HpaP modulates type III effector secretion in Ralstonia solanacearum and harbours a substrate specificity switch domain essential for virulence

Abstract

Many pathogenic bacteria have evolved a type III secretion system (T3SS) to successfully invade their host. This extracellular apparatus allows the translocation of proteins, called type III effectors (T3Es), directly into the host cells. T3Es are virulence factors that have been shown to interfere with the host's immunity or to provide nutrients from the host to the bacteria. The Gram-negative bacterium Ralstonia solanacearum is a worldwide major crop pest whose virulence strongly relies on the T3SS. In R. solanacearum, transcriptional regulation has been extensively studied. However, very few data are available concerning the role played by type III-associated regulators, such as type III chaperones and T3SS control proteins. Here, we characterized HpaP, a putative type III secretion substrate specificity switch (T3S4) protein of R. solanacearum which is not secreted by the bacterium or translocated in the plant cells. HpaP self-interacts and interacts with the PopP1 T3E. HpaP modulates the secretion of early (HrpY pilin) and late (AvrA and PopP1 T3Es) type III substrates. HpaP is dispensable for the translocation of T3Es into the host cells. Finally, we identified two regions of five amino acids in the T3S4 domain that are essential for efficient PopP1 secretion and for HpaP's role in virulence on tomato and Arabidopsis thaliana, but not required for HpaP-HpaP and HpaP-PopP1 interactions. Taken together, our results indicate that HpaP is a putative R. solanacearum T3S4 protein important for full pathogenicity on several hosts, acting as a helper for PopP1 secretion, and repressing AvrA and HrpY secretion.

Comparative Secretome Analysis of Ralstonia solanacearum Type 3 Secretion-Associated Mutants Reveals a Fine Control of Effector Delivery, Essential for Bacterial Pathogenicity

“…However, no difference could be observed between the wild-type strain and the ripBJ mutant in pathogenicity assays, probably because of functional redundancy among Rip effectors (15)(16)(17). In a former study (23), we provided evidence of a post-translational control of T3E secretion in R. solanacearum by the HpaP protein. In this work, we combined proteomic and phenotypic analyses studying three additional hpa mutants, hpaB, hpaD, and hpaG.…”

“…pAM5 (18) was added by electroporation into these 40 strains. Then, secretion assays were performed as previously described (23). Proteins from an equal amount of cultures (normalized by OD 600 ) were loaded in each lane.…”

“…The sensitivity of the method incited us to explore and compare the secretomes of potential T3S regulators. Some of them were characterized in phytopathogenic bacteria for their participation to T3E delivery, by promoting or repressing secretion through protein-protein interactions (21,23). In R. solanacearum strain GMI1000, four potential Hpa proteins were identified: HpaB (RSp0853), HpaD (RSp0848), HpaG (RSp0842), and HpaP (RSp0862).…”

“…HpaB and HpaD have features of molecular chaperones (20, 42) with small sizes (16.8 kDa and 13.9 kDa, respectively) and acidic pI (4.4 and 4.2, respectively). HpaP was recently shown to modulate T3S (23) and HpaG (also known as LrpE) is a leucine-rich repeats (LRR) containing protein that negatively regulates the production of Hrp pili (43). We conducted secretome analyses of the hpaB, hpaD, and hpaG mutants using a similar experimental procedure as described above (supplemental Fig.…”

“…Some secretion helper proteins have been well described, like the T3Cs (type 3 chaperones), or the T3S4 (type 3 secretion substrate specificity switch) proteins (6,22). Our previous work on R. solanacearum HpaP, a putative T3S4 protein, revealed that this regulatory protein could act as a modulator of the secretion level, targeting some T3Es, notably via direct protein interactions (23).…”