title: 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.
authors.affiliation: From the ‡INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441, Castanet-Tolosan, F-31326, France; §CNRS, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR2594, Castanet-Tolosan, F-31326, France;
authors.forename: Fabien
authors.initials: F
authors.lastname: Lonjon
authors.affiliation: From the ‡INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441, Castanet-Tolosan, F-31326, France; §CNRS, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR2594, Castanet-Tolosan, F-31326, France;
authors.forename: Marie
authors.initials: M
authors.lastname: Turner
authors.affiliation: ¶PAPPSO, Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France;
authors.forename: Céline
authors.initials: C
authors.lastname: Henry
authors.affiliation: From the ‡INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441, Castanet-Tolosan, F-31326, France; §CNRS, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR2594, Castanet-Tolosan, F-31326, France;
authors.forename: David
authors.initials: D
authors.lastname: Rengel
authors.affiliation: From the ‡INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441, Castanet-Tolosan, F-31326, France; §CNRS, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR2594, Castanet-Tolosan, F-31326, France;
authors.forename: David
authors.initials: D
authors.lastname: Lohou
authors.affiliation: From the ‡INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441, Castanet-Tolosan, F-31326, France; §CNRS, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR2594, Castanet-Tolosan, F-31326, France;
authors.forename: Quitterie
authors.initials: Q
authors.lastname: van de Kerkhove
authors.affiliation: From the ‡INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441, Castanet-Tolosan, F-31326, France; §CNRS, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR2594, Castanet-Tolosan, F-31326, France;
authors.forename: Anne-Claire
authors.initials: AC
authors.lastname: Cazalé
authors.affiliation: From the ‡INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441, Castanet-Tolosan, F-31326, France; §CNRS, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR2594, Castanet-Tolosan, F-31326, France;
authors.forename: Nemo
authors.initials: N
authors.lastname: Peeters
authors.affiliation: From the ‡INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441, Castanet-Tolosan, F-31326, France; §CNRS, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR2594, Castanet-Tolosan, F-31326, France;
authors.forename: Stéphane
authors.initials: S
authors.lastname: Genin
authors.affiliation: From the ‡INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441, Castanet-Tolosan, F-31326, France; §CNRS, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR2594, Castanet-Tolosan, F-31326, France; ‖Université de Toulouse; INP; ENSAT; 18 chemin de Borde Rouge, Castanet Tolosan, 31326, France fabienne.vailleau@toulouse.inra.fr.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Vailleau
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D018832:Molecular Chaperones / Q000235:genetics; D009154:Mutation; D010935:Plant Diseases / Q000235:genetics / Q000382:microbiology*; D010944:Plants / Q000382:microbiology; D040901:Proteomics; D043368:Ralstonia solanacearum / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones