Insertion of Epitope Tag into NB-LRR Class Plant Virus Resistance Protein.

It is prerequisite to detect plant disease resistance proteins for studying the function of the proteins. Numerous studies have used epitope tags fused to either N- or C-terminus for the detection of resistance proteins. However, some resistance proteins do not tolerate the terminal fusions of epitope tags. In this chapter, we provide a protocol for searching the protein regions in which the inserted epitope tag does not affect the protein function. In the protocol, we first perform an in silico search to select the insertion site candidates and then insert there a short sequence containing restriction sites to find out the sites, in which the insertion does not affect the protein function. Epitope tags are inserted into the experimentally selected sites to produce a functional protein with an epitope tag.