Bacterial strains and plasmids used in this study are listed in Supplementary Table S1. Escherichia coli strains were cultivated at 37°C in lysogeny broth (LB) medium, X. campestris pv. vesicatoria strains at 30°C in nutrient-yeast extract-glycerol (NYG) medium or minimal medium A (MA, pH 7.0) supplemented with sucrose (10mM) and casamino acids (0.3%; Daniels et al., 1984; Ausubel et al., 1996). Plasmids were introduced into E. coli by electroporation and into X. campestris pv. vesicatoria by electroporation or conjugation. Antibiotics were added to the media at the following final concentrations: ampicillin, 100μg/ml; kanamycin, 25μg/ml; rifampicin, 100μg/ml; spectinomycin, 100μg/ml, gentamycin, 15μg/ml; streptomycin, 25μg/ml; and nalidixic acid, 15μg/ml.

For infection studies, X. campestris pv. vesicatoria bacteria were resuspended in 1mM MgCl₂ and infiltrated at densities of 1×10⁸ colony-forming units (CFU) ml⁻¹ into leaves of the near-isogenic pepper cultivars Early Cal Wonder (ECW), ECW-10R and ECW-30R using a needle-less syringe (Minsavage et al., 1990; Kousik and Ritchie, 1998). Infected plants were incubated in growth chambers for 16h of light at 28°C and 8h of darkness at 22°C. The HR was documented 1–2 dpi (days post inoculation) after destaining of the leaves in 70% ethanol. Disease symptoms were photographed 1–9 dpi. The results of infection experiments were reproduced at least two times with different transformants.

For the generation of hrcQ expression constructs with mutations in possible start codons, construct pB-PStophrcQ containing hrcQ downstream of the native promoter was used as a template for PCR reactions with primers amplifying the whole plasmid and annealing to each other. Mutations of putative start codons from GTG (V1) or TTG to GCG encoding alanine were introduced by the primer sequences. The PCR amplicons were transferred into E. coli after a DpnI digest, and constructs with single mutations were used as templates to introduce additional mutations.

To generate an expression construct encoding HrcQ-sfGFP under control of the native promoter, the annotated hrcQ coding sequence and 299bp upstream region were amplified from X. campestris pv. vesicatoria strain 85–10 by PCR and subcloned into pICH41021 as blunt-end fragment using SmaI and ligase. Subsequent ligation with sfgfp (construct pEX-A-sfgfp) into the BsaI sites of pBRM-P by Golden Gate cloning (Engler et al., 2008) resulted in construct pB-PhrcQ-sfGFP. The mutation in codon 203 was introduced by PCR using primers which annealed back-to-back to pB-PhrcQ-sfGFP and contained BpiI sites for Golden Gate-based religation of the PCR product. The M211A mutation was inserted using complementary primers annealing to pB-PhrcQ-sfGFP, and the PCR amplicon was transferred into E. coli after DpnI digest. Using a similar strategy, mutations leading to the M203A and M211A amino acid exchanges were introduced into pB-PhrcQ which encodes HrcQ-c-Myc under control of the native promoter.

For the generation of expression constructs containing hrcQ_M211A-c-myc downstream of the lac promoter, hrcQ_M211A was amplified by PCR using pB-PhrcQ_M211A as a template. The PCR product was subcloned into pICH41021 as blunt-end fragment after SmaI digest and ligation. hrcQ_M211A was ligated into the Golden Gate-compatible pBRM and into the BsaI sites of the bacterial adenylate cyclase-based two-hybrid (BACTH) vectors pUT18_GG, pUT18C_GG, pKT25_GG, and pKNT25_GG.

For in cis expression of hrcQ_M211A in X. campestris pv. vesicatoria, the gene was amplified by PCR using pB-PhrcQ_M211A as template and assembled with a module containing the native hrcQ promoter in pLAND-P, in frame with a C-terminal 3×c-Myc epitope-encoding sequence. To insert hrcQ_M211A-c-myc into the genome of X. campestris pv. vesicatoria, pLAND-PhrcQ_M211A was conjugated into strains 85–10∆hrcQ and 85*∆hrcQ. Double homologous recombination events led to the insertion of hrcQ_M211A-c-myc into the hpaFG region and were selected as described previously (Huguet et al., 1998).

To generate hrcQ_C expression constructs, the corresponding region of hrcQ without stop codon was amplified by PCR, subcloned as blunt-end fragment into pICH41021, and ligated into the BsaI sites of pBRM and the Golden Gate-compatible BACTH vectors. Additionally, hrcQ_C was ligated with a PCR amplicon corresponding to the native hrcQ promoter into the BsaI sites of pBRM-P. To generate a glutathione S-transferase (GST)-HrcQ_C expression construct, two modules corresponding to hrcQ_C and ptac-gst (encodes GST under control of the ptac promoter) were inserted into the BsaI sites of pBRM-P-stop, resulting in pB-P-ptacGST-hrcQ_C.

Modular T3S gene cluster constructs were generated as described in the Supplementary Material and are summarized in Supplementary Figure S6 and Supplementary Table S1 (Hausner et al., 2019).

Proteins were analyzed by SDS-PAGE and immunoblotting, using antibodies directed against the c-Myc and FLAG epitope, GST, HrcJ, HrcQ, AvrBs3, and HrpB1, respectively (Knoop et al., 1991; Rossier et al., 2000). Horseradish peroxidase-labeled anti-mouse, anti-rabbit, or anti-goat antibodies were used as secondary antibodies. Binding of antibodies was visualized by enhanced chemiluminescence.

In vitro T3S assays were performed as described (Rossier et al., 2000). Briefly, bacteria were grown overnight in MA medium (pH 7.0) supplemented with sucrose (10mM) and casamino acids (0.3%) and resuspended in MA medium (pH 5.3) containing 50μg/ml BSA (bovine serum albumin) and 10μM thiamine at an OD_600nm of 0.15. The cultures were incubated on a rotary shaker overnight at 30°C, and the bacterial cells and secreted proteins were separated by filtration using low protein binding filters. Proteins in the culture supernatant were precipitated by the addition of trichloroacetic acid and resuspended in 20μl of Laemmli buffer. Total cell extracts and culture supernatants were analyzed by SDS-PAGE and immunoblotting.

BACTH assays were performed using the EUROMEDEX BACTH system kit. Expression constructs were transformed into JM109 E. coli cells to analyze protein synthesis. For this, bacterial cultures were induced with IPTG (isopropyl-β-D-thiogalactopyranoside; 2mM final concentration) at an OD₆₀₀ of 0.6–0.8 and incubated on a rotary shaker for 2h at 37°C. Bacterial cells were collected by centrifugation, resuspended in Laemmli buffer, and analyzed by immunoblotting, using a FLAG epitope-specific antibody.

For protein-protein interaction studies, expression constructs encoding T18 and T25 fusion proteins were cotransformed into chemically competent DHM1 or BTH101 E. coli strains, and transformants were plated on LB plates containing kanamycin and gentamicin. Four colonies per transformation were used to inoculate LB overnight cultures with appropriate antibiotics, which were incubated overnight at 30°C on a rotary shaker. Two μl of the overnight cultures was spotted on selective LB plates containing X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; 40μg/ml) and 2mM IPTG. The plates were incubated at 22°C, and the color of the colonies was monitored over a period of three to 5days. The experiments were performed at least three times with four different transformants from independent cotransformations.

For GST pull-down assays, expression constructs encoding GST, GST-HrcQ_C, HrcQ-c-Myc, and HrcQ_M211A-c-Myc interaction partners were introduced into E. coli BL21(DE3) cells and grown in LB medium until OD₆₀₀ 0.6–0.8. Gene expression was induced by 2mM IPTG (final concentration). After 2h of incubation at 37°C, bacterial cells were harvested by centrifugation, resuspended in PBS (phosphate-buffered saline), and lysed using a French press. The lysates were centrifuged to remove cell debris, and soluble GST and GST-HrcQ_C proteins were immobilized on a glutathione sepharose matrix according to the manufacturer’s instructions (GE Healthcare). The matrix with immobilized GST and GST-HrcQ_C proteins was washed and incubated with bacterial lysates containing HrcQ-c-Myc or HrcQ_M211A-c-Myc for 2h at 4°C on an overhead shaker. After washing of the matrix, bound proteins were eluted with Laemmli buffer. Cell lysates and eluted proteins were analyzed by SDS-PAGE and immunoblotting, using c-Myc epitope- and GST-specific antibodies.

For fluorescence microscopy studies, X. campestris pv. vesicatoria strains were grown overnight in MA medium (pH 7.0) supplemented with sucrose (10mM) and casamino acids (0.3%). Cells were resuspended in MA medium (pH 5.3) supplemented with BSA and thiamine as described above at an OD_600nm of 0.15 and incubated on a tube rotator at 30°C for 1h. Bacteria were transferred onto a microscopy slide on top of a pad of 1% agarose dissolved in MA medium (pH 5.3) as described (Hausner et al., 2019). Fluorescence was inspected with a confocal laser scanning microscope (Leica STELLARIS 8) with a 60× magnification objective and 5× digital magnification. Specific filter sets were used for sfGFP (excitation at 485nm; emission at 510nm) and mKOκ (excitation at 551nm; emission at 563nm). Fluorescent foci were counted in approximately 300 cells of three transconjugants for each strain.

The predicted C ring component HrcQ is encoded by the first gene of the hrpD operon in the hrp gene cluster from X. campestris pv. vesicatoria and is conserved in Xanthomonas spp. (73–93% amino acid identity; Figure 1; Supplementary Figures S1, S2). The C-terminal region of HrcQ also shares 27–34% amino acid identity with corresponding regions of HrcQ proteins from plant pathogens and SctQ proteins from the animal pathogens Yersinia spp. and Salmonella spp. In contrast, no significant homology was detected between HrcQ and the SctQ proteins EscQ from E. coli and Spa33 from S. flexneri, suggesting that these proteins are not conserved in every species (Figure 1; Table 1).

Several sctQ genes from animal-pathogenic bacteria contain internal translation initiation sites which lead to the separate synthesis of the C-terminal protein regions (SctQ_C; Yu et al., 2011; Bzymek et al., 2012; McDowell et al., 2016; Lara-Tejero et al., 2019). In hrcQ from X. campestris pv. vesicatoria, there are two ATG codons at positions 203 and 211 which could serve as additional internal translation start sites for the separate synthesis of the C-terminal region of HrcQ (HrcQ_C) and are conserved in hrcQ genes from Xanthomonas spp. (Supplementary Figure S1). A potential Shine Dalgarno sequence (GAGG) is present 11 nucleotides upstream of codon 211 (Figure 1). To investigate a possible internal translation initiation in hrcQ transcripts, we generated expression constructs encoding HrcQ derivatives with M203A or M211A mutations (mutation of the ATG to GCG) under control of the native promoter and in fusion with a C-terminal 3×c-Myc epitope. For complementation studies, expression constructs were transferred to X. campestris pv. vesicatoria hrcQ deletion mutants, and transformants were infiltrated into leaves of susceptible and resistant pepper plants. When analyzed in X. campestris pv. vesicatoria strain 85–10∆hrcQ, HrcQ_M203A-c-Myc but not HrcQ_M211A-c-Myc restored the wild-type phenotype with respect to disease symptoms in susceptible and the HR induction in resistant plants (Figure 2A). Both HrcQ derivatives complemented the mutant phenotype of strain 85–10hrpG*∆hrcQ (85*∆hrcQ), which contains HrpG*, a constitutively active version of the key regulator HrpG (Figure 2A). HrpG* leads to constitutive expression of T3S genes and accelerated plant reactions (Wengelnik et al., 1999). We, therefore, assume that the negative effect of the M211A mutation was compensated by overexpression of the T3S genes in the presence of HrpG*.

Immunoblot analysis of bacterial cell extracts containing HrcQ-c-Myc or HrcQ_M203A-c-Myc led to the detection of HrcQ and a derivative thereof at a size of approximately 20kDa corresponding to the size of the predicted internal translation product (Figure 2A). In contrast, significantly reduced amounts of the full-length protein and no internal translation product were detectable in bacterial cell extracts containing HrcQ_M211A-c-Myc (Figure 2A), suggesting that the ATG codon at position 211 serves as additional internal translation start site and also contributes to the stability of HrcQ.

Given the low levels of HrcQ_M211A-c-Myc, we also analyzed HrcQ-sfGFP fusions containing M203A or M211A mutations. We previously showed that HrcQ-sfGFP complements the hrcQ mutant phenotype and forms fluorescent foci in bacterial cells in the presence of a functional T3S system (Hausner et al., 2019). Plant infection studies showed that HrcQ-sfGFP and HrcQ_M203A-sfGFP complemented the mutant phenotypes of strain 85–10∆hrcQ with respect to HR induction and disease symptoms, whereas no complementation was observed for HrcQ_M211A-sfGFP (Figure 2B). When analyzed in strain 85*∆hrcQ, however, HrcQ_M211A-sfGFP restored pathogenicity and HR induction in susceptible and resistant plants, respectively, suggesting that the negative effect of the M211A mutation was compensated by the overexpression of the T3S genes as was observed for HrcQ-c-Myc derivatives (see above; Figures 2A,B). Immunoblot analyses led to the detection of HrcQ-sfGFP and truncated derivatives thereof at sizes of approximately 30 and 40kDa, which likely correspond to cleaved sfGFP and the predicted additional translation product of HrcQ-sfGFP, respectively (Figure 2B). The M211A but not the M203A mutation abolished the detection of the truncated 40kDa HrcQ-sfGFP derivative (Figure 2B). Notably, the M211A mutation did not significantly affect the levels of full-length HrcQ-sfGFP, suggesting that the sfGFP fusion partner stabilizes HrcQ in the presence of the M211A mutation when expressed in trans (Figure 2B). Taken together, we conclude that the ATG codon at position 211 of hrcQ contributes to protein function and serves as an additional translation start site of hrcQ. This is supported by the presence of a potential Shine Dalgarno sequence upstream of codon 211 which is conserved in hrcQ genes from Xanthomonas spp. (Figure 1; Supplementary Figure S1).

Next, we investigated a possible effect of the copy number on HrcQ_M211A function. For this, we inserted hrcQ_M211A-c-myc including the native hrcQ promoter into the hpaFG region of strains 85–10∆hrcQ and 85*∆hrcQ. The hpaFG region is located adjacent to the hrp gene cluster and serves as a landing platform for gene insertion (Tamir-Ariel et al., 2007; Lorenz et al., 2012). We previously showed that the genomic insertion of hrcQ-c-myc under control of the native promoter restores pathogenicity in hrcQ deletion mutants (Lorenz et al., 2012; Figure 3A). In contrast, expression of hrcQ_M211A-c-myc in cis did not complement the phenotypes of strains 85–10∆hrcQ and 85*∆hrcQ, suggesting that the ATG codon at position 211 is essential for pathogenicity when hrcQ is present as a single copy in the chromosome (Figure 3A).

To analyze whether pathogenicity of genomic hrcQ_M211A mutants could be restored by ectopic expression of hrcQ_c in trans, we introduced expression constructs containing hrcQ_c-c-myc under control of the native or the lac promoter into strains 85–10hrcQ::hrcQ_M211A and 85*∆hrcQ::hrcQ_M211A. Immunoblot analysis of bacterial cell extracts showed that HrcQ_C-c-Myc was stably synthesized (Figure 3B; Supplementary Figure S3; see also below). When bacteria were infiltrated into leaves of susceptible and AvrBs1-responsive resistant pepper plants, ectopic expression of hrcQ_C-c-myc under control of the native promoter restored pathogenicity, whereas expression of hrcQ_C-c-myc under control of the lac promoter only partially complemented the mutant phenotype of strain 85*∆hrcQ::hrcQ_M211A but not of strain 85–10∆hrcQ::hrcQ_M211A (Figure 3B; Supplementary Figure S3). Ectopic expression of hrcQ_C under control of the lac or the native promoter in strain 85*∆hrcQ::hrcQ_M211A, however, restored HR induction in ECW-30R pepper plants which recognize the effector protein AvrBs3 (Römer et al., 2009; Figure 3B). For these experiments, an avrBs3 expression construct was additionally introduced into strain 85*∆hrcQ::hrcQ_M211A and derivatives thereof. When analyzed in the wild-type strain 85–10, ectopic expression of hrcQ_C-c-myc under control of the lac promoter interfered with pathogenicity, suggesting that it exerts a dominant-negative effect (Figure 3C). No complementation by HrcQ_C was observed in the hrcQ deletion mutant 85*∆hrcQ, suggesting that the N-terminal region of HrcQ is required for protein function (Supplementary Figure S3).

To investigate the effect of the internal translation start site in hrcQ on in vitro T3S, bacteria were grown in secretion medium. Total cell extracts and culture supernatants were analyzed by immunoblotting using antibodies specific for the effector protein AvrBs3 and the IM ring component HrcJ. As reported previously, deletion of hrcQ abolished the detectable secretion of AvrBs3 (Figure 3B; Rossier et al., 2000). T3S was restored by expression of hrcQ-c-myc in cis, albeit not to wild-type levels (Figure 3B). No complementation was observed upon in cis expression of hrcQ_M211A-c-myc, whereas expression of hrcQ_C in trans under control of the lac or the native promoter partially restored T3S in strain 85*∆hrcQ::hrcQ_M211A (Figure 3B). Taken together, these results suggest that HrcQ_C can act in trans and that the loss of pathogenicity of genomic hrcQ_M211A mutants is, therefore, likely caused by the lack of hrcQ_C expression and not by a negative effect of the M211A mutation per se on HrcQ stability or folding.

Analysis of the additional HrcQ translation product revealed that HrcQ_C-c-Myc migrated at a slightly higher molecular weight when encoded under control of the native instead of the lac promoter (Figure 3B; Supplementary Figure S3). This suggests the presence of a translation start site upstream of the annotated start codon of hrcQ. Inspection of the hrcQ promoter sequence revealed two TTG codons, 13 and 30 codons, respectively, upstream of the annotated GTG start codon, which are also present in the upstream regions of annotated hrcQ genes from other Xanthomonas spp. (Figure 1; Supplementary Figure S1). TTG codons can serve as alternative translation start sites and presumably lead to reduced gene expression (Belinky et al., 2017). Given that the translation start site of hrcQ has not yet been experimentally confirmed, we investigated the importance of the annotated start codon and, therefore, mutated the GTG codon to GCG in an expression construct encoding HrcQ under control of the native promoter. Complementation studies showed that the resulting HrcQ_V1A protein complemented the phenotype of strain 85*∆hrcQ with respect to disease symptoms and the HR in susceptible and resistant pepper plants (Figure 4).

To investigate a possible translation initiation upstream of the annotated start codon, we generated additional expression constructs with mutations in the TTG codons upstream of the annotated start of hrcQ (hereafter referred to as codon positions +13 and +30; Figure 1) leading to L-to-A (mutation of TTG to GCG) amino acid exchanges. All HrcQ derivatives containing single, double, or triple mutations complemented the mutant phenotype of strain 85*∆hrcQ (Figure 4). Complementation of the hrcQ mutant phenotype by HrcQ_{V1A/L+13A/L+30A} was confirmed in strain 85–10∆hrcQ (Supplementary Figure S4). Immunoblot analysis of bacterial protein extracts revealed that L+13A mutations led to reduced HrcQ levels irrespective of the presence of the annotated start codon (Figure 4). L+30A mutations further decreased HrcQ levels, whereas mutation of all three putative start codons abolished the detection of the corresponding HrcQ derivative (Figure 4). The presence of a putative Shine Dalgarno sequence eight nucleotides upstream the TTG codon at position +30, which is also conserved in the upstream regions of annotated hrcQ genes from other Xanthomonas spp. (Supplementary Figure S1) and the fact that the L+30A mutation had the most severe effect on HrcQ protein levels strongly suggests the contribution of this codon to translation initiation of hrcQ. Given the presence of a stop codon 72 nucleotides upstream (see Figure 1), mutation of all three potential start codons in a hrcQ expression construct presumably led to translation initiation downstream of the GTG codon, thus resulting in a low-level synthesis of an N-terminally truncated but functional HrcQ derivative which is not detectable by immunoblot analysis (Figure 4). An ATG codon 105 nucleotides downstream of the annotated GTG codon might serve as translation start in the construct encoding HrcQ_{V1A/L+13A/L+30A} (Figure 1). Taken together, we conclude that translation of the native hrcQ gene is initiated at the TTG codon located 30 codons upstream of the annotated GTG start site. Notably, the annotated start site of hrcQ from X. campestris pv. campestris is located immediately upstream of the TTG codon which is conserved in hrcQ and upstream sequences of Xanthomonas spp. (Supplementary Figure S1).

To investigate whether HrcQ and HrcQ_C interact with each other as reported for SctQ and SctQ_C proteins from animal-pathogenic bacteria (Diepold, 2020), we used a BACTH assay, which depends on the reconstitution of the catalytic domain of the adenylate cyclase (Cya) from two subdomains (T18 and T25). The interaction of T18 and T25 fusion proteins leads to the assembly of functional Cya and thus to the synthesis of cAMP, which activates the expression of the lac operon in E. coli reporter strains lacking the native cya gene (Karimova et al., 1998; Battesti and Bouveret, 2012).

For BACTH assays, we generated expression constructs encoding HrcQ, HrcQ_C, and HrcQ_M211A as N- or C-terminal fusion partners of the T18 and T25 domains. Immunoblot analysis of bacterial cell extracts revealed that all HrcQ fusion proteins were stably synthesized (Supplementary Figure S5). Protein-protein interactions were analyzed in the E. coli reporter strain DHM1, and bacteria were grown on indicator plates containing X-Gal and IPTG. We detected an interaction of HrcQ with itself and with HrcQ_C in all possible combinations (Figure 5A). Given that the self-interaction of the full-length protein was detected with N-terminal T18 and T25 fusion partners, it likely did not result from the interaction of two HrcQ_C derivatives. When compared with the HrcQ self-interaction, the interaction of HrcQ with HrcQ_M211A and the self-interaction of HrcQ_M211A appeared to be reduced for several combinations (Figure 5A). To confirm the results of the BACTH studies, we performed in vitro GST pull-down assays. For this, GST and GST-HrcQ_C were immobilized on a glutathione sepharose matrix and incubated with bacterial lysates containing C-terminally 3×c-Myc epitope-tagged HrcQ and HrcQ_M211A, respectively. HrcQ-c-Myc and HrcQ_M211A-c-Myc coeluted with GST-HrcQ_C but not with GST, which confirms the interaction between the full-length HrcQ protein and HrcQ_C (Figure 5B).

We also investigated whether HrcQ_C interacts with the IM ring component HrcD and the cytoplasmic HrpB4 protein. We previously identified HrcD and HrpB4 as interaction partners of HrcQ and reported that HrpB4 likely acts similarly to SctK proteins from animal pathogens as a linker between HrcQ and the cytoplasmic domain of HrcD (Otten and Büttner, 2021). In the present study, the results of BACTH assays showed that HrcQ_C interacts with both HrcD and HrpB4 when analyzed as T18 or T25 fusion, suggesting a possible contribution of HrcQ_C to the docking of HrcQ complexes to the IM ring (Figures 6A,B).

Next, we analyzed the contribution of the internal translation start site on complex formation by a HrcQ-sfGFP fusion protein. Previous fluorescence microscopy studies showed that a HrcQ-sfGFP reporter fusion assembles into complexes (Hausner et al., 2019). For these experiments, we used a modular expression construct containing the hrp gene cluster, regulatory (hrpG* and hrpX) and accessory (hpaH and xopA) genes (Hausner et al., 2019). The accessory genes encoding HpaH and XopA contribute to the assembly of the T3S system in the periplasm and to efficient effector translocation, respectively (Noël et al., 2002; Hausner et al., 2017). The modular design of the construct, which is referred to as hrp_HAGX and was assembled by Golden Gate cloning from single gene and promoter modules, allows the insertion of reporter genes and the deletion of single or multiple genes of the T3S gene cluster (Hausner et al., 2019). hrcQ-sfgfp was inserted at position 6 of a modular T3S gene cluster lacking the native hrcQ gene (Figure 7A; Supplementary Figure S6). The resulting construct was analyzed in the hrp-deficient X. campestris pv. vesicatoria strain 85*∆hrp_fsHAGX, which lacks the T3S gene cluster and functional hrpG, hrpX, xopA, and hpaH genes. As reported previously, the modular construct restored pathogenicity in strain 85*∆hrp_fsHAGX, suggesting that it was functional and that HrcQ-sfGFP complemented the hrcQ mutant phenotype (Hausner et al., 2019; Figure 7B).

To analyze the influence of the internal translation start site in hrcQ-sfgfp, we generated modular T3S gene cluster constructs lacking the native hrcQ gene and containing hrcQ_M211A-sfgfp, hrcQ_C-sfgfp or a combination of hrcQ_M211A-sfgfp and hrcQ_C-mKOκ (mKusabira Orange kappa; Tsutsui et al., 2008). mKOκ is an orange fluorescent protein, used here for colocalization of HrcQ and HrcQ_C. sfgfp and mKOκ fusions were inserted adjacent to the hrp gene clusters at positions 6 and 2′ of the modular constructs (Figure 7A). Plant infection experiments revealed that the hrcQ mutant phenotype was complemented by HrcQ-sfGFP but not by HrcQ_M211A-sfGFP with respect to disease symptoms and the HR (Figure 7B). This is in line with the results obtained for the genomic hrcQ_M211A mutation and confirms the finding that the M211A mutation interferes with HrcQ function (see above). Coexpression of HrcQ_M211A-sfGFP with HrcQ_C-mKOκ restored pathogenicity in the absence of the native hrcQ gene, suggesting that HrcQ_C can act in trans as described above (Figure 7B). Immunoblot analysis revealed that all proteins were stably synthesized (Figure 7C). The M211A mutation abolished the detection of the HrcQ_C-sfGFP derivative and led to reduced levels of the full-length HrcQ-sfGFP protein (Figure 7C). Notably, wild-type protein levels of HrcQ_M211A-sfGFP were restored in the presence of HrcQ_C-mKOκ, suggesting that reduced stability was not caused by the M211A mutation per se (Figure 7C).

For localization studies, X. campestris pv. vesicatoria bacteria were grown under T3S-permissive conditions in minimal medium (pH 5.3) and inspected with a confocal laser scanning microscope. As observed previously, HrcQ-sfGFP formed one to four fluorescent foci per cell (Figure 8A; Hausner et al., 2019; Otten and Büttner, 2021). No foci were detected when the hrpA-hpaB operons had been replaced by dummy modules, confirming that the assembly of HrcQ-sfGFP depends on the T3S system (Hausner et al., 2019). The presence of the M211A mutation reduced foci formation by HrcQ-sfGFP (Figure 8A). HrcQ_C-sfGFP itself did not form foci in the absence of the full-length HrcQ protein (Figure 8A). The analysis of sfGFP and mKOκ fluorescence revealed that HrcQ_M211A-sfGFP and HrcQ_C-mKOκ colocalize, suggesting that HrcQ and HrcQ_C are part of the same protein complex (Figure 8A). We, therefore, assume that HrcQ_C is a component of the predicted sorting platform and promotes complex formation by HrcQ.

We previously showed that efficient foci formation by HrcQ-sfGFP in X. campestris pv. vesicatoria depends on the IM ring component HrcD. Thus, in the absence of HrcD, HrcQ-sfGFP forms one bright fluorescent spot which presumably corresponds to a cytoplasmic HrcQ-containing protein complex (Otten and Büttner, 2021). HrcQ is likely attached via the SctK-like protein HrpB4 to the cytoplasmic N-terminal domain of the IM ring component HrcD (Otten and Büttner, 2021). To confirm this hypothesis, we deleted codons 2–92 of hrcD in the modular T3S gene cluster construct and analyzed the effect of this mutation on bacterial pathogenicity and on the localization of HrcQ-sfGFP, HrcQ_M211A-sfGFP and HrcQ_C-mKOκ.

As expected, deletion of the N-terminal domain of HrcD led to a loss of pathogenicity of strain 85*∆hrp_fsHAGX containing corresponding modular T3S gene cluster expression constructs but did not affect the stability of HrcQ derivatives (Supplementary Figure S7). Fluorescence microscopy studies showed that HrcQ-sfGFP forms one bright fluorescent spot in hrcD deletion and hrcD_∆2–92 mutant strains, suggesting that the cytoplasmic domain of HrcD is required for efficient foci formation by HrcQ-sfGFP (Figure 8B). To investigate whether HrcQ_C is part of the cytoplasmic protein complex, we performed colocalization studies as described above with HrcQ_M211A-sfGFP and HrcQ_C-mKOκ. Both proteins were stably synthesized and colocalized in the absence of the cytoplasmic domain of HrcD in one fluorescent spot per cell (Figure 8B; Supplementary Figure S7). This is in agreement with the hypothesis that HrcQ_C associates with HrcQ-containing protein complexes and shows that the colocalization of HrcQ and HrcQ_C does not depend on the docking of the HrcQ complex to the cytoplasmic domain of HrcD.