In Vitro and In Vivo Secretion/Translocation Assays to Identify Novel Ralstonia solanacearum Type 3 Effectors
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Phytopathogenic bacteria have evolved multiple strategies to infect plants. Like many gram-negative bacteria, Ralstonia solanacearum, the causal agent of bacterial wilt, possesses a specialized protein secretion machinery to deliver effector proteins directly into the host cells. This type 3 secretion system (T3SS) and the bacterial proteins translocated, called type 3 effectors (T3Es), constitute the main pathogenicity determinants of the R. solanacearum species complex (RSSC). Up to 113 orthologous groups defining T3E genes have been identified among the RSSC strains sequenced to date. The increasing number of R. solanacearum genomic sequences available still expands the number of T3E candidates which require experimental validation. Here, we describe in vitro (type 3 secretion) and in vivo (type 3 translocation based on CyaA′ reporter gene) methods to identify and validate type 3-dependent delivery of proteins of interest highlighted as candidate T3Es. We also present protocols to generate dedicated vectors and R. solanacearum transformation to perform these experiments.
Key wordsSecretion Translocation Ralstonia solanacearum Type 3 secretion system Type 3 effector CyaA′
Fabien Lonjon was funded by a grant from the French Ministry of National Education and Research. Fabienne Vailleau work was supported by a French Agence Nationale de la Recherche grant (ANR-2010-JCJC-1710-01). Our work is performed at the LIPM that is part of the Laboratoire d’Excellence (LABEX) entitled TULIP (ANR-10-LABX-41).
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