Functional, c‐myc‐tagged Cf‐9 resistance gene products are plasma‐membrane localized and glycosylated

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Abstract
Summary: 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.