1.14.7
STUDY OF NEMATODE-INDUCIBLE PLANT PROMOTERS IN HAIRY ROOTS OF DIFFERENT PLANT SPECIES

M KARIMI1, E GOELEVEN1, N BARTHELS1, M VAN MONTAGU1 and G GHEYSEN1,2

1Laboratorium voor Genetica, Departement Genetica, Viaams lnteruniversitair lnstituut voor Biotechnologie (VIB), Universiteit Gent, KL Ledeganckstraat 35, B-9000 Gent, Belgium; 2Vakgroep Plantaardige Productie, Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen, Coupure Links 653, B-9000 Gent, Belgium

Background and objectives
Plant-parasitic nematodes are obligate sedentary parasites of higher plants. They are amongst the most destructive pests of agricultural plants worldwide. They infect plants and establish complex feeding sites inside the root tissues. Induction and maintenance of feeding sites are accompanied by changes in host gene expression. Plant-parasitic nematodes are controlled to some extent by using conventional methods. These methods are either not efficient or cause environmental hazards. An alternative and promising approach is making nematode-resistant plants by introducing engineered genes. The identification of plant genes/promoters that are induced by the nematodes is an essential step in this study.

Results and conclusions
Using the promoter-tagging approach [1] and differential screening of cDNA library, we have identified several nematode-inducible plant promoters. After isolation of these promoters, they have been fused to the beta-glucuronidase (gus) marker. We have introduced the promoter-gus fusions into different plant species via Agrobacterium tumefaciens or Agrobacterium rhizogenes. Because plant-parasitic nematodes invade the plant root, we found re-introduction of plant promoters using A. rhizogenes relatively quick and easy. To get a high frequency of transformation and hairy root regeneration, plant media and hormone compositions were optimized. To see whether the presence of the Ri T-DNA changes the expression patterns of the transgenes, hairy roots were produced from some transgenic Arabidopsis lines which harboured nematode inducible-gus fusions. GUS histochemical analyses revealed the same patterns of gus expression as observed in the original lines. Then a number of plant species were transformed using A. rhizogenes pRi 1 5834. In the series of experiments aimed at the production of hairy roots transgenic for a reporter-gus fusion, the A. rhizogenes used additionally harboured a binary T-DNA vector. This vector contained a T-DNA composed of a nematode-inducible promoter-gus fusion and neomycin phosphotransferase II (nptII) gene. Integration of the T-DNAs from the binary vector and the Ri plasmid resulted in the generation of kanamycin-resistant hairy roots. Independent hairy root lines were inoculated with the root-knot nematode Meloidogyne incognita and the cyst nematode Heterodera schachtii. To analyse the expression patterns of the promoters, a gus histochemical assay was performed at different times after inoculation.

Reference
1. Barthels N, van der Lee FM, Klap J et al., 1997. Plant Cell 9, 2119-2134.