Over-expression and production of plant allergens by molecular farming strategies.

Recombinant allergens have become a valuable tool for diagnosis and may also be used for therapy in the near future. To supply the required large amounts of functional recombinant proteins on a cost-effective basis, the production of allergens in plants by molecular farming is an alternative to microbial expression systems. Especially as post-translational modifications of the allergens, e.g., phosphorylation and glycosylation, may be important for recognition by the human immune system, the plant-based production of recombinant allergens enables the correct folding, glycosylation, and other modifications of the recombinant allergen. An introduction to the methods for plant transformation via the tumor-inducing bacterium, Agrobacterium tumefaciens, is given in this paper.

Evidence for RNA-mediated defence effects on the accumulation of Potato leafroll virus.

In plants infected with Potato leafroll virus (PLRV), or other luteoviruses, infection is very largely confined to cells in the vascular system. Even in tobacco plants transformed with PLRV full-length cDNA, in which all mesophyll cells should synthesize infectious PLRV RNA transcripts, only a minority of the mesophyll cells accumulate detectable amounts of virus. We have explored this phenomenon further by transforming a better PLRV host, Nicotiana benthamiana, with the same transgene, by superinfecting transformed plants with Potato virus Y and by producing tobacco plants in which cells contained both PLRV cDNA and DNA encoding the P1/HC-Pro genes of the potyvirus Tobacco etch virus. A greater proportion of cells in superinfected plants or in doubly transgenic plants accumulated PLRV than did in singly transgenic tobacco plants. However, most cells in these plants did not accumulate virus. To investigate restriction of the multiplication of viruses containing PLRV sequences, transgenic plants were infected with a chimeric virus that consisted of Tobacco mosaic virus (TMV) containing genes for either the coat protein (CP) of PLRV or jellyfish green fluorescent protein (GFP) in place of the TMV coat protein. The virus that encoded PLRV CP spread more slowly and accumulated less extensively than did the virus that expressed GFP. The results support the suggestion that an RNA-mediated form of resistance that resembles post-transcriptional gene silencing operates in non-vascular cells and may be part of the mechanism that restricts PLRV to vascular tissue in conventionally infected plants.

Transformation of tobacco and potato with cDNA encoding the full-length genome of potato leafroll virus: evidence for a novel virus distribution and host effects on virus multiplication.

A full-length cDNA copy of the genome of Potato leafroll virus (PLRV) was introduced into the genome of tobacco and potato plants by Agrobacterium tumefaciens-mediated transformation. Transgenic lines were obtained in which the transgene was readily detected by PCR with DNA extracted from T(1) tobacco seedlings and clonally multiplied potato plants. PLRV-specific genomic and sub- genomic RNAs, coat protein antigen and virus particles were detected in transgenic plants. Aphids fed on the transgenic tobacco plants readily transmitted PLRV to test plants. Infected transgenic tobacco plants, like non-transgenic (WT) PLRV-infected plants, displayed no symptoms of the infection but transgenic plants of potato were severely stunted. In parallel tests, the mean PLRV titres in WT tobacco plants and transgenic tobacco plants were 600 and 630 ng virus/g leaf, respectively, although differences in PLRV titres among transgenic plants were much greater than those among infected WT plants. In similar tests with potato, the mean PLRV titre of WT plants was 50 ng virus/g leaf whereas higher concentrations (up to 3400 ng virus/g leaf) accumulated in transgenic potato plants. In tissue prints of stems, PLRV was detected in similar proportions of phloem cells in transgenic and infected WT plants. In transgenic tobacco and potato plants, but not in infected WT plants, a few stem epidermal cells also contained virus. From tissue prints of transgenic tobacco leaves, it was estimated that about one in 40000 mesophyll cells contained virus, but in transgenic potato, a greater proportion of mesophyll cells was infected.