title: Molecular characterization of XopAG effector AvrGf2 from Xanthomonas fuscans ssp. aurantifolii in grapefruit.
abstract: Xanthomonas fuscans ssp. aurantifolii group C strains exhibit host specificity on different citrus species. The strains possess a type III effector, AvrGf2, belonging to the XopAG effector gene family, which restricts host range on citrus. We dissected the modular nature and mode of action of AvrGf2 in grapefruit resistance. XopAG effectors possess characteristic features, such as a chloroplast localization signal, a cyclophilin-binding domain characteristic amino acid sequence motif (GPLL) and a C-terminal domain-containing CLNAxYD. Mutation of GPLL to AASL in AvrGf2 abolished the elicitation of the hypersensitive response (HR), whereas mutation of only the first amino acid to SPLL delayed the HR in grapefruit. Yeast two-hybrid experiments showed strong interaction of AvrGf2 with grapefruit cyclophilin (GfCyp), whereas AvrGf2-SPLL and AvrGf2-AASL mutants showed weak and no interaction, respectively. Molecular modelling and in silico docking studies for the cyclophilin-AvrGf2 interaction predicted the binding of citrus cyclophilins (CsCyp, GfCyp) to hexameric peptides spanning the cyclophilin-binding domain of AvrGf2 and AvrGf2 mutants (VAGPLL, VASPLL and VAAASL) with affinities equivalent to or better than a positive control peptide (YSPSA) previously demonstrated to bind CsCyp. In addition, the C-terminal domain of XopAG family effectors contains a highly conserved motif, CLNAxYD, which was identified to be crucial for the induction of HR based on site-directed mutagenesis (CLNAxYD to CASAxYD). Our results suggest a model in which grapefruit cyclophilin promotes a conformational change in AvrGf2, thereby triggering the resistance response.
authors.affiliation: Citrus Pathology, EEA INTA Bella Vista, CC5 Bella Vista, Corrientes, Argentina 3432.|Department of Plant Pathology, University of Florida, Gainesville, FL, USA 32611.
authors.forename: Alberto M
authors.initials: AM
authors.lastname: Gochez
authors.affiliation: Department of Plant Pathology, University of Florida, Gainesville, FL, USA 32611.
authors.forename: Deepak
authors.initials: D
authors.lastname: Shantharaj
authors.affiliation: Department of Plant Pathology, University of Florida, Gainesville, FL, USA 32611.
authors.forename: Neha
authors.initials: N
authors.lastname: Potnis
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Lake Alfred, FL, USA 33850.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Department of Plant Pathology, University of Florida, Gainesville, FL, USA 32611.
authors.forename: Gerald V
authors.initials: GV
authors.lastname: Minsavage
authors.affiliation: Department of Plant Pathology, University of Florida, Gainesville, FL, USA 32611.
authors.forename: Frank F
authors.initials: FF
authors.lastname: White
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Lake Alfred, FL, USA 33850.
authors.forename: Nian
authors.initials: N
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Physics and Geology, Citrus Research and Education Center, Winthrop University, Rock Hill, SC, USA 29733.
authors.forename: Jason C
authors.initials: JC
authors.lastname: Hurlbert
authors.affiliation: Department of Plant Pathology, University of Florida, Gainesville, FL, USA 32611.
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Jones
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002736:Chloroplasts / Q000378:metabolism; D032083:Citrus paradisi / Q000276:immunology / Q000382:microbiology*; D019295:Computational Biology; D021983:Cyclophilins / Q000378:metabolism; D020868:Gene Silencing; D018613:Microscopy, Confocal; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D000072417:Protein Domains; D021382:Protein Sorting Signals; D016415:Sequence Alignment; D020798:Two-Hybrid System Techniques; D014974:Xanthomonas / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D021983:Cyclophilins
keywords: citrus canker; citrus cyclophilin; citrus resistance; hypersensitive response