Functional, c-myc-tagged Cf-9 resistance gene products are plasma-membrane localized and glycosylated.
The Cf-9 resistance gene from tomato confers resistance to races of the fungal 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- or carboxy-terminus of the mature protein. Tobacco plants carrying these constructs activate a defence response to Avr9 peptide. The Cf-9 sequence predicts a protein of 94 kDa, with 22 glycosylation sites. Using c-myc antibodies, c-myc : Cf-9 protein was detected as a unique band with a molecular size of 160 kDa. The band shifted to approximately 105 kDa after glucosidase treatment, indicating that Cf-9 protein is highly glycosylated. Plasma membranes were isolated using two-phase partitioning, and c-myc : Cf-9 was enriched in these fractions, indicating that Cf-9 is a plasma membrane protein. This was confirmed by silver-enhanced immunogold labelling of tobacco protoplasts carrying the amino-terminal c-myc tag; a higher labelling density was 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 membrane is consistent with its role in conferring recognition of the extracellular Avr9 peptide.
Piedras
P
P
The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK.
Rivas
S
S
Dröge
S
S
Hillmer
S
S
Jones
J D
JD
D000483:Alleles; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000378:metabolism; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D010947:Plants, Toxic; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics
D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D010940:Plant Proteins; D016271:Proto-Oncogene Proteins c-myc; C073605:AVR9 protein, Cladosporium fulvum