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

Abstract

Ralstonia solanacearum, the causal agent of bacterial wilt, exerts its pathogenicity through more than a hundred secreted proteins, many of them depending directly on the functionality of a type 3 secretion system. To date, only few type 3 effectors have been identified as required for bacterial pathogenicity, notably because of redundancy among the large R. solanacearum effector repertoire. In order to identify groups of effectors collectively promoting disease on susceptible hosts, we investigated the role of putative post-translational regulators in the control of type 3 secretion. A shotgun secretome analysis with label-free quantification using tandem mass spectrometry was performed on the R. solanacearum GMI1000 strain. There were 228 proteins identified, among which a large proportion of type 3 effectors, called Rip (Ralstonia injected proteins). Thanks to this proteomic approach, RipBJ was identified as a new effector specifically secreted through type 3 secretion system and translocated into plant cells. A focused Rip secretome analysis using hpa (hypersensitive response and pathogenicity associated) mutants revealed a fine secretion regulation and specific subsets of Rips with different secretion patterns. We showed that a set of Rips (RipF1, RipW, RipX, RipAB, and RipAM) are secreted in an Hpa-independent manner. We hypothesize that these Rips could be preferentially involved in the first stages of type 3 secretion. In addition, the secretion of about thirty other Rips is controlled by HpaB and HpaG. HpaB, a candidate chaperone was shown to positively control secretion of numerous Rips, whereas HpaG was shown to act as a negative regulator of secretion. To evaluate the impact of altered type 3 effectors secretion on plant pathogenesis, the hpa mutants were assayed on several host plants. HpaB was required for bacterial pathogenicity on multiple hosts whereas HpaG was found to be specifically required for full R. solanacearum pathogenicity on the legume plant Medicago truncatula. Molecular & Cellular Proteomics 15: 10.1074/ mcp.M115.051078, 598-613, 2016.The soil-borne vascular bacterium R. solanacearum is described as one of the most destructive plant pathogenic bacterium worldwide (1), mainly because of its broad host range and wide geographic distribution. Indeed, R. solanacearum attacks more than 250 plant species, distributed in more than 50 botanical families (2). R. solanacearum causes dramatic crop losses, more specifically in the tropics, notably affecting emerging countries. Durable protection strategies against this bacterium are lacking. R. solanacearum penetrates into the plant via the roots, and then colonizes the xylem vessels. The bacterium reaches the aerial parts of the plant, causes wilting symptoms leading to the death of the plant, and will eventually return to the soil, completing the cycle (3). To achieve these first colonization steps, the bacterium uses a molecular syringe called the type 3 secretion system (T3SS), 1 which deFrom the ‡INRA, Laboratoire des Interactions Plantes-...

A Resource Allocation Trade-Off between Virulence and Proliferation Drives Metabolic Versatility in the Plant Pathogen Ralstonia solanacearum

“…Moreover, this reconstruction associates a metabolic network with a macromolecule network to account for the production and secretion of virulence factors since R . solanacearum is known to produce hundreds of extracellular proteins involved in pathogenesis [ 40 – 41 ]. R .…”

Bacterial pathogenesis of plants: future challenges from a microbial perspective

“…Bacterial plant pathogens also employ hrp‐associated ( hpa ) proteins, such as chaperones and export control proteins, for coordinated delivery of T3Es into host cells (Lohou et al ., ). Recently, a proteomics approach to the R. solanacearum secretome identified differential T3E secretion patterns in hpaB , hpaD and hpaG mutant strains, which affected bacterial virulence and supports the idea of the hierarchical delivery of T3Es (Lonjon et al ., ). Mechanistically, it has been suggested that the global T3E export chaperone HpaB interacts with HpaA to select and guide T3Es to the T3SS‐associated ATPase HrcN for translocation (Büttner et al ., ; Chang et al ., ).…”

Analysis of the Proteins Secreted from the Oryza meyeriana Suspension-Cultured Cells Induced by Xanthomonas oryzae pv. oryzae

“…Secreted proteins play important roles in a number of pathological processes, such as signal transduction [ 40 ] and defense [ 41 ]. Suspension-culture is a model system for studying the secreted proteins [ 42 ] and has been widely used for secretome analysis of interactions between plants and their pathogens including bacteria [ 43 ] and fungi [ 44 ]. In this study, we used this method to investigate the altered protein expression in resistant rice cells in response to Xoo inoculation.…”