HrpB7 from Xanthomonas campestris pv. vesicatoria is an essential component of the type III secretion system and shares features of HrpO/FliJ/YscO family members

Abstract

The Gram‐negative bacterium Xanthomonas campestris pv. vesicatoria translocates effector proteins via a type III secretion system (T3SS) into eukaryotic cells. The T3SS spans both bacterial membranes and consists of more than 20 proteins, 9 of which are conserved in plant and animal pathogens and constitute the core subunits of the secretion apparatus. T3S in X. campestris pv. vesicatoria also depends on nonconserved proteins with yet unknown function including HrpB7, which contains predicted N‐ and C‐terminal coiled‐coil regions. In the present study, we provide experimental evidence that HrpB7 forms stable oligomeric complexes. Interaction and localisation studies suggest that HrpB7 interacts with inner membrane and predicted cytoplasmic (C) ring components of the T3SS but is dispensable for the assembly of the C ring. Additional interaction partners of HrpB7 include the cytoplasmic adenosinetriphosphatase HrcN and the T3S chaperone HpaB. The interaction of HrpB7 with T3SS components as well as complex formation by HrpB7 depends on the presence of leucine heptad motifs, which are part of the predicted N‐ and C‐terminal coiled‐coil structures. Our data suggest that HrpB7 forms multimeric complexes that associate with the T3SS and might serve as a docking site for the general T3S chaperone HpaB.

HrpB4 from Xanthomonas campestris pv . vesicatoria acts similarly to SctK proteins and promotes the docking of the predicted sorting platform to the type III secretion system

“…A similar T3S system‐independent membrane localization was previously described for EscK from E. coli as well as for HrpB7 from X. campestris pv. vesicatoria which is a predicted member of the HrpO/FliJ/YscO family and associates with the IM and predicted C ring components (Drehkopf, Otten, Hausner, Seifert, & Büttner, 2020; Soto et al, 2017). The results of our in vivo and in vitro interaction studies revealed that HrpB4 interacts with the putative C ring protein HrcQ, which is homologous to SctQ proteins from animal‐pathogenic bacteria and might thus be part of the predicted sorting platform of the T3S system (Figure 3).…”

“…We have previously shown that HrcQ interacts with the ATPase HrcN and its predicted regulator HrcL (Lorenz et al, 2012). HrcQ and HrcN both interact with the predicted coiled‐coil protein HrpB7, which is a member of the HrpO/FliJ/YscO family and also binds to the IM ring of the T3S system (Drehkopf et al, 2020) (Figure 9). However, HrpB7 presumably does not act as an essential linker between the sorting platform and the membrane‐spanning secretion apparatus because it is dispensable for the formation of fluorescent HrcQ‐sfGFP foci (Drehkopf et al, 2020).…”

“…HrcQ and HrcN both interact with the predicted coiled-coil protein HrpB7, which is a member of the HrpO/FliJ/YscO family and also binds to the IM ring of the T3S system (Drehkopf et al, 2020) (Figure 9). However, HrpB7 presumably does not act as an essential linker between the sorting platform and the membrane-spanning secretion apparatus because it is dispensable for the formation of fluorescent HrcQ-sfGFP foci (Drehkopf et al, 2020). In future experiments, we will, therefore, analyse the contribution of the ATPase complex to HrcQ-sfGFP localization in wild-type and hrpB4 mutant strains.…”

The C-terminal domain of the type III secretion chaperone HpaB contributes to dissociation of chaperone-effector complex in Xanthomonas campestris pv. campestris

“…targets to the cytoplasmic and/or inner components of T3SS, which presumably is used to regulate secretion and/or translocation of T3SS substrates [11,12,14,22,48,49]. Given that chaperone HpaB may contribute to effector targeting to T3SS, we examined possible interactions of HpaB and its C-terminally truncated variant with secretion platform components of T3SS, such as, HrcN, HrcQ, and HrcU using the GST-pulldown assays.…”

“…The T3SS of Xanthomonas is encoded by a chromosomal hrp [hypersensitive response (HR) and pathogenicity] cluster regulated by two master regulators, HrpG and HrpX, whose expression is highly elevated in planta or in particular minimal media [4][5][6][7]. The T3SS of Xanthomonas is a molecular nanomachine and consists of peripheral cytoplasm components (HrcQNL and HrpB7), inner membrane (IM) components (HrcUVRST), periplasm or associated IM components (HrcJ, HrpB1, and HrpB2), outer membrane (OM) component (HrcC), extracellular Hrp pili (HrpE), and translocon (HrpF) [1,[8][9][10][11][12][13][14].…”