RNA Interference-Based Gene Silencing as an Efficient Tool for Functional Genomics in Hexaploid Bread Wheat

Abstract

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. 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.

RNA interference for wheat functional gene analysis

“…Three recent wheat studies shed new light on the potential of RNAi silencing to simultaneously knockdown transcripts of homoeologous genes in polyploid wheat (Travella et al 2006;Uauy et al 2006b;Yue et al 2007). In the first study, Travella et al (2006) targeted the phytoene desaturase (PDS) and ethylene insensitive 2 (EIN2) genes, which both have three homoeologous copies that are expressed in wheat.…”

“…In the first study, Travella et al (2006) targeted the phytoene desaturase (PDS) and ethylene insensitive 2 (EIN2) genes, which both have three homoeologous copies that are expressed in wheat. Silencing of these genes generates phenotypes that are easy to visualize making them ideal targets for evaluating silencing of individual homoeologous genes.…”

“…EIN2 is a central component of the ethylene signaling pathway (Alonso et al 1999) B2 C....C.......A...T...A.............C...........G....................G..C........A.---....T.C..C...........CC..... D2 C............A...T.................C...........A....................G..C........A.---....T.C..C...........CC For the PDS genes, Travella et al (2006) used a 480-bp trigger fragment from the wheat D genome that shares over 96% nucleotide identity with the other two homoeologous copies. Using particle bombardment, they generated 64 independent transgenic lines showing the photobleaching phenotype at the seedling stage.…”

“…For the EIN2 gene, a 518-bp fragment from the EIN2 B genome allele (96% nucleotide identity to homoeologous copies) was used as the RNAi trigger (Travella et al 2006). Six independent transgenic T 0 lines displayed a significant reduction in endogenous EIN2 expression levels.…”

“…Using genome-specific primers and qRT-PCR, Travella et al (2006) measured the endogenous PDS or EIN2 mRNA levels of the individual homoeologous copies, as well as the total transcript levels for all three homoeologues. They found that all three copies of each gene were equally affected by the RNAi and that the transcript levels detected by the consensus primers (which amplify all three copies) were indicative of the three individual genomes.…”