LAUSR

Protein-protein interactions discovered by yeast two-hybrid systems must be confirmed in vivo in a homologous system. In the case of plants, one of the easiest and fastest methods to validate protein interactions in vivo is the transient expression of the proteins in Nicotiana benthamiana leaves followed by coimmunoprecipitation. This method consists of the following steps: growth of the appropriate Agrobacterium tumefaciens cultures, preparation of the infiltration mixtures, infiltration into N. benthamiana leaves, protein extraction and immunoprecipitation. The utilization of epitope tags to immunoprecipitate and detect the proteins of interest is very useful in this procedure. In this chapter we describe a standard protocol to coimmunoprecipitate proteins expressed in N. benthamiana leaves.