Expression of a β-1,3-glucanase from a biocontrol fungus in transgenic pearl millet

Abstract

Sclerospora graminicola is an oomycete (heterotrophic Stramenopiles), fungal-like obligate phytopathogen, the causal agent of downy mildew in pearl millet (Pennisetum glaucum [L.] R. Br.), and a major constraint in the production of this cereal crop. In this study a hydrolytic enzyme, β-1,3-glucanase (gluc78), from the biocontrol fungus Trichoderma atroviride, was introduced into the genome of a pearl millet breeding line, 842B, by particle bombardment. Constructs were prepared containing the gluc78 gene, encoding the 78 kDa β-1,3-glucanase protein, downstream of either the constitutive ubiquitin promoter or the wound inducible potato proteinase inhibitor IIK gene promoter (pin2). The positive selectable marker gene, manA, encoding mannose-6-phosphate isomerase (phosphomannose isomerase) under the control of the ubiquitin promoter, was used for co-transformation. Transgenic plants were obtained harbouring the manA selectable marker gene and the antifungal gene gluc78 downstream of either the ubiquitin or pin2 promoter. Full constructs or minimal transgene expression cassettes containing the genes of interest were successfully introduced into the genome of pearl millet. Progeny of stably transformed plants, harbouring the gluc78 transgene which is driven by the pin2 promoter and followed by the rice Act1 intron sequences, was subjected to pathogenicity trials. One transgenic event exhibited a reduction of 58% in the incidence of S. graminicola infection, however other transgenic pearl millet events showed no resistance to this phytopathogen. The event conferring decreased susceptibility to S. graminicola had high levels of the glucanase transcript especially in transgenic plants showing higher levels of downy mildew infection.