Summary

Introduction Plant pathogenic nematodes form one of the most serious threats to modern agricultural practice. Almost all major crop species can be attacked by nematodes, and the world wide losses caused by these pathogens exceed one hundred billion US dollars every year.

The most severe damage is inflicted by the root-knot nematodes (Meloidogyne spp.) and the cyst nematodes (Heterodera and Globodera spp.), which penetrate the roots of the host plants and establish a parasitic relationship with the plant. While penetrating the plant, the nematodes cause considerable damage to the roots which leads to a malfunctioning of the entire root system. This in turn leads to a poor performance of the crop plants in the fields. There are not many possibilities to solve the nematode problem. Applying a wide interval crop rotation in which the attacked crop is only grown once in five years will keep the nematode populations in the soil at a relatively low level, but this solution is not feasible from an economic point of view. An other solution is the chemical control of the nematode populations, and at present each year more than 29,000 tons of nematicides (active compounds) are applied by the European farmers. These nematicides, especially methylbromide, belong to the severest classes of pesticides that are in use in agriculture, and many countries will ban these chemicals or will strictly reduce the use of nematicides due to environmental concerns.

The very best solution would be the growing of nematode resistant crop plants. In general, nematode resistance can be found in many wild relatives of crop plants, but is not present in the crops themselves. The introduction of nematode resistance genes into the crop species by classical breeding probably will take decades to accomplish. A much quicker method to supply crop plants with nematode resistance would be the introduction of resistance genes by genetic engineering. However, at the time of the project was initiated, no nematode resistance gene had been isolated from any plant species.

Objectives The cloning of such a resistance gene was the principle objective of the project. Further aims of the project were the construction of nematode resistant crop plants by the introduction of a cloned resistance gene, and the detailed analysis of such a gene to broaden understanding of how plants defend themselves against plant pathogenic nematodes.

Activities The nematode resistance gene that was aimed at in this research project is the Hs1 gene from the wild beet species Beta procumbens. This gene is known to confer full resistance to the beet cyst nematode Heterodera schachtii. As starting material, sugar beet plant B883 was used which harbours a large piece of B. procumbens DNA on which the Hs1 gene is located. (This plant itself is not very useful for sugar beet breeding as the nematode resistance trait is frequently lost during crossings).

The strategy that was followed to clone the Hs1 gene is referred to as "map based cloning" For this, first a detailed map of the B. procumbens DNA in plant B883 was constructed. This map was subsequently used for the cloning of large pieces of the B. procumbens DNA in so-called YAC-vectors. The resulting YAC clones were transferred to the yeast Saccharomyces cerevisiea, where they were analysed one by one for the presence of potential nematode resistance genes. Identified candidate nematode resistance genes were transferred to the roots of sugar beet plants. Next, these roots were infected with the beet cyst nematode and the infection of the roots by the nematodes was assayed. In this way one gene was identified, the Hs1 gene, that rendered the otherwise susceptible sugar beet roots completely resistant to nematode infection. Transgenic sugar beet plants were tested in the greenhouse, and plants were identified showing high levels of nematode resistance under greenhouse conditions.

The analysis of the Hs1 gene showed that this gene is a novel gene with little resemblance to any plant gene cloned before. The resistance gene was transferred to Arabidopsis thaliana and showed, also in this heterologous background, to be functional against the beet cyst nematode H. schachtii. However, when the Hs1 gene was transferred to potato, no resistance against the potato cyst nematode Globodera pallida was obtained. This shows that the nematicidal activity of the Hs1 gene probably is confined to the beet cyst nematode only.

A detailed genetic analysis showed that at least three additional nematode resistance genes are present in the wild beet B. procumbens. Furthermore, genes resembling the Hs1 gene were found in the wild beet species B. patellaris and B. webbiana, in susceptible sugar beet and in A. thaliana.

Exploitation The analysis of the Hs1 gene and the locus that holds this gene has yielded valuable scientific data which have been published in thirteen articles in international scientific journals. Furthermore, the work has been presented in over 25 presentations in various international and national scientific meetings. Also, the outcome of this research project has been communicated to the end-users of the results, such as European farmers and plant breeders, in more than 10 informational publications. The intellectual property rights on the outcome of the project have been secured by the filing of an international patent application. In addition, an exploitation plan is currently executed by which major European breeding companies, as well as the international scientific community, are supplied with the Hs1 resistance gene and other relevant output from the project.

Deliverables The deliverables resulting from this project are in full accordance with the expected deliverables as formulated in the Technical Annex:

• DNA-constructs containing the cloned beet cyst nematode resistance gene Hs1^pro-1
• nematode resistant sugar beet
• insight in the working mechanism of nematode resistance in plants

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Third Consolidated Report

The principal aim of the project is the isolation of a gene from one of the wild beet species B. procumbens or B. patellans conferring resistance against the beet cyst nematode Heterodera schachtii and the introduction of this gene into sugar beet and rape seed in order to obtain nematode resistant varieties

The strategy which is followed to clone the resistance gene is referred to as "map-based cloning". In this strategy, four different phased can be discerned: First, molecular markers closely linked to the gene of interest must be obtained. Then, in the second phase, genetic maps and detailed physical maps of the locus encompassing the resistance gene must be constructed, which can be used in chromosome walking towards the gene. This chromosome walking, which is carried out in the third phase, is accomplished by assembling YAC (Yeast Artificial Chromosome) contigs which connect the molecular markers linked to the gene. In the final phase, candidate genes are isolated from the YAC clones spanning the nematode resistance locus. These genes can be tested in plants for nematocidal activity, which will result in the identification of the resistance gene.

During the period covered by this report research was focused on chromosome walking and on the identification of candidate resistance genes. The chromosome walking resulted in the isolation of 4 novel YAC clones, so that at present approximately 1 Mb of DNA from the nematode resistance locus Hs1(pro-1) has been cloned in a total of 10 YACs. These YACs are arranged into 4 unlinked contigs and cover an estimated two-third of the resistance locus as present in the nematode resistant B. procumbens translocation lines A906001, AN1-65-2 and B883. By using the cloned ends of the various YAC clones, the two different maps that were constructed previously could be integrated into a single comprehensive map of the Hs1(pro-1) locus.

The experiments aiming at the isolation of candidate resistance genes yielded a large number of genes, isolated from resistant sugar beet lines, that show a high homology to plant resistance genes conferring resistance against pathogenic fungi, bacteria and viruses. However, it remains to be proven that the nematode resistance gene is amongst these isolated genes.

A very promising candidate nematode resistance gene was isolated from one of the isolated YAC clones. This gene, referred to as 1832, was shown to confer full nematode resistance when expressed under the control of the CAMV 35S promoter in hairy roots of susceptible sugar beet. Since nematode resistant sugar beet plants are available which lack the 1832 gene, it must be concluded that probably more than one gene conferring nematode resistance can be found in the Hs1(pro-1) locus as present in the B. procumbens translocation lines.