A series of tobacco mosaic virus (TMV)-based hybrid vectors for transient gene expression were constructed with similar designs but differing in the source of heterologous tobamovirus sequence: Odontoglossum ringspot virus, tobacco mild green mosaic virus variants U2 and U5, tomato mosaic virus, and sunn-hemp mosaic virus. These vectors contained a heterologous coat protein subgenomic mRNA promoter and coat protein open reading frame (ORF) and either TMV or heterologous 3' nontranslated region. The foreign ORF, from the jellyfish green fluorescent protein (GFP) gene, was transcribed from the native TMV coat protein subgenomic mRNA promoter, which extended into the coat protein ORF. The presence of an in-frame stop codon within the GFP mRNA leader and the choice of sequence of GFP ORFs substantially affected translational efficiency. However, the major regulatory component of gene expression in these vectors appeared to be transcriptional rather than translational. There was an inverse relationship between expression of GFP and the heterologous coat protein genes that was reflected in accumulation of the respective mRNAs and proteins. The most effective vector in this series (30B) contained sequences encoding the coat protein subgenomic mRNA promoter, coat protein ORF, and 3' nontranslated region from tobacco mild green mosaic virus U5. Expressed from 30B, GFP accumulated up to 10% of total soluble protein in leaves.
No abstract
Alfalfa mosaic virus (AlMV) coat protein is involved in systemic infection of host plants, and a specific mutation in this gene prevents the virus from moving into the upper uninoculated leaves. The coat protein also is required for different viral functions during early and late infection. To study the role of the coat protein in long-distance movement of AlMV independent of other vital functions during virus infection, we cloned the gene encoding the coat protein of AlMV into a tobacco mosaic virus (TMV)-based vector Av. This vector is deficient in long-distance movement and is limited to locally inoculated leaves because of the lack of native TMV coat protein. Expression of AlMV coat protein, directed by the subgenomic promoter of TMV coat protein in Av, supported systemic infection with the chimeric virus in Nicotiana benthamiana, Nicotiana tabacum MD609, and Spinacia oleracea. The host range of TMV was extended to include spinach as a permissive host. Here we report the alteration of a host range by incorporating genetic determinants from another virus.The interaction between virus and plant proteins determines the capability of the virus to multiply and systemically infect the host plant (1). Systemic infection with plant viruses requires cell-to-cell and long-distance movement of viral genomic RNA (2, 3). Many plant viruses find access into cells through wounds. Upon initial entry into a plant cell, the virus multiplies and moves locally from cell to cell (local infection). In most cases, the transfer of viral RNA between cells is supported by a virus-encoded movement protein(s) (4-7), whereas in some viruses the capsid protein is a primary determinant of cell-to-cell movement (8-10). The movement protein interacts with the plasmodesmata and transfers the viral RNA into a neighboring uninfected cell (11-13). During systemic infection, the virus moves through the vascular system and multiplies in upper uninoculated leaves. Movement into the upper uninoculated tissue is a critical step in the infection by many plant viruses, and prevention of this step can result in significant protection of the host(s) from virus invasion (14). Coat proteins (CPs) of several plant viruses are essential for long-distance movement (15-23). Moreover, some RNA plant viruses, such as tobacco mosaic virus (TMV) (16,17,24), cowpea mosaic virus (25), alfalfa mosaic virus (AlMV) (18), tobacco etch potyvirus (26), and red clover necrotic mosaic virus (27) require functional CP for long-distance movement.Here, to study the role of AlMV CP in long distance movement, we designed a hybrid virus consisting of TMV, the type member of the tobamovirus group, and the CP of AlMV. The TMV genome consists of a single plus-sense RNA (6,395 nt) encapsidated with a 17.5-kDa CP, which results in rodshaped particles (300 nm in length). In addition to CP, TMV has three nonstructural proteins. Proteins (183 and 126 kDa) are translated from genomic RNA and are required for virus replication (28). The 30-kDa protein is a movement protein and pro...
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