Double‐stranded RNA interferes with gene function at the single‐cell level in cereals

Abstract

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.

RNA Interference-Mediated Repression of Cell Wall Invertase Impairs Defense in Source Leaves of Tobacco

“…AB055500]). RNAi is homology dependent, and earlier studies revealed that 90% sequence identity between two nucleotide sequences is sufficient to trigger effective RNA degradation (Schweizer et al, 2000;Le et al, 2006;Chen et al, 2008).…”

Host-Induced Gene Silencing in Barley Powdery Mildew Reveals a Class of Ribonuclease-Like Effectors

“…To determine if the contributions of any of the eight candidate effectors to infection related to an ability to suppress host PCD, we took advantage of the observation that transient expression of maize transcription factor genes B-Peru and C1 in barley leads to accumulation of anthocyanins and that this only occurs in intact vacuoles of viable cells (Schweizer et al 2000). To assess effects of silencing the BEC on the fractions of host cells that die during the early stages of infection, we co-bombarded each of the RNAi BEC constructs with the B-Peru/C1-expression plasmid pBC17 and pUbiGUS (as cell-death insensitive marker for transformation efficiency).…”

“…For examination of cellular viability after HIGS, we delivered 7 μg per shot of vector pTA30BEC (for BEC silencing) together with 7 μg of the B-Peru/C1-expression plasmid pBC17 (Schweizer et al 2000), for anthocyanin production, and pUbiGUS, as transformation marker, into epidermal cells of barley seedling primary leaves by particle bombardment. Half of the leaves were inoculated 1 day after transformation.…”

RNA interference for wheat functional gene analysis

“…The earliest report of RNAi in wheat was a single cell based transient RNA-silencing assay using wheat epidermal cells (Schweizer et al 2000). In this study, in vitro synthesized dsRNA was cotransformed through particle bombardment with either the gusA (GUS) reporter gene or with the fusion of a germinlike protein (GLP) and reporter gene green fluorescence protein (GFP) (TaGLP2a::GFP).…”

“…In this study, in vitro synthesized dsRNA was cotransformed through particle bombardment with either the gusA (GUS) reporter gene or with the fusion of a germinlike protein (GLP) and reporter gene green fluorescence protein (GFP) (TaGLP2a::GFP). Through phenotypic analysis, Schweizer et al (2000) showed that dsRNA of GUS, TaGLP2a, and GFP were all able to interfere with their respective target genes in a sequence specific manner. In another transient RNAi study in wheat, Christensen et al (2004) used a hairpin construct to target TaGLP2a (renamed TaGLP4 in this later study) to determine the effect of this gene on plant defense responses.…”