title: The Cf-9 disease resistance protein is present in an approximately 420-kilodalton heteromultimeric membrane-associated complex at one molecule per complex.
abstract: The tomato Cf-9 gene confers race-specific resistance to the fungal pathogen Cladosporium fulvum expressing the corresponding avirulence gene Avr9. In tobacco, Cf-9 confers a hypersensitive response to the Avr9 peptide. To investigate Cf-9 protein function in initiating defense signaling, we engineered a functional C-terminal fusion of the Cf-9 gene with the TAP (Tandem Affinity Purification) tag. In addition, we established a transient expression assay in Nicotiana benthamiana leaves for the production of functional Cf-9:myc and Cf-9:TAP. Transiently expressed Cf-9:myc and Cf-9:TAP proteins induced an Avr9-dependent hypersensitive response, consistent with previous results with stably transformed tobacco plants and derived cell suspension cultures expressing c-myc-tagged Cf-9. Gel filtration of microsomal fractions solubilized with octylglucoside revealed that the Cf-9 protein, either as c-myc or TAP fusions, migrated at a molecular mass of 350 to 475 kD. By using blue native gel electrophoresis, the molecular size was confirmed to be approximately 420 kD. Our results suggest that only one Cf-9 protein molecule is present in the Cf-9 complex and that Cf-9 is part of a membrane complex consisting of an additional glycoprotein partner(s). The high structural similarity between Cf proteins and Clavata2 (CLV2) of Arabidopsis, together with the similarity of molecular mass between Cf-9 and CLV complexes (420 and 450 kD, respectively), led us to investigate whether Cf-9 is integrated into membrane-associated protein complexes like those formed by CLV1 and CLV2. Unlike CLV2, the Cf-9 protein did not form disulfide-linked heterodimers, no ligand (Avr9)-dependent shift in the molecular mass of the Cf-9 complex was detected, and no Rho-GTPase-related proteins were found associated with Cf-9 under the conditions tested. Thus, Cf-9-dependent defense signaling and CLV2-dependent regulation of meristem development seem to be accomplished via distinct mechanisms, despite the structural similarity of their key components Cf-9 and CLV2.
authors.affiliation: The Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, United Kingdom.
authors.forename: Susana
authors.initials: S
authors.lastname: Rivas
authors.forename: Tina
authors.initials: T
authors.lastname: Romeis
authors.forename: Jonathan D G
authors.initials: JD
authors.lastname: Jones
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D029681:Arabidopsis Proteins; D002352:Carrier Proteins / Q000378:metabolism; D002850:Chromatography, Gel; D002963:Cladosporium / Q000235:genetics / Q000254:growth & development* / Q000472:pathogenicity; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics; D015870:Gene Expression; D006160:Guanosine Triphosphate / Q000378:metabolism; D007113:Immunity, Innate / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D014774:Virulence / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; C401621:CLV2 protein, Arabidopsis; D002352:Carrier Proteins; D004220:Disulfides; D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010940:Plant Proteins; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; C073605:AVR9 protein, Cladosporium fulvum; D006160:Guanosine Triphosphate