A high-throughput inducible RNAi vector for plants

Abstract

We describe here a vector system that allows dexamethasone-inducible RNAi against plant genes. The system utilizes a modified pHELLSGATE vector, under the control of the pOp6 promoter, and the synthetic transcription factor, LhGR. We demonstrate that the production of RNAi-inducing hairpin RNA from this system can be regulated by the application and removal of dexamethasone. Silencing of a target gene encoding phytoene desaturase was highly effective 24 h after application of dexamethasone. In the presence of the hormone silencing was maintained for at least 5 days while removal of the inducer resulted in significant recovery within 24 h. A transgene encoding luciferase was silenced with similar speed and efficiency following application of dexamethasone but unlike phytoene desaturase, mRNA levels did not recover within 10 days after dexamethasone was removed. Insertion of target gene sequences into this vector is mediated by Gateway™ recombination, facilitating its use for high-throughput applications, such as gene discovery or validation. The inducibility of RNAi from this system may be useful in helping to identify the functions of genes which when constitutively silenced give embryo lethality or pleiotropic phenotypes. A modified version of this system may also be used for tissue-specific hairpin RNA expression. © 2005 Blackwell Publishing Ltd.