Abstract
Insertional mutagenesis and gene silencing are efficient tools for the determination of gene function. In contrast to gain- or loss-of-function approaches, RNA interference (RNAi)-induced gene silencing can possibly silence multigene families and homoeologous genes in polyploids. This is of great importance for functional studies in hexaploid wheat (Triticum aestivum), where most of the genes are present in at least three homoeologous copies and conventional insertional mutagenesis is not effective. We have introduced into bread wheat double-stranded RNA-expressing constructs containing fragments of genes encoding Phytoene Desaturase (PDS) or the signal transducer of ethylene, Ethylene Insensitive 2 (EIN2). Transformed plants showed phenotypic changes that were stably inherited over at least two generations. These changes were very similar to mutant phenotypes of the two genes in diploid model plants. Quantitative real-time polymerase chain reaction revealed a good correlation between decreasing mRNA levels and increasingly severe phenotypes. RNAi silencing had the same quantitative effect on all three homoeologous genes. The most severe phenotypes were observed in homozygous plants that showed the strongest mRNA reduction and, interestingly, produced around 2-fold the amount of small RNAs compared to heterozygous plants. This suggests that the effect of RNAi in hexaploid wheat is gene-dosage dependent. Wheat seedlings with low mRNA levels for EIN2 were ethylene insensitive. Thus, EIN2 is a positive regulator of the ethylene-signaling pathway in wheat, very similar to its homologs in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). Our data show that RNAi results in stably inherited phenotypes and therefore represents an efficient tool for functional genomic studies in polyploid wheat.
Highlights
Insertional mutagenesis and gene silencing are efficient tools for the determination of gene function
The same reverse transcription (RT)-PCR fragment was used as a probe to estimate the number of copies of wheat PDS (wPDS) in the wheat genome by hybridization to hexaploid and diploid (Triticum monococcum cv DV92) wheat DNA digested with several restriction enzymes (Supplemental Fig. 2A)
We have demonstrated that RNA interference (RNAi)-mediated gene silencing is effective in hexaploid wheat and can efficiently induce reduction of mRNA levels of three homoeologous genes
Summary
Insertional mutagenesis and gene silencing are efficient tools for the determination of gene function. In contrast to gain- or lossof-function approaches, RNA interference (RNAi)-induced gene silencing can possibly silence multigene families and homoeologous genes in polyploids This is of great importance for functional studies in hexaploid wheat (Triticum aestivum), where most of the genes are present in at least three homoeologous copies and conventional insertional mutagenesis is not effective. The most severe phenotypes were observed in homozygous plants that showed the strongest mRNA reduction and, interestingly, produced around 2-fold the amount of small RNAs compared to heterozygous plants This suggests that the effect of RNAi in hexaploid wheat is gene-dosage dependent. Yan et al (2004) and Loukoianov et al (2005) used RNAi to stably transform wheat and demonstrate that reduction of VRN2 and VRN1 transcript levels, respectively, accelerated and delayed flowering initiation in winter wheat In both studies, only one independent transgenic plant showed the expected silenced phenotype. To develop RNAi technology for functional genomics in wheat, there is a need to characterize in molecular detail the silencing of homoeologous genes as well as the inheritance of RNAi-induced phenotype
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