Synthetic chitinase gene driven by root-specific LjNRT2 and AtNRT2.1 promoters confers resistance to Fusarium oxysporum in transgenic tobacco and tomato

Abstract

Fusarium wilt is a soil-borne disease causing substantial yield losses in various crops and vegetables. We have previously reported the synthetic chitinase (NIC) gene (1.2 kb), in which codon usage of fungus, replaced with that of plant, conferred resistance against Botrytis cinerea. In this study, the NIC or GUS gene was linked to two root-specific promoters, LjNRT2 or AtNRT2.1 (nitrate transporter 2), derived from Lotus japonica and Arabidopsis thaliana, respectively. Transgenic tobacco lines expressing LjNRT2-GUS and LjNRT2-NIC, and tomato lines expressing AtNRT2.1-NIC, were produced by Agrobacterium-mediated transformation. GUS histochemical staining was observed in vascular regions of the roots but was conspicuously absent in the leaves of transgenic plants. Western blot analysis showed the production of NIC proteins in the roots but not in the leaves of transgenic tobacco and tomato lines. These results indicate that LjNRT2 and AtNRT2.1 promoters expressed transgenes in a root-specific manner. When in vitro whole plant resistance assay against Fusarium oxysporum was conducted, transgenic plants showed increased levels of resistance compared to non-transgenic plants. Antifungal activities of the root extract against spore germination of F. oxysporum showed lower CFU (colony-forming unit) than those of the leaf extract. Root colonization assay against F. oxysporum showed much lower CFU values in the roots of transgenic plants than in those of non-transgenic plants. These results suggest that NIC gene triggered by the root-specific promoters successfully expressed only in the roots and conferred increased levels of resistance against the root pathogen, F. oxysporum.