The cyst nematode Heterodera schachtii is an important pest for the sugarbeet crop in Europe.
Very few effective nematicides are on the market and increasing environmental concerns will most probably result in restricting these toxic chemicals in Europe by the end of the century. Therefore, the introduction of cyst nematode resistance into @ugarbeet is a priority target for the sugarbeet companies. Since interspecific crossing-in natural resistance genes causes tremendous problems in sugarbeet, we propose the design and evaluation of a novel genetic basis for nematode resistance in plants.
The proposed strategy for genetic engineering resistance to cyst nematodes in sugarbeet aims at destroying selectively the nematode-induced feeding sites (syncytia) in sugarbeet roots. Sedentary nematodes (cyst and root knot nematodes) are absolutely dependent on the formation and maintenance of plant feeding sites. The strategy requires a chimeric construct consisting of a nematode-induced plant promoter, more or less specifically active in the feeding site, and a cytotoxic gene.
Several plant promoters that are activated (or down-regulated) in the feeding sites have been identified (in Arabidopsis, tomato and potato). These promoters will be tested by fusion to a reporter gene and analysis of their expression in transgenic sugarbeet roots. This will allow to confirm if the isolated promoters are strongly induced (or down-regulated) in the feeding sites of H. schachtii in sugarbeet roots.
As a cytotoxic gene, the barnase gene, a natural ribonuclease gene, will be fused to the nematode-induced promoters. A second chimeric gene containing a promoter which is down-regulated fused to the barstar (an inhibitor of barnase) gene will be used to inactivate unwanted expression of the barnase gene outside of the feeding structure. The barnase/barstar system has been successfully applied for the selective destruction of anther tissue resulting in the engineering of male sterility in oilseed rape, tobacco and corn.
The proposed project encompasses the cloning and transformation into sugarbeet of the corresponding "nematode resistance" (NR) constructs using the promoters which are induced or down-regulated in syncytia. A first nematological evaluation will be done on Agrobacterium rhizogenes-transformed sugarbeet root cultures that will allow a rapid selection of those chimeric gene combinations that confer nematode resistance. The effect of the engineered resistance will be studied on a cellular level using electron microscopy.
The work will be complemented by detailed molecular and functional analyses of the induced promoters to allow the design of an improved construct that has more specific feeding-site expression and less background expression in other tissues.
Finally, on basis of the obtained data, optimized constructs will be transformed into sugarbeet and the resulting transgenic plants will be evaluated for nematode resistance in vitro and in the greenhouse.
Funding SchemeCSC - Cost-sharing contracts
2333 CB Leiden