To improve the basic technology to express and target `single chain variable antibody fragments' (scFv) of antibody molecules in plants. This technology offers the possibility to design and alter metabolic routes (catalytic antibodies), to engineer disease resistance and to study plant growth and development by anti-sense like approaches.
-Resistance mediated by intracellularly expressed single chain antibody fragments was obtained against artichoke mottled crinkle virus (AMCV) and tobacco mosaic virus (TMV).
-Full secreted antibodies against TMV also protected Nicotiana tabacum plants.
-Stable expression of scFv's was obtained in different subcellular compartments of plant cells.
MAJOR SCIENTIFIC BREAKTHROUGHS:
-Resistance against pathogens
Resistance against plant viruses by intracellular expression of scFv antibodies against AMCV was shown in the first year of the project and with full size secreted antibodies against TMV in the second year. In both cases the expression levels were relatively high (>0.1% antibodies of total soluble protein). It was recently shown that intracellular expression of scFv's around the detection level (<0.02%) also mediates resistance against plant viruses i.e. TMV. These results show different mechanisms for resistance. Intracellular expression results in interference with both the uncoating of the virus after infection and assembly after replication. Extracellular expression of full size antibodies results in decoration of the virus particles and, thus, infection is prevented.
Proteins secreted from the subventral esophageal gland of the root knot and cyst nematodes are likely to be involved in the induction of feeding cells in the roots of host plants. Various scFv's directed against the subventral gland of the root knot nematode Meloidogyne incognita have been cloned. Transformation is underway and will be followed by screening for resistance.
-Expression of scFv's and subcellular targeting
Adding a KDEL endoplasmic reticulum retention signal to the anti-beta-cutinase 21C5-scFv as well as to several other scFv's resulted in retention in the ER when preceded by a murine k-chain ER translocation signal. When a KDEL retention signal was fused to scFv's lacking an ER translocation signal the expression levels increased from almost undetectable to 0.2%. Preliminary analysis by immunocytolocalization of this phenomenon suggested that in the latter case the scFv's are still in the cytoplasm. The ER targeted version could be found retained in the ER, the cytoplasmic version was not at all detectable although the expression levels in both analysed plants were similar (0.2%).
Furthermore an effect has become apparent of the linker sequence, fusing the two antibody variable domains. The (G4S)3 linker results in polypeptides which are most stable in the cytosol. This is clearly a posttranslational effect as mRNA levels are abundant regardless the linker sequence used.
Secreted full size antibodies as well as Fab fragments could be visualised in the intercellular spaces of leaf mesophyll cells. Single chain fragments to which an KDEL sequence was added were shown to be present in the endoplasmic reticulum.
Funding SchemeCSC - Cost-sharing contracts
6700 AA Wageningen
00060 Santa Maria Di Galeria Roma