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.

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Summary Double‐stranded RNA (dsRNA) has been shown to specifically interfere with gene function in several organisms including tobacco and the model plant Arabidopsis. Here, we report on rapid and sequence‐specific interference of dsRNA with gene function in cereals. Delivery of cognate dsRNA into single epidermal cells of maize, barley or wheat by particle bombardment interfered with the function of co‐bombarded UidA (GUS) and TaGLP2a::GFP reporter genes. Cognate dsRNA was also found to specifically interfere with the function of the endogenous genes A1 and Ant18 encoding dihydroflavonol‐4‐reductase in maize and barley, respectively. Dihydroflavonol‐4‐reductase is an essential enzyme of the anthocyanin biosynthetic pathway in maize and barley. This pathway can be induced by transient expression of the C1‐ and b‐Peru genes that encode transcription factors. In the presence of dsRNA corresponding to the dihydroflavonol‐4‐reductase gene, C1‐ and b‐Peru‐dependent, cell‐autonomous accumulation of red anthocyanin pigments in bombarded cells of maize and barley was reduced. dsRNA was also demonstrated to negatively interfere with Mlo, which encodes a negative regulator of race non‐specific resistance to the powdery mildew fungus in barley. In the presence of Mlo dsRNA, transformed cells became more resistant, thereby phenocopying plants that carry a heritable loss‐of function mlo resistance allele. The results suggest that direct delivery of dsRNA to cereals leads to a rapid and sequence‐specific interference with gene function at the single‐cell level.

Double-stranded RNA (dsRNA) has been shown to specifically interfere with gene function in several organisms including tobacco and the model plant Arabidopsis. Here, we report on rapid and sequence-specific interference of dsRNA with gene function in cereals. Delivery of cognate dsRNA into single epidermal cells of maize, barley or wheat by particle bombardment interfered with the function of co-bombarded UidA (GUS) and TaGLP2a:GFP reporter genes. Cognate dsRNA was also found to specifically interfere with the function of the endogenous genes A1 and Ant18 encoding dihydroflavonol-4-reductase in maize and barley, respectively. Dihydroflavonol-4-reductase is an essential enzyme of the anthocyanin biosynthetic pathway in maize and barley. This pathway can be induced by transient expression of the C1- and b-Peru genes that encode transcription factors. In the presence of dsRNA corresponding to the dihydroflavonol-4-reductase gene, C1- and b-Peru-dependent, cell-autonomous accumulation of red anthocyanin pigments in bombarded cells of maize and barley was reduced. dsRNA was also demonstrated to negatively interfere with Mlo, which encodes a negative regulator of race non-specific resistance to the powdery mildew fungus in barley. In the presence of Mlo dsRNA, transformed cells became more resistant, thereby phenocopying plants that carry a heritable loss-of function mlo resistance allele. The results suggest that direct delivery of dsRNA to cereals leads to a rapid and sequence-specific interference with gene function at the single-cell level.