We have initiated a project to identify protein-protein interactions involved in the pathogenicity of the bacterial plant pathogen Xanthomonas axonopodis pv . citri . Using a yeast two-hybrid system based on Gal4 DNA-binding and activation domains, we have focusedon identifying interactions involving subunits, regulators,and substrates of the type III secretion system coded by thehrp [for hypersensitive response and pathogenicity], hrc [for hrp conserved], and hpa [for hrp associated] genes . We have identified several previously uncharacterized interactions involving [i] HrpG, a two-component system response regulator responsible for the expression of X . axonopodis pv . citri hrp operons, and XAC0095, a previously uncharacterized protein encountered only in Xanthomonas spp.; [ii] HpaA, a protein secreted by the type III secretion system, HpaB, and the C-terminal domain of HrcV;[iii] HrpB1, HrpD6, and HrpW; and [iv] HrpB2 and HrcU . Homotropic interactions were also identified for the ATPase HrcN . Thesenewly identified protein-protein interactions increase our understandingof the functional integration of phytopathogen-specific typeIII secretion system components and suggest new hypotheses regardingthe molecular mechanisms underlying Xanthomonas pathogenicity.

 
A number of species of gram-negative bacteria are able to infectand cause disease in, or establish symbiotic relationships with,specific plant hosts [2] . These phytopathogenic bacteria use a full barrage of molecular strategies by which to enter andcolonize host tissues . This invasion eventually modifies, andin many cases compromises, plant homeostasis at the tissue levelor at the level of the entire plant . Biochemical, genetic, andcellular studies of phytopathogenic bacteria have revealed thatthese mechanisms involve a variety of factors such as adhesins,pili, bacterial signaling factors, receptors of external andplant-derived factors, proteins involved in signal transduction,specialized transcription factors, alternate sigma factors,and proteins which generate, assemble, and regulate specificmacromolecular secretion systems that transport bacterial macromolecularpathogenicity factors [1, 2, 8, 15, 25, 32, 53, 67] . Only afew of these processes are understood fully at the molecular level and, in the few cases where they have been elucidatedfor more than one system, significant differences have beenobserved [32, 39]—differences that must eventually beattributed to the specific biologies of the interacting bacterium-plantpair.

During the infective process, a large variety of pathogenic gram-negative bacteria inject macromolecular pathogenic factorsinto their animal or plant host cells . Bacteria may use oneof two systems to accomplish this: the type III [6, 12, 20,25, 26, 32, 46] and type IV [3, 10, 11, 14, 18, 19, 60, 65,69] secretion systems . Recently, the genomes of a number ofbacterial phytopathogens have been sequenced, including Xylellaspp . [52, 58], Ralstonia solanacearum [49], Xanthomonas spp.[21], Agrobacterium tumefaciens [66], and Pseudomonas syringae [9] . These sequences have revealed the existence of severalgene clusters that code for putative macromolecule secretion systems that are demonstrably or possibly involved in pathogenesis. The type III secretion system [TTSS] is coded for by a groupof ca . 25 genes, most of which are localized to a single chromosomal locus [hrp] . A subgroup of products encoded by these genes consists of homologues of the core flagellar secretory components, which has led to the conclusion that the TTSS and flagellar machinesare evolutionarily related [6, 42, 57] . In contrast, the typeIV secretion systems [TFSS] responsible for secreting pathogenicityfactors are related to the machines responsible for transferof nucleic acid-protein complexes during bacterial conjugation[19].

We identify here new protein-protein interactions involving components, substrates, and regulators of the TTSS of the phytopathogen Xanthomonas axonopodis pv . citri, the causal agent of citrus canker . In the plant pathogens Xanthomonas, Ralstonia, Erwinia,and Pseudomonas spp., a subset of hrp genes are induced uponcontact with the plant in response to a variety of diffusibleor nondiffusible plant-derived factors [8, 25, 32, 51] . Beyond their requirement for pathogenicity, the mechanisms of action of most of the components of this secretion machine are notwell understood at the molecular level . This is especially truefor phytopathogen-specific components with no homologs in animalTTSS or in flagella . Interactions among some components of TTSSfrom several bacterial pathogens have been elucidated, and inmany cases these interactions have revealed possible functionalroles of specific components [34] . However, the fact that severalof the gene products of these clusters are only distantly related,if at all, to other proteins of known function places limitson the homology-based approach for deduction of function . Thislimitation is further highlighted by the fact that the X . axonopodispv . citri hrp cluster [Fig . 1] possesses open reading frames[ORFs] that code for previously uncharacterized proteins, in several cases specific to xanthomonads and in some cases specific to X . axonopodis pv . citri . To understand the specific mechanisms by which X . axonopodis pv . citri uses its TTSS to interact with and modify the metabolism of its host, it is of utmost importance to delimit the protein-protein interactions involving X . axonopodis pv . citri TTSS components and associated proteins . To this end, we have used a yeast two-hybrid system [17] to perform genome-scaleprotein-protein interaction screens by using specific X . axonopodispv . citri hrp, hrc, and hpa proteins as baits . Our results reveala number of previously uncharacterized interactions involvingthese proteins that may be of importance to the processes ofhrp gene expression and TTSS function.

 
X . axonopodis pv . citri [strain 306] genomic DNA library. X . axonopodis pv . citri strain 306 genomic DNA was nebulized under nitrogen, repaired, and size separated in agarose gels. Fragments of 500 to 1,500 bp and 1,500 to 3,000 bp were purifiedand cloned into the plasmid vector pOAD [56] previously linearizedwith PvuII and dephosphorylated with calf intestine alkalinephosphatase . Then, 1-µl aliquots of the ligation reactions were used to transform competent Escherichia coli DH10B cells by electroporation . Cells from each set of 10 transformations were pooled and, after the addition of 1 volume of 50% glycerol, stored at –70°C . Before freezing, 20-µl aliquotswere separated, frozen at –70°C, thawed, and platedto determine the total number of independent clones in thatpool, which varied from 2,000 to 20,000 . Transformations wereperformed until each library [500 to 1,500 bp and 1,500 to 3,000bp] contained >10⁶ independent clones.

Transformants were thawed and pooled into groups containing between 100,000 and 200,000 independent clones . These were diluted into 16 2-liter flasks containing a total of ca . 10 liters of2xTY medium supplemented with 200 µg of carbenicillin/ml.After growth at 37°C for 8 h, 10-ml aliquots were removedfrom each flask, combined, diluted with 1 volume of 50% glycerol,and stored at –70°C . The rest of the culture was incubatedat 37° until it reached an optical density at 600 nm of1.2, at which point the cells were collection by centrifugation,and the plasmid DNA was purified [50] . HindIII digests of eachpreparation were analyzed by agarose gel electrophoresis . DNAconcentrations were determined, and preparations from all ofthe libraries were pooled in amounts proportional to the numberof independent clones per unit mass of DNA . This pOAD-library mixture was used to transform yeast cells in two-hybrid screens.

Cloning of baits for two-hybrid screens. X . axonopodis pv . citri DNA sequences coding proteins for useas baits were amplified by PCR by using X . axonopodis pv . citrigenomic DNA and primers designed based on the X . axonopodispv . citri genome sequence [21] . The primers also contained unique restriction sites [usually NcoI and XhoI] to facilitate cloning into the NcoI and SalI sites of the pOBD vector [56] downstreamof and in frame with the Gal4 DNA-binding domain . After transformationinto DH10B E . coli cells, individual colonies were picked forplasmid isolation and confirmation by DNA sequencing . Most baitscorresponded to the full-length proteins as annotated in theX . axonopodis pv . citri genome sequence [21] . In some cases,analysis of the protein sequence by using the PSORT algorithm[43] indicated the presence of domains containing one or moreputative transmembrane helices and protein fragments lackingthe transmembrane helices were used . These cases are describedin the text and in Table 1 . In the case of XAC0095, the baitused contained a 12-amino-acid N-terminal extension encodedby the 36 nucleotides upstream of the annotated start codon.This 85-amino-acid bait is therefore coded for by nucleotides111185 to 111442 of the X . axonopodis pv . citri chromosome.

 
Growth of yeast strains and transformation. Saccharomyces cerevisae strain PJ694-a [MATa trp1-901 leu2-3 112 ura3-52 his3-200 gal4 gal80 LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ][35] was grown at 30°C in YAPD medium [1% yeast extract,2% peptone, 2% glucose, 0.008% adenine] or in SC medium [0.66%nitrogen base [without amino acids], 2% glucose, 0.008% adenine,0.8% amino acid mixture adjusted to pH 5.6] as described previously[45] . Where indicated, SC medium was prepared lacking one ormore specific components: adenine [–Ade], histidine [–His],tryptophan [–Trp], and leucine [–Leu] . In the caseof growth on solid media, 1.6% Bacto Agar and 3-aminotriazole[3AT] [see below] was added . Rapid transformations with pOBD-baitplasmids were carried out by using the PEG3350-lithium acetateprotocol described by Gietz et al . [28] and selected on SC–Trpplates at 30°C for 2 to 4 days . These cells were then usedin high-efficiency transformations with the pOAD-library using30 µg of plasmid DNA and the 30x scale-up procedure describedby Gietz and Woods [27], which resulted in, on average, 0.5x 10⁷ to 1 x 10⁷ transformants on SC–Trp–Leu plates.

Tests for autoactivation and false-positive results. To determine the amount of 3AT to be used for each bait, ca.1,000 yeast cells transformed with the pOBD-bait plasmid wereplated on SC–Trp–His medium containing 0, 1, 5,10, 25, or 50 mM 3AT and incubated for 5 days at 30°C . Similarly,the small number of baits that autoactivate the GAL2-ADE2 reporterwere identified by growth on SC–Trp–Ade plates.The pOAD-library was shown to lack any clones that autoactivatethe His or Ade gene reporters on their own or simultaneously.On the other hand, when transformed into PJ694-a cells previouslytransformed with the pOBD vector [lacking a bait insert], ca.1,000 colonies were found to grow in the presence of up to 3mM 3AT . Sequencing of the preys in 66 of these colonies subsequentlyshowed them to be derived from only a few specific X . axonopodispv . citri genes [see Results].

Yeast two-hybrid assays and DNA sequencing. After transformation with the pOAD-library, cells were resuspendedin 5 ml of sterile water and spread on 10 plates with SC–Trp–Leu–His–Adeplus 3AT [150 by 15 mm] . The amount of 3AT used varied between5 and 50 mM, depending on the bait: 5 mM for all baits exceptfor HpaA [10 mM] and HrcN [50 mM] . Plates were incubated at30°C for up to 14 days . Colonies that grew in the absenceof His and Ade were transferred to fresh plates with SC–Trp–Leu–His–Adeplus 3AT . Plasmid DNA was isolated from yeast colonies thatwere able to grow under the selection of both reporters by usinga method [50] scaled up and modified for execution in 96-wellplates . Purified plasmid DNA mixtures were used to transformDH10B E . coli cells . After overnight growth at 37°C, plasmidDNA [a mixture of pOAD- and pOBD-derived vectors] was purified.The prey or bait DNA sequences were sequenced by using pOAD-or pOBD-specific primers, respectively . Sequences were analyzedby comparison with the X . axonopodis pv . citri genome database[21].

 
X . axonopodis pv . citri two-hybrid library construction. We constructed a X . axonopodis pv . citri total genomic DNA library [X . axonopodis pv . citri chromosome plus plasmids] containing fragments of 500 to 3,000 bp cloned into the PvuII site of the vector pOAD [56] . The quality of the library was tested in a number of independent ways . [i] HindIII digests of the sublibraries after amplification but before pooling revealed a broad population of inserts of the expected sizes [500 to 1,500 bp or 1,500 to 3,000 bp] . [ii] Initial sequencing of ca . 1,000 independentlychosen clones did not reveal any obvious bias in the libraryand that, of the 3.0 million independent clones in the combinedlibrary, 78% [2.35 million] contained X . axonopodis pv . citrigenomic DNA inserts . In the final pooled library, fragmentsin the size ranges of 500 to 1,500 bp and 1,500 to 3,000 bpwere present in about equal proportions [1.2 and 1.15 million,respectively] . If we assume a random distribution of fragmentationsites in the genome, the library contains fragments initiatingevery 2.2 bp or every 13 bp in a correct reading frame . Althoughsome in-frame clones code for full-length proteins with N-terminalextensions, most fragments code a protein with an N-terminaldeletion . [iii] Subsequent analysis of two-hybrid results revealedthat, except for some special cases due to yeast-based recombinationevents [see below], clones of preys derived from a specificgene initiated at many unique sites within that gene . [iv] TheX . axonopodis pv . citri genomic library in pOAD does not containany clones that on their own activate the HIS3 and ADE2 reportergenes individually . However, in the presence of the empty pOBDvector, some library clones did lead to activation . In an experimentof a total of 2 x 10⁷ transformations, ca . 1,000 colonies wereobserved to grow on plates with SC–Trp–Leu–His–Ade plus 3AT . Sequencing of the pOAD-derived vector from 62 of these colonies revealed that all contained DNA inserts derived from only a small number of X . axonopodis pv . citri genes: 89% of the preys coded for fragments of the TolC protein, 6.5% of thepreys coded for fragments of the RibA protein, and single cloneswere found that were derived from the phoQ, glnA, and piuB genes. TolC-derived fragments seem to be promiscuous interactors in two-hybrid assays and should be considered false positives,as shown below . RibA preys have thus far only been detectedwhen empty pOBD is used as bait.

Identification of false positives. In using this X . axonopodis pv . citri genomic DNA library, weidentified two classes of prey that we consider to be falsepositives . One class is composed of specific preys that appearto interact with a great number of apparently unrelated baitswith a frequency that does not appear to be of physiologicalrelevance, at least in the context of the physiological roleof the baits . Of more than 100 baits tested thus far with thislibrary [data not shown], 40% interacted with at least one preyderived from the TolC protein and, for several baits, the majorfraction of interactors was derived from TolC . Due to the ubiquityof these sequences in the two-hybrid screens, these sequenceswere considered to be false positives and therefore disregarded.TolC tends to interact with the Gal4 DNA-binding domain [seeabove], and this tendency may be inhibited or enhanced by baitfusions . In addition to TolC, several other proteins were detectedas preys at a frequency to warrant suspicion of their physiologicalsignificance . These proteins included [i] members of the largeTonB-dependent receptor family, [ii] products of the two wapAgenes [XAC1866 and XAC1305], [iii] the XAC3515 protein, and [iv] the plasmid-encoded PthA and KfrA proteins . For this reason, these preys were also considered to be false positives and are not considered further.

The majority of baits interacted with a large number of preys, most of which were derived from a small number of X . axonopodis pv . citri proteins [Table 1] . In the case of some baits, however[for example, HrpF, Hpa2, and the N-terminal fragment of HpaB],no clear preference for any specific X . axonopodis pv . citriprotein was observed, and almost all of the preys sequenced were derived from a different protein [data not shown] . The apparent lack of specificity of these baits suggests that the majority of their preys do not reflect physiologically relevant interactions . In such a case, even if a small number of thepreys of such a bait do in fact represent a true interactor,this fact is not immediately apparent from these data aloneand must be confirmed by other experimental approaches.

Positive controls. Three proteins known to participate in well-characterized protein-proteininteractions were initially used as positive controls for thetwo-hybrid screen itself, as well as to further test the qualityof the library . All three baits tested—GroES, the ' subunitof DNA polymerase III, and FtsZ—were found to interact with previously identified interacting partners: GroES with itself in 22 of 24 clones sequenced, the ' subunit of DNA polymeraseIII [36] with the product of the gene coding for the and subunitsof the polymerase in one of four clones sequenced, and FtsZ [29] with itself in 11 of 13 preys sequenced [Table 1].

Prey-bait vector recombination events. Detailed sequencing analysis of the Gal4 activation domain-preyjunction in the prey vector could demonstrate that for somebaits, including FtsZ and GroES controls, a fraction of thepreys were derived from recombination events in which the baitgene was transferred into the pOAD vector [3 of 11 for FtsZand 21 of 22 for GroES] . Recombination is facilitated by thefact that pOBD and pOAD vectors [56] have identical sequencesbefore [60 bp] and after [2,840 bp] their multiple cloning sites.To test whether these in vivo recombinations necessitate a specificlibrary clone or could occur even with the empty pOAD vector,we transformed PJ694-a cells with both the empty pOAD plasmidand the pOBD-XAC0095 plasmid . XAC0095 is an X . axonopodis pv.citri conserved hypothetical protein that was observed to sufferthe same type of recombination event [see below] . Of a totalof 5 x 10⁶ transformants, 25 colonies were observed to growon plates with SC–Trp–Leu–Hi–Ade plus3AT . Sequencing of the pOAD vector in 16 of these clones revealedthat they all now contained the full-length XACb0095 bait inthe specific site in which it was cloned in the pOBD vector[distinct from the PvuII site in which the genomic DNA library was cloned in pOAD] . Therefore, full-length copies of the bait in both vectors can be obtained via recombination of the full-length bait with, in principle, any library clone, including the emptypOAD vector if the bait protein can interact with itself formingdimers or higher-order complexes [as is in fact known to bethe case for FtsZ and GroES] . This phenomenon was observed fora few other TTSS-related proteins [see below].

Two-hybrid assays of proteins involved in the type III secretory pathways. Two-hybrid assays were performed by using simultaneous screeningof two reporter genes under the control of different induciblepromoters [GAL1-HIS3 and GAL2-ADE2] . This simultaneous screeningin combination with the high stringency of the GAL2-ADE2 reportersignificantly reduced the number of false positives [35] . Onlybaits that did not simultaneously autoactivate these two reporterswere tested in screens against the prey library . Table 1 liststhe TTSS-related proteins analyzed and summarizes the resultsobtained in two-hybrid assays in which they were used as baits.The table presents the total number of positive interactionsin which each prey was identified . False positives, as definedabove, are not indicated . For some baits capable of homotropicinteractions, the number of preys derived from recombinationof the bait gene into the pOAD vector is indicated . In assaysnecessitating the simultaneous activation of the GAL1-HIS3 andGAL2-ADE2 gene reporters, interactions observed at least twotimes with preys derived from the same X . axonopodis pv . citriprotein were considered to be of potential significance, especiallyif the physiological significance of the interaction was apparent.What follows is a detailed description of these results andtheir significance.

Interactions involving HrpG. HrpG is a DNA-binding transcriptional regulatory protein thatfunctions at the top of the hrp gene cluster regulatory cascade,controlling the expression of the hrpA gene in X . campestrispv . vesicatoria [hrcC in X . axonopodis pv . citri], as well as the gene for the downstream transcription regulator HrpX [62, 63] . HrpX in turn activates expression of the hrpB-F loci, aswell as of a number of Xanthomonas outer protein [xop] genesin X . campestris pv . vesicatoria [61] . The 263-residue HrpGprotein of X . axonopodis pv . citri, which belongs to the OmpRfamily of two-component system response regulators, containsan N-terminal response regulator receiver [RR] domain and aC-terminal DNA-binding motif [62] . Although the means by whichthe Xanthomonas HrpG protein is regulated remains unknown, itshomolog in R . solanacearum has been shown to be regulated bya cell-contact signal transduction cascade involving PrhA [aprobable receptor for insoluble host factors], PrhR [a transmembraneprotein that receives the signal from PrhA], PrhI [an ECF sigmafactor], and PrhJ [a LuxR/UhpA family transcription regulator][1, 7, 8] . PrhA, -R, and -I are homologues of the FecA, -R, and -I proteins, which interact in the signaling pathway that controls genes involved in ferric citrate transport in E . coli [22, 54] . In spite of this, no direct protein-protein interactionsinvolving HrpG in Xanthomonas or Ralstonia spp . have as yetbeen identified . Furthermore, no candidate two-component systemhistidine kinase has been identified which could in principleregulate HrpG via phosphorylation of its RR domain.

When the full-length HrpG protein was used as bait, all preyswere derived from one of four proteins [Table 1 and Fig. 2].Just over half of the preys [48 of 90] were derived from identicalclones that code for the full-length HrpG protein . Sequencinganalysis of the sequence coding for the junction between theGal4 activation domain and the HrpG coding sequence demonstratedthat all of these clones were derived from recombination eventsbetween the pOBD-HrpG bait plasmid and pOAD in which the full-lengthHrpG gene plus 25 nucleotides of the pOBD polylinker was transferredto the pOAD vector . HrpG-HrpG interactions have not been observedbefore . However, OmpR family proteins are known to bind to DNAas dimers, although dimerization has not been observed in solution[31, 37].

 
The second largest set of preys [36 of 90] obtained using HrpGas bait were derived from the hypothetical protein [XAC0095],whose only known homologs are coded by three ORFs in the X.campestris pv . campestris genome [Fig . 3] [21] . XAC0095 is a73-residue protein [Fig . 3], and all of these clones coded forat least residues 4 to 73 [Fig. 2B] . The HrpG-XAC0095 interactionwas confirmed when we used the XAC0095 protein with a 12-residueN-terminal extension as bait: 3 of 71 prey clones coded fora fragment of either the full-length HrpG protein or a nearfull-length fragment beginning at amino acid residue 6 [Fig.2A] . Two factors may contribute to the relatively low fractionof preys mapping to HrpG: [i] the HrpG RR domain is locatedin the N-terminal portion of the protein [residues 11 to 130]and may be required for interaction with XAC0095, and [ii] thepresence of an in-frame stop codon immediately upstream fromthe start codon [TAAATG] eliminates all clones containing insertsthat begin upstream from the start codon . If HrpG-HrpG interactionsare also mediated through N-terminal domain contacts, this secondfactor could also explain the predominance of recombination-derivedfull-length HrpG preys when HrpG is used as bait [above].

 
Five HrpG preys were derived from XAC1568, a 157-residue conserved hypothetical protein [Table 1] . All five preys began at either residue 64 or 65, indicating that the C-terminal domain of XAC1568 mediates its interactions with HrpG . Interestingly, the N-terminal domain contains several well-conserved cysteine residues found in all homologs from a large number of bacteria, whereas the C-terminal domain is present in only a much smaller group ofproteins found in X . campestris pv . campestris, A . tumefaciens, Mesorhizobium spp., Sinorhizobium spp., Caulobacter crescentus, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Rhodospirillumrubrum, Rhodobacter sphaeroides, and P . aeruginosa . None ofthese homologs, however, have known functions.

Finally, HrpG was also found to interact with a single prey derived from the XAC3683 gene that encodes a two-component system composite sensor-histidine kinase/response regulator [Table 1] . This result is consistent with a probable interaction betweenHrpG and a two-component system histidine kinase phosphoacceptordomain . The domain structure of XAC3683 includes a N-terminalPAS and PAC motifs frequently associated with signal sensordomains [47], central histidine kinase and phosphoacceptor domains,and a C-terminal RR receiver domain [44, 55] . The fragment ofXAC3683 detected as a positive prey begins at residue 96 locatedjust before the first PAS domain [residues 110 to 172] . Thisis the first time that HrpG has been shown to interact witha specific sensor protein and opens up the way to our understandingof the complete transmission pathway from external effectorto hrp gene expression . Since only 1 of 90 HrpG preys was derivedfrom XAC3683, the significance of this interaction will haveto be confirmed in other biochemical tests.

Since 40% of HrpG preys were derived from XAC0095, we decidedto use this protein as a bait in two-hybrid screens againstthe X . axonopodis pv . citri genomic prey library . The bait weused contains a 12-amino-acid N-terminal extension [Fig . 2B]. The vast majority [62 of 72] of XAC0095 preys were mapped to the XAC0095 gene [Table 1 and Fig . 2] . Of these, all clonescoded for the whole XAC0095 ORF . Sequencing analysis could demonstratethat all but one of these preys are derived from recombinationevents in which the XAC0095 bait gene was transferred into thepOAD vector [Table 1 and see above] and that a single prey wasderived from the library . This library-derived clone codes forthe full-length XAC0095 protein plus a 23-residue N-terminusextension.

The XAC0095 bait also interacted with a single prey that iscoded for by a previously unannotated ORF that has 45% identitywith XAC0095 . This ORF, which we name XAC0095b, is located between nucleotides 1331630 and 1331896 of the X . axonopodis pv . citri chromosome and codes for an 88-amino-acid protein [Fig . 3], residues 8 to 88 of which are coded by the XAC0095 prey . Interestingly, further analysis of the X . axonopodis pv . citri genome sequence allowed us to identify a third, also previously unidentified, X . axonopodis pv . citri ORF [which we name XAC0095c] between nucleotides 5030863 and 5031120 that codes for an 85-residuehomolog with 35% identity with XAC0095 and 40% identity withXAC0095b [Fig. 3] . Thus, both X . axonopodis pv . citri and X. campestris pv . campestris [see above] seem to have three XAC0095 homologs . An alignment of these three putative X . axonopodis pv . citri proteins and their corresponding homologs in X . campestris pv . campestris is shown in Fig . 3 . Some specific features ofthe alignment are of interest . [i] There appear to be two blocksof particularly well conserved residues: one 29-residue block in the N-terminal half of the protein and one 23-residue block in the C-terminal half [indicated by thin horizontal lines inFig. 3] . Within these two blocks, 19 residues are absolutely conserved in all six homologs . [ii] Each N-terminal block is flanked by proline residues on both sides . The C-terminal blocksin all six proteins are also flanked by prolines on their N-terminal sides, and three are flanked by prolines on their C-terminalsides as well [Fig . 3] . These conserved prolines may correspond to turns in specific structural features in these homologs. [iii] All six homologs have a conserved cysteine residue intheir C-terminal block . [iv] Both blocks contain conserved heptad pseudo-repeats [abcdefg] in which each a and d position is almostinvariably a hydrophobic residue [Fig . 3] . In fact, the C-terminalblock has two hydrophobic heptad repeats superimposed on oneanother [Fig . 3] . Such heptad repeats mediate side-by-side hydrophobicinteractions between amphipathic alpha-helices in coiled-coilproteins [41].

Finally, XAC0095 baits interacted with six preys derived fromthe XAC0524 protein [Table 1 and Fig . 2C] . This uncharacterized125-residue protein contains a helix-turn-helix DNA-bindingdomain in its N-terminal [residues 8 to 60] that is found ina number of transcription regulators, including the bacteriophage repressor proteins Cro, C1, and C2 . The C-terminal half of the protein, however, has only a few homologs in the public databases.Of these, only the E . coli DicA protein [30% identity with XAC0524], derived from a cryptic prophage, has been characterized [5]. The minimal XAC0524 domain observed to interact with XAC0095is delimited by residues 63 to 125, the C-terminal half of theprotein that follows the putative DNA-binding domain [Fig . 2C].

The function of the XAC0095 protein and its homologs is notknown at the moment, but its interaction with HrpG and XAC0524strongly suggest a role in the control of gene expression, particularlyhrp gene expression . Since HrpG is a bifunctional protein withboth a receiver/response regulator and DNA-binding domains,its interaction with XAC0095 could serve to modulate its interactionswith downstream factors associated with the hrpX and hrcC/hrpAgene promoters, as observed in X . campestris pv . vesicatoria[62], or with upstream effectors such as a two-component systemsensor or histidine kinase domains . XAC0095 could stabilizeHrpG or stabilize the phosphoryated or dephosphorylated stateof specific aspartic acid residues in its RR receiver domain[Asp60 in X . axonopodis pv . citri HrpG] . Phosphorylation ofRR receiver domain Asp residues is known to modulate the functionsof neighboring effector domains in two-component signal transductionpathways [44, 55].

HrpB2 interacts with a C-terminal domain of HrcU that is proteolytically cleaved in HrcU homologs. HrpB2 is a substrate of the type III secretion system: in X.campestris pv . vesicatoria, it is secreted to the exterior ofthe bacterial cell and is necessary for secretion of other proteinsvia the TTSS [48] . HrpB2 homologs are found only in Xanthomonasspp . and R . solanacearum and are essential for pathogenesisin X . campestris pv . vesicatoria [48] . When HrpB2 was used as bait in the two-hybrid assay, 4 of 10 clones sequenced were found to map to HrcU, a conserved member of the TTSS superfamily associated with the bacterial inner membrane [Table 1] . All four clones code for HrcU fragments beginning between residues 222 and 256, and all terminate downstream of the terminationcodon [Fig . 2D] . Interestingly, the HrcU paralog FlhB has been shown to have a direct role [along with FliK] in the switch that determines which substrates [hook versus filament subunits]are secreted by the type III secretion systems responsible forflagellar assembly [64] . Another HrcU paralog, YscU is necessary for the secretion of Yersinia anti-host factors [Yops] [40]. Both FlhB and YscU undergo site-specific proteolytic cleavage at a conserved Asn-Pro-Thr-His sequence, also found in X . axonopodis pv . citri HrcU [residues 264 to 267] . In fact, three of fourof the preys found to interact with HrpB2 map almost perfectlyto this putative C-terminal cleavage fragment in HrcU [Fig.2D] . In Yersinia, overexpression of a full-length uncleavableform of YscU inhibits growth, whereas overexpression of theC-terminal cleavage fragment results in increased Yop secretion[40] . It is therefore intriguing that a HrcU fragment possiblyinvolved in the control of type III secretion system substratespecificity interacts physically with a known substrate [HrpB2]that, in turn, is required for the secretion of other proteinsby this pathway [48] . This direct physical interaction has notbeen demonstrated previously.

Interactions between HrpB1, HrpD6, and HrpW. Preys obtained when HrpB1 is used as bait mapped to a numberof different X . axonopodis pv . citri proteins [Table 1 and data not shown] . Of 29 sequenced preys, 10 were derived from theHrpD6 protein, also coded for by the hrp locus . Both HrpB1 andHrpD6 are small [130 and 80 residues, respectively] cytoplasmicproteins whose homologs are essential for pathogenicity in X.campestris pv . vesicatoria [48] . However, neither of the proteins has homologs in the flagellar apparatus or in the TTSSs of animal pathogens, and no functional analyses of these two proteinsare available in the literature . In fact, HrpD6 homologs havebeen identified thus far only in Xanthomonas spp., whereas HrpB1 homologs are only found in Xanthomonas spp., as well as in R. solanacearum and Burkholderia pseudomallei . The smallest HrpD6 fragment found to interact with HrpB1 corresponded to residues 3 to 80 [Fig . 2E] . The interactions between these two proteins,identified here for the first time, may indicate that they acttogether in a common function . Unfortunately, when HrpD6 was used as a bait in two-hybrid screens, all eight preys were derived from the false-positive TolC protein [data not shown].

Two other HrpB1 preys were derived from clones coding for a full-length X . axonopodis pv . citri HrpW protein [XAC2922] [Table 1] . In spite of its name, the X . axonopodis pv . citri hrpW geneis not situated in the hrp locus . However, in X . campestrispv . campestris, the hrpW gene is located within the hrp locus,adjacent to the hpaB gene [21] . In P . syringae and Erwinia amylovora, the >41-kDa HrpW protein binds to pectate, and its TTSS-dependent secretion can elicit the plant hypersensitive response [16, 38] . The harpin domain in HrpW of P . syringae has seven glycine-richrepeats between residues 119 and 188 . However, HrpW from X.axonopodis pv . citri [33 kDa] and X . campestris pv . campestris[36 kDa], which share 46% identity and 59% similarity, do notpossess complete harpin domains and contain fewer glycine-richrepeats . The pectate lyase domain in X . axonopodis pv . citriand X . campestris pv . campestris HrpW seem complete howeverand shows >30% identity with the corresponding domain inthe Pseudomonas and Erwinia HrpW proteins, as well as with pectatelyase of Bacillus spp . The interaction between HrpB1, a cytosolicprotein, and HrpW, a homolog of known TTSS substrates, may indicatethat HrpB1 [and perhaps HrpD6] are involved in directing HrpWand other substrates to the TTSS apparatus [see Fig . 5, below].Finally, we note that six of the HrpB1 preys were derived froma protein [XAC2047, 407 amino acids] with only very little identity[23% identity, 40% similarity] with a putative polyhydroxyalkanoatesynthase subunit PhaE from Ectothiorhodospira shaposhnikovii[unpublished, gi|11096253] [Table 1 and see below].

 
HrpB4. HrpB4 is a 209-residue protein highly conserved [>90% identity]in Xanthomonas spp . Its only other known homolog is the HrpHprotein of R . solanacearum . HrpB4 has been shown to fractionatemostly to the soluble fraction of X . campestris pv . vesicatorialysates [48], although primary structure analysis has detectedputative transmembrane helices [13] . When used as bait, HrpB4was found to interact six times [out of a total of ten] withXAC2054 . XAC2054 is a two-component system composite sensor/histidinekinase/response regulator . The domain architecture of XAC2054includes multiple N-terminal PAS, PAC, and GAF domains frequentlyassociated with signal sensors, central histidine kinase, andphosphoacceptor domains and a C-terminal RR receiver domain.The smallest HrpB4-interacting fragment begins at residue 313of this 1,127-amino-acid protein . Interestingly, the XAC2054gene is located 3.6 kb from the XAC2047 gene whose product wasobserved to interact with HrpB1 [see above] . These observationspoint to the involvement of HrpB4 and HrpB1 in interactionsthat may be integrating the TTSS with other X . axonopodis pv.citri proteins.

HrcN. HrcN is a conserved component of all type III secretion systems,localized to the bacterial cytoplasm, and possibly associatedwith inner membrane components of the TTSS [46] . HrcN is highlysimilar to FliI, which is essential for bacterial flagellarassembly [64] . HrcN is also similar to InvC, Spa47 [MxiB], HrpB6,and YscN, all components of the type III protein secretion systemsin Salmonella spp., Shigella flexneri, X . campestris, and Yersinia,respectively [6, 46] . All of these proteins are homologs ofthe catalytic beta [and alpha] subunit of the F₀F₁-ATPase . WhenHrcN is used as a bait, four of the six preys sequenced were derived from the HrcN protein itself, the minimal prey fragment corresponding to residues 30 to 442 . [Interestingly, in thiscase, no preys were derived from recombination events from thebait plasmid.] This may reflect a hexameric ring state for theHrcN protein in vivo similar to the a₃b₃ ring structure in theF₀F₁-ATPase, as proposed recently for the HrcN homolog Spa47in Shigella sp . by Blocker et al . [6].

HpaA-HpaB-HrcV interactions. HpaA and HpaB are two proteins coded by genes located withinthe Xanthomonas hrp locus [Fig. 1] . HpaA homologs are foundonly in Xanthomonas spp . and R . solanacearum, and HpaA has beenshown to be secreted by X . campestris pv . vesicatoria [33]. HpaA may function as an effector molecule in X . campestris pv. vesicatoria as disruptions of the hpaA gene eliminate disease symptoms in tomato and pepper plants without affecting the ability to elicit hypersensitive response [33] . Furthermore, the HpaAprimary sequence contains two nuclear localization signals, one in the N-terminal and one in the C-terminal half of the protein, and HpaA protein, transiently expressed in onion cells,has been shown to localize to the nucleus [33] . Less is known about the putative function of HpaB, except that it may be localized to the bacterial inner membrane due to a single putative transmembrane helix [43] . Tn3-gus insertion mutagenesis of the hpaB gene ofX . axonopodis pv . glycines reduced bacterial pathogenicity [39].HpaB homologs have been found only in Xanthomonas spp., R . solanacearum, and B . pseudomallei.

Both HpaA and HpaB were used as baits in two-hybrid screens.When HpaA [271 amino acids] was used as a bait, 100% of the40 preys sequenced were found to be HpaB [Table 1] . Interestingly, all of these clones coded for polypeptides containing at least all except the first six amino acids of HpaB, suggesting thatthe HpaA-HpaB interaction requires an almost complete N-terminalregion of the HpaB polypeptide chain [Fig . 4] . We also tested two baits derived from the hpaB gene: one coding for the full-lengthsequence of 156 residues and a second coding for residues 1to 92 . This N-terminal fragment was used since residues 92 to 108 are predicted to form a transmembrane helix by the PSORT algorithm [43] . Each of the 16 preys sequenced when the HpaB N-terminal fragment was used was derived from a different gene in the X . axonopodis pv . citri genome . This result indicates that this fragment, on its own, does not make specific interactions with other peptides in the X . axonopodis pv . citri proteome. On the other hand, the results obtained with the full-lengthHpaB sequence were much more specific . Of 49 clones sequenced,37 were derived from the hpaA gene [Fig . 4] . The smallest HpaA fragment found to interact with HpaB corresponded to residues 126 to 271, the C-terminal half of the protein . Other than the presence of one of the nuclear localization signals, no functional information is available regarding this or any other regionof HpaA.

 
The remaining 12 preys obtained when the full-length HpaB proteinwas used as bait were derived from the HrcV protein, a componentof the type-III secretion machinery . The minimal fragment ofHrcV found to interact with full-length HpaB corresponded toresidues 360 to 645 [Fig . 4] . This observation is consistentwith the prediction that the N-terminal domain of HrcV [up toresidue 316] codes for at least six transmembrane segments [43], whereas the C-terminal half of the protein forms a soluble cytosolic domain . When the HrcV C-terminal domain [residues 325 to 646] was used as a bait, 14 of 18 preys were derived from the HrcV protein . All of these preys were derived from recombinationevents with the bait vector; therefore, the minimal domain necessaryfor this interaction could not be determined . Furthermore, oneprey was derived from the full-length HpaB protein, confirmingthe HpaB-HrcV interaction observed when HpaB was used as bait.A summary of the interactions observed between HpaA, HpaB, andHrcV is shown in Fig. 4 . Since HrcV [and possibly HpaB] is localizedto the inner membrane, whereas HpaA is a soluble protein withdomains indicative of activity in the host cell nucleus [seeabove], a functional role for these interactions becomes immediatelyapparent: HpaA, HpaB, and the C-terminal cytosolic domain ofHrcV may interact in a manner that results in the targetingof HpaA to the type III secretion machinery and its subsequenttranslocation into the host cell [Fig . 5] . The above resultsseem to point to HpaB possibly functioning as a chaperone orprotein usher for HpaA, perhaps facilitating its interactionwith HrcV at the cytoplasmic entrance of the TTSS channel [Fig.5].

Concluding remarks. The yeast two-hybrid system has been used to identify protein-proteininteractions in protein complexes such as the yeast pheromone-responsepathway complex [23] and yeast RNA polymerase III [24] and between Drosophila cyclin-dependent protein kinase interactors and cyclin-dependent kinases involved in cell cycle regulation [30] . It has alsobeen used to elucidate an interaction map of proteins involved in Caenorhabditis elegans vulval development [59], to screenan oligopeptide expression library [68], and to identify protein-proteininteractions on the proteome-scale [4, 56] . Ward et al . [60]recently used this methodology to delimit interactions betweencomponents of the type IV secretion system of A . tumefaciens.The two-hybrid system has only been used in one other studyto investigate interactions between TTSS components in Yersiniapestis [34], responsible for the export of 12 Yersinia outerproteins . In that study, specific interactions were observedfor YscQ with YscK and YscL and for YscL with YscQ and YscN.Those authors suggested that YscKQLN may form a complex peripherallyassociated with the inner membrane in a manner similar to theF₁ and V₁ multiprotein complexes of the F₀F₁ and V₀V₁ proton-translocatingATPases . YscQ and YscN are homologs of X . axonopodis pv . citriproteins HrcQ and HrcN, whereas YscK and YscL have no X . axonopodispv . citri homologs . The interactors and interactions observedin the Yersinia study [34] were therefore different from thoseobserved in the present study.

In the present study, we have identified a number of potentially physiologically relevant interactions between subunits, regulators, and substrates of the X . axonopodis pv . citri type III secretion system . We have identified interactions involving proteins previously known or suspected to be involved in X . axonopodis pv . citri pathogenicity, including HrpG, HpaA, HpaB, HrcV, HrpB1, HrpD6, HrpB2, HrcU, HrpW, HrpB4, and HrcN . The fact that our prey library consists of whole genomic X . axonopodis pv . citri DNA significantly increased the possibility of observing so-called "false-positive" interactions . In spite of this, relatively few and in many cases easily identifiable false-positives were observed, and it is highly significant that the majority of the interactions observedin these assays "make sense" physiologically . In fact, the multiple interactions observed between known Hrp proteins when a wholegenomic DNA prey library was used is a strong confirmation ofthe physiological relevance of these interactions . A similarhigh degree of internal consistency has been observed by usin two-hybrid assays in which the baits were derived from X.axonopodis pv . citri type IV secretion system components [M.C . Alegria et al., unpublished data].

The protein-protein interactions identified here have clear implications for our understanding of the molecular mechanisms underlying Xanthomonas pathogenicity in general and the workings and regulation of the TTSS in particular . Figure 5 presentsa summary of the interactions observed in the present study and their integration into a functional model of the Xanthomonas spp . TTSS that has emerged from the important contributionsof many other laboratories . What is particularly interestingis that we now have subsets of protein-protein interactionsthat point to a relationship between specific TTSS substrates[HpaA and HrpB2] and specific conserved components of the TTSSmachinery [HrcV and HrcU, respectively] . In the case of HpaAand HrcV, their association appears to be mediated via HpaB.These results point to more specific studies that should becarried out in the near future, including investigations into[i] the molecular interactions important for HpaA secretion,including those involving HpaB and HrcV; [ii] the possible occurrenceand role of HrcU proteolytic cleavage in TTSS assembly and regulationof TTSS substrate specificity, in particular the secretion ofHrpB2; and [iii] specific interactions between HrpB1, HrpD6, and the pectate lysase harpin homolog HrpW and the possibility of their functional integration with HrpB4 and the conserved hypothetical proteins XAC2047 and XAC2054 . Finally, of special interest for future studies are the specific molecular interactions between HrpG and its upstream and downstream regulators, including members of the newly identified XAC0095 family of proteins, transcription factors, and two-component system sensor histidine kinases.

 
We are deeply grateful for the excellent technical assistanceof Izaura Nabuko Toma, Ilda de Souza Costa, and Elizabeth S.N.Mandetta . We thank Stanley Fields for kindly providing the pOBDand pOAD plasmids . We also thank Phillip James for kindly providingthe PJ694-a yeast cells.

This study was supported by the Fundação de Amparoà Pesquisa do Estado de São Paulo [FAPESP] andthe Conselho Nacional de Pesquisa of Brazil . M.C.A., C.D., andL.K . are graduate fellows of FAPESP.

 
* Corresponding author . Mailing address: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av . Prof . Lineu Prestes, 748, CEP 05599-970 São Paulo-SP, Brazil . Phone: 55-11-3091-3312 . Fax: 55-11-3815-5579 . E-mail: chsfarah@iq.usp.br.

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