The 5' terminal sequence of alfalfa mosaic virus RNA 3 is dispensable for replication and contains a determinant for symptom formation

Abstract

Transgenic P12 tobacco plants, transformed with the replicase genes P1 and P2 of alfalfa mosaic virus (AIMV), can be infected with RNA 3 of the tripartitite AIMV genome or with a DNA copy of RNA 3 fused to the CaMV 35S promoter and nos terminator. The effect of various modifications on the infectivity of the 35S/cDNA 3 construct to P12 plants was studied. When nonviral sequences ranging from 11 to 200 bp were inserted between the 35S promoter and cDNA 3, the infection became dependent on addition of coat protein (CP) to the inoculum. About 80% of the progeny RNAs resulting from these infections were full-length and had lost the nonviral sequence, whereas 20% were truncated by a deletion of the 5' terminal 79 nucleotides (nt). When the sequence corresponding to the 5' terminal 22 nt of RNA 3 was deleted from the 35S/cDNA 3 construct, the clone was as infectious as the wild type (wt), provided that CP was added to the inoculum, but only progeny RNA with a 5' terminal deletion of 79 nt was produced. The 5' truncated RNA 3 molecules induced necrotic ringspot-like symptoms on P12 tobacco plants, whereas wt RNA 3 did not induce detectable symptoms on these plants. It is proposed that in the infections with the modified 35S/cDNA 3 clones, CP is required in the inoculum to permit internal initiation of plus-strand RNA 3 synthesis on 3'-extended or 3'-truncated minus-strand RNA templates. Evidence was obtained that minus-strand RNA 3 synthesized under the control of the 35S promoter was not infectious to P12 plants.