Engineering Cowpea Mosaic Virus RNA-2 into a Vector to Express Heterologous Proteins in Plants

AbstractA series of new cowpea mosaic virus (CPMV) RNA-2-based expression vectors were designed. The jellyfish green fluorescent protein (GFP) was introduced between the movement protein (MP) and the large (L) coat protein or downstream of the small (S) coat protein. Release of the GFP inserted between the MP and L proteins was achieved by creating artificial processing sites each side of the insert, either by duplicating the MP-L cleavage site or by introducing a sequence encoding the foot-and-mouth disease virus (FMDV) 2A catalytic peptide. Eight amino acids derived from the C-terminus of the MP and 14–19 amino acids from the N-terminus of the L coat protein were necessary for efficient processing of the artificial Gln/Met sites. Insertion of the FMDV 2A sequence at the C-terminus of the GFP resulted in a genetically stable construct, which produced particles containing about 10 GFP-2A-L fusion proteins. Immunocapture experiments indicated that some of the GFP is present on the virion surface. Direct fusion of GFP to the C-terminus of the S coat protein resulted in a virus which was barely viable. However, when the sequence of GFP was linked to the C-terminus by an active FMDV 2A sequence, a highly infectious construct was obtained

Detection of T-DNA transfer to plant cells by A. tumefaciens virulence mutants using agroinfection

To test whether virulence mutants of Agrobacterium tumefaciens are capable of promoting T-DNA transfer into plant cells, a tandem array of Cauliflower Mosaic Virus (CaMV) DNA was cloned between T-region border sequences on a wide host range plasmid and introduced into various virulence mutants. The resulting strains were used to infect Brassica rapa cv. Just Right. This assay, recently referred to as agroinfection, is based on the appearance of viral symptoms following transfer of T-DNA to plant cells, and is shown to be at least 100 times more sensitive in detecting T-DNA transfer than tumour formation. Mutants in the loci vir A, B and G, which were avirulent on turnip, failed to induce virus symptoms. Of the two vir D mutants tested, neither induced turnouts, but one was capable of inducing virus symptoms. Mutants in vir E, C and F, which induced respectively no, small and normal tumours on turnip, all induced virus symptoms.

N-Acetylglucosamine and Glucosamine-Containing Arabinogalactan Proteins Control Somatic Embryogenesis

In plants, complete embryos can develop not only from the zygote, but also from somatic cells in tissue culture. How somatic cells undergo the change in fate to become embryogenic is largely unknown. Proteins, secreted into the culture medium such as endochitinases and arabinogalactan proteins (AGPs) are required for somatic embryogenesis. Here we show that carrot (Daucus carota) AGPs can contain glucosamine and N-acetyl-d-glucosaminyl and are sensitive to endochitinase cleavage. To determine the relevance of this observation for embryogenesis, an assay was developed based on the enzymatic removal of the cell wall from cultured cells. The resulting protoplasts had a reduced capacity for somatic embryogenesis, which could be partially restored by adding endochitinases to the protoplasts. AGPs from culture medium or from immature seeds could fully restore or even increase embryogenesis. AGPs pretreated with chitinases were more active than untreated molecules and required an intact carbohydrate constituent for activity. AGPs were only capable of promoting embryogenesis from protoplasts in a short period preceding cell wall reformation. Apart from the increase in embryogenesis, AGPs can reinitiate cell division in a subpopulation of otherwise non-dividing protoplasts. These results show that chitinase-modified AGPs are extracellular matrix molecules able to control or maintain plant cell fate