Virus-derived ssDNA vectors for the expression of foreign proteins in plants.

Plant viruses with ssRNA genomes provide a unique opportunity for generating expression vehicles for biopharming in plants, as constructs containing only the replication origin, with the replication-associated protein (Rep) gene provided in cis or in trans, can be replicationally amplified in vivo by several orders of magnitude, with significant accompanying increases in transcription and expression of gene(s) of interest. Appropriate replicating vectors or replicons may be derived from several different generic geminiviruses (family Geminiviridae) or nanoviruses (family Nanoviridae), for potential expression of a wide range of single or even multiple products in a wide range of plant families. The use of vacuum or other infiltration of whole plants by Agrobacterium tumefaciens suspensions has allowed the development of a set of expression vectors that rival the deconstructed RNA virus vectors in their yield and application, with some potential advantages over the latter that still need to be explored. Several modern applications of ssDNA plant vectors and their future potential will be discussed.

Identification of long intergenic region sequences involved in maize streak virus replication.

The main cis-acting control regions for replication of the single-stranded DNA genome of maize streak virus (MSV) are believed to reside within an approximately 310 nt long intergenic region (LIR). However, neither the minimum LIR sequence required nor the sequence determinants of replication specificity have been determined experimentally. There are iterated sequences, or iterons, both within the conserved inverted-repeat sequences with the potential to form a stem-loop structure at the origin of virion-strand replication, and upstream of the rep gene TATA box (the rep-proximal iteron or RPI). Based on experimental analyses of similar iterons in viruses from other geminivirus genera and their proximity to known Rep-binding sites in the distantly related mastrevirus wheat dwarf virus, it has been hypothesized that the iterons may be Rep-binding and/or -recognition sequences. Here, a series of LIR deletion mutants was used to define the upper bounds of the LIR sequence required for replication. After identifying MSV strains and distinct mastreviruses with incompatible replication-specificity determinants (RSDs), LIR chimaeras were used to map the primary MSV RSD to a 67 nt sequence containing the RPI. Although the results generally support the prevailing hypothesis that MSV iterons are functional analogues of those found in other geminivirus genera, it is demonstrated that neither the inverted-repeat nor RPI sequences are absolute determinants of replication specificity. Moreover, widely divergent mastreviruses can trans-replicate one another. These results also suggest that sequences in the 67 nt region surrounding the RPI interact in a sequence-specific manner with those of the inverted repeat.