5.3.25
IMMUNIZING PLANTS - DEVELOPING SINGLE-CHAIN ANTIBODY FRAGMENTS AGAINST THE CYLINDRICAL INCLUSION PROTEIN OF TAMARILLO MOSAIC POTYVIRUS FOR VIRUS RESISTANCE

D WEBSTER1, P GUY1 and R FORSTER2

1Department of Botany, University of Otago, PO Box 56, Dunedin, New Zealand; 2Hort Research, Private Bag 92169, Auckland, New Zealand

Background and objectives
Potyviruses are the largest group of plant viruses. They infect most of the world's major crop plants, often causing significant yield losses. A number of approaches have resulted in plants resistant to individual potyviruses. However, more than one potyvirus may infect individual plants. Few resistance mechanisms are available that combat a broad spectrum of potyviruses. Used in conjunction with existing control methods, a generic resistance gene may be a valuable front-line or back-up tool in the control of potyviruses. This research aims to obtain resistance to a broad spectrum of potyviruses by investigating the use of antibody fragments against highly conserved proteins to disable virus function.

When expressed in plants, single-chain antibody fragments can reduce and delay symptoms of a tombusvirus infection [1]. This single-chain antibody fragment was specific to the tombusvirus coat protein. We propose to produce single-chain antibody fragments that bind to the potyvirus cylindrical inclusion (Cl) protein. The Cl protein is a replication protein with well characterized helicase properties [2]. Our aim is to find a single-chain antibody fragment that is able to bind to a highly conserved helicase motif, such as the NTP binding domain. Such an antibody fragment has the potential to confer resistance to a broad spectrum of potyviruses. By targeting a functional replication protein rather than structural proteins, we hope to improve the level of resistance that can be obtained using single-chain antibody fragments.

Results and conclusions
The Cl protein of tamarillo mosaic potyvirus was purified from Nicotiana benthamiana and identified by N-terminal peptide sequencing. Pure Cl protein was used to immunize BALB/c mice. Total spleen RNA was extracted and antibody variable genes were amplified and assembled by RT-PCR. The assembled single-chain antibody fragments were then cloned into the phagemid vector pHEN. The resulting combinatorial library was of adequate size and diversity, containing an acceptable proportion of full-size single-chain antibody fragments.

The library is currently being screened for Cl-specific single-chain antibody fragments by phage display. This Cl-specific subset will be analysed for their ability to successfully interfere with the helicase activity of the potyvirus Cl protein. Future research projects will investigate the ability of specific single-chain antibody fragments to generate both individual and broad-spectrum potyvirus resistance.

References
1. Taviadoraki P, Benvenuto E, Trinca S et al., 1993. Nature 366, 469-472.
2. Eagles RM, Balmori-Melian E, Beck D et al., 1994. European Journal of Biochemistry 224, 677-684.