protein, strains T7174R and 74HrpXo were cultivated in the hrp-inducing medium XOM2, and the transcription of cysP2 and cysP3 in each strain was analyzed by RT-PCR. We used specific primers for RT to distinguish transcriptional products of cysP2 and cysP3 (Fig. 5A). A specific DNA fragment corresponding to the internal sequence of cysP2 was amplified from T7174R, whereas it was not amplified from 74HrpXo (Fig. 5A). Although a specific fragment derived from cysP3 mRNA that was dependent on the presence of HrpXo was also detected, the signal intensity of cysP3 was much lower than that of cysP2. These results suggest that the 30-kDa protein is CysP2 and that transcription of cysP2 is regulated by HrpXo.

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FIG. 4. Gene map of a region containing three copies of a cysteine protease homolog and the nucleotide sequence of cysP2 and the promoter region. The deduced amino acid sequence of CysP2 is given in the one-letter code below the nucleotide sequence. Restriction enzymes are abbreviated as E (EcoRI) and A (ApaI) on the gene map. The amino acid sequence determined in this study is underlined. The putative start codon and the termination codon are in boldface type. An imperfect PIP box, TTCGC(N12)TTCGC, is double underlined. Boxed sequences represent the deduced signal peptide of CysP2. An open triangle represents the transposon insertion site in 74CysP2. A closed triangle indicates the position at which the gus gene was fused in pHMCysP2GUS. Arrows show the primers used for RT-PCR.

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FIG. 5. (A) Transcriptional regulation of the cysP2 and cysP3 genes by HrpXo. RT-PCR was performed to analyze the expression of cysP2 and cysP3. To distinguish the mRNA of cysP2 from that of cysP3, specific primers were used for RT. The same reverse primers were used for PCR as for RT, and a common forward primer was used for the amplification of the internal sequence of cysP2 or cysP3 (351 bp in common). Primer sets are shown. PCR products (upper gel) and rRNA (lower gel) were separated by agarose gel electrophoresis and stained with ethidium bromide. Asterisks indicate sequences common to the cysP2 and cysP3 reverse primers. (B) HrpXo-dependent expression of CysP2. Strains T7174R and 74HrpXo transformed with an empty vector pHM1 (C) or with pHMCysP2GUS (S) were incubated in XOM2 for 1 day, and GUS activity was measured. Similar results were obtained from three independent experiments.

HrpXo-dependent expression of a cysP2::gus fusion gene. To confirm HrpXo-dependent expression of CysP2, we constructed pHMCysP2GUS, which expresses a cysP2::gus fusion gene, and introduced it into X. oryzae pv. oryzae T7174R and 74HrpXo. Each transformant was cultured in XOM2, and GUS activities were measured after a 1-day incubation. The transformant T7174R(pHMCysP2GUS) showed remarkable GUS activity, while T7174R transformed with the vector plasmid pHM1 and 74HrpXo(pHMCysP2GUS) showed no activity (Fig. 5B). These results support the idea that HrpXo regulates the expression of CysP2.

Protein secretion in a cysP2 mutant, 74Cysp2. To clarify that the 30-kDa secretory protein which was not detected in mutants deficient in HrpXo and in the type II secretion system is CysP2, we generated the mutant 74CysP2, in which an EZ::TN transposon was inserted in cysP2 (Fig. 4), and analyzed it for secretory protein by culturing it in XOM2. The mutant was incubated in XOM2 for 2 days, and secretory proteins were compared with those from T7174R. The 30-kDa protein was specifically missing from the culture supernatant of 74CysP2 (Fig. 6). These results indicate that HrpXo regulates the expression of not only the components of a TTSS and type III secretory proteins but also some type II secretory proteins.

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FIG. 6. Detection of secretory proteins in 74CysP2. Proteins in 14 ml of culture supernatant incubated with T7174R (WT), 74CysP2 (CysP2), and 74HrpXo (HrpXo) were separated on a Tricine-SDS-17.5% PAGE gel and transferred to the polyvinylidene difluoride membrane. The membrane was stained with 0.025% Coomassie brilliant blue R-250. The asterisk and arrow indicate CysP2 and Hpa1, respectively.

Top Abstract Introduction Materials and Methods Results Discussion References

 
hrpX of xanthomonads has been thought to be a regulatory gene of other hrp genes which encode the components of a TTSS and effector proteins secreted via the TTSS (23, 36). For X. oryzae pv. oryzae, we have demonstrated that Hpa1, a harpin-like protein (19) whose expression is regulated by HrpXo, is secreted via a TTSS using an efficient hrp-inducing medium, XOM2 (14). However, there had been no reports of other secretory proteins. In this report, we newly identified HrpE1 and HrpF as HrpXo-regulated type III secretory proteins. Moreover, we indicated that HrpXo also regulates the expression of some type II secretory proteins and identified one of them as a cysteine protease homolog (CysP2).

By comparing the secretory proteins in culture incubated with wild-type strain T7174R and an hrcV mutant which lacks a TTSS, it was found that there were not many HrpXo-regulated type III secretory proteins in XOM2. By using mutants lacking a type II secretion system, many of the HrpXo-dependent proteins secreted from the wild-type strain were suggested to be secreted via such a system. In the culture of mutants deficient in a type II secretion system, more large-molecular-size proteins were detected than in that of the wild-type strain. It is likely that there are some products digested by proteases which are secreted via a type II secretion system in the culture of strains possessing this secretion system. On the other hand, in type II secretion system-deficient mutants, such protein digestion might not occur, and as a result, only large intact proteins might be detected. However, it is unlikely that all of the proteins detected in the culture of strains with a type II secretion system are products of digestion by proteases because the amounts of proteins detected were greater in those strains than in mutant strains lacking the secretion system. In fact, the size of the 30-kDa protein that we identified as a homolog of a cysteine protease from X. axonopodis pv. citri and X. campestris pv. campestris almost corresponded to that deduced from the nucleotide sequence.

N-terminal amino acid sequence analysis of the 30-kDa protein, which was detected in culture supernatants of T7174R and 74HrcV but not in those of 74HrpXo and 74XpsL, revealed that this protein is a homolog of cysteine protease. We found that at least three copies of this cysteine protease homolog are present in the genomic DNA of X. oryzae pv. oryzae and that these copies (products of cysP1, cysP2, and cysP3) are tandemly located in an approximately 10-kb region. Detailed nucleotide sequence analysis of cysP genes revealed that they are highly homologous (95 to 97%) and that their deduced products have a signal peptide at the N terminus. The amino acid sequence that we determined starts just after the most likely cleavage site by signal peptidase (Fig. 4 [CysP2] and data not shown [CysP1 and CysP3]), suggesting that the 30-kDa protein is secreted via the type II system.

Among three cysteine protease homologs, we considered CysP2 to be the most probable candidate for the 30-kDa secretory protein. The reasons we considered CysP2 are (i) the amino acid sequence was not completely consistent with the corresponding sequence of CysP1 and (ii) the transcriptional level was much higher in cysP2 than in cysP3 according to RT-PCR with specific primers. Actually, the mutant that had a transposon insertion in cysP2 did not secrete the 30-kDa protein.

Nucleotide sequence analysis of cysP genes revealed that all of them, not only cysP2 but also cysP1 and cysP3, have an imperfect PIP box, TTCGC(N₁₂)TTCGC, upstream of each open reading frame (Fig. 4 [cysP2] and data not shown [cysP1 and cysP3]). The PIP box, a consensus sequence consisting of TTCGC(N₁₅)TTCGC, is reported to be located upstream of HrpX regulons such as hrp genes and some avirulence genes and is required for the transcription of the regulons in xanthomonads (12). We show HrpXo-dependent transcription of cysP2 and cysP3 in Fig. 5A. By using a plasmid harboring a cysP1::gus fusion gene, HrpXo-dependent expression was also observed, although the GUS activity was extremely weak (data not shown). These results imply the importance of the imperfect PIP boxes. However, we do not have any experimental evidence that the imperfect PIP boxes upstream of cysP genes function as cis elements for the transcription activator. There might be some unknown sequence recognized by HrpXo or other regulatory genes that mediate between HrpXo and each cysP gene. Anyway, although the secretion of CysP1 and CysP3 via a type II secretion system was unclear, at least for CysP2, we obtained the first evidence that HrpXo regulates the expression of a type II secretory protein. Besides CysP2, we detected some proteins secreted from the wild type and the TTSS mutant but not from the HrpXo mutant and the type II secretion system-deficient mutant. Some of these genes must also be HrpXo regulons secreted via the type II secretion system.

The genomic sequences of X. axonopodis pv. citri and X. campestris pv. campestris have now been completely determined (9). By detecting the PIP box or a sequence similar to it, da Silva et al. (9) have provided candidates for hrpX regulons. The cysteine protease of X. campestris pv. campestris (XCC2693), which is homologous to CysP2, is one of the candidates for HrpX regulons (9). Like CysP2, some of the candidates might be regulated in their transcription by HrpXo. The candidates of the regulons contain hrp gene products and effector proteins which are secreted via a TTSS. They also include some proteins with amino-terminal type II signal peptide sequences and are, therefore, likely to be secreted via a type II secretion system. On the other hand, genes that are unlikely encoding components of the secretion system or secretory proteins are contained in the candidates, suggesting that HrpXo is some sort of a global regulatory factor.

In this study, we also identified two HrpXo-dependent type III secretory proteins. The function of HrpE1 remains unclear, and HrpF is suggested to play a role at the bacterium-plant interface as part of a bacterial translocon which mediates effector protein delivery across the host cell membrane in X. campestris pv. vesicatoria (8, 16). Both proteins are required for pathogenicity on host plants and hypersensitive-response induction on nonhost plants. Many effector proteins have been shown to be secreted via a TTSS both in animals and in plant-pathogenic bacteria. There have been few reports regarding type III secretory effector proteins from X. oryzae pv. oryzae (14). By comparing secretory protein profiles between the wild-type and the type III-defective strains, effector proteins from the bacterium could be detected and identified.

 
This work was supported by Grants-in-Aid for Scientific Research B (no. 13460024) and C (no. 14560043) from the Ministry of Education, Science, Sports and Culture, Japan.

 
* Corresponding author. Mailing address: Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto Prefectural University, Kyoto 606-8522, Japan. Phone: 81 75 703 5614. Fax: 81 75 703 5614. E-mail: s_tsuge{at}love.kpu.ac.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, March 2004, p. 1374-1380, Vol. 186, No. 5
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.5.1374-1380.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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