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-...

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