HpaC Controls Substrate Specificity of the Xanthomonas Type III Secretion System

The Gram-negative bacterial plant pathogen Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to inject bacterial effector proteins into the host cell cytoplasm. One essential pathogenicity factor is HrpB2, which is secreted by the T3S system. We show that secretion of HrpB2 is suppressed by HpaC, which was previously identified as a T3S control protein. Since HpaC promotes secretion of translocon and effector proteins but inhibits secretion of HrpB2, HpaC presumably acts as a T3S substrate specificity switch protein. Protein–protein interaction studies revealed that HpaC interacts with HrpB2 and the C-terminal domain of HrcU, a conserved inner membrane component of the T3S system. However, no interaction was observed between HpaC and the full-length HrcU protein. Analysis of HpaC deletion derivatives revealed that the binding site for the C-terminal domain of HrcU is essential for HpaC function. This suggests that HpaC binding to the HrcU C terminus is key for the control of T3S. The C terminus of HrcU also provides a binding site for HrpB2; however, no interaction was observed with other T3S substrates including pilus, translocon and effector proteins. This is in contrast to HrcU homologs from animal pathogenic bacteria suggesting evolution of distinct mechanisms in plant and animal pathogenic bacteria for T3S substrate recognition.


Introduction
Many Gram-negative bacterial pathogens of plants and animals depend on a type III secretion (T3S) system to successfully infect their hosts [1]. The term ''T3S system'' refers to both translocation-associated and flagellar T3S systems that evolved from a common ancestor [2]. Eleven components of the membrane-spanning basal body are conserved, suggesting a similar overall architecture of the secretion apparatus [1,3]. Main structural differences are found in the extracellular appendages associated with the basal body. The flagellar T3S apparatus is connected via an extracellular hook to the filament, the key bacterial motility organelle [4]. By contrast, the basal body of translocation-associated T3S systems is associated with an extracellular pilus (plant pathogens) or needle (animal pathogens), which serve as conduits for secreted proteins to the host-pathogen interface [1,5]. Pilus and needle are proposed to be linked to the T3S translocon, a channel-like protein complex that is inserted into the eukaryotic plasma membrane and allows protein translocation into the host cell cytosol [6,7].
Translocation-associated T3S systems secrete two types of proteins, i.e., extracellular components of the secretion apparatus such as needle/pilus and translocon proteins, and effectors that are translocated into the host cell [3]. Efficient secretion and/or translocation of T3S substrates depends on a signal in the N terminus, which is not conserved on the amino acid level [1,8,9]. In many cases, specific T3S chaperones bind to one or several homologous T3S substrates in the bacterial cytoplasm and promote stability and/or secretion of their respective binding partners. T3S chaperones are small, acidic and leucine-rich proteins that presumably guide secreted proteins to the secretion apparatus at the inner membrane [1,10,11].
Given the architecture of the T3S system, it is conceivable that secretion of extracellular components of the secretion apparatus precedes effector protein translocation. In translocation-associated and flagellar T3S systems from animal pathogenic bacteria, experimental evidence suggests that substrate specificity is altered by so-called T3S substrate specificity switch (T3S4) proteins, e.g., YscP from Yersinia spp. and the flagellar homolog FliK [12][13][14]. The substrate specificity switch depends on the C-terminal cytoplasmic domain of a conserved inner membrane protein of the FlhB/YscU family [12,13]. T3S4 proteins are not highly conserved among different pathogens and have so far only been identified in animal pathogenic bacteria [14,15]. It therefore remained enigmatic whether plant pathogenic bacteria employ similar mechanisms to orchestrate secretion of different T3S substrates.
In our laboratory, we study T3S of the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria, the causal agent of bacterial spot disease in pepper and tomato. The T3S system of X. campestris pv. vesicatoria is essential for bacterial growth and disease symptom formation in susceptible host plants and the induction of the hypersensitive response (HR) in resistant plants. The HR is a rapid programmed cell death at the infection site that is triggered upon recognition of individual effector proteins, also termed avirulence (Avr) proteins, in plants that carry a cognate disease resistance gene [16,17]. In susceptible plants, effector proteins presumably modulate host cellular pathways to the pathogen's benefit and thus primarily act as virulence factors [18,19].
The T3S system from X. campestris pv. vesicatoria is encoded by the 23-kb chromosomal hrp (hypersensitive response and pathogenicity) gene cluster, which is organized in eight operons and contains 25 genes [20][21][22][23]. Eleven genes (termed hrc for hrp conserved) encode proteins that are conserved among plant and animal pathogenic bacteria [24]. Most hrp genes are essential for bacterial pathogenicity [25]. Several Hrp proteins are secreted and thus constitute extracellular components of the T3S system such as the pilus protein HrpE and the translocon protein HrpF [25][26][27]. T3S of extracellular components of the secretion apparatus and effector proteins is presumably controlled by the products of hpa (hrp-associated) genes that are encoded in the hrp gene cluster and contribute to pathogenicity [28][29][30][31]. Examples are the export control protein HpaC, which is required for the efficient secretion of translocon and some effector proteins, and the global T3S chaperone HpaB, which promotes secretion and translocation of multiple effector proteins [30,31].
In this study, we analyzed HrpB2, which is an essential pathogenicity factor of X. campestris pv. vesicatoria. HrpB2 is a 13.7-kDa protein that is encoded by the second gene of the hrpB operon and is secreted by the T3S system [25,32]. Homologous proteins are present in Xanthomonas spp., Burkholderia spp. and Ralstonia solanacearum, suggesting that HrpB2 also plays an important role in other pathogens. In X. campestris pv. vesicatoria, HrpB2 is essential for pilus formation and T3S and is therefore presumably one of the first proteins that travel the T3S apparatus [25,26]. However, the mode of HrpB2 action is unknown because HrpB2 does not share significant sequence or structural similarity with a protein of known function. Here, we provide experimental evidence that secretion of HrpB2 is required for bacterial pathogenicity. Secretion of HrpB2 is significantly enhanced in the absence of the export control protein HpaC. Protein-protein interaction studies showed that HrpB2 binds to HpaC and to the C-terminal domain of the conserved inner membrane protein HrcU, which also interacts with HpaC. Our data suggest that the interaction between HpaC and the C-terminal domain of HrcU promotes a switch in substrate specificity of the T3S system from HrpB2 secretion to secretion of translocon and effector proteins.

HrpB2 is essential for bacterial pathogenicity and T3S
Previously, we identified HrpB2 as a T3S substrate of X. campestris pv. vesicatoria strain 85-10 [25]. Infection studies with hrpB2 deletion mutant strains revealed that HrpB2 is crucial for disease symptoms in susceptible and the HR induction in resistant pepper plants [25]. Similar results were obtained with strains 85* and 85*DhrpB2, which carry hrpG*, a mutated version of the key regulatory gene hrpG in the bacterial chromosome ( Fig 1A). hrpG* leads to constitutive expression of the T3S system and is key for the analysis of in vitro T3S [33]. It is noteworthy that in planta growth of hrpG* strains is like wild-type [34]. The hrpB2 mutant phenotype could be complemented by ectopic expression of hrpB2, suggesting that loss of pathogenicity was specifically due to the deletion of hrpB2 and did not result from a polar effect of the mutation on expression of other genes in the hrpB operon (Fig. 1A

Author Summary
The Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease in pepper and tomato. Pathogenicity of X. campestris pv. vesicatoria depends on a type III protein secretion (T3S) system that injects bacterial effector proteins directly into the host cell cytosol. The T3S system is a highly complex nanomachine that spans both bacterial membranes and is associated with an extracellular pilus and a translocon that inserts into the host cell membrane. Given the architecture of the secretion apparatus, it is conceivable that pilus formation precedes effector protein secretion. The pilus presumably consists of two components, i.e., the major pilus subunit HrpE and HrpB2, which is required for pilus assembly. Secretion of HrpB2 is suppressed by HpaC that switches substrate specificity of the T3S system from secretion of HrpB2 to secretion of translocon and effector proteins. The substrate specificity switch depends on the cytoplasmic domain of HrcU, which is a conserved inner membrane protein of the T3S apparatus that interacts with HrpB2 and HpaC.
The fact that secretion of the effector protein AvrBs3 is abolished in hrpB2 deletion mutants suggested that HrpB2 is involved in T3S [25]. To investigate the contribution of HrpB2 to secretion of additional T3S substrates, strains 85* and 85*DhrpB2 were incubated in secretion medium, and total cell extracts and culture supernatants were analyzed by immunoblotting using specific polyclonal antibodies. We tested secretion of the putative translocon proteins HrpF and XopA, and the pilus protein HrpE. For technical reasons, HrpE was studied as a fusion protein consisting of the N-terminal 50 amino acids of HrpE and the reporter protein AvrBs3D2, which is a derivative of AvrBs3. AvrBs3D2 lacks the first 152 amino acids and thus the secretion and translocation signal [35]. It was previously demonstrated that the N-terminal 50 amino acids of HrpE restore secretion but not translocation of AvrBs3D2, indicating that they contain a functional T3S signal [36]. Fig. 1B shows that HrpF, XopA and HrpE 1-50 -AvrBs3D2 were present in the culture supernatant of the wild-type strain but were not detectable in the supernatant of the hrpB2 deletion mutant, suggesting that HrpB2 is essential for secretion of these proteins.

Secretion of HrpB2 is inhibited by HpaC
Since HrpB2 is secreted and is also required for T3S, it is presumably one of the first substrates that travel the T3S apparatus [25]. Notably, the amount of HrpB2 present in the culture supernatant of strain 85* is at the detection limit of the HrpB2specific antibody [25]. Similar results were observed for a Cterminally c-Myc epitope-tagged version of HrpB2, suggesting that HrpB2 is only weakly secreted by the T3S system ( Fig. 2A). To investigate whether HrpB2 secretion is regulated by the known export control proteins HpaB and HpaC, we performed in vitro T3S assays with strains 85*, the hpaB deletion mutant 85*DhpaB and the hpaC deletion mutant 85*DhpaC. We did not detect any influence of the global T3S chaperone HpaB on secretion of HrpB2. Interestingly, however, significantly increased amounts of HrpB2 were secreted by strain 85*DhpaC (Fig. 2). This was not due to a general increase of T3S in strain 85*DhpaC since secretion of the translocon protein HrpF was reduced when compared to the wildtype strain 85* ( Fig. 2A and C). This is in agreement with the previous finding that HpaC is required for the efficient secretion of translocon and some effector proteins [31]. Oversecretion of HrpB2 in strain 85*DhpaC was specifically due to deletion of hpaC since the secretion phenotype was complemented by ectopic expression of hpaC-c-myc (Fig. 2B). We did not detect HrpB2 in the culture supernatant of the T3S double mutant 85*DhpaCDhrpE, which additionally lacks the Hrp pilus gene hrpE (Fig. 2C). We therefore conclude that increased HrpB2 secretion in strain 85*DhpaC was mediated by the translocation-associated T3S system.

The N terminus of HrpB2 is crucial for protein function
Next, we investigated whether secretion of HrpB2 is crucial for protein function. For this, we analyzed N-terminal HrpB2 deletion derivatives. Surprisingly, deletion of the N-terminal 10 amino acids of HrpB2 did not abolish its secretion in wild-type and hpaC deletion mutant strains (data not shown). By contrast, secretion of a HrpB2 deletion derivative lacking amino acids 10 to 25 was severely reduced in strain 85*DhpaC, suggesting that amino acids 10 to 25 harbour at least part of the secretion signal (Fig. 2D). Notably, HrpB2 D10-25 did not complement the hrpB2 mutant phenotype with respect to disease symptom formation in susceptible and HR induction in resistant pepper plants (Fig. 1A). This was not due to the presence of the C-terminal c-Myc epitope since complementation studies were performed with untagged HrpB2 and derivatives. Immunoblot analysis of bacterial total cell extracts revealed that HrpB2 D10-25 was stably synthesized in strain 85*DhrpB2 (Fig. 2D). We therefore conclude that amino acids 10 to 25 are crucial for efficient secretion of HrpB2 and that HrpB2 secretion is presumably required for protein function.

HrpB2 interacts with HpaC
To investigate whether oversecretion of HrpB2 in the hpaC deletion mutant was due to increased hrpB2 transcript levels, we performed reverse transcriptase (RT)-PCR analysis of strains 85* and 85*DhpaC grown under secretion-permissive conditions. Fig. 3A shows that hrpB2 transcript levels were comparable in both strains, suggesting that deletion of hpaC did not affect the transcriptional regulation of hrpB2.
We therefore studied whether there is an interaction between HrpB2 and HpaC proteins using glutathione S-transferase (GST) pull-down assays. For this, GST and a GST-HpaC fusion protein were synthesized in Escherichia coli, immobilized on glutathione sepharose matrix and incubated with an E. coli lysate containing HrpB2-c-Myc. Bound proteins were eluted from the matrix and analyzed by immunoblotting using c-Myc epitope-and GSTspecific antibodies. HrpB2-c-Myc specifically eluted with GST-HpaC but not with GST alone, indicating that HrpB2 interacts with HpaC (Fig. 3B). Similar results were obtained with an Nterminal HrpB2 deletion derivative that lacks the first 26 amino acids and thus at least part of the T3S signal ( Fig. 3B; see above). The interaction between HpaC and HrpB2 is reminiscent of our previous finding that HpaC binds to different T3S substrates including translocon and effector proteins [31]. We did not observe an interaction between HrpB2 and the global T3S chaperone HpaB (Fig. 3C), which is in line with the fact that HpaB does not control HrpB2 secretion (see above).

HrpB2 binds to the C-terminal domain of HrcU that is proteolytically cleaved
In animal pathogenic bacteria T3S substrate recognition is mediated by members of the conserved FlhB/YscU family of inner membrane proteins [37][38][39]. YscU, FlhB and their homologs contain four predicted transmembrane domains and a C-terminal cytoplasmic protein region that is cleaved between the asparagine and proline residues of the conserved NPTH motif [39][40][41][42][43]. To investigate a possible cleavage of the YscU/FlhB homolog HrcU from X. campestris pv. vesicatoria, we synthesized a C-terminally c-Myc epitope-tagged HrcU derivative in both E. coli and X. campestris pv. vesicatoria and analyzed protein extracts by immunoblotting using a c-Myc-specific antibody. We detected two proteins of approximately 50 kDa and 20 kDa in E. coli and X. campestris pv. vesicatoria extracts irrespective of the growth medium (Fig. 4A). Both proteins presumably correspond to full-length HrcU (39 kDa+5 kDa epitope tag) and the predicted C-terminal cleavage product (10 kDa+5 kDa epitope tag). The HrcU proteins migrate slower than predicted, which was previously also reported for other T3S system-associated proteins from X. campestris pv. vesicatoria [27,29].
Because yeast two-hybrid-based interaction studies of proteins from Xanthomonas axonopodis pv. citri suggested an interaction between HrpB2 and the C-terminal domain of HrcU [44], we performed GST pull-down assays with HrpB2 and HrcU from X. campestris pv. vesicatoria. For this, we generated expression constructs encoding GST-HrcU, GST-HrcU-c-Myc and GST-HrcU 255-357 , the latter of corresponds to the C-terminal cytoplasmic domain of HrcU. To test for proteolytic cleavage, GST-HrcU and GST-HrcU-c-Myc were analyzed by immunoblotting of E. coli protein extracts, using GST-and c-Myc-specific antibodies. Both proteins and several degradation products were visualized by a GST-specific antibody (Fig. 4B). Furthermore, GST-HrcU-c-Myc and a smaller protein of approximately 20 kDa were also detected by a c-Myc specific antibody. The smaller protein presumably corresponds to the C-terminal cleavage product of HrcU (see Fig. 4A), indicating that GST-HrcU fusions are proteolytically cleaved (Fig. 4B).
For protein-protein interaction studies, GST-HrcU and GST-HrcU 255-357 (HrcU C-terminal domain), immobilized on glutathione sepharose, were incubated with HrpB2-c-Myc. HrpB2-c-Myc eluted with GST-HrcU and GST-HrcU 255-357 , but not with GST alone, suggesting that HrpB2 interacts with the C-terminal domain of HrcU ( Fig. 5A and B).   HpaC interacts with the C-terminal domain of HrcU but not with the full-length protein HrcU homologs from animal pathogenic bacteria are involved in the T3S substrate specificity switch [37,39]. We therefore tested a possible interaction between HrcU and HpaC, which presumably acts as a T3S4 protein (see also below). When GST-HrcU was immobilized on glutathione sepharose and incubated with HpaC-c-Myc, we did not detect HpaC-c-Myc in the eluate (Fig. 5C). By contrast, HpaC-c-Myc coeluted with GST-HrcU 255-357 , suggesting that it interacts with the C-terminal domain of HrcU but not with the full-length protein (Fig. 5D). Since GST-HrcU is proteolytically cleaved (see Fig. 4B), we assume that the protein is correctly folded. Our data therefore suggest that the interaction between HpaC and HrcU depends on a certain conformation of the HrcU C terminus that is altered in the context of the full-length protein.

The C-terminal domain of HrcU does not interact with HrpE, XopA and XopF1
Next, we investigated whether the C-terminal domain of HrcU also interacts with other secreted proteins, e.g., the putative translocon protein XopA, the pilus protein HrpE and the effector protein XopF1. For this, GST, GST-HpaC, GST-XopA and GST-HrpE were immobilized on glutathione sepharose and incubated with HrcU-c-Myc. Fig. 6A shows that the C-terminal cleavage product of HrcU (see above) was detected in the eluate of GST-HpaC but not of GST-XopA or GST-HrpE. This suggests that the C-terminal domain of HrcU interacts with HpaC but not with HrpE and XopA. We did not detect full-length HrcU-c-Myc in the eluate of GST-HpaC (Fig. 6A), which confirms our previous observation that HpaC specifically interacts with the C-terminal domain of HrcU but not with the full-length protein (see Fig. 5C and D). To investigate a possible interaction between HrcU and the effector protein XopF1, we expressed XopF1 as a C-terminally c-Myc epitope-tagged derivative because a GST-XopF1 fusion protein was unstable in E. coli. XopF1-c-Myc was incubated with GST-HrcU, GST-HrcU 255-357 and GST-HpaB, which was used as a positive control for the interaction assay. GST-HpaB was previously shown to interact with XopF1 [31]. As expected, XopF1-c-Myc was detected in the eluate of GST-HpaB but did not coelute with GST-HrcU and GST-HrcU 255-357 (Fig. 6B). Taken together, our results suggest that the C-terminal domain of HrcU does not interact with the T3S substrates XopA, HrpE and XopF1. This is in contrast to the C-terminal region of the flagellar HrcU homolog FlhB, which interacts with several secreted proteins and is therefore presumably involved in substrate recognition [38].

HpaC contains a putative T3S4 domain that is crucial for the interaction with the C-terminal domain of HrcU
The finding that HpaC is involved in control of T3S substrate specificity and interacts with the C-terminal domain of HrcU suggests that it acts similarly to T3S4 proteins that were identified in translocation-associated and flagellar T3S systems from animal pathogenic bacteria. Despite limited sequence conservation, known T3S4 proteins harbour a structurally conserved T3S4 domain in the C terminus, which is responsible for the substrate specificity switch [15,45]. PSI-BLAST searches and hydrophobic cluster analysis showed that the T3S4 domain is not only present in proteins from animal pathogenic bacteria but also shares weak sequence similarity with the C terminus of HpaP from Ralstonia solanacearum [15]. HpaP is 27% sequence-identical to HpaC. A pairwise sequence alignment of HpaP and HpaC revealed that most conserved amino acids in the predicted T3S4 domain of HpaP are also present in HpaC or are substituted by amino acids with similar chemical properties (Fig. S1).
To investigate whether the predicted T3S4 domain of HpaC participates in the interaction with the C terminus of HrcU, we performed GST pull-down assays with C-terminal HpaC deletion derivatives, which are shown in Fig. 7A. HpaC 1-182 -c-Myc, which is deleted in the C-terminal 30 amino acids and thus lacks part of the predicted T3S4 domain, coeluted with GST-HrcU 255-357 , but not with GST alone (Fig. 7B). However, when compared to the fulllength HpaC protein, which has a strong affinity for HrcU 255-357 , the interaction between HpaC 1-182 -c-Myc and GST-HrcU 255-357 was significantly reduced (Fig. 7B). By contrast, binding of HpaC 1-182 -c-Myc to other known HpaC interaction partners such as HpaB, XopF1, XopA, HrcV and also the HpaC self-interaction was not affected (Fig. 7B and C) [31]. Next, we analyzed a HpaC deletion derivative, HpaC 1-118 -c-Myc, which lacks the C-terminal 94 amino acids and thus the complete T3S4 domain. The fact that HpaC 1-118c-Myc was not detectable in the eluate of GST-HrcU 255-357 suggests that the predicted T3S4 domain of HpaC is important for the interaction with the C terminus of HrcU (Fig. 7D).

The predicted T3S4 domain of HpaC is crucial for protein function
To address whether the predicted T3S4 domain of HpaC is also important for protein function, we expressed HpaC and deletion  (Fig. 8A and B). Furthermore, HpaC 1-118 -c-Myc and HpaC 1-182 -c-Myc did not restore the deficiency in HrpF secretion in strain 85*DhpaC (Fig. 8B). We therefore speculate that the T3S4 domain of HpaC and thus the interaction with the Cterminal domain of HrcU is essential for the HpaC-dependent substrate specificity switch.

Discussion
In this study, we analyzed the pathogenicity factors HrpB2 and HpaC from X. campestris pv. vesicatoria. We discovered that HrpB2 is not only crucial for secretion of effectors but also of extracellular components of the secretion apparatus, i.e., the putative translocon proteins XopA and HrpF and the pilus protein HrpE. Since HrpB2 is itself secreted by the T3S system, it is presumably one of the first substrates that travels the secretion apparatus [25]. The analysis of N-terminal HrpB2 deletion derivatives revealed that the secretion signal of HrpB2 is located between amino acids 10 to 25 and is crucial for protein function. It is therefore possible that HrpB2 is an extracellular component of the secretion apparatus that promotes pilus assembly. However, HrpB2 is probably not a major pilus subunit since only low amounts of HrpB2 are secreted by the T3S system. Notably, the pilus protein HrpE is required for HrpB2 secretion and vice versa, suggesting that HrpB2 is not part of an extracellular needle-like structure below the pilus. An analogous finding was recently reported for the symbiotic bacterium Rhizobium strain NGR234. Pilus assembly and T3S in strain NGR234 depends on the secreted protein NopB that presumably associates with NopA, which is the major pilus subunit [46,47].
The second important finding is that secretion of HrpB2 from X. campestris pv. vesicatoria is suppressed by the export control protein HpaC, which promotes secretion of translocon and effector proteins [31]. Proteins that differentially regulate secretion of different T3S substrates were described for flagellar or translocation-associated T3S systems and include, e.g., the flagellar chaperone FliS and T3S4 proteins from animal pathogenic bacteria [12][13][14]48,49]. We speculate that HpaC acts similarly to T3S4 proteins and alters the specificity of the secretion apparatus from early (HrpB2) to later (translocon and effector proteins) T3S substrates. We believe that this substrate specificity switch takes place at the protein level because HpaC interacts with HrpB2 and with the C-terminal domain of HrcU, which belongs to the FlhB/YscU family of inner membrane proteins [3,12,13]. This is in agreement with our previous finding that HpaC binds to different T3S substrates including translocon and effector proteins and also interacts with conserved inner membrane components of the T3S system such as HrcV [31]. It was therefore proposed that HpaC acts as a linker between secreted proteins and the secretion apparatus [31]. However, HpaC is dispensable for secretion of HrpB2. Targeting of HrpB2 to the secretion apparatus is presumably mediated by the C-terminal domain of HrcU, which interacts with both HrpB2 and HpaC. The latter interaction presumably depends on a certain conformation of the HrcU C terminus since we did not detect binding of HpaC to full-length HrcU. The analysis of HpaC deletion derivatives revealed that the HrcU-binding site is located in the C terminus of HpaC, which contains the putative T3S4 domain [15] and is required for protein function. This observation suggests that the interaction between HpaC and the C-terminal domain of HrcU is required for HpaC-mediated suppression of HrpB2 secretion. Our data are reminiscent of the finding that the T3S4 protein FliK from Salmonella spp. interacts with the C-terminal domain of the HrcU homolog FlhB [37,38]. It was proposed that binding of FliK induces a conformational change in the C-terminal cytoplasmic domain of FlhB and thus alters the substrate specificity of the flagellar T3S system from secretion of hook components to filament proteins [12,50]. Since the C-terminal domain of FlhB interacts with several secreted proteins, it presumably serves as a docking point for T3S substrates [37,38]. This clearly differs from the FlhB homolog HrcU from X. campestris pv. vesicatoria since the C-terminal domain of HrcU does not interact with translocon and effector proteins that were tested in this study. It is conceivable that T3S substrate binding in X. campestris pv. vesicatoria is mediated by other conserved inner membrane components of the T3S system such as HrcV or the putative ATPase HrcN [3].
The precise mechanism underlying the HpaC/HrcU-mediated substrate specificity switch in X. campestris pv. vesicatoria remains to be determined. We speculate that after activation of the T3S system, binding of HpaC to the C-terminal domain of HrcU inhibits the interaction between this domain and HrpB2 and thus blocks secretion of HrpB2. Preliminary GST pull-down assays revealed that HrpB2-c-Myc coelutes with GST-HrcU 255-357 irrespective of the presence of HpaC, suggesting that all three proteins can form a complex. It still remains to be investigated whether both proteins simultaneously bind to the C-terminal domain of HrcU or whether they are both present in the eluate because they interact with each other. Taken together, our data suggest that plant and animal pathogenic bacteria share similar mechanisms to switch the substrate specificity of the T3S system but that they differ in the components that recognize T3S substrates. Another important difference between plant and animal pathogenic bacteria concerns the length control of extracellular structures associated with the membrane-spanning secretion apparatus. In translocation-associated and flagellar T3S systems from animal pathogenic bacteria, the substrate specificity switch is coupled to length control of needle and hook structures. In the flagellar T3S system from Salmonella spp., for instance, FliK activates secretion of filament proteins after hook formation. Deletion of fliK leads to elongated hook structures, suggesting that FliK is required for hook length control [51][52][53]. Similarly to FliK, the T3S4 protein YscP from Yersinia spp. determines needle length in the translocationassociated T3S system [12]. Since YscP is itself secreted it was proposed that the N terminus of YscP anchors to the tip of the growing needle while the C terminus of the protein remains attached to the secretion apparatus and activates the substrate specificity switch [54]. According to this model, T3S4 proteins act as molecular rulers that are coupled to a substrate specificity switch [12,15]. The molecular ruler model was challenged by the finding that the T3S4 protein InvJ from Salmonella typhimurium is required for formation of the inner rod of the T3S apparatus. It was suggested that formation of the inner rod triggers a conformational change in the secretion apparatus that leads to the substrate specificity switch [55]. This model is supported by the recent finding that the T3S4 protein YscP from Yersinia controls secretion of the predicted inner rod protein YscI [56]. Wood et al. identified YscI point mutants that allow effector secretion in the absence of a detectable needle structure, suggesting that the needle is not required for the substrate specificity switch.
The future challenge is to investigate the molecular mechanisms underlying the HpaC-mediated T3S substrate specificity switch in X. campestris pv. vesicatoria. Since HpaC is not secreted by the T3S system, it presumably does not act as a molecular ruler protein [31]. Electron microscopy studies have suggested that pilus length is not controlled by HpaC [26]. Furthermore, it should be emphasized that secretion of the Hrp pilus subunit HrpE is not affected in hpaC mutants [31]. In contrast to the relatively short (approximately 50 nm) T3S needle from animal pathogenic bacteria, the Hrp pilus from plant pathogens can reach a length of up to 2 mm that cannot be bridged by a single proteinaceous molecular ruler. We therefore speculate that HpaC acts as a T3S4 protein that is not involved in length control of extracellular structures of the T3S system. This hypothesis is supported by the fact that secretion-deficient derivatives of the T3S4 proteins YscP and FliK are still active, indicating that length control and substrate specificity switch functions can be uncoupled [54,57].

Bacterial strains and growth conditions
Bacterial strains and plasmids used in this study are listed in Table 1. E. coli cells were cultivated at 37uC in lysogeny broth (LB). X. campestris pv. vesicatoria strains were grown at 30uC in NYG medium [58] or in minimal medium A [59] supplemented with sucrose (10 mM) and casamino acids (0.3%). Plasmids were introduced into E. coli by electroporation and into X. campestris pv. vesicatoria by conjugation, using pRK2013 as a helper plasmid in triparental matings [60]. For the generation of strain 85*DhpaCDhrpE, pOK-hrpED9-93, which is a derivative of the suicide plasmid pOK1 (see Table 1), was introduced into the genome of X. campestris pv. vesicatoria strain 85*DhpaC by conjugation. Double cross-overs resulted in deletion mutants that were selected as described [28].

Plant material and plant inoculations
The near-isogenic pepper cultivars Early Cal Wonder (ECW), ECW-10R and ECW-30R [61] were grown and inoculated with X. campestris pv. vesicatoria as described previously [22]. Bacteria were hand-infiltrated into the intercellular spaces of leaves at concentrations of 2610 8 cfu/ml in 1 mM MgCl 2 if not stated otherwise. The appearance of disease symptoms and the HR were scored over a period of three to five days after inoculation. For better visualization of the HR, leaves were bleached in 70% ethanol.

RNA analyses
For RT-PCR analysis, bacteria were grown in secretion medium. RNA extraction and cDNA synthesis were performed as described [62] and hrpB2 transcripts were amplified by PCR. To exclude that RNA preparations contained genomic DNA, total RNA was used as a template in a control PCR using hrpB2-specific primers. The lack of detectable hrpB2 amounts suggested that the RNA preparations were DNA-free (data not shown). Sequences of primers used in this study are available upon request.

Generation of hrpB2 expression constructs
For the generation of hrpB2 expression constructs, hrpB2 and Nterminal deletion derivatives were amplified by PCR from X. campestris pv. vesicatoria strain 85-10 and cloned into the EcoRI and HindIII sites of pDSK602. To create c-Myc epitope-tagged derivatives of HrpB2, hrpB2 and truncated gene fragments were subcloned into the EcoRI/SacI sites of pC3003, in frame with a triple-c-myc epitope-encoding sequence, and the resulting inserts were introduced into the EcoRI/HindIII sites of pDSK602. For the generation of expression constructs encoding HrpB2 D10-25 and HrpB2 D10-25 -c-Myc, full-length hrpB2 cloned into pUC119 or pC3003 was used as template for a PCR. PCR products were religated and the respective inserts were cloned into pDSK602.

Generation of hrcU expression constructs
For the generation of GST fusion proteins, full-length hrcU and a fragment encoding amino acids 255 to 357, respectively, were amplified by PCR and cloned into the EcoRI/XhoI sites of pGEX-2TKM, respectively, downstream and in frame with the GSTencoding sequence. To construct a C-terminally c-Myc epitopetagged HrcU derivative, hrcU was amplified by PCR, inserted into pENTR/D-TOPO and recombined into pDGW4M using Gateway technology (Invitrogen, Carlsbad, Calif.). pDGW4M is a Gatewaycompatible derivative of pDSK602 containing attR sites, chloramphenicol resistance and ccdB genes and the 46 c-Myc-encoding sequence of vector pGWB16 inserted into the EcoRI/HindIII sites.
To generate a GST-HrcU-c-Myc expression construct, hrcU was amplified by PCR, subcloned by SacI and partial EcoRI digest in pC3003, which contains a triple c-myc-encoding sequence and introduced into the EcoRI/SacI sites of pGEX-6P-1, in frame with a gst-encoding sequence.

Secretion experiments and protein analysis
In vitro secretion assays were performed as described [27]. Total cell extracts and culture supernatants were analyzed by SDS-PAGE and immunoblotting. We used polyclonal antibodies specific for HrpF [27], XopA [29], AvrBs3 [63] and HrpB2 [25], respectively, and monoclonal anti-c-Myc and anti-GST antibodies (Amersham Pharmacia Biotech, Freiburg, Germany). Horseradish peroxidase-labelled anti-rabbit, anti-mouse and antigoat antibodies (Amersham Pharmacia Biotech) were used as secondary antibodies. Antibody reactions were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). To ensure that no bacterial lysis had occurred, blots were routinely reacted with an antibody specific for the intracellular protein HrcN (data not shown) [25].

GST pull-down assays
GST pull-down assays were performed as described previously [31]. Briefly, GST and GST fusion proteins were expressed in E. coli and bacterial cells from 50 ml cultures were broken with a French press. GST and GST fusions were immobilized on glutathione sepharose and incubated with a c-Myc epitope-tagged derivative of the putative interaction partner. Bound proteins were eluted with 10 mM reduced glutathione. 5 ml total protein lysates  [67] pGWB16 binary expression vector, contains attR1-CmR-ccdB-attR2 upstream of 46c-myc epitope-encoding sequence [68] pGEX-2TKM GST expression vector; p tac GST lacI q pBR322 Stratagene ori; Ap r , derivative of pGEX-2TK with polylinker of pDSK604 [69] pGEX-6P-1 GST expression vector Stratagene pOK1 suicide vector; sacB sacQ mobRK2 oriR6K; Sm r [28] pOK-hrpED9-93 pOK1 derivative containing hrpE with a stop codon insertion after codon 8 [26] pRK2013 and 20 ml eluted proteins were analyzed by SDS-PAGE and immunoblotting. For the generation of GST-HrpE, hrpE was amplified by PCR and cloned into the EcoRI/XhoI sites of pGEX-2TKM. The same blot was always incubated with an anti-c-Myc and an anti-GST antibody, respectively. Figure S1 The C terminus of HpaC contains a predicted T3S4 domain. Sequence alignment of HpaC from X. campestris pv. vesicatoria strain 85-10 (accession number CAJ22055) and HpaP from R. solanacearum (accession number CAB58249). Amino acid sequences were aligned using CLUSTAL W (http://www.ebi.ac. uk/clustalw/). Conserved amino acids are shaded black, similar amino acids are shaded grey. The black bar indicates the predicted T3S4 domain in HpaP, stars refer to amino acids that are conserved among HpaP and T3S4 domain-containing proteins [15].