The predicted sorting platform dynamically associates with the type III secretion system from Xanthomonas euvesicatoria in response to the external pH

Otten, Christian; Büttner, Daniela

doi:10.1186/s12866-025-04227-6

Research
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Published: 25 August 2025

The predicted sorting platform dynamically associates with the type III secretion system from Xanthomonas euvesicatoria in response to the external pH

BMC Microbiology volume 25, Article number: 545 (2025) Cite this article

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Abstract

The Gram-negative bacterium Xanthomonas euvesicatoria (Xe) causes bacterial spot disease in pepper and tomato plants, and employs a type III secretion (T3S) system to translocate effector proteins into plant cells. The T3S system is a highly complex nanomachine which spans both bacterial membranes and is conserved in different bacterial species. In animal-pathogenic bacteria, structural and functional studies showed that the membrane-spanning secretion apparatus dynamically associates with a cytoplasmic sorting platform for substrate docking. The sorting platform is a wheel-like structure and contains the central ATPase SctN, which is connected via SctL to peripheral pod-like SctQ complexes. The architecture and function of a similar substructure of the T3S system in plant-pathogenic bacteria is still unknown. We previously reported that the SctQ homolog HrcQ from Xe forms complexes with its alternative translation initiation product HrcQ_C, which stabilizes HrcQ and contributes to T3S. In the present study, we analysed the protein-protein interaction network of predicted sorting platform components from Xe and performed localization studies with HrcQ and the ATPase HrcN by fluorescence microscopy. We show that HrcQ complex formation depends on the SctL homolog HrcL which interacts with HrcQ and HrcN via its N- and C-terminal regions, respectively. Both HrcQ and HrcL contribute to HrcN stability, suggesting that a preassembled HrcQ-HrcL complex associates with and stabilizes the ATPase. Colocalization studies revealed that the HrcN-HrcL-HrcQ complex preferentially assembles at the membranes under T3S-permissive conditions when bacteria are cultivated at a low external pH. In contrast, at pH 7.0, HrcN and HrcQ form large cytoplasmic aggregates. Our data indicate that the interaction of the predicted sorting platform with the T3S system is influenced by external pH levels. These dynamic protein-protein interactions likely control the initiation of effector delivery in response to environmental stimuli, a strategy that might be also used by other plant pathogens.

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Background

Many Gram-negative bacterial pathogens translocate effector proteins into eukaryotic host cells and thus manipulate cellular pathways such as defence responses to promote bacterial survival and proliferation [1,2,3]. Effector protein delivery often depends on a type III secretion (T3S) system which is present in many plant- and animal-pathogenic bacteria and is evolutionarily related to the bacterial flagellum [4, 5]. T3S systems span both bacterial membranes and are associated with an extracellular pilus-like appendage which serves as a transport channel for type III effectors (T3Es) to the host-pathogen interface [6]. Translocation of T3Es into target cells is mediated by a bacterial translocon which inserts into the eukaryotic plasma membrane [7]. At least nine components of T3S systems are conserved in plant- and animal-pathogenic bacteria, suggesting a similar architecture of the secretion apparatus in different bacterial species [8, 9]. Conserved core components of T3S systems from animal pathogens are designated Sct (secretion and cellular translocation) followed by a letter referring to the nomenclature of T3S system components from Yersinia spp [9,10,11]. Cryo-electron microscopy (cryo-EM) and in situ cryo-electron tomography studies of single core components and isolated T3S systems revealed that Sct proteins are involved in the formation of oligomeric rings in the inner membrane (IM) and outer membrane (OM) [12, 13]. The OM ring is assembled by the secretin protein SctC, which is connected to 24mer oligomeric IM rings consisting of SctD on the outer and SctJ on the inner side [13]. The IM rings surround the export apparatus which is essential for protein transport across the IM. The export apparatus consists of a periplasmic SctR₅-SctS₄-SctT₁ complex which associates with the transmembrane protein SctU and is embedded in a nonameric ring of SctV proteins [6, 14,15,16,17,18,19]. SctU and SctV span the IM and contain large cytoplasmic domains which are involved in substrate recruitment [13].

Additional substrate binding sites are provided by the cytoplasmic sorting platform which is a wheel-like protein complex and likely involved in the establishment of a T3S hierarchy [20]. Given its dynamic nature, the sorting platform from animal pathogens did not copurify with the T3S system and was, therefore, visualized by in situ cryo-electron tomography and superresolution microscopy [21,22,23,24,25,26,27]. Situated in the centre of the sorting platform is the hexameric ATPase SctN which is connected via a stalk-like structure consisting of SctO to the cytoplasmic domains of SctV proteins. Furthermore, SctN is linked via spoke-like structures formed by homodimers of SctL to six peripheral pods assembled by tetramers of SctQ proteins [13, 20, 28]. SctQ proteins were also designated C ring proteins due to their homology to FliM and FliN from the bacterial flagellum which form a ring-like cytoplasmic structure [29]. The C-terminal region of SctQ proteins corresponds to FliN and is often translated as separate protein (SctQ_C) from an internal alternative translation start site [30,31,32,33,34]. SctQ_C proteins interact with and thus stabilize SctQ and were identified as essential structural components of the sorting platform in Yersinia spp. and Shigella flexneri [30,31,32,33,34,35].

The sorting platform attaches to the IM ring of the T3S system via SctK which links each of the six SctQ pods to the cytoplasmic domains of the 24mer IM ring component SctD [25,26,27,28, 36, 37]. In Salmonella spp., docking of the sorting platform leads to a conformational rearrangement of the cytoplasmic domain of SctD into six discrete patches [25]. Imaging in live bacteria by fluorescence microscopy revealed that the attachment of the sorting platform to SctD is a dynamic process, which is likely controlled by environmental conditions. Components of the sorting platform presumably preassemble in the cytoplasm and associate with SctD to activate T3S [20, 27, 34, 35, 38, 39]. In plant-pathogenic bacteria, however, the presence and function of a similar structure of the T3S system is still unknown.

In the present study, we analysed predicted sorting platform components from the plant-pathogenic bacterium Xanthomonas euvesicatoria (Xe, formerly known as Xanthomonas campestris pv. vesicatoria), which is the causal agent of bacterial spot disease in pepper and tomato and a model organism for the analysis of bacterial virulence strategies in plants [40, 41]. The T3S system from Xe translocates at least 36 T3Es into plant cells where they interfere with various cellular processes to promote bacterial invasion and multiplication in the plant apoplast [3, 42, 43]. In resistant plants, which contain corresponding resistance genes, individual T3Es are recognized by the plant immune system and often trigger the hypersensitive response (HR), a local defence response at the infection site [44, 45]. Given the essential contribution of T3S genes to T3E delivery and thus to the interaction with susceptible and resistant plants, they were designated hrp (hypersensitive response and pathogenicity) [46]. hrp genes from Xe comprise 25 genes organized in eight operons in a chromosomal gene cluster. Two regulators, HrpG and HrpX, are encoded outside the T3S gene cluster and activate hrp gene expression when the bacteria enter the plant or are cultivated in special minimal media [46,47,48]. Eleven hrp gene products are homologous to corresponding T3S system components from plant- and/or animal-pathogenic bacteria and were, therefore, named Hrc for Hrp conserved followed by a letter which refers to the corresponding Sct proteins from animal pathogens [9]. In addition to Hrc proteins, the function and assembly of the T3S system from Xe depends on non-conserved Hrp proteins, which are only present in Xe or related plant pathogens. Several Hrp proteins are themselves secreted and constitute extracellular components of the T3S system such as the pilus protein HrpE and the translocon protein HrpF [49, 50]. Additional Hpa (Hrp associated) proteins encoded in the hrp gene cluster contribute to pathogenicity and include the T3S chaperone HpaB, which is required for efficient T3E delivery [51, 52].

The mechanisms underlying the recognition and docking of T3S substrates to the secretion apparatus are largely unknown. Targeting of substrates to the T3S system often depends on N-terminal export signals which are part of unstructured protein regions and are not conserved on the amino acid level [10, 53,54,55,56]. In Xe, possible T3S substrate docking sites include the ATPase HrcN and the SctQ homolog HrcQ, which are both components of the predicted sorting platform [57, 58]. We previously showed that HrcQ from Xe is stabilized by its C-terminal region, HrcQ_C, which is translated as separate protein from an internal alternative translation initiation codon as was reported for SctQ_C proteins from animal-pathogenic bacteria [30,31,32,33,34, 59, 60]. A complex of HrcQ_C and HrcQ associates with the SctK-like protein HrpB4 which acts as a linker to the cytoplasmic domain of the IM ring component HrcD [37, 59].

HrcQ complex formation was visualized in Xe by fluorescence microscopy analysis of a HrcQ-sfGFP (superfolder green fluorescent protein) fusion protein which forms fluorescent foci at the cell periphery [59, 61]. Genetic approaches showed that foci formation depends on a functional T3S system and on the cytoplasmic domain of the IM ring component HrcD which provides the docking sites for the predicted sorting platform [37, 59]. In the absence of HrcD, HrcQ-sfGFP forms large fluorescent complexes in the bacterial cytoplasm [59]. The formation of cytoplasmic but not of membrane-associated HrcQ complexes was independent of HrpB4, suggesting that HrpB4 is specifically required for the attachment of HrcQ to the T3S system [37]. It is, however, still unknown whether HrcQ is part of the predicted T3S sorting platform and associates with the ATPase complex in the bacterial cytoplasm. In the present study, we, therefore, examined potential interactions of HrcQ with the ATPase HrcN and its predicted regulator HrcL and evaluated their roles in HrcQ complex formation using fluorescence microscopy. Our experimental findings and the results of structural modeling indicate that the N- and C-terminal regions of HrcL link the HrcQ/HrcQ_C complexes to the ATPase HrcN. Furthermore, our data suggest that a preassembled HrcQ-HrcL complex is required for the recruitment and stabilization of HrcN. Notably, the predicted sorting platform specifically associates with the T3S system under T3S-permissive conditions. We, therefore, propose that the HrcN-HrcL-HrcQ complex dynamically interacts with the T3S system in response to environmental conditions to regulate T3S in Xe.

Methods

Bacterial strains and growth conditions

Plasmids were introduced into Escherichia coli and Xe using either electroporation or triparental conjugation and pRK2013 as a helper plasmid. E. coli strains were cultivated at 37 °C in lysogeny broth (LB) medium and Xe strains at 30 °C in nutrient-yeast extract-glycerol (NYG) medium or in minimal medium A (MA; 12 mM K₂HPO₄, 6.6 mM KH₂PO₄, 1.52 mM (NH₄)₂SO₄, 0.34 mM Na-citrate*2H₂O, 1 mM MgSO₄). MA medium was supplemented with sucrose (10 mM) and casamino acids (0.3%) at pH 5.3 or pH 7.0 [62] and antibiotics were added to the media at the following final concentrations: ampicillin, 100 µg/ml; kanamycin, 25 µg/ml; rifampicin, 100 µg/ml; spectinomycin, 100 µg/ml, gentamycin, 15 µg/ml and streptomycin, 25 µg/ml. All plasmids and bacterial strains, which were used in this study, are summarized in Table S1.

Plant material and plant infections

Bacteria were infiltrated with a needle-free syringe into leaves of the near-isogenic pepper cultivars Early Cal Wonder (ECW) and ECW-10R. If not stated otherwise, bacterial solutions were adjusted in 1 mM MgCl₂ to an optical density at 600 nm of 0.1 (corresponding to 1 × 10⁸ colony-forming units (CFU) ml^-1). After infection, plants were incubated in growth chambers for 16 h of light at 28 °C and 8 hours of darkness at 22 °C. Disease symptoms and the HR were photographed over a period of one to seven days post inoculation (dpi). While disease symptoms including water-soaking and yellowing are best observed in photographs, infected leaves of resistant plants were bleached over night at 65 °C in 70% ethanol to enhance HR visualization. All infection experiments were performed at least three times with consistent results, and one representative image is shown.

Protein-protein interaction studies using the BACTH system

For BACTH (bacterial adenylate cyclase-based two-hybrid) assays, expression constructs encoding T25 and T18 fusion proteins were introduced into chemically competent cells of the E. coli reporter strain DHM1. To select cells with both plasmids, transformants were plated on LB medium containing kanamycin and gentamycin. For each plasmid combination, three transformants were grown in 500 µl of LB medium containing both antibiotics. Cultures were incubated in 96 well plates overnight at 30 °C with shaking. Subsequently, 2 µl of each culture were spotted on LB indicator plates containing kanamycin, gentamycin, X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside; 40 µg/ml) and IPTG (isopropyl-β-D-thiogalactopyranoside, final concentration of 2 mM). The indicator plates were incubated for five to six days at room temperature in the dark before the colonies were photographed. As negative control, every fusion protein was tested against the T25 or the T18 domain alone. Each interaction assay was conducted at least three times with transformants derived from independent transformations. One representative colony is shown.

For protein detection, T18 and T25 expression constructs were transferred into the cya⁺ E. coli strain JM109, in which recombination is minimized (Table S1). JM109 cultures were grown in LB medium and gene expression was induced with IPTG at an optical density at 600 nm (OD₆₀₀) of 0.6–0.8. After two hours of induction at 37 °C, cells from 500 µl of the cultures were collected by centrifugation, resuspended in Laemmli buffer and analysed by immunoblotting, using a FLAG epitope-specific antibody. Since cAMP/CAP is required for optimal activation of the lac promoter which drives the expression of T25 and T18 reporter fusions, protein synthesis is analysed in cya⁺ E. coli strains instead of the cya mutant and reporter strain DHM1, which is used for the BACTH assays.

Immunoblot analyses of Xe cell extracts

To detect HrcQ, HrcN and HrcJ in Xe strains, bacteria were grown overnight at 30 °C in minimal medium A (pH 7.0) without antibiotics on a rotary shaker. Cells were harvested from 500 µl of the cultures by centrifugation and resuspended in 50 µl of Laemmli buffer. Equal amounts of cell extracts adjusted according to the OD₆₀₀ were subjected to SDS-PAGE and analysed by immunoblotting, using antibodies directed against HrcN, HrcQ, the IM ring component HrcJ and the c-Myc epitope (polyclonal antibody from rabbit, Sigma-Aldrich) [63]. Horseradish peroxidase-labelled anti-rabbit antibodies served as secondary antibodies and binding of antibodies was visualized with a chemiluminescence imager (Vilber Fusion FX Edge). HrcN- and HrcQ-specific signals were quantified using the open-source image processing tool Fiji/ImageJ2 [64]. For this, the immunoblot images were transformed via Affinity Photo into “Grayscale” and JPEG files. For each blot, in which the analysed proteins (HrcQ and HrcN) and the loading control (HrcJ) were detected, an ROI (region of interest) frame covering the minimal area of the largest band was defined and the intensity of each signal was measured (the “Set measurements” were set to “Grey Mean Value”). With the same ROI frames, a background measurement was performed by moving the frames into areas without any signals above or below the detected proteins. All data were exported to Excel and the relative values of the signals were calculated. For this, the pixel density was inverted using the formula 255– X, in which X is the value recorded by Fiji. For the determination of the net value of each signal, the inverted background value was deducted. The final relative values were calculated as ratio of the net values of the respective HrcQ and HrcN signal and the HrcJ loading control.

Fluorescence microscopy analyses

For the analysis of fluorescent reporter fusions, Xe strains containing the respective modular T3S gene cluster constructs were grown in MA medium (pH 7.0) supplemented with sucrose (10 mM) and casamino acids (0.3%) without antibiotics. Following overnight cultivation, the cells were resuspended at an OD₆₀₀ of 0.15 in MA medium containing BSA and thiamine, adjusted to pH 5.3 or pH 7.0. The cultures were then incubated at 30 °C on a tube rotator for at least 1 h. For microscopy, bacterial cultures were subsequently spotted onto a slide overlaid with a 1.5% agarose pad prepared with MA medium at pH 5.3 or pH 7.0, following a previously described protocol [61]. Fluorescence analysis was performed using a confocal laser scanning microscope (Leica STELLARIS 8) equipped with a 60 x magnification objective and 5 x digital magnification. Fluorophores were excited using a white light laser at wavelengths specific for each fluorophore (485 nm for sfGFP and 551 nm for mKOκ), and fluorescence signals were captured using HyD S detectors with emission detection windows optimized for the fluorophores (sfGFP at 510 nm and mKOκ at 563 nm). Pictures were taken from approximately 500–1000 cells of each of the three transconjugants for every strain. Foci were counted in a single z-plane. Image analysis across multiple z-planes confirmed that minimal to no foci were lost, ensuring that restricting the analysis to one z-plane would not affect the results (Fig. S14). To enhance or sharpen fine structures and details of sfGFP-HrcN in the colocalization studies, the images were processed using the Leica LIGTHNING detection concept. Fig. S12 exemplifies for each fluorescence image the foci with the weakest fluorescence which were included in the quantification. Foci were manually counted using the Multi-Point-Tool of the ImageJ2 processing package Fiji, thus ensuring accuracy and preventing errors like missed or miscounted foci that could arise from manual note-taking [64]. For the analysis of protein synthesis, bacteria incubated in MA medium as described above were harvested by centrifugation and resuspended in Laemmli buffer. Equal amounts of the cell extracts adjusted according to the OD₆₀₀ were analysed by SDS-PAGE and immunoblotting using GFP- (Roche Applied Science), FLAG- (Sigma-Aldrich), HrpB1- and HrcC-specific antibodies [62, 65].

Generation of expression constructs by Golden Gate cloning

For the generation of sfgfp, hrcL, hrcN and hrcQ expression constructs, the corresponding genes or deletion derivatives thereof were amplified by PCR and respective primers (listed in Table S2), and the amplicons were inserted via blunt-end cloning using SmaI and ligase into vector pICH41021. After Sanger sequencing, individual inserts were cloned downstream of the native hrpB promoter comprising 113 bp upstream of the ATG start codon of hrpB1 into vector pBRM-P or downstream of the lac promoter into vector pBRM or the BACTH vectors pUT18_GG, pUT18C _GG, pKT25_GG and pKNT25 _GG using the Golden Gate cloning technique. For this, cut-ligation reactions containing vector and insert were incubated with BsaI and ligase in a thermocycler for 55 cycles at 37 °C for 2 min and 16 °C for 5 min followed by 10 min incubation at 50 °C and 20 min incubation at 65 °C [66]. E. coli Top10 or JM109 cells were used for subsequent transformation of the reactions and transformants were analysed by test restriction of isolated plasmids.

Generation of modular T3S gene cluster constructs

Modular T3S gene cluster constructs were generated by Golden Gate-based modular cloning (MoClo) as described previously [61, 67]. For this, promoter and ORF modules were assembled using a series of MoClo vectors (designated level − 2, -1, 0, 1, M and P) which allow the stepwise cloning of multigene constructs by alternate use of the type IIs enzymes BsaI and BpiI [67]. The MoClo system was previously used to assemble the T3S gene cluster, the regulatory and accessory genes hrpG*, hrpX, xopA and hpaH as well as dummy modules, which can be replaced by reporter fusions [61].

Mutation of single or multiple T3S genes in the modular T3S gene cluster expression constructs

In the present study, we introduced deletions into single T3S genes by replacing the corresponding gene modules in the modular T3S gene cluster constructs. To generate a deletion in hrcL, the 5’ and 3’ regions of hrcL were amplified by PCR using construct pAGB192 (hrcL) as template and primer pairs hrcL-MoClo-F1/hrcL-Del-MoClo-R1 and hrcL-Del-MoClo-F2/hrcL-MoClo-R2. The resulting amplicons were assembled in vector pAGM9121 using BpiI and ligase, thus generating the level − 2 construct pAGB957 (∆hrcL). The corresponding insert was subsequently assembled with the inserts of constructs pAGB193 (hrcN), pAGB194 (hrpB7), pAGB195 (hrcT) and pAGB196 (hrcC) in vector pAGM1311, using BsaI and ligase, thus leading to the level − 1 construct pAGB961. The inserts of pAGB961 and pAGB197 (hrpB operon promoter + hrpB1 to hrpB4) were then cloned into vector pICH41331 using BpiI and ligase, thus generating the level 0 construct pAGB965 (hrpA and hrpB operon + ∆hrcL). Next, the insert of pAGB965 was transferred into the BsaI sites of pICH47811 to generate the level 1 construct pAGB969. For the assembly of the modular level M T3S gene cluster construct pAGB979 containing the deletion in hrcL, the inserts of pICH54011 (dummy pos. 1), pAGB969 (hpaA to hrpB operons and ∆hrcL fragment), pAGB155 (hrpC to hpaB operons), pAGB156 (hrpF operon) and pICH50900 (end linker 4) were cloned into vector pAGM8031 using BpiI and ligase. For localization studies with HrcQ-sfGFP, an additional deletion in hrcQ was introduced during the assembly by replacing construct pAGB155 with construct pAGB275 (hrpC to hpaB operons + ∆hrcQ), thus resulting in the level M construct pAGB973.

For the introduction of a deletion in hrcN, the 5’ and 3’ regions flanking the deletion were amplified by PCR using phosphorylated primers (hrcN-Del-MoClo-F/hrcN-Del-MoClo-R) and the level − 2 plasmid pAGB193 (hrcN) as template. The amplicon was ligated to generate the level − 2 plasmid pAGB514 (∆hrcN). The corresponding insert was subsequently assembled with the inserts from constructs pAGB192 (hrcL), pAGB194 (hrpB7), pAGB195 (hrcT) and pAGB196 (hrcC) in vector pAGM1311, using BsaI and ligase. The resulting level − 1 construct pAGB674 was ligated together with the insert of pAGB197 (containing the hrpB operon promoter as well as hrpB1 to hrpB4) using BpiI and ligase to generate the level 0 construct pAGB678 (hrpA and hrpB operon including ∆hrcN). The insert of pAGB678 was ligated into the BsaI sites of vector pICH47811 to generate the level 1 construct pAGB681. The final level M construct pAGB737 containing the T3S gene cluster with a deletion in hrcN was generated by assembly of constructs pICH54011 (dummy pos. 1), pAGB681 (hpaA to hrpB operons including ∆hrcN), pAGB155 (hrpC to hpaB operons), pAGB156 (hrpF operon) and pICH50900 (end-linker 4) in vector pAGM8031 using BpiI and ligase. To introduce an additional deletion in hrcQ, which was necessary for the localization studies with HrcQ-sfGFP, the insert of pAGB155 was replaced by the insert of pAGB275 (hrpC to hpaB operons including ∆hrcQ).

To introduce deletions in both hrcN and hrcL, the last 46 bp of hrcN were amplified by PCR using primers hrcNL-Del-MoClo-F and hrcNL-Del-MoClo-R and pAGB193 (hrcN) as template. The resulting amplicon was cloned into vector pAGM9121 using BpiI and ligase to obtain the level − 2 construct pAGB659 (∆hrcN/hrcL). The insert of pAGB659 was assembled with the inserts of constructs pAGB194 (hrpB7), pAGB195 (hrcT) and pAGB196 (hrcC) into vector pAGM1311 using BsaI and ligase, thus resulting in the level − 1 construct pAGB660. Subsequent assembly of pAGB660 with pAGB197 (hrpB operon promoter and hrpB1 to hrpB4) in vector pICH41331 using BpiI and ligase led to the level 0 construct pAGB661. The insert of pAGB661 was transferred to vector pICH47811 using BsaI and ligase to generate the level 1 construct pAGB662. The final level M construct pAGB666 containing deletions in hrcN and hrcL resulted from assembly of constructs pICH54011 (dummy pos. 1), pAGB662 (hpaA to hrpB operons including ∆hrcN/hrcL), pAGB155 (hrpC to hpaB operons), pAGB156 (hrpF operon) and pICH50900 (end-linker 4) with BpiI and ligase in vector pAGM8031. For the additional deletion of hrcQ, construct pAGB155 was replaced by construct pAGB275 (hrpC to hpaB operons including ∆hrcQ).

To mutate both hrcN and hrpB4 in the modular T3S gene cluster, the inserts of constructs pAGB776 (contains the hrpB operon promoter and hrpB1 to hrpB4 with a frameshift mutation in hrpB4) and pAGB674 (contains hrcC and hrcL to hrcT including ∆hrcN) were assembled using BpiI and ligase to generate the level 0 construct pAGB1270 and were subsequently transferred to vector pICH47811 using BsaI and ligase, leading to the level 1 construct pAGB1273. A level M construct (pAGB1275) containing the T3S gene cluster with mutations in hrcN and hrpB4 was generated by assembly of pAGB1275 with pICH54011 (dummy pos. 1), pAGB155 (hrpC to hpaB operons), pAGB156 (hrpF operon) and pICH50900 (end-linker 4) using BpiI and ligase in vector pAGM8031. For the generation of the hrcN, hrcL and hrpB4 triple mutant, the inserts of pAGB776 (hrpB operon promoter and hrpB1 to hrpB4 including fshrpB4) and pAGB660 (hrcC and hrcL to hrcT including ∆hrcN/hrcL) were cloned into vector pICH41331 using BpiI and ligase to obtain the level 0 construct pAGB1201 (hrpA and hrpB operon + ∆hrcN/hrcL/fshrpB4). The insert of pAGB1201 was transferred into vector pICH47811 using BsaI and ligase to generate the level 1 construct pAGB1202 and subsequently assembled with the inserts of pICH54011 (dummy pos. 1), pAGB1202 (hpaA to hrpB operons including ∆hrcN/hrcL/fshrpB4), pAGB155 (hrpC to hpaB operons), pAGB156 (hrpF operon) and pICH50900 (end linker 4) using BpiI and ligase in vector pAGM8031, leading to the level M construct pAGB1394. To introduce an additional deletion in hrcQ, pAGB155 was replaced by pAGB275 (hrpC to hpaB operons including ∆hrcQ) to generate the level M construct pAGB1203. All constructs are listed in Table S1. Sequences with the following GenBank accession numbers were used to generate the expression constructs and gene modules listed in Table S1: hrcC (APO92110), hrpB1 (AAB08456), hrpB2 (AAB08457), hrcJ (NEK90484), hrpB4 (AAB08459), hrcL (CAJ22062), hrcN (MG847397), hrpB7 (AAB08462), hrcT (NEL30510), hrcU (NEK90481), hrcV (KLA79882), hpaC (CAJ22055), hrcQ (AAD21320), hrcR (AAD21321), hrcS (AAD21322), hpaA (AAD21323), hrcD (CAJ22050), hrpD6 (AAD21324), hrpE (DQ286694), hpaB (AAD21328), hpaE (CAJ22046), hrpF (AAB86527), xopA (AAL78294), hpaH (AAL78295), hrpG (U57625) and hrpX (AB026466).

Generation of reporter fusions

The hrcQ-sfgfp fusion was generated as described previously and assembled with xopA, hpaH, hrpX and hrpG* in the level M vector pAGM8079 to generate the level M construct pAGB322 [61]. For localization studies, pAGB322 was assembled with the level M constructs pAGB762 (hrp gene cluster + ∆hrcN/∆hrcQ), pAGB973 (hrp gene cluster + ∆hrcL/∆hrcQ), pAGB764 (hrp gene cluster + ∆hrcN/L/∆hrcQ) or pAGB1203 (hrp gene cluster + ∆hrcN/L/∆hrcQ/fshrpB4) (see above) to generate the corresponding level P constructs pAGB765, pAGB990, pAGB767 and pAGB1205, respectively (see Table S1). For the generation of a hrcQ-mKOκ expression cassette, hrcQ was amplified by PCR using primers hrcQ-NTM-MoClo-F and hrcQ-NTM-MoClo-R and the resulting amplicon was cloned into vector pICH41021 using SmaI and ligase, resulting in the level − 1 construct pAGB805 (NTM [N-terminal]-hrcQ). NTM-hrcQ was subsequently assembled with the inserts of pAGB1000 (2 x AKLEGPAGL), which encodes a two-fold linker sequence, and pAGB1153 (CTM [C-terminal]-mKOκ) in pICH41308 using BpiI and ligase, thus generating the level 0 construct pAGB1172. Assembly of pAGB1172 (hrcQ-2 x AKLEGPAGL-mKOκ) with pAGB249 (hrpD operon promoter) and pAGB231 (Xe term1) in the level 1 destination vector pICH4778 using BsaI and ligase led to the level 1 construct pAGB1173. In the following cloning step, the inserts of pAGB1173, pAGB157 (xopA + hpaH), pAGB160 (hrpX), pAGB163 (hrpG*), pICH54022 (dummy pos. 2’) and pICH50881 (end linker 2) were cloned into the level M vector pAGM8079 using BsaI and ligase, thus generating the level M construct pAGB1184. In the final step, assembly of pAGB1184 with pAGB273 (T3S gene cluster including ∆hrcQ) and pICH79264 (level P end-linker 2) in the BsaI sites of pICH75322 led to the level P construct pAGB1185.

To generate N-terminal translational fusions to HrcN, the corresponding gene was amplified by PCR using primers hrcN-CTM-MoClo-F and hrcN-CTM-MoClo-R and the amplicon was cloned into vector pAGM9121 to generate the level − 2 construct pAGB608 (CTM-hrcN). The insert of pAGB608 was cloned into vector pAGM1311 using BsaI and ligase to generate the level − 1 construct pAGB478 (CTM-hrcN). pAGB478 was then assembled with pAGB488 (NTM-sfgfp, includes the linker AKLEGPAGL) in vector pICH41308 using BpiI and ligase to generate the level 0 construct pAGB831. The insert of pAGB831 (sfgfp-hrcN) was subsequently ligated with the inserts of pAGB512 (hrpB operon promoter) and pAGB231 (Xe term1) into the level 1 destination vector pICH47781, using BsaI and ligase to generate the level 1 construct pAGB839. For the generation of the level 0 construct pAGB232 (Xe term2), which encodes a native Xe translational terminator, the corresponding terminator sequence was amplified by PCR using primers termX2-MoClo-F and termX2-MoClo-R and chromosomal DNA from Xe as template. The resulting amplicon was cloned into vector pICH41276 using BpiI and ligase to generate the level 0 construct pAGB232. The level 1 construct pAGB839 containing the hrpB operon promoter, sfgfp-hrcN and Xe term2 was assembled with pAGB157 (xopA + hpaH), pAGB160 (hrpX), pAGB163 (hrpG*), pICH54022 (dummy pos. 2’) and pICH50881 (end-linker 2) in the level M vector pAGM8079 using BpiI and ligase. The resulting level M construct pAGB847 was subsequently assembled with pAGB737 (hrp gene cluster + ∆hrcN) and pICH79264 (level P end linker 2) in the BsaI site of pICH75322 to generate the level P construct pAGB855.

For colocalization studies with sfGFP-HrcN and HrcQ-mKOκ, pAGB831 (sfgfp-hrcN), pAGB512 (hrpB operon promoter) and pAGB232 (Xe term2) were assembled in the additional level 1 destination vector pICH47742, thus generating the level 1 construct pAGB1342, which was subsequently assembled with the inserts of pAGB1173 (hrcQ-2 x AKLEGPAGL-mKOκ), pAGB157 (xopA + hpaH), pAGB160 (hrpX), pAGB163 (hrpG*) and pICH50881 (end-linker 2) in vector pAGM8079 using BpiI and ligase, resulting in the level M construct pAGB1343. To generate the final level P constructs, the inserts of pAGB1343 (hrcQ-2 x AKLEGPAGL-mKOκ + sfgfp-hrcN) and pICH79264 (level P end linker 2) were assembled with pAGB762 (hrp gene cluster + ∆hrcN/∆hrcQ) or with pAGB764 (hrp gene cluster + ∆hrcN/L/∆hrcQ) in the BsaI site of vector pICH75322 to obtain the level P constructs pAGB1344 and pAGB1376, respectively.

To generate an N-terminal translational fusion to HrcL, hrcL was amplified by PCR using primers hrcL-CTM-MoClo-F and hrcL-CTM-MoClo-R and the resulting amplicon was cloned into vector pICH41021 using SmaI and ligase to obtain the level − 2 plasmid pAGB1118 (CTM-hrcL). The insert of pAGB1118 was transferred into the BsaI sites of vector pAGM1311 to generate the level − 1 construct pAGB1119 (CTM-hrcL). The module for an N-terminal translational fusion of sfGFP to the target protein, sfgfp was amplified (sfGFP-NTM-woLink-F/ sfGFP-NTM-woLink-R) and the PCR product cloned into pICH41021 with SmaI and ligase to obtain the level − 1 plasmid pAGB1047 (NTM-sfgfp). For the generation of the linker (2 x GGAGGAGG) for the next cloning step, the two primers MoClo-LinkDiepold-5-3 and MoClo-LinkDiepold-3-5 were annealed and cloned with SmaI and ligase into vector pICH41021 to generate the level − 1 construct pAGB1001. The inserts of constructs pAGB1001 (2 x GGAGGAGG), pAGB1119 (CTM-hrcL) and pAGB1047 (NTM-sfgfp) were assembled in vector pICH41308, using BpiI and ligase and generating the level 0 construct pAGB1121. The inserts of pAGB1121 (sfgfp- 2x GGAGGAGG-hrcL), pAGB512 (hrpB operon promoter) and pAGB232 (Xe term2) were subsequently assembled in the level 1 destination vector pICH47781 using BsaI and ligase, leading to the level 1 construct pAGB1122. The corresponding insert was ligated with the insert of pAGB157 (xopA + hpaH), pAGB160 (hrpX), pAGB163 (hrpG*), pICH54022 (dummy pos. 2’) and pICH50881 (end-linker 2) into the BpiI sites of the level M vector pAGM8079, resulting in the level M construct pAGB1124. The final level P construct for the complementation studies was assembled by cloning the inserts of pAGB1124, pAGB979 (hrp gene cluster + ∆hrcL) and pICH79264 (level P end-linker 2) into the level P vector pICH1126, using BsaI and ligase, thus obtaining the level P construct pAGB1126.

Structure predictions using AlphaFold 2

For the generation of a Xe sorting platform model, single structures and complex predictions were performed using the AlphaFold2 notebook from ColabFold with default parameters [68]. The predictions were individually performed for HrcD_N (HrcD_{1 − 100}), HrpB4, HrcQ, HrcQ_C, HrcL, HrcN and HrpB7. To generate the hexameric ATPase complex consisting of HrcN and the predicted stalk protein HrpB7, the predicted models were superimposed onto the hexameric EscN structure with the associated EscO stalk (PDB: 6NJP) [69] using UCSF ChimeraX [70,71,72,73]. For the modelling of the remaining components of the sorting platform, the multimeric complexes 2×HrcD_N-HrpB4, HrpB4-HrcQ, HrcQ-2×HrcQ_C, HrcQ-HrcL, HrcL-HrcN were individually predicted using AlphaFold2 and the overlapping protein structures were subsequently superimposed (e.g. 2×HrcD_N-HrpB4, HrpB4-HrcQ) to assemble a pod structure subunit (2×HrcD_N-HrpB4-HrcQ-2×HrcQ_C-HrcL-HrcN). This subunit was then superimposed onto the predicted HrcN₆-HrpB7 complex using the structure of a HrcN monomer to generate the wheel-like sorting platform. The amino acids which form the interaction interfaces between HrcQ-HrcL and HrcL-HrcN were predicted with the “select → contact” command in UCSF ChimeraX and labelled with a different colour. The structures of deletion derivatives of HrcN and HrcL were predicted accordingly. The Xe proteins with the following accession numbers were analysed: HrcD (CAJ22050), HrpB4 (AAB08459), HrcQ (AAD21320), HrcN (CAJ22063), HrcL (CAJ22062) and HrpB7 (AAB08462).

Results

Predicted structure of the sorting platform from Xe

Structural studies in animal-pathogenic bacteria showed that SctQ proteins assemble at the periphery of the sorting platform [13, 20, 23]. In situ visualization by cryo-electron tomography in Salmonella and Shigella spp. revealed that the sorting platform is a hexagonal cage-like structure and consists of the central ATPase SctN which is connected through SctL with pod-like structures containing SctQ and dimers of SctQ_C [24, 25, 28, 74]. A similar architecture is predicted for the T3S system and the corresponding Hrc proteins from Xe (Fig. 1). Our previous in vitro pull-down experiments with corresponding proteins from Xe revealed that the ATPase HrcN interacts with itself as well as with HrcL and HrcQ [57, 58]. HrcN is conserved in different bacterial species while HrcL and HrcQ only share weak sequence similarities with SctL and SctQ from animal-pathogenic bacteria (0–35% amino acid identity). To predict the structure of the HrcN-HrcL-HrcQ complex in association with the SctO-like protein HrpB7 and the SctK-like protein HrpB4, we used the AlphaFold 2 algorithm and the molecular visualization program UCSF ChimeraX [70, 71]. Superimposition of the structural models of HrcN and HrpB7 onto the EscN₆-EscO complex from EPEC suggests that a HrcN hexamer is attached to the stalk-like HrpB7 protein [69, 75] (Fig. S1 and Fig. 2A). Furthermore, the AlphaFold 2 model and the assembly of protein structures by ChimeraX predict a hexagonal protein complex, in which the central HrcN hexamer is linked via HrcL to pod-like structures assembled by HrcQ and dimers of HrcQ_C (Fig. 2A, Fig. S2 and Fig. S3). A similar hexagonal arrangement was shown by cryo-electron microscopy of T3S system from Shigella and Salmonella spp [24, 25]. The N- and C-terminal regions of HrcQ are separated by a flexible linker and HrcQ_C adopts a saddle-shaped structure as was also predicted for SctQ_C proteins from animal pathogens [31, 32]. A 1:2 complex as the minimal unit in pod-like structures of sorting platforms was also reported for SctQ and SctQ_C proteins from S. flexneri and Yersinia [31, 32]. Additionally, a similar arrangement was described for the flagellar C ring components FliM and FliN, which share similarities with SctQ and SctQ_C proteins [76].

The N-terminal region of HrcQ likely interacts with the SctK-like protein HrpB4 which connects HrcQ to the cytoplasmic domains of the IM ring component HrcD [37]. Structural modelling predicts the binding sites for HrcQ and the cytoplasmic N-terminal domain of HrcD (HrcD_Nt) at opposite sides of HrpB4 (Fig. 2A and Fig. S2). This confirms the potential role of HrpB4 as a linker between HrcQ and the IM ring of the T3S system [37]. Interestingly, AlphaFold 2 indicates that HrpB4 interacts with a dimer of HrcD_nt (Fig. 2 and Fig. S2). This observation aligns with earlier findings showing that the SctK protein from Salmonella spp. interacts with a tetramer of the cytoplasmic domain of SctD, leading to structural rearrangements in the IM ring upon activation of the T3S system [74]. Likewise, the association of six HrpB4 proteins with the IM ring may induce rearrangements in the cytoplasmic domains of HrcD, thus possibly facilitating the attachment of the predicted sorting platform. To characterize the impact of the proposed protein assemblies on T3S and to validate the AlphaFold 2 model of the predicted sorting platform from Xe, we investigated the interactions of HrcN, HrcL and HrcQ by genetic approaches, interaction and localization studies as is described below.

Interactions of the predicted sorting platform components HrcQ, HrcL, HrcN and HrpB4

Previous in vitro pull-down studies suggested that HrcD, HrpB4, HrcQ, HrcN and HrcL interact with themselves as well as with each other as is summarized in Fig. S4. To validate these interactions and to experimentally confirm the AlphaFold 2-predicted associations of HrcQ, HrcL, HrcN and HrpB4, we performed additional protein-protein interaction studies using the BACTH system, which is based on the reconstitution of the catalytic domain of the adenylate cyclase (Cya) from two subdomains (T18 and T25) [77, 78]. The interaction of T18 and T25 fusions leads to cAMP synthesis and the expression of the lac operon. In E. coli reporter strains lacking the native cya gene, LacZ activity results in blue colonies on indicator plates containing X-Gal [77, 78]. For BACTH assays, we used expression constructs encoding HrcQ, HrcL, HrcN and HrpB4 as N- or C-terminal fusion partners of T18 and T25 domains. hrcQ expression constructs contained the annotated hrcQ coding sequence and 30 additional codons with a predicted TTG translational start codon upstream of the annotated GTG start site [59]. We previously reported that translation of hrcQ presumably starts at the TTG codon, which is located downstream of a predicted Shine Dalgarno sequence and is required for wild-type HrcQ levels [59]. The annotated HrcQ protein, which was used in previous interaction studies, is hereafter designated “HrcQ_ATG” and presumably lacks the N-terminal 30 amino acids [59]. BACTH assays revealed that HrcQ weakly interacts with HrcN when analysed as fusion partner of the T18 but not the T25 domain (Fig. 3A). However, both T18 and T25 fusions of HrcQ interacted with HrcL and HrpB4 in all possible combinations, suggesting that HrcQ binds to both HrcL and HrpB4 (Fig. 3A and B). Unlike HrcQ, the annotated HrcQ_ATG only weakly interacted with HrpB4, indicating that the extra 30 amino acids at the N-terminus of HrcQ enhance its association with HrpB4 (Fig. 3B). This is also supported by the structural model, which shows that the N-terminal region of HrcQ is closely associated with HrpB4, while the C-terminal region interacts with HrcL (see above, Fig. 2A and C; Fig. S2 and Fig. S3). We also performed interaction studies with HrcQ_C, which likely acts as a chaperone for HrcQ and binds to both HrcQ and HrpB4 [37, 59] (Fig. S4). Similarly to HrcQ, HrcQ_C interacted with HrcN and HrcL, suggesting that the C-terminal region of HrcQ is involved in the association with the ATPase complex (Fig. 3A). All proteins were stably synthesized in E. coli as was shown by immunoblot analyses of bacterial cell extracts (Fig. S5 and S6) [37, 59]. Weak protein-protein interactions were, therefore, likely not caused by protein instabilities. Taken together, we conclude from these results that the N-terminal region of HrcQ contributes to the interaction with HrpB4 whereas the C-terminal region is involved in the binding of HrcQ to HrcN and HrcL as predicted by AlphaFold 2.

The N- and C-terminal regions of HrcL interact with HrcQ and HrcN, respectively

According to the AlphaFold 2 predictions, HrcL forms spoke-like structures in the predicted sorting platform and interacts via its N-terminal region with HrcQ complexes and via the C-terminal region with HrcN (Fig. 2B and C, Fig. S3). Homo- and heterooligomerization of HrcN and HrcL was previously shown by in vitro pull-down assays [58] and was confirmed in the present study by the results of BACTH assays with T18 and T25 fusions of HrcN and HrcL (Fig. S4 and Fig. S5). According to AlphaFold 2 predictions, the N-terminal region of HrcN comprising the first 95 amino acids provides the binding site for HrcL (Fig. 2B and Fig. S3). In agreement with this model, deletion of the N-terminal 95 amino acids of HrcN abolished the detectable interaction with HrcL but not the self-interaction of HrcN_∆2−95 in BACTH assays (Fig. 4A). To analyse the contribution of the N-terminal region of HrcN to protein function, we performed complementation studies in Xe hrcN mutants. For this, expression constructs encoding C-terminally c-Myc epitope-Please correct the line break.tagged derivatives of HrcN and HrcN_∆2−95 were introduced into the Xe hrcN deletion mutant strain 85*∆hrcN. Strain 85* (85-10hrpG*) is a derivative of the wild-type strain 85 − 10 and contains HrpG*, a constitutively active version of the key regulator HrpG. HrpG* activates hrp gene expression under non-inducing conditions and leads to enhanced plant reactions [79]. For complementation studies, strains 85* and 85*∆hrcN with or without HrcN-c-Myc or HrcN_∆2−95-c-Myc were infiltrated into leaves of susceptible ECW and resistant ECW-10R pepper plants. ECW-10R plants express the Bs1 resistance gene and activate the HR upon recognition of the T3E AvrBs1 [80]. As expected, strain 85* induced water soaking and the HR in ECW and ECW-10R plants, respectively, whereas no plant reactions were detected after infiltration of strain 85*∆hrcN (Fig. 4B) [63]. The hrcN mutant phenotype was complemented by HrcN-c-Myc but not by the HrcN deletion derivative, suggesting that the N-terminal protein region is required for HrcN function (Fig. 4B). Similar results were observed with strain 85 − 10∆hrcN which contains the wild-type HrpG regulator and does not overexpress the T3S genes (Fig. S7). Notably, immunoblot analyses revealed reduced protein levels of HrcN_∆2−95-c-Myc when compared with the wild-type protein, suggesting that the N-terminal region contributes to stability of HrcN (Fig. 4B and Fig. S8).

To investigate the contribution of the N- and C-terminal regions of HrcL to the interaction with HrcN and HrcQ, we performed additional BACTH assays with HrcL derivatives lacking the N-terminal 40 (HrcL_∆2−40) or the C-terminal 83 amino acids (HrcL_∆150−233). T18 and T25 fusions of HrcL_∆2−40 interacted with corresponding T25 and T18 fusions of HrcN whereas the interaction with HrcQ was reduced in some but not all combinations (Fig. 5A). In contrast, deletion of the C-terminal region of HrcL abolished the detectable interaction with HrcN (Fig. 5A). However, HrcL_∆150−233 interacted with HrcQ in three out of four possible combinations, suggesting that the C-terminal region of HrcL is dispensable for the binding of HrcQ as was predicted by structural modelling (Figs. 2A and 5A). Immunoblot analyses confirmed that all fusion proteins were stably synthesized (Fig. S5 and S6). When analysed by in vivo complementation studies, HrcL deletion derivatives did not restore the wild-type phenotype in strains 85*∆hrcL and 85 − 10∆hrcL, suggesting that the N- and C-terminal protein regions are essential for HrcL function (Fig. 5B and Fig. S7). Lack of complementation was presumably not caused by a possible dominant-negative effect of HrcL and HrcN deletion derivatives on pathogenicity because ectopic expression of the corresponding genes in strain 85 − 10 did not alter the wild-type phenotype (Fig. S7). Notably, as observed for HrcN, deletion of the N-terminal region led to reduced HrcL levels (Fig. 5B and Fig. S9). It is possible that the N-terminal regions of HrcN and HrcL contribute to protein folding and thus stability. Alternatively, they might be required for stabilization of HrcN and HrcL in the context of the sorting platform. Taken together, we assume that the N- and C-terminal regions of HrcL contribute to pathogenicity and the interaction with HrcQ and HrcN, respectively, as predicted by AlphaFold 2.

HrcL contributes to HrcQ complex formation

We previously described that a HrcQ-sfGFP fusion forms complexes which likely represent the formation of the predicted sorting platform and were visualized by fluorescence microscopy. For these studies, we used Xe strains with a modular T3S gene cluster which was assembled from single promoter and gene modules and contains all necessary structural, regulatory and accessory genes [61] (Fig. 6A and Fig. S10). The modular design facilitates genetic approaches for functional studies as well as the insertion of reporter fusions by Golden Gate-based modular cloning [61]. The modular T3S gene cluster is functional in Xe strain 85*∆hrp_fsHAGX, which lacks the native T3S gene cluster and contains frameshift mutations in the regulatory and accessory genes hpaH, xopA, hrpG and hrpX [61]. hpaH and xopA encode a predicted lytic transglycosylase and a putative translocon protein, respectively, and thus likely contribute to the assembly of the T3S system and to T3E delivery [81, 82]. HrpG and HrpX are the key regulators, which activate T3S gene expression when the bacteria enter the plant apoplast or are cultivated in specific minimal media [47, 48, 79]. As reported previously, HrcQ-sfGFP restored pathogenicity when encoded by the modular T3S gene cluster lacking the native hrcQ gene, suggesting that the sfGFP fusion partner did not interfere with HrcQ function [61] (Fig. S11). When bacteria were incubated under T3S-permissive conditions at pH 5.3 in minimal medium and analysed by fluorescence microscopy, HrcQ-sfGFP formed one to five fluorescent foci at the cell periphery, suggesting that it assembles in complexes in the presence of a functional T3S system [61] (Fig. 6B and Fig. S12). Foci formation was specifically caused by HrcQ, as sfGFP without fusion partner localized in the bacterial cytoplasm and did not form fluorescent foci (Fig. S12).

We previously showed that HrcQ-sfGFP complex formation depends on the IM ring component HrcD [37, 59]. In the present study, we analysed the contribution of HrcL and HrcN to HrcQ-sfGFP foci formation. For this, the corresponding genes were deleted individually or in combination from the modular T3S gene cluster (Fig. S10). Infection studies with derivatives of strain 85*∆hrp_fsHAGX containing the respective modular T3S gene cluster constructs revealed that deletion of hrcL or hrcN led to a loss of pathogenicity as expected (Fig. 7A and Fig. S11) [58, 61]. Complementation studies confirmed that the hrcN mutant phenotype is specifically caused by the absence of HrcN and not by a polar effect of the deletion on downstream genes [58, 61] (Fig. 7A). Similarly, the hrcL mutant phenotype was complemented by ectopic expression of hrcL (Fig. S11). When bacteria were analysed by fluorescence microscopy, deletion of hrcL or hrcN led to reduced HrcQ-sfGFP foci (Fig. 6B). The reduced foci formation observed in the hrcL mutant remained almost unaffected by the additional deletion of hrcN, indicating that HrcN and HrcL do not have an additive effect on HrcQ complex assembly (Fig. 6B). In the absence of HrcL, HrcN and HrpB4, HrcQ-sfGFP foci formation was nearly abolished (Fig. 6B and C). Differences in foci formation were not caused by alterations in protein stabilities because HrcQ-sfGFP was stably synthesized in all strains (Fig. S11 and Fig. S13). Our findings support the previous observation that HrpB4 acts as a linker between HrcQ and the IM ring component HrcD [37]. Additionally, we conclude that HrpB4 is essential for HrcQ docking to the T3S system in the absence of HrcL and HrcN.

HrcQ and HrcN colocalize at the T3S system

To investigate a possible association of HrcN with HrcQ complexes, we performed colocalization studies. For this, HrcQ and HrcN were analysed as fusion partners of the fluorescent reporter proteins mKOκ and sfGFP, respectively [83, 84]. The corresponding gene fusions were inserted individually or in combination into the flanking region of modular T3S gene clusters deleted in the native hrcQ and/or hrcN genes and the resulting expression constructs were transferred into strain 85*∆hrp_fsHAGX. When bacteria were infiltrated into susceptible ECW and resistant ECW-10R pepper plants, HrcQ-mKO_κ and sfGFP-HrcN complemented the hrcQ and hrcN mutant phenotypes with respect to disease symptoms and HR induction, respectively, suggesting that both proteins were functional (Fig. 7A). Similarly, simultaneous expression of hrcQ-mKO_κ and sfgfp-hrcN restored pathogenicity of a hrcQ/hrcN double deletion mutant (Fig. 7A). Both proteins were stably synthesized as was shown by immunoblot analyses of bacterial cell extracts (Fig. 7B). Fluorescence microscopy revealed that HrcQ-mKO_κ and sfGFP-HrcN assembled in fluorescent foci, which colocalized in the presence of an active T3S system, suggesting that HrcQ and HrcN are part of a common protein complex (Fig. 7C and Fig. S12). Foci formation by HrcQ-mKO_κ was specifically attributed to HrcQ as a fusion between mKO_κ and the type III effector protein XopB localized to the cytoplasm (Fig. S14). Lack of HrcL led to a significant reduction in the number of HrcQ/HrcN-containing foci (Fig. 7C). This indicates that HrcL promotes complex formation by both HrcQ (see above, Fig. 6) and HrcN which is in agreement with the predicted architecture of the sorting platform and the role of HrcL as linker protein. For additional colocalization studies, we generated fluorescent fusions of HrpB4, HrcL, the stalk-like protein HrpB7, the IM ring components HrcJ and HrcD as well as the OM secretin HrcC. Proteins were analysed in fusion with different flexible linkers and fluorescent reporters including sfGFP, mCherry, mKOκ, TagBFP and sfTq2oc as is summarized in Fig. S15 and Table S3. Localization studies, however, were not possible because the fusion proteins were either unstable or the fluorescent reporters interfered with protein function as was shown by complementation studies (data not shown).

Stability of the ATPase HrcN depends on HrcL and HrcQ

Considering that HrcL plays a role in the assembly of HrcQ and HrcN complexes, we wondered whether it also promotes the stability of both proteins. Consistent with our earlier discovery that HrcL stabilizes HrcN in Xe [58], we observed reduced levels of sfGFP-HrcN in a hrcL mutant (Fig. 7B). To investigate whether HrcN stability depends on additional sorting platform components, we performed immunoblot analyses of cell extracts from strain 85*∆hrp with modular T3S gene cluster constructs lacking hrcQ, hrcN, hrcL or hrpB4. HrcN was stably synthesized in the presence of the wild-type T3S gene cluster and in the hrpB4 mutant whereas significantly reduced levels of HrcN were detected in the absence of hrcQ or hrcL (Fig. 8A and Fig. S16). In contrast to HrcN, the levels of HrcQ were unaffected in the analysed deletion mutants. The detection of equal amounts of the IM ring component HrcJ in all samples confirmed equal loading (Fig. 8A and Fig. S16).

To further investigate the contribution of HrcQ to HrcN stability, we analysed HrcN levels in the hrcQ deletion mutant strain 85*∆hrcQ, in which hrcQ or a derivative thereof containing a mutation in the internal translation start site (M241A mutation) were reinserted in cis into the flanking region of the T3S gene cluster [59]. Wild-type levels of HrcN were detected upon in cis expression of hrcQ but not of hrcQ_M241A in the hrcQ deletion mutant, suggesting that the alternative translation product HrcQ_C contributes to HrcN stability (Fig. 8B and Fig. S17). In trans expression of hrcQ_C in the hrcQ_M241A mutant restored wild-type levels of HrcN, indicating that reduced HrcN stability in strain 85*∆hrcQ::hrcQ_M241A was specifically caused by the absence of HrcQ_C (Fig. 8B and Fig. S17). Our findings suggest that the stability of the ATPase HrcN depends on HrcL, HrcQ and HrcQ_C, and thus possibly on the association of HrcN with a HrcL-HrcQ/HrcQ_C complex. In agreement with this hypothesis, deletion of the N-terminal region of HrcN, which provides the binding site for HrcL, led to reduced HrcN stability (see above, Fig. 4B).

The association of HrcQ and HrcN with the T3S system depends on the external pH

The sorting platform from animal-pathogenic bacteria was previously described as dynamic structure which changes its localization from the bacterial cytoplasm to the T3S system and thus possibly promotes substrate loading into the secretion channel [27, 34, 35]. To investigate a similar dynamic localization of HrcN-HrcL-HrcQ complexes in Xe, we analysed HrcQ-sfGFP complex formation in bacteria which were incubated in minimal medium at pH 5.3 or pH 7.0. T3S in Xe is activated in minimal medium at pH 5.3, leading to the detection of secreted proteins in bacterial culture supernatants whereas T3S is abolished in minimal medium at pH 7.0 [62]. As shown previously, HrcQ-sfGFP formed one to five fluorescent foci when bacteria were incubated at pH 5.3 under T3S-permissive conditions [61] (Fig. 9A). In contrast, at pH 7.0, only one bright fluorescent spot was detected which likely reflects a cytoplasmic HrcQ-sfGFP complex (Fig. 9A). The cytoplasmic localization of HrcQ-sfGFP complexes at pH 7.0 remained unchanged in the absence of the IM ring component HrcD, suggesting that it was not caused by the docking of HrcQ to the T3S system (Fig. S18 and Fig. S19). As reported previously, the cytoplasmic domain of HrcD is required for the localization of HrcQ complexes at the T3S system at pH 5.3 [59].

To investigate whether cytoplasmic HrcQ-sfGFP complexes represent the predicted assembled sorting platform, we performed colocalization studies with HrcQ-mKO_κ and sfGFP-HrcN fusions encoded by corresponding modular expression constructs as described above (Fig. 7C and Fig. S10). Both HrcQ-mKO_κ and sfGFP-HrcN were detected in one bright fluorescent spot when bacteria were incubated under T3S non-permissive conditions at pH 7.0 (Fig. 9B and Fig. S20). This suggests that HrcQ and HrcN integrate into the same cytoplasmic protein complex, which presumably corresponds to the sorting platform and detaches from the T3S system under T3S non-permissive conditions. Additional deletion of hrcL in the modular T3S gene cluster reduced the number of bacteria with one fluorescent spot (Fig. 9B and Fig. S20). This supports the above observation that HrcL stabilizes HrcQ and HrcN complexes. Taken together, we conclude that HrcN, HrcL and HrcQ assemble in a common dynamic protein complex which presumably corresponds to the sorting platform and changes its localization from the cytoplasm to the T3S system in response to the external pH (Fig. 10).

Discussion

The existence, architecture and assembly of the sorting platform in plant-pathogenic bacteria has long been enigmatic because the low copy number of the T3S system made its isolation and in situ visualization challenging. In the present study, we identified an interaction network of the predicted sorting platform components HrcN, HrcL and HrcQ from Xe and showed that they dynamically associate with the T3S system in response to the external pH level. According to structural modelling, HrcN, HrcL and HrcQ form a hexagonal cage-like complex which resembles the sorting platform from animal pathogens and consists of the central hexameric ATPase HrcN which is linked via HrcL to six peripheral HrcQ/HrcQ_C complexes. The C-terminal region of HrcN faces the IM and interacts with HrpB7, a small SctO-like protein which forms highly stable complexes. HrpB7 presumably connects the ATPase with the cytoplasmic domain of the export apparatus component HrcV as was reported for homologous Sct proteins from animal-pathogenic bacteria [6, 75, 85]. Structure predictions, in vivo interaction assays and complementation studies with HrcN and HrcL deletion derivatives indicate that the N-terminal region of HrcN interacts with the C-terminal region of HrcL. This interaction presumably maintains HrcN function and stability. HrcL likely forms spoke-like structures between HrcN and HrcQ as is suggested by structural modelling and our interaction studies. Given that HrcL can interact with itself, it might be present as a homo-dimer in the HrcN-HrcL-HrcQ complex as was shown for SctL proteins from animal-pathogenic bacteria [24, 25, 86, 87].

We previously studied the assembly of the predicted sorting platform in Xe by the analysis of a HrcQ-sfGFP fusion protein using fluorescence microscopy and a modular T3S gene cluster [59, 61]. Prerequisite for localization studies with fluorescent reporter fusions is their stability and functionality in Xe. To date, HrcQ and HrcN are the only suitable candidates for localization studies in Xe because other T3S system components were either unstable or not functional when analysed as fusion partners of fluorescent reporters. As shown previously, efficient foci formation by HrcQ-sfGFP depends on its C-terminal alternative translation product, HrcQ_C, which interacts with HrcQ and likely acts as a chaperone [59]. The finding that HrcQ_C also associates with the ATPase complex, the linker protein HrpB4 and the IM ring protein HrcD suggests that it contributes to the assembly and the docking of the predicted sorting platform to the T3S system [59]. We assume that the broad binding specificity of HrcQ_C is due to a SPOA (surface presentation of antigen) domain which is also present in SctQ_C proteins from animal-pathogenic bacteria and is likely involved in protein-protein interactions [33, 59, 60, 88]. Structural modelling suggests that HrcQ_C dimerizes and associates with HrcQ in pod-like structures at the periphery of the predicted sorting platform. Interestingly, the HrcQ/HrcQ_C complex shares structural similarities with corresponding SctQ/SctQ_C complexes from animal pathogens and the corresponding FliM-FliN₃ complex of the flagellar C ring [32, 76].

According to structure predictions and interaction studies, the C-terminal region of HrcQ associates with HrcL whereas its N-terminal region binds to the SctK-like protein HrpB4. We previously reported that HrpB4 connects the HrcQ/HrcQ_C pod-like structures to the cytoplasmic domain of the IM ring component HrcD and thus acts similarly to SctK proteins from animal-pathogenic bacteria [37]. Notably, deletion of hrpB4 significantly reduced but did not completely abolish foci formation by HrcQ-sfGFP, suggesting that HrcQ attaches to the IM rings of the T3S system in the absence of HrpB4 [37]. Lack of the IM ring component HrcD or its cytoplasmic domain, however, leads to the formation of cytoplasmic HrcQ-sfGFP complexes as expected [37]. HrcQ stability and complex formation in the cytoplasm is independent of HrpB4, indicating that HrpB4 is dispensable for the assembly of the predicted sorting platform in the cytoplasm [37] (Fig. 10). It is yet unknown whether HrpB4 detaches together with the HrcQ-HrcL-HrcN complex from the membranes or whether its association with HrcQ depends on HrcD as was reported for SctQ and SctK proteins from Salmonella spp [74]. Future studies in Xe will, therefore, focus on the analysis of interactions between HrcQ, HrpB4 and HrcD in different mutant backgrounds using in vivo photocrosslinking approaches.

Our fluorescence microscopy studies showed that the efficient docking of HrcQ complexes to the T3S system requires HrcL which connects HrcQ to the ATPase HrcN. This is reminiscent of the finding that complex formation between SctQ and SctL in Salmonella spp. is a prerequisite for the interaction of SctQ with the linker protein SctK. It was, therefore, proposed that binding of SctL induces a conformational change in SctQ which promotes the SctQ-SctK interaction [74]. In S. flexneri, however, SctL is dispensable for the incorporation of the SctQ and SctK proteins Spa33 and MxiK into pod-like structures [89], suggesting a different assembly of the sorting platform. In Xe, the formation of HrcQ-HrcL complexes presumably precedes the attachment of the ATPase HrcN which plays a minor role in the formation of fluorescent HrcQ-sfGFP foci (Fig. 10). Given that HrcQ, HrcQ_C and HrcL are required to maintain wild-type HrcN levels, binding to the HrcQ-HrcL complex likely stabilizes HrcN. Similar findings were reported for the ATPases from Salmonella spp. and S. flexneri which depend on the assembly of the sorting platform for oligomerization and stabilization [24, 25, 27, 33, 74]. In Yersinia spp., however, the ATPase YscN stabilizes YscQ complexes and promotes interactions of sorting platform components which presumably form a variety of dynamic cytoplasmic complexes [34, 36, 60, 86]. Fluorescence recovery after photobleaching measurements and single molecule tracking microscopy showed a dynamic localization of YscQ between the T3S system and the cytoplasm with increased exchange rates under secretion-permissive conditions, suggesting a continuous release and attachment of the sorting platform to the T3S system [34, 35, 90]. A similar dynamic localization of the sorting platform was reported in Salmonella spp. and was proposed to shuttle T3S substrates to the secretion apparatus [20, 27, 60, 91]. In Yersinia spp. and S. flexneri, sorting platform components dissociate from the T3S system at a low external pH and thus likely suppress T3S when the bacteria pass the stomach [39]. Interestingly, we also observed a dynamic localization of the predicted sorting platform from Xe in response to changes in the external pH level. Thus, under T3S-permissive conditions at pH 5.3, HrcQ and HrcN colocalize in fluorescent foci at the cell periphery in the presence of a functional T3S system [57, 59, 61]. Under T3S non-permissive conditions at pH 7.0, however, both HrcQ and HrcN were present in a common complex in the bacterial cytoplasm, suggesting that the change in pH leads to the detachment of the entire HrcQ/HrcQ_C-HrcL-HrcN complex from the membranes. This is in line with our previous observation that HrcQ predominantly localizes to the cytoplasm at pH 7.0 whereas an acidic pH promotes its membrane association [57].

The dynamic attachment of the sorting platform to the T3S system in response to environmental conditions such as changes in the external pH possibly increases the local concentration of T3S cargo proteins and thus activates T3S [34, 39]. Given that the sorting platform associates with T3S substrates, it might provide a reservoir for secreted proteins in the cytoplasm [91]. A recent study in Yersinia spp. revealed a direct interaction of T3Es with SctQ and SctL in the bacterial cytoplasm and suggests that the binding of one T3E to one SctQ protein and the exchange rates of SctQ between the cytoplasm and the T3S system accounts for the delivery rate of T3Es [92]. In Xe, potential docking sites for T3S substrates in the predicted sorting platform are provided by HrcQ and HrcN [57, 58]. We assume that the acidic pH in the plant apoplast, possibly together with an additional yet unknown plant-derived signal, is required for T3S and thus leads to the attachment of the predicted sorting platform to the T3S system [62, 93, 94]. The mechanisms underlying pH sensing, recognition of T3S substrates and the activation of the T3S system are not yet understood. In Yersinia spp. and S. flexneri, the external pH is presumably sensed by the IM ring component SctD, which dissociates from the T3S system at an acidic pH [39]. Similarly, a rearrangement of SctD proteins from a 24mer oligomeric transmembrane ring to tetrameric complexes was described in Salmonella spp. and occurs upon docking of the sorting platform [25]. In Xe, a potential role of HrcD in the pH-mediated docking of the predicted sorting platform remains to be investigated. Furthermore, future in vivo approaches in Xe including localization studies and photocrosslinking are required for an in-depth analysis of the mechanisms leading to the recruitment of the HrcN-HrcL-HrcQ complex to the T3S system, the binding of cargo proteins and the activation of T3S. Unravelling the interplay between T3S substrates and structural components of the T3S system will elucidate the mechanisms underlying T3E delivery and might also help to develop strategies for the management of bacterial diseases in crop plants.

Conclusions

Taken together, our study provides the first detailed analysis of a predicted sorting platform from a plant pathogen. Structural modelling suggests that HrcQ, HrcL and HrcN form a hexagonal cage-like structure, similar to sorting platforms found in animal pathogens, which is crucial for the function of the T3S system. As revealed by fluorescence microscopy studies, the predicted sorting platform dynamically associates with the T3S system in response to environmental stimuli and thus presumably functions as reservoir for T3S substrates in the cytoplasm. The proposed dynamics in protein-protein interactions shed light on T3S regulation in plant-pathogenic bacteria, which is still poorly understood. Our findings draw parallels between the sorting platforms of plant and animal pathogens, suggesting that similar mechanisms may be employed across different bacterial species.

Data availability

Data generated or analysed during this study are included within the article and its additional files. The Xe proteins with the following accession numbers were analysed: HrcD (CAJ22050), HrpB4 (AAB08459), HrcQ (AAD21320), HrcN (CAJ22063), HrcL (CAJ22062) and HrpB7 (AAB08462). The datasets underlying the analysis of fluorescent foci formation, which are summarized in this article, are available from the corresponding author on reasonable request.

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Acknowledgements

We are grateful to M. Jordan for excellent technical assistance.

Funding

Open Access funding enabled and organized by Projekt DEAL. This work was funded by grants from the Deutsche Forschungsgemeinschaft to DB (BU 2145/10 − 1 and BU 2145/9 − 2). We acknowledge the financial support of the Open Access Publication Fund of the Martin-Luther-University Halle-Wittenberg.

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Christian Otten
Present address: Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT, USA

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Institute of Biology, Department of Genetics, Martin-Luther University Halle- Wittenberg, Halle (Saale), Germany
Christian Otten & Daniela Büttner

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Christian Otten
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Daniela Büttner
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CO and DB designed the experiments, CO performed the experiments, CO and DB analysed the data, DB wrote the manuscript, CO contributed to the writing of the manuscript. Both authors approved the manuscript.

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Correspondence to Daniela Büttner.

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Otten, C., Büttner, D. The predicted sorting platform dynamically associates with the type III secretion system from Xanthomonas euvesicatoria in response to the external pH. BMC Microbiol 25, 545 (2025). https://doi.org/10.1186/s12866-025-04227-6

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Received: 09 December 2024
Accepted: 14 July 2025
Published: 25 August 2025
DOI: https://doi.org/10.1186/s12866-025-04227-6

The predicted sorting platform dynamically associates with the type III secretion system from Xanthomonas euvesicatoria in response to the external pH

Abstract

Background

Methods

Bacterial strains and growth conditions

Plant material and plant infections

Protein-protein interaction studies using the BACTH system

Immunoblot analyses of Xe cell extracts

Fluorescence microscopy analyses

Generation of expression constructs by Golden Gate cloning

Generation of modular T3S gene cluster constructs

Mutation of single or multiple T3S genes in the modular T3S gene cluster expression constructs

Generation of reporter fusions

Structure predictions using AlphaFold 2

Results

Predicted structure of the sorting platform from Xe

Interactions of the predicted sorting platform components HrcQ, HrcL, HrcN and HrpB4

The N- and C-terminal regions of HrcL interact with HrcQ and HrcN, respectively

HrcL contributes to HrcQ complex formation

HrcQ and HrcN colocalize at the T3S system

Stability of the ATPase HrcN depends on HrcL and HrcQ

The association of HrcQ and HrcN with the T3S system depends on the external pH

Discussion

Conclusions

Data availability

References

Acknowledgements

Funding

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Additional information

Publisher’s note

Electronic supplementary material

Supplementary Material 1

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About this article

Cite this article

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Keywords

BMC Microbiology

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