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

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.