title: Cre Reporter Assay for Translocation (CRAfT): a tool for the study of protein translocation into host cells.
abstract: Many pathogenic bacteria introduce virulence proteins, also called effector proteins, into host cells to accomplish infection. Such effector proteins are often translocated into host cells by bacterial type III (T3SS) or type IV secretion systems (T4SS). To better understand the molecular mechanisms underlying virulence, it is essential to identify the effector proteins and determine their functions. Several reporter assays have been established to identify translocated effector proteins and verify T3SS- or T4SS-dependent transport into host cells. Here we describe a protocol to monitor the translocation of candidate effector proteins using Cre recombinase as a reporter, and more specifically how this Cre Reporter Assay for Translocation (CRAfT) can be used to detect translocation of Vir proteins from Agrobacterium tumefaciens into yeast and plant cells. The assay can be adapted for the study of the T3SS or T4SS of human pathogens.
authors.affiliation: Sylvius Laboratory, Institute of Biology Leiden, Leiden University, Sylviusweg 72, Leiden, The Netherlands.
authors.forename: Amke
authors.initials: A
authors.lastname: den Dulk-Ras
authors.forename: Annette C
authors.initials: AC
authors.lastname: Vergunst
authors.forename: Paul J J
authors.initials: PJ
authors.lastname: Hooykaas
mesh_terms: D016960:Agrobacterium tumefaciens / Q000378:metabolism / Q000502:physiology; D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology; D058947:Bacterial Secretion Systems / Q000502:physiology; D006801:Humans; D019426:Integrases / Q000378:metabolism; D021381:Protein Transport / Q000235:genetics / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; C045073:Cre recombinase; D019426:Integrases