Engineering of Alfalfa mosaic virus RNA 3 into an expression vector

RNA 3 of alfalfa mosaic virus (AMV) encodes the 5'-proximal movement protei n (MP) gene and the 3'-proximal coat protein (CP) gene which is expressed f rom a subgenomic RNA. Several strategies were explored to use this RNA as a vector for expression of the green fluorescent protein (GFP) in Nicotiana tabaccum plants expressing the viral polymerase proteins P1 and P2 (P12 pla nts). Insertion of a subgenomic promoter (sgp)-GFP cassette between the CP gene and the 3'-untranslated region (UTR) interfered with RNA accumulation in protoplasts, indicating that cis-acting sequences required for replicati on were disrupted. When GFP was fused to the N-terminus of MP or CP, the ch imeric RNAs accumulated in protoplasts but cell-to-cell movement in plants was blocked. Insertion of a GFP-sgp cassette immediately upstream of the CP gene caused a hypersensitive host response. However, insertion of a GFP-sg p cassette upstream of the MP gene did not affect symptom formation and yie lded a vector that expressed GFP in inoculated but not in the systemic leav es of both P12 tobacco and non-transgenic N. benthamina plants. When the si ze of the GFP gene was reduced from 700 to 300 nucleotides, virus infection was observed in the noninoculated leaves. Analysis of the progeny of some chimera revealed novel data on replication, encapsidation and recombination of AMV RNA 3.