title: An approximately 400 kDa membrane-associated complex that contains one molecule of the resistance protein Cf-4.
abstract: Despite sharing more than 91% sequence identity, the tomato Cf-4 and Cf-9 proteins discriminate between two Cladosporium-encoded avirulence determinants, Avr4 and Avr9. Comparative studies between Cf-4 and Cf-9 are thus of particular interest. To investigate Cf-4 protein function in initiating defence signalling, we established transgenic tobacco lines and derived cell suspension cultures expressing c-myc-tagged Cf-4. Cf-4:myc encodes a membrane-localized glycoprotein of approximately 145 kDa, which confers recognition of Avr4. Elicitation of Cf-4:myc and Cf-9:myc tobacco cell cultures with Avr4 and Avr9, respectively, triggered the synthesis of active oxygen species and MAP kinase activation. Additionally, an Agrobacterium-mediated transient assay was used to express Cf-4:myc and a newly engineered fusion protein Cf-4:TAP. Both transiently expressed proteins were found to be functional in an in vivo assay, conferring a hypersensitive response (HR) to Avr4. Consistent with previous observations that Cf-9 is present in a protein complex, gel filtration analysis of microsomal fractions solubilized with octylglucoside revealed that epitope-tagged Cf-4 proteins migrated at a molecular mass of 350-475 kDa. Using blue native gel electrophoresis, the molecular size was confirmed to be approximately 400 kDa. Significantly, this complex appeared to contain only one Cf-4 molecule, supporting the idea that, as previously described for Cf-9, additional glycoprotein partners participate with Cf-4 in the perception of the Avr4 protein. Intriguingly, Cf proteins and Clavata2 (CLV2) of Arabidopsis are highly similar in structure, and the molecular mass of Cf-4 and CLV complexes is also very similar (400 and 450 kDa, respectively). However, extensive characterization of the Cf-4 complex revealed essentially identical characteristics to the Cf-9 complex and significant differences from the CLV2 complex.
authors.affiliation: The Sainsbury Laboratory, John Innes Centre, Norwich Research Park, UK.
authors.forename: Susana
authors.initials: S
authors.lastname: Rivas
authors.forename: Tatiana
authors.initials: T
authors.lastname: Mucyn
authors.forename: Harrold A
authors.initials: HA
authors.lastname: van den Burg
authors.forename: Jacques
authors.initials: J
authors.lastname: Vervoort
authors.forename: Jonathan D G
authors.initials: JD
authors.lastname: Jones
mesh_terms: D002478:Cells, Cultured; D002963:Cladosporium / Q000235:genetics / Q000254:growth & development; D015180:Electrophoresis, Gel, Two-Dimensional; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D007113:Immunity, Innate / Q000235:genetics; D018551:Solanum lycopersicum / Q000235:genetics; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008968:Molecular Conformation; D010935:Plant Diseases / Q000382:microbiology; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D030821:Plants, Genetically Modified; D015398:Signal Transduction / Q000235:genetics; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism
chemical_list: C085987:AVR4 protein, Cladosporium fulvum; C109609:Cf protein, Lycopersicon esculentum; D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010940:Plant Proteins; C073605:AVR9 protein, Cladosporium fulvum; D019204:GTP-Binding Proteins