A Transient Assay System for the Functional Assessment of Defense-Related Genes in Wheat

Abstract

The generation and characterization of transgenic wheat plants is a tedious and time-consuming process that limits the number of putatively important transgenes that can be tested. We therefore established a transient assay system based on wheat leaves to study the effect of transiently expressed genes on the interaction with the wheat powdery mildew fungus Erysiphe (syn. Blumeria) graminis f. sp. tritici. Young wheat leaves were bombarded with tungsten particles coated with a mixture of plasmids carrying the β-glucuronidase (GUS) reporter gene and a test gene. Leaves were subsequently challenge inoculated with E. graminis and the fungus was allowed to develop for 40 h. After being stained for GUS enzymatic activity as well as for epiphytic fungal structures, the phenotype of transformed epidermal cells was evaluated by bright-field microscopy. The fungus was routinely found to penetrate cells transiently expressing GUS with an efficiency of approximately 35%, which should suffice to detect putative transgene effects. Transgenes encoding a low-molecular-weight cell-wall protein of wheat (WIR1), a thaumatin-like protein, and a glucanase had no effect on fungal penetration of transformed epidermal cells. On the other hand, trans-genes encoding a pathogen-induced wheat protein of unknown function (WCI5), a chitinase, a glucose oxidase, and a putative peroxidase significantly reduced fungal penetration.