5.3.6S
GENETICALLY ENGINEERED RESISTANCE TO NEMATODES

HJ ATKINSON, PE URWIN, CJ LILLEY and MJ McPHERSON

Centre for Plant Biochemistry and Biotechnology, University of Leeds, Leeds LS2 9JT, UK

Background and objectives
Nematodes are often controlled by environmentally unacceptable crop protection chemicals. Transgenic resistance offers a new and powerful means of lessening this dependence on nematicides. Approaches centred on modifying natural resistance and on introducing other foreign genes have been demonstrated. Progress towards effective resistance with the latter approach will be reviewed for both developed and developing world crops.

Results and conclusions
Transgenic resistance requires a gene promoter that provides an appropriate expression pattern plus a novel effector that disrupts some aspect of the host-parasite interaction. The required pattern of promoter activity depends upon the design of transgenic resistance that is envisaged. Key promoter groupings include those that respond to wounding of cells or to their modification by nematodes. A third distinct promoter group is characterized by extensive root-specific activity. They underpin design of a standing defence against nematodes. The different types of promoters have been defined following differential library screening, gene tagging and by research in other fields [1].

Many nematodes modify plant cells into feeding sites that are essential for their prolonged, biotrophic interaction with the host. Disrupting this process is an attractive target for nematode control. Proof of principle demonstrations have been achieved using feeding cell-specific promoters plus either antisense RNA or an RNase that attenuates or destroys the pathogen-modified plant cells. Success requires avoidance of unwanted cell death due to leaky promoters that may cause intolerance or other harmful consequences for the plant. This can be overcome in several ways including use of a bipartite effector. In this case, each component is under control of a distinct promoter with overlapping activity in feeding cells only. Ideally, anti-feeding cell approaches provide a highly specific defence activated only by those nematodes that induce activity of the responsive promoter.

A second basis for transgenic resistance offers the potential of a generic defence against a wide range of nematodes. It involves expression of anti-nematode proteins in plants. In this case, highly specific promoter activity is unimportant and root-specific promoters can provide a standing defence against a wide range of nematodes. Hairy root transformation using Agrobacterium rhizogenes provides a rapid basis for preliminary evaluation of potential anti-nematode proteins. Such proteins can be identified on a rational basis from other work including study of Caenorhabditis elegans. The oral route for uptake is favoured, but the maximum molecular size of the anti-nematode protein is limited by the pathogen's feeding tube. This structure is secreted into a plant cell and feeding involves fluid uptake through its walls. Progress on a proteinase inhibitor (Pi)-based defence demonstrates key steps in development of this approach. The proteinases of targeted nematodes have been cloned and characterized to underpin protein engineering of Pis with enhanced efficacy against targeted nematodes. Differences in proteinase expression during parasitic development correlate with stage-specific effects of Pis on cyst nematodes. Meloidogyne and cyst nematodes have both been controlled by one Pi [2]. These results indicate a simple and effective approach to nematode control. Durable resistance is required to enable molecular biologists to keep pace with resistance-breaking abilities of nematode populations. Durability is favoured by combining effects of different effectors. Additive resistance has been achieved and a novel linker technology has been developed to enhance delivery of effectors. Adapting durable and safe technology to developing world needs for royalty free-distribution in staple crops will help lessen the high impact of nematodes in subsistence agriculture.

References
1. Atkinson HJ, Urwin PE, Hansen, E, McPherson MJ, 1995. Trends in Biotechnology 13, 369-374.
2. Urwin PE, Lilley CJ, McPherson MJ, Atkinson HJ, 1997. Plant Journal 12, 455-461.