Functional, c-myc-tagged Cf-9 resistance gene products are plasma-membranelocalized and glycosylated

The Cf-9 resistance gene from tomato confers resistance to races of the fun gal pathogen Cladosporium fulvum that express the corresponding avirulence gene, Avr9. Avr9 encodes a secreted peptide. To investigate Cf-9 function, we tagged the Cf-9 protein with a triple myc epitope at either the amino- o r carboxy-terminus of the mature protein. Tobacco plants carrying these con structs activate a defence response to Avr9 peptide. The Cf-9 sequence pred icts a protein of 94 kDa, with 22 glycosylation sites. Using c-myc antibodi es, c-myc : Cf-9 protein was detected as a unique band with a molecular siz e of 160 kDa. The band shifted to approximately 105 kDa after glucosidase t reatment, indicating that Cf-9 protein is highly glycosylated. Plasma membr anes were isolated using two-phase partitioning, and c-myc : Cf-9 was enric hed in these fractions, indicating that Cf-9 is a plasma membrane protein. This was confirmed by silver-enhanced immunogold labelling of tobacco proto plasts carrying the amino-terminal c-myc tag; a higher labelling density wa s observed on the surface of protoplasts derived from c-myc : Cf-9 tobacco compared to untransformed control. The presence of Cf-9 in the plasma membr ane is consistent with its role in conferring recognition of the extracellu lar Avr9 peptide.