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Engineering and evaluation of cyst nematode resistance in sugarbeet

Contract nr: FAIR-CT96-1714
Project nr: 1714
Project type: SC
Starting date: 01/03/1997
Duration: 48 months
Total cost: 1,393,319 EUR
EC Contribution: 1,215,000 EUR
Scientific Officer: Richard HARDWICK
Research topic: Plant health
Acronym: NEMATODE RESISTANT SUGARBEET

Background:
The cyst nematode Heterodera schachtii is a major sugarbeet crop pest 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 in the next few years. Therefore, the introduction of cyst nematode resistance into sugarbeet is a priority target for the sugarbeet companies.

Objectives:
Since interspecific crossing-in of 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 selectively destroying 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 that is more or less specifically active in the feeding site, and a cytotoxic gene.

Description:
Several plant promoters that are activated (or down-regulated) in the nematode feeding sites (NFS) have been identified. These promoters will be tested in sugarbeet by fusion to a reporter gene and analysis of their expression in transgenic roots. This will allow a confirmation that 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 another tissue resulting in the engineering of male sterility in e.g. oilseed rape 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 carried out on A. rhizogenes-transformed sugarbeet root cultures, that will allow a rapid selection of the chimeric gene combinations that confer nematode resistance. 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. Finally, on the basis of the obtained data, optimised constructs will be transformed into sugarbeet and the resulting transgenic plants will be evaluated for nematode resistance in vitro and in the greenhouse.

Current situation/results:
Promoters that are up- or downregulated in feeding sites induced by sedentary nematodes have been isolated. Most of these promoters have been identified in the model system A. thaliana/H. schachtii and one in the tomato/M. incognita interaction. To determine if these promoters retain their tissue-specificity and responsiveness to nematode infection in the sugarbeet-H. schachtii interaction, chimeric gus constructs have been made and introduced into sugarbeet hairy roots. The following nematode-responsive promoters have been used:

  • the tomato Lemmi9 promoter, upregulated in NFS;
  • the ARM1 promoter from the A. thaliana line Att0001, the promoter from the A. thaliana line Att1164 and a promoter from the line Att0025, all three upregulated in NFS;
  • the RolC and RolD promoters from Agrobacterium rhizogenes and the A. tumefaciens nopaline synthase (nos) promoter, downregulated in NFS;
  • the 35S promoter, downregulated in NFS.

A detailed GUS-analysis (including sections) has been performed on the sugarbeet root cultures transformed with these constructs, at several time points after infection with stage 2 juveniles of H. schachtii. The results indicate that ARM1 and Att1164 are upregulated in syncytia and the nos promoter is quickly down-regulated. The 35S, RolC and RolD promoters are down-regulated to a lesser extent or at a later time point. For the other up-regulated promoters, more data are needed to draw a conclusion.
Molecular analysis of the promoters that are upregulated in NFS has started with the identification (and modification) of discrete sequences that are specific to nematode induction. Some of these promoters have been analysed in other better understood plant-nematode systems. We have shown that a DNA fragment containing the proximal part of the Lemmi9 promoter (ca. 200 bp) specifically binds a protein from infected tomato roots. In vivo footprinting assays in infected tomato roots have identified two possible sequences in this 100 fragment. Further experiments with synthetic oligonucleotides confirmed the involvement of one of the elements in the protein binding activity. Functional assays will determine its possible role in nematode-induced gene expression, and hence its potential for engineering resistance in sugarbeet. In the ARM1 and Att1164 promoters, regions that are most likely involved in transcription regulation have also been identified by in vivo footprinting. Thus far, constructs with deletions of the Lemmi9, ARM1, Att1164 and Att0025 promoters have been made and transformed (Arabidopsis) plants have been analysed.
The four up-regulated promoters have been cloned in barnase/barstar constructs. As a first test system, these constructs have been introduced into sugarbeet hairy roots. It is well established that nematodes can infect and propagate on sugarbeet hairy roots, furthermore A. rhizogenes transformation was used to confirm by complementation the cloning of the nematode resistance gene Hs1 from Beta procumbens. Nevertheless, in our first resistance tests we have seen high variability in susceptibility to nematode infection, even between hairy root lines without NR construct. Therefore, in collaboration with SES Tienen, part of the constructs have been introduced into sugarbeet and transgenic lines are ready to be analysed now.

Website: http://www.plantgenetics.rug.ac.be/public/research/groups/lighe/fair.html


Coordinator
Godelieve GHEYSEN
Flanders Interuniversity
Institute for Biotechnology vzw
K.L. Ledeganckstraat 35
B-9000 Gent
Tel.: +32 9 264 51 82
Fax: +32 9 264 53 49
E-mail: lighe@gengenp.rug.ac.be


Partners

  • Carmen FENOLL
    Universidad Autonoma de Madrid
    Cantoblanco
    E-28049 Madrid
    Tel.: +34 91 397 81 98
    Fax: +34 91 397 83 44
    E-mail: Carmen.Fenoll@uam.es

  • Florian M.W. GRUNDLER
    Christian-Albrechts-Universität zu Kiel
    Hermann-Rodewald Straße 9
    D-24118 Kiel
    Tel.: +49 431 880 46 69
    Fax: +49 431 880 15 83
    E-mail: fgrundler@phytomed.uni-kiel.de

  • Stephan Andreas OHL
    Zeneca Mogen
    Einsteinweg 97
    NL-2333 CB Leiden
    Tel.: +31 715 25 82 82
    Fax: +31 715 22 14 71
    E-mail: Stephan.Ohl@AGEUROPE.zeneca.com
 
 
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