A Spontaneous Red Clover Necrotic Mosaic Virus Mutant with a Truncated Movement Protein

Abstract

A spontaneous red clover necrotic mosaic virus mutant, TpM-341, was isolated by multiple passage of Czechoslovakian isolate TpM-34 in beans, followed by three cycles of single lesion isolation in cowpea and in Chenopodium quinoa. The symptoms induced in cowpea by TpM-34 and TpM-341 differed. TpM-34 gave rise to chlorotic lesions which expanded with time, often becoming confluent with adjacent lesions, and developed necrotic margins; the plants became systemically infected. TpM-341 induced necrotic lesions which, once developed, did not expand further; plants did not become systemically infected. Analysis of pseudorecombinants formed between the RNAs of TpM-34 and TpM-341 showed that RNA 2 determined the difference in symptoms. Comparison of the nucleotide sequence of the open reading frames (ORFs) encoding the P2 movement proteins of the two isolates revealed only one difference, a deletion of an A residue in a sequence of four A residues (nucleotides 790 to 793). Construction of full-length TpM-34 and TpM-341 RNA 2 cDNA clones, from which infectious RNA 2 could be transcribed in vitro, and in vitro mutagenesis of a cDNA clone of TpM-341, confirmed that the difference in the symptoms induced by TpM-341 was caused by the loss of this A residue. This single base deletion was predicted to cause a translational frameshift in the P20RF causing the loss of 88 amino acids at the C terminus which were replaced by a sequence of 34 different amino acids, producing a truncated P2 protein. In vitro translation ofRNA 2 transcribed from cDNA clones showed that RNA with four or three A residues starting at nucleotide 790 produced proteins of Mr 36K and 30K respectively, in agreement with the predictions based on the nucleotide sequence.

Spontaneous deletion enhances movement of a cucumber necrosis virus based chimera expressing the red clover necrotic mosaic virus movement protein gene†

“…, 1997). Osman et al . (1991) have previously shown that a naturally arising RCNMV mutant, TpM‐341, was capable of cell‐to‐cell and systemic movement in cowpea after deletion of the C‐terminal 88 amino acids of the MP.…”

“…Local lesions were produced on cowpea, but were smaller than those produced by either CNV or RCNMV. The overall similarity in disease symptoms between ClM5/RM2dd1 and RCNMV supports the notion that the RCNMV MP contains symptom determinants (Osman et al. , 1991; Solovyev et al.…”

“…Osman et al . (1991) reported the spontaneous occurrence of an RCNMV mutant (TpM‐341) that produced a truncated MP lacking a large portion (88 amino acids) of the carboxyl‐terminus.…”

“…These chimeras are capable of both cell‐to‐cell and systemic movement in N. benthamiana and therefore we conclude that the terminal 103 amino acids of the RCNMV MP are not required for local or systemic movement of chimeric virus in N. benthamiana . Studies in cowpea showed that ClM5/RM2dd1 produces small necrotic lesions (Table 1) similar to those observed by Osman et al . (1991) with TpM‐341.…”

“…In vivo , a high order of specificity exists but the RCNMV MP is still capable of supporting the movement of a number of taxonomically diverse plant viruses (Giesman-Cookmeyer et al , 1995;Morozov et al , 1997;Rao et al , 1998;Solovyev et al , 1997). Osman et al . (1991) have previously shown that a naturally arising RCNMV mutant, TpM-341, was capable of cell-to-cell and systemic movement in cowpea after deletion of the C-terminal 88 amino acids of the MP.…”