Identification of Arabidopsis proteins that interact with the cauliflower mosaic virus (CaMV) movement protein

Gene I of cauliflower mosaic virus (CaMV) encodes a protein that is require d for virus movement. The CaMV movement protein (MP) was used in a yeast 2- hybrid system to screen an Arabidopsis cDNA library for cDNAs encoding MP-i nteracting (MPI) proteins. Three different clones were found encoding prote ins (MPI1, -2 and -7) that interact with the N-terminal third of the CaMV M P. The interaction in the 2-hybrid system between MPI7 and CaMV MP mutants correlated with the infectivity of the mutants. A non-infectious MP mutant, ER2A, with two amino acid changes in the N-terminal third of the MP failed to interact with MPI7, while an infectious second-site mutant, that differ ed from ER2A by only a single amino acid change, interacted in the 2-hybrid system. MPI7 is encoded by a member of a large, but diverse gene family in Arabidopsis. MPI7 is related in sequence, size and hydropathy profile to m ammalian proteins (such as rat PRA1) described as a rab acceptor. The gene encoding MPI7 is expressed widely is Arabidopsis plants, and in transgenic plants the MPI7:GFP fusion protein is localized in the cytoplasm, concentra ted in punctate spots. In protoplasts transfected with CFP:MP and MPI7:YFP, CFP:MP colocalized to some of the sites where MPI7:YFP is expressed. At th ese sites, fluorescence resonance energy transfer (FRET) between fluorophor es was observed indicating an interaction in planta between the CaMV MP and MPI7.