An Approximately 400 KDa Membrane-associated Complex That Contains One Molecule of the Resistance Protein Cf-4

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.