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NONEMA
Making plants resistant to plant parasitic nematodes: no access - no feeding

This project will build on preceding EU projects (BI04-CT96-0318, FAIRl-CT95-0905), by generating potato and tomato plants with robust and durable resistance to root knot and cyst nematodes. The resistance will be based on the creation of multiple barriers. As the basic mechanisms of plant-nematode interactions will be studied and disrupted, the outcome of this project will also provide the means to engineer resistance against other plant parasitic nematodes. To increase the chance of public acceptance, the engineered resistance will be highly specific for the target organisms, and the expression of the anti-nematode genes will be reduced to a minimum outside the areas of interest.

Objectives
The objective of this proposal is to identify crucial elements in the interaction between plant parasitic nematodes and plants, and to exploit these elements for engineering broad and durable resistance against root knot and cyst nematodes by creating multiple barriers. Firstly, the nematode will be prevented from migrating to the site where it begins the initiation of a feeding cell. Secondly, if initiation had begun, the nematode will be prevented from forming a feeding cell. Proper targeting will be guaranteed, either by using nematode-responsive elements from available promoters, or sets of promoters with common activity only at the site of interest. Finally, these promoters will be used to drive a very localised expression of multiple nematode-disturbing genes, and the resulting plants will be tested for cyst and root knot nematode resistance.

Results
Within WP 1, the number of putative pathogenicity factors identified from cyst and root knot nematodes was higher than expected. The characterisation of the expression patterns of nematode-affected cell cycle genes is on schedule and this holds true as well for the WP3, the characterisation of plant cell wall-degrading enzymes recruited by nematodes. In WP4, the identification of (sets of) plant promoters that are activated early during syncytium and giant cell induction with minimal expression in other plant tissues is more difficult than expected. Promoter deletion studies are ongoing to see what elements are nematode responsive.
In WP1, Additional cell wall-degrading enzymes and nematode-defense related proteins were identified from root knot and cyst nematodes. Moreover, a family of at least 20 members of a RanBPM-like proteins were cloned from a potato cyst nematode. A protocol was developed that allows for the delivery of dsRNAs to the oesophageal glands of cyst nematodes. The applicability of this protocol for root knot nematodes will be investigated in the near future.
In WP2, Detailed expression profiles of cell cycle genes were generated including CDC7 and WEE1. ARM-1 driven anti-sense inhibition of cell cycle genes resulted in several transgenic plant lines with a reduced infection especially with M. incognita. One of the interesting lines, S266, shows a non-significant reduction of H. schachtii infection as well. However, M.incognita infection is highly significantly lower in this line compared to control lines and this in repeated assays.
WP3 Two cellulases and two expansins were shown to be recruited by cyst nematodes. This phenomenon has been investigated in great detail at the transcriptional (in situ hybridisation) and the translational (immuno-localisation) level. In the meantime, post-transcriptional gene silencing-based tomato transformation experiments have been started to exactly establish the importance of these four protein in syncytium proliferation.
WP4 includes the analysis of promoters identified in the preceding ARENA project ((BIO4-CT96-0318) and the identification of new promoters. Progress was made in both fields. The promoters are currently being studied in considerable detail in potato/ tomato. Resources and PMs that could be redistributed as a result of the withdrawal of P8 were - in part - used to pinpoint the nematode-affected part of the tomato root transcriptome, and corresponding promoters regions will isolated in YR4.
WP5 included the generation of an intra- and an internet site and the generation of additional information packages to communicate scientific results to the general public.

Classified in ARABLE CROPS, HORTICULTURE, GENOMICS

Scientist responsible for the project

Mr HANS HELDER
Binnenhaven 10 Box 8123
6700 ES Wageningen
Netherlands (The) - NL

Phone: +31 317 483136
Fax: +31 317 484254
E-mail: Hans.Helder@wur.nl

References

Project ID QLRT-1999-01501
Organisation Wageningen University - Laboratory of nematology
Area 5.1.1
Start date 01 February 2000
Duration (months) 48
Total cost 3 546 538 €
Total EC contribution   2 978 656 €
Status Completed
Web address of the project   http://nonema.uni-kiel.de/

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