Analysis of nematode-responsive promoters in sugar beet hairy roots

One of the strategies to make crops resistant to the beet cyst nematode Heterodera schachtii is the destruction of the feeding site or syncytium. This can be achieved by local expression of the cytotoxic barnase gene under control of a nematode-inducible plant promoter that is active in the syncytium. Expression of barnase outside the feeding site has to be neutralized by its inhibitor barstar driven from a constitutive promoter that is downregulated in the syncytium. Several promoters that are upregulated in feeding structures were identified using the promoter tagging strategy in Arabidopsis thaliana (Barthels et al., 1997) or by differential cDNA screening in tomato (Van der Eycken et al., 1996). Nematode downregulated promoters in Arabidopsis were described by Goddijn et al. (1993). Five nematode-induced promoters (ARM1, 1164, 728, 25 and Lemmi9) and four downregulated promoters (CaMV35S, the nopaline synthase promoter (nos) and the rooting loci promoters RolC and RolD) fused to the beta-glucuronidase (gus) reporter gene were introduced into sugar beet hairy roots by transformation with Agrobacterium rhizogenes to evaluate their expression pattern. All upregulated promoters were found to be active at the base of lateral roots. The 728 and 25 promoter were as well active in root tips. In the 25-gus lines GUS could also be detected in the vascular tissue, while the ARM1 promoter was also active in sugar beet callus. The Lemmi9 promoter and the 4 constitutive promoters were active in the entire root. The transgenic hairy roots were inoculated with Heterodera schachtii and at different time-points (4, 8, 15, 22 days after inoculation; dpi) GUS analysis was performed on the infected roots. For the ARM1, 1164 and 728 promoter the highest gus expression level in syncytia was observed at 8 dpi. In 4 days old syncytia of the 25-gus lines the intensity of the GUS signal was of the same extent as the non-specific vascular signal. In later stages it even disappeared from the feeding sites. The gus expression level in syncytia of Lemmi9-gus hairy roots was equal to that in control roots. The RolC and 35S promoter were found to be downregulated at 8 dpi, the RolD promoter at 15 dpi and the nos promoter already at 4 dpi.