Journal of Bacteriology, March 2004, p . 1374-1380, Vol . 186, No . 5

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

 
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 ofX . oryzae pv . oryzae . Many of these proteins disappeared or decreased in amount in culture on XOM2 when incubated with thestrain 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 byhrp genes, was similar to that of the wild-type strain exceptthat a few proteins had disappeared . This finding suggests thatmany HrpXo-dependent secretory proteins are secreted via systemsother than the TTSS . By isolating mutant strains lacking a typeII secretion system, we examined this hypothesis . As expected,many of the HrpXo-dependent secretory proteins disappeared ordecreased when the mutant was cultured in XOM2 . By determiningthe N-terminal amino acid sequence, we identified one of thetype II secretory proteins as a cysteine protease homolog, CysP2.Nucleotide sequence analysis revealed that cysP2 has an imperfectplant-inducible-promoter box, a consensus sequence which HrpXoregulons possess in the promoter region, and a deduced signalpeptide sequence at the N terminus . By reverse transcription-PCRanalysis and examination of the expression of CysP2 by usinga plasmid harboring a cysP2::gus fusion gene, HrpXo-dependentexpression of CysP2 was confirmed . Here, we reveal that thehrp regulatory gene hrpXo is also involved in the expressionof not only hrp genes and type III secretory proteins but alsosome type II secretory proteins.

 
In general, plant-pathogenic bacteria possess hypersensitiveresponse and pathogenicity [hrp] genes, which are clusteredin their chromosomes . hrp genes encode a type III secretionsystem [TTSS] that delivers virulence and avirulence factorsfrom the bacteria to plant cells and are required for pathogenesisin host plants and for triggering a hypersensitive responsein nonhost plants [1, 38] . Transcriptional regulation of hrpgenes 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, hrpAto hrpF, which are all required for full pathogenicity, andtheir expression is regulated by two genes, hrpX and hrpG, whichare 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] . Ithas also been suggested that HrpX controls some effector proteins [5] . Several genes that are regulated in a HrpX-dependent mannerpossess the consensus nucleotide sequence TTCGC[N₁₅]TTCGC, whichhas 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] . Usingthis medium, we have identified Hpa1 as one of the HrpXo-regulatedtype III secretory proteins in X . oryzae pv . oryzae [14] . Hpa1is encoded by an hrp cluster with a PIP box, and its requirementfor disease development in rice plants has been reported [14, 41] . We also detected HrpXo-regulated secretory proteins otherthan HpaI, none of which have been identified [14] . Some ofthese 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 deficientin 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 asone of the HrpXo-regulated type II secretory proteins.

 
Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listedin Table 1. Escherichia coli DH5MCR [Stratagene] was grown at37°C in Luria-Bertani medium [25]. X . oryzae pv . oryzaestrains were usually grown at 28°C in nutrient broth-yeastextract [NBY] medium [32] or in an hrp-inducing medium, XOM2[31] . Xanthomonas axonopodis pv . citri was grown at 28°Cin NBY medium . All media were supplemented with the followingantibiotics 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 forE . coli; spectinomycin, 25 µg/ml for X . oryzae pv . oryzaeand 50 µg/ml for E . coli.

 
Recombinant DNA techniques. DNA manipulations were performed by standard procedures [25].

Sequence analysis. A dye terminator cycle sequencing reaction was performed witha DNA sequencing kit [Applied Biosystems, Piscataway, N.J.]according to the manufacturer's instructions followed by electrophoresisand 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 byPSORT [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 transposonEZ::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 secretedby the type II secretion system [10, 14, 15] . Assays for theseenzymatic activities were done according to the procedures describedby Tsuchiya et al . [29] and Keen et al . [18], respectively.Sequence analysis of the regions flanking the transposon revealedthat 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 componentof 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 mediumfor 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 centrifugationat 10,000 x g for 5 min and filtration, and the supernatantwas 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 resuspendedin 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] . Proteinswere 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 secretoryproteins from 14 ml of XOM2 culture medium, proteins were separatedon 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-sizeImmun-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 extractedwith an RNeasy kit [QIAGEN, Valencia, Calif.] . cDNA synthesisand 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 . oryzaepv . 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 byPCR with genomic DNA of X . axonopodis pv . citri MAFF302104 usedas a template and the primers 5'-ATGGGCCTGAAGCCTTCGTC-3' and5'-TCGGCGCCGATCACATCCTT-3' . An approximately 5-kb EcoRI-HindIIIfragment 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-rangevector, pHM1 [17], to obtain pHMCysP2PIP . An approximately 1.8-kbSacI-KpnI fragment containing the gus gene from pBSGUS [31] was then inserted into pHMCysP2PIP, and the plasmid obtainedwas named pHMCysP2GUS . Plasmid pHMCysp2GUS was then introducedinto X . oryzae pv . oryzae.

Assay of GUS activity. ß-Glucuronidase [GUS] activity was assayed as describedpreviously [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] wasselected by Southern blot analysis and sequence analysis . Theplasmid was introduced into strain T7174R by electroporation,and kanamycin-resistant clones were selected . Marker exchangemutagenesis was confirmed by genomic Southern blot analysis[data not shown], and one of the clones was named 74CysP2.

 
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 . oryzaeT7174R in the hrp-inducing medium XOM2 were compared with thosefrom strain 74HrpXo, in which an hrp-regulatory gene, hrpXo,is disrupted . Many of the proteins which were detected in theculture of T7174R disappeared or decreased in that of 74HrpXo [Fig . 1] . To clarify whether those proteins were secreted viaa TTSS, secretory proteins in 74HrcV, which has a transposoninsertion in a conserved TTSS component gene and cannot secreteHpa1, a type III secretory protein [13], were investigated.Interestingly, most of the proteins detected in the cultureof T7174R were also detected in that of 74HrcV, although a fewproteins containing Hpa1 were not detected [Fig. 1] . These resultssuggest that many of the secretory proteins from the wild-typestrain detected in XOM2 are secreted not via a TTSS but viaother systems . To examine the involvement of the type II systemin the secretion of these proteins, mutants lacking this systemwere isolated [74XpsE, 74XpsL, and 74XpsN; see Materials andMethods] . A deficiency in secretion in these mutants was confirmedby a decrease of extracellular cellulase and xylanase activities[Fig . 2] . The secretory proteins of 74XpsL were compared withthose of 74HrcV, wild-type strain T7174R, and 74HrpXo [Fig.1] . Several signals detected in strains T7174R and 74HrcV haddisappeared or weakened in 74XpsL along with 74HrpXo . Proteinprofiles 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 enzymaticactivities, the hrpXo mutant 74HrpXo showed both activitiesto the same extent as the wild-type strain did . These resultssuggest that HrpXo regulates not only the expression of typeIII secretory proteins but also that of some type II secretoryproteins.

 
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 norin a type II secretion system-deficient mutant [Fig. 3A] . TheN-terminal sequence [EVHGKGLKPS] of the 30-kDa protein was almostidentical to the internal sequence of X . axonopodis pv . citricysteine protease [AVHGMGLKPS, amino acids [aa] 25 to 34; XAC2853[refer to GenBank accession no. AE011926]] and that of X . campestrispv . campestris [AMHGMGLKPS, aa 25 to 34; XCC2693 [refer to GenBankaccession no. AE012381]] . The PSORT program predicted the presenceof a signal peptide sequence at the N terminus [aa 1 to 24]of these proteins . These cysteine proteases are predicted tobe secreted via the type II secretion system, and the N terminusof the mature form must start from aa 25.

 
We also identified three type III secretory proteins whose molecular masses were approximately 50, 7, and 6 kDa [Fig . 3] . The N-terminalsequence [NDEFNPKDIKGS] of the 50-kDa protein was perfectlyconsistent with the internal sequence of HrpF of X . oryzae pv.oryzae . The predicted size of HrpF is 84.9 kDa, and the consistencyof the sequence determined with HrpF started from aa 223 . Therefore,the 50-kDa protein is likely to be a processed or degraded productof HrpF . The N-terminal sequences of the 7- and 6-kDa proteinswere MEILPQISSL and SLNSRFQQGM, respectively, perfectly consistentwith the start and internal [aa 9 to 18] sequences of HrpE1of X . oryzae pv . oryzae, whose molecular mass is predicted tobe 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 95to 97%, and all encoded 271 amino acid residues with a predictedmolecular mass of 29.1 to 29.5 kDa . These homologs possesseda deduced signal sequence at the N terminus . The amino acidsof the homologs at positions 25 to 34 were EVHAKGLKPS for CysP1and EVHGKGLKPS for CysP2 and CysP3, indicating that the 30-kDaprotein was CysP2 or CysP3 . To determine which of these wasthe 30-kDa secretory protein, strains T7174R and 74HrpXo werecultivated in the hrp-inducing medium XOM2, and the transcriptionof cysP2 and cysP3 in each strain was analyzed by RT-PCR . Weused specific primers for RT to distinguish transcriptionalproducts of cysP2 and cysP3 [Fig. 5A] . A specific DNA fragmentcorresponding to the internal sequence of cysP2 was amplifiedfrom T7174R, whereas it was not amplified from 74HrpXo [Fig.5A] . Although a specific fragment derived from cysP3 mRNA thatwas dependent on the presence of HrpXo was also detected, thesignal intensity of cysP3 was much lower than that of cysP2.These results suggest that the 30-kDa protein is CysP2 and thattranscription of cysP2 is regulated by HrpXo.

 
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 introducedit into X . oryzae pv . oryzae T7174R and 74HrpXo . Each transformantwas cultured in XOM2, and GUS activities were measured aftera 1-day incubation . The transformant T7174R[pHMCysP2GUS] showedremarkable GUS activity, while T7174R transformed with the vectorplasmid pHM1 and 74HrpXo[pHMCysP2GUS] showed no activity [Fig.5B] . These results support the idea that HrpXo regulates theexpression 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 systemis CysP2, we generated the mutant 74CysP2, in which an EZ::TNtransposon was inserted in cysP2 [Fig. 4], and analyzed it forsecretory protein by culturing it in XOM2 . The mutant was incubatedin XOM2 for 2 days, and secretory proteins were compared withthose from T7174R . The 30-kDa protein was specifically missingfrom the culture supernatant of 74CysP2 [Fig . 6] . These resultsindicate that HrpXo regulates the expression of not only thecomponents of a TTSS and type III secretory proteins but alsosome type II secretory proteins.

 
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 . oryzaepv . oryzae, we have demonstrated that Hpa1, a harpin-like protein[19] whose expression is regulated by HrpXo, is secreted viaa TTSS using an efficient hrp-inducing medium, XOM2 [14] . However,there had been no reports of other secretory proteins . In thisreport, we newly identified HrpE1 and HrpF as HrpXo-regulatedtype III secretory proteins . Moreover, we indicated that HrpXoalso regulates the expression of some type II secretory proteinsand 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 typeII secretion system, many of the HrpXo-dependent proteins secretedfrom the wild-type strain were suggested to be secreted viasuch a system . In the culture of mutants deficient in a typeII secretion system, more large-molecular-size proteins weredetected than in that of the wild-type strain . It is likelythat there are some products digested by proteases which aresecreted via a type II secretion system in the culture of strainspossessing this secretion system . On the other hand, in typeII secretion system-deficient mutants, such protein digestionmight not occur, and as a result, only large intact proteinsmight be detected . However, it is unlikely that all of the proteinsdetected 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 cysteineprotease 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 proteinis a homolog of cysteine protease . We found that at least threecopies of this cysteine protease homolog are present in thegenomic DNA of X . oryzae pv . oryzae and that these copies [productsof cysP1, cysP2, and cysP3] are tandemly located in an approximately10-kb region . Detailed nucleotide sequence analysis of cysPgenes revealed that they are highly homologous [95 to 97%] andthat their deduced products have a signal peptide at the N terminus.The amino acid sequence that we determined starts just afterthe 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 CysP2to be the most probable candidate for the 30-kDa secretory protein.The reasons we considered CysP2 are [i] the amino acid sequencewas not completely consistent with the corresponding sequenceof CysP1 and [ii] the transcriptional level was much higherin cysP2 than in cysP3 according to RT-PCR with specific primers.Actually, the mutant that had a transposon insertion in cysP2did 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 imperfectPIP box, TTCGC[N₁₂]TTCGC, upstream of each open reading frame[Fig . 4 [cysP2] and data not shown [cysP1 and cysP3]] . The PIPbox, 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 plasmidharboring a cysP1::gus fusion gene, HrpXo-dependent expressionwas also observed, although the GUS activity was extremely weak[data not shown] . These results imply the importance of theimperfect PIP boxes . However, we do not have any experimentalevidence that the imperfect PIP boxes upstream of cysP genesfunction as cis elements for the transcription activator . Theremight be some unknown sequence recognized by HrpXo or otherregulatory 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 fromthe HrpXo mutant and the type II secretion system-deficientmutant . Some of these genes must also be HrpXo regulons secretedvia 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 detectingthe PIP box or a sequence similar to it, da Silva et al . [9]have provided candidates for hrpX regulons . The cysteine proteaseof X . campestris pv . campestris [XCC2693], which is homologousto CysP2, is one of the candidates for HrpX regulons [9] . LikeCysP2, some of the candidates might be regulated in their transcriptionby HrpXo . The candidates of the regulons contain hrp gene productsand effector proteins which are secreted via a TTSS . They alsoinclude some proteins with amino-terminal type II signal peptidesequences and are, therefore, likely to be secreted via a typeII secretion system . On the other hand, genes that are unlikelyencoding components of the secretion system or secretory proteinsare contained in the candidates, suggesting that HrpXo is somesort of a global regulatory factor.

In this study, we also identified two HrpXo-dependent type III secretory proteins . The function of HrpE1 remains unclear, andHrpF is suggested to play a role at the bacterium-plant interfaceas part of a bacterial translocon which mediates effector proteindelivery across the host cell membrane in X . campestris pv.vesicatoria [8, 16] . Both proteins are required for pathogenicityon host plants and hypersensitive-response induction on nonhostplants . 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 secretoryprotein profiles between the wild-type and the type III-defectivestrains, effector proteins from the bacterium could be detectedand identified.

 
This work was supported by Grants-in-Aid for Scientific ResearchB [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@love.kpu.ac.jp.

1. Alfano, J . R., and A . Collmer. 1997 . The type III [Hrp] secretion pathway of plant pathogenic bacteria: trafficking harpins, Avr proteins, and death . J . Bacteriol . 179:5655-5662.
2. Altschul, S . F., W . Gish, W . Miller, E . W . Myers, and D . J . Lipman. 1990 . Basic local alignment search tool . J . Mol . Biol . 215:403-410.
3. Angadi, C . V. 1978 . Extra-cellular slime of Xanthomonas oryzae in bacterial leaf blight of rice . Phytopathol . Z . 93:170-180.
4. Angadi, C . V. 1978 . In vivo production of slime toxin in bacterial blight infected rice plants . Phytopathol . Z . 92:193-201.
5. Astua-Monge, G., G . V . Minsavage, R . E . Stall, M . J . Davis, U . Bonas, and J . B . Jones. 2000 . Resistance of tomato and pepper to T3 strains of Xanthomonas campestris pv . vesicatoria is specified by a plant-inducible avirulence gene . Mol . Plant-Microbe Interact . 13:911-921.
6. Bonas, U. 1994 . hrp genes of phytopathogenic bacteria . Curr . Top . Microbiol . Immunol . 192:79-98.
7. Bonas, U., R . Schulete, S . Fenselau, G . V . Minsavage, B . J . Staskawicz, and R . E . Stall. 1991 . Isolation of a gene cluster from Xanthomonas campestris pv . vesicatoria that determines pathogenicity and the hypersensitive response on pepper and tomato . Mol . Plant-Microbe Interact . 4:81-88.
8. Büttner, D., D . Nennstiel, B . Klüsener, and U . Bonas. 2002 . Functional analysis of HrpF, a putative type III translocon protein from Xanthomonas campestris pv . vesicatoria. J . Bacteriol . 184:2389-2398 .
9. da Silva, A . C . R., J . A . Ferro, F . C . Reinach, C . S . Farah, L . R . Furlan, R . B . Quaggio, C . B . Monteiro-Vitorello, M . A . Van Sluys, N . F . Almeida, L . M . C . Alves, A . M . Do Amaral, M . C . Bertolini, L . E . A . Camargo, G . Camarotte, F . Cannavan, J . Cardozo, F . Chambergo, L . P . Ciapina, R . M . B . Cicarelli, L . L . Coutinho, J . R . Cursino-Santos, H . El-Dorry, J . B . Faria, A . J . S . Ferreira, R . C . C . Ferreira, M . I . T . Ferro, E . F . Formighieri, M . C . Franco, C . C . Greggio, A . Gruber, A . M . Katsuyama, L . T . Kishi, R . P . Leite, E . G . M . Lemos, M . V . F . Lemos, E . C . Locali, M . A . Machado, A . M . B . N . Madeira, N . M . Martinez-Rossi, E . C . Martins, J . Meidanis, C . F . M . Menck, C . Y . Miyaki, D . H . Moon, L . M . Moreira, M . T . M . Novo, V . K . Okura, M . C . Oliveira, V . R . Oliveira, H . A . Pereira, A . Rossi, J . A . D . Sena, C . Silva, R . F . de Souza, L . A . F . Spinola, M . A . Takita, R . E . Tamura, E . C . Teixeira, R . I . D . Tezza, M . Trindade dos Santos, D . Truffi, S . M . Tsai, F . F . White, J . C . Setubal, and J . P . Kitajima. 2002 . Comparison of the genomes of two Xanthomonas pathogens with differing host specificities . Nature 417:459-463.
10. Dow, J . M., D . E . Miligan, L . Jaison, C . E . Barber, and M . J . Daniels. 1987 . A gene cluster in Xanthomonas campestris required for pathogenicity controls the excretion of polygalacturonate lyase and other enzymes . Physiol . Mol . Plant Pathol . 31:261-271.
11. Ezuka, A., and O . Horino. 1974 . Classification of rice varieties and Xanthomonas oryzae strains on the basis of their differential interactions . Bull . Tokai Kinki Natl . Agric . Exp . Stn . 27:1-19.
12. Fenselau, S., and U . Bonas. 1995 . Sequence and expression analysis of the hrpB pathogenicity operon of Xanthomonas campestris pv . vesicatoria which encodes eight proteins with similarity to components of the Hrp, Ysc, Spa, and Fli secretion system . Mol . Plant-Microbe Interact . 8:845-854.
13. Furutani, A., S . Tsuge, T . Oku, K . Tsuno, Y . Inoue, H . Ochiai, H . Kaku, and Y . Kubo. 2003 . Hpa1 secretion via type III secretion system in Xanthomonas oryzae pv . oryzae. J . Gen . Plant Pathol . 69:271-278.
14. He, S . Y., M . Lindeberg, A . K . Chatterjee, and A . Collmer. 1991 . Cloned Erwinia chrysanthemi out genes enable Escherichia coli to selectively secrete a diverse family of heterologous proteins to its milieu . Proc . Natl . Acad . Sci . USA 88:1079-1083.
15. Hu, N.-T., M.-N . Hung, S.-J . Chiou, F . Tang, D.-C . Chiang, H.-Y . Huang, and C.-Y . Wu. 1992 . Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv . campestris. J . Bacteriol . 174:2679-2687.
16. Huguet, E., and U . Bonas. 1997 . hrpF of Xanthomonas campestris pv . vesicatoria encodes an 87-kDa protein with homology to NolX of Rhizobium fredii. Mol . Plant-Microbe Interact. 10:488-498.
17. Innes, R . W., M . A . Hirose, and P . L . Kuempel. 1988 . Induction of nitrogen-fixing nodules on clover requires only 32 kilobase pairs of DNA from the Rhizobium trifolii symbiosis plasmid . J . Bacteriol . 170:3793-3802.
18. Keen, N . T., C . Boyd, and B . Henrissat. 1996 . Cloning and characterization of a xylanase gene from corn strains of Erwinia chrysanthemi. Mol . Plant-Microbe Interact . 9:651-657.
19. Kim, J.-G., B . K . Park, C.-H . Yoo, E . Jeon, J . Oh, and I . Hwang. 2003 . Characterization of the Xanthomonas axonopodis pv . glycines Hrp pathogenicity island . J . Bacteriol . 185:3155-3166 .
20. Laemmli, U . K. 1970 . Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227:680-685.
21. Nakai, K., and M . Kanehisa. 1991 . Expert system for predicting protein localization sites in Gram-negative bacteria . Proteins Struct . Funct . Genet . 11:95-110.
22. Noda, T., Z . Sato, H . Kobayashi, S . Iwasaki, and S . Okuda. 1989 . Isolation and structural elucidation of phytotoxic substances produced by Xanthomonas campestris pv . oryzae [Ishiyama] Dye . Ann . Phytopathol . Soc . Jpn . 46:663-666.
23. Oku, T., A . M . Alvarez, and C . I . Kado. 1995 . Conservation of the hypersensitivity-pathogenicity regulatory gene hrpX of Xanthomonas campestris and X . oryzae. DNA Sequence 5:245-249.
24. Rahme, L . G., M . N . Mindrinos, and N . J . Panopoulos. 1991 . Genetic and transcriptional organization of the hrp cluster of Pseudomonas syringae pv . phaseolicola . J . Bacteriol . 173:575-586 . [Erratum, 174:3840.]
25. Sambrook, J., F . F . Fritsch, and T . Maniatis. 1989 . Molecular cloning: a laboratory manual, 2nd ed . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
26. Sandkvist, M. 2001 . Biology of type II secretion . Mol . Microbiol . 40:271-283.
27. Suvendra, K . R., R . Rajeshwari, and R . V . Sonti. 2000 . Mutants of Xanthomonas oryzae pv . oryzae deficient in general secretory pathway are virulence deficient and unable to secrete xylanase . Mol . Plant-Microbe Interact . 13:394-401.
28. Swings, J., M . Van den Mooter, L . Vauterin, B . Hoste, M . Gillis, T . W . Mew, and K . Kersters. 1990 . Reclassification of causal agents of bacterial blight [Xanthomonas campestris pv . oryzae] and bacterial leaf streak [Xanthomonas campestris pv . oryzicola] of rice as pathovars of Xanthomonas oryzae [ex Ishiyama 1922] sp . nov., nom . rev . Int . J . Syst . Bacteriol . 40:309-311.
29. Tsuchiya, K., T . W . Mew, and S . Wakimoto. 1982 . Bacteriological and pathological characteristics of wild types and induced mutants of Xanthomonas campestris pv . oryzae. Phytopathology 72:43-46.
30. Tsuge, S., A . Furutani, R . Fukunaka, Y . Kubo, and O . Horino. 2001 . Growth complementation of hrpXo mutants of Xanthomonas oryzae pv . oryzae by virulent strains in rice cultivars resistant and susceptible to the parental strain . J . Gen . Plant Pathol . 67:51-57.
31. Tsuge, S., A . Furutani, R . Fukunaka, T . Oku, K . Tsuno, H . Ochiai, Y . Inoue, H . Kaku, and Y . Kubo. 2002 . Expression of Xanthomonas oryzae pv . oryzae hrp genes in a novel synthetic medium, XOM2 . J . Gen . Plant Pathol . 68:363-371.
32. Vidaver, A . K. 1967 . Synthetic and complex media for the rapid detection of phytopathogenic pseudomonds: effect of the carbon source . Appl . Microbiol . 15:1523-1524.
33. Vieira, J., and J . Messing. 1987 . Production of single-stranded plasmid DNA . Methods Enzymol . 153:3-11.
34. Watabe, M., M . Yamaguchi, I . Furusawa, and O . Horino. 1993 . Virulence, and bacterial multiplication and movement in rice leaves of Xanthomonas campestris pv . oryzae mutants impaired in productivity of extracellular polysaccharide . Ann . Phytopathol . Soc . Jpn . 59:544-550.
35. Wei, Z . M., R . J . Laby, C . H . Zumoff, D . W . Bauer, Y . He, A . Collmer, and S . V . Beer. 1992 . Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science 257:85-88.
36. Wengelnik, K., and U . Bonas. 1996 . HrpXv, an AraC-type regulator, activates expression of five out of six loci in the hrp cluster of Xanthomonas campestris pv . vesicatoria essential for pathogenicity and induction of the hypersensitive reaction . J . Bacteriol . 178:3462-3469.
37. Wengelnik, K., G . Van den Ackerveken, and U . Bonas. 1996 . HrpG, a key hrp regulatory protein of Xanthomonas campestris pv . vesicatoria is homologous to two-component response regulators . Mol . Plant-Microbe Interact . 9:704-712.
38. Willis, D . K., J . J . Rich, and E . M . Hrabak. 1991 . hrp genes of phytopathogenic bacteria . Mol . Plant-Microbe . Interact . 4:132-138.
39. Xiao, Y., Y . Lu, S . Heu, and S . W . Hutcheson. 1992 . Organization and environmental regulation of the Pseudomonas syringae pv . syringae 61 hrp cluster . J . Bacteriol . 174:1734-1741.
40. Xu, G . W., and C . F . Gonzalez. 1989 . Evaluation of TN4431-induced protease mutants of Xanthomonas campestris pv . oryzae for growth in plants and pathogenicity . Phytopathology 79:1210-1215.
41. Zhu, W., M . M . Magbanua, and F . F . White. 2000 . Identification of two novel hrp-associated genes in the hrp gene cluster of Xanthomonas oryzae pv . oryzae. J . Bacteriol . 182:1844-1853 .

Free Online Full-text Article

What Is Functional Genomics?, What Is Bioengineering?, What Is Activated Sludge?, What Is Antibiotic?, What Is Anthrax?, o, Bacteria, a, Microorganisms, n, Microbe, c, Bacterium, n, Bacteriology, n, Antimicrobial, r, Suspension cells, c, Bacteriological, n, Escherichia coli, a, Prokaryotes, n, Pseudomonas aeruginosa, e, Microorganism, r, Antibiotics, n, Yeasts, c, Bacillus, r, Kluyveromyces, n, Bacillus subtilis, i, Pasteurella, n, Streptomycin, o, Bacillus, a, Pichia, c, Streptococci, o, Ciprofloxacin, a, Antibiotic treatment, s, S. cerevisiae, o, Meningococcus




 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005