THE 5' TERMINAL SEQUENCE OF ALFALFA MOSAIC-VIRUS RNA 3 IS DISPENSABLEFOR REPLICATION AND CONTAINS A DETERMINANT FOR SYMPTOM FORMATION

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 modificati ons on the infectivity of the 35S/cDNA 3 construct to P12 plants was s tudied. When nonviral sequences ranging from 11 to 200 bp were inserte d between the 35S promoter and cDNA 3, the infection became dependent on addition of coat protein (CP) to the inoculum. About 80% of the pro geny RNAs resulting from these infections were full-length and had los t the nonviral sequence, whereas 20% were truncated by a deletion of t he 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 constr uct, 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 induc ed necrotic ringspot-like symptoms on P12 tobacco plants, whereas wt R NA 3 did not induce detectable symptoms on these plants. It is propose d that in the infections with the modified 35S/cDNA 3 clones, CP is re quired in the inoculum to permit internal initiation of plus-strand RN A 3 synthesis on 3'-extended or 3'-truncated minus-strand RNA template s. Evidence was obtained that minus-strand RNA 3 synthesized under the control of the 355 promoter was not infectious to P12 plants. (C) 199 6 Academic Press, Inc.