Conundrum of the Lack of Defective RNAs (dRNAs) Associated with Tobamovirus Infections: dRNAs That Can Move Are Not Replicated by the Wild-Type Virus; dRNAs That Are Replicated by the Wild-Type Virus Do Not Move

Abstract

Two classes of artificially constructed defective RNAs (dRNAs) of Tobacco mosaic virus (TMV) were examined in planta with helper viruses that expressed one (183 kDa) or both (126 and 183 kDa) of the replicaseassociated proteins. The first class of artificially constructed dRNAs had the helicase and polymerase (POL) domains deleted; the second had an intact 126-kDa protein open reading frame (ORF). Despite extremely high levels of replication in protoplasts, the first class of dRNAs did not accumulate in plants. The dRNAs with an intact 126-kDa protein ORF were replicated at moderate levels in protoplasts and in planta when supported by a TMV mutant that expressed the 183-kDa protein but not the 126-kDa protein (183F). These dRNAs were not supported by helper viruses expressing both replicase-associated proteins. De novo dRNAs were generated in plants infected by 183F but not in plants infected with virus with the wild-type replicase. These novel dRNAs each contained a new stop codon near the location of the wild-type stop codon for the 126-kDa protein and had most of the POL domain deleted. The fact that only dRNAs that contained a complete 126-kDa protein ORF moved systemically suggests that expression of a functional 126-kDa protein or the presence of certain sequences and/or structures within this ORF is required for movement of dRNAs. At least two factors may contribute to the lack of naturally occurring dRNAs in association with wild-type TMV infections: an inability of TMV to support dRNAs that can move in plants and the inability of dRNAs that can be replicated by TMV to move in plants.

Tobacco Mosaic Virus Movement Protein Functions as a Structural Microtubule-Associated Protein

“…Although ER aggregation appears to be correlated with the presence of MP (66,86), the accumulation of MP in these inclusions (42) is not required for replication and vRNA movement (13,69). Nevertheless, evidence for a role of the viral replicase in cell-to-cell movement (44,45,47,54,55) suggests that the processes of virus replication and movement are functionally linked. This view is in agreement with observations implicating large membrane-associated complexes that contain components of the replication machinery in vRNA movement within and between cells (6,51).…”

A Cryohistological Protocol for Preparation of Large Plant Tissue Sections for Screening Intracellular Fluorescent Protein Expression

“…All TMV derivatives were made from the infectious wild-type TMV cDNA clone (22). Cla151, a TMV defective RNA (23), was PCR-amplified to attach a Stu I site to the 5 untranslated region (UTR) and a Kpn I site to the 3 UTR. The amplified fragment was digested with Stu I and Kpn I and inserted into the similarly digested pCASS4Rz plasmid to create pCASSCla151 (E. Knapp, unpublished data), which was used for further cloning.…”

Tobacco mosaic virus, not just a single component virus anymore

“…A TMV mutant expressing the 183 kDa protein, but not the 126 kDa protein, moved cell‐to‐cell and long distances in plants (Fig. 2B), although less efficiently compared to wild‐type TMV (Knapp et al ., 2001; Lewandowski and Dawson, 2000).…”

“…Some dRNAs have been shown to move in plants (Raffo and Dawson, 1991; Wu and Shaw, 1997). However, a series of dRNAs with a truncated replication protein ORF that replicated to high levels in protoplasts failed to move in plants (Knapp et al ., 2001). In contrast, artificially constructed dRNAs encoding a functional 126 kDa protein were replicated by and moved in plants with mutant helper viruses deficient in production of the 126 kDa protein (Knapp et al ., 2001).…”