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Published on July 21, 2006; 10.1104/pp.106.084517

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Received June 2, 2006
Accepted July 12, 2006

RNAi based gene silencing as an efficient tool for functional genomics in hexaploid bread wheat (Triticum aestivum L.)

Silvia Travella , Theres E. Klimm , and Beat Keller *

Institute of Plant Biology, University of Zürich, Zollikerstrasse 107, 8008 Zürich, Switzerland

* Corresponding author; email: bkeller{at}botinst.unizh.ch.

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 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 dsRNA-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 PCR 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 which showed the strongest mRNA reduction and, interestingly, produced around two-fold the amount of small RNAs compared to heterozygous plants. This suggests that the effect of RNA interference 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 and rice. Our data show that RNA interference results in stably inherited phenotypes and therefore represents an efficient tool for functional genomic studies in polyploid wheat.




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