High-Throughput RNAi for Rapid Gene Discovery in Grasses.

In plants, the most effective RNA interference (RNAi) strategy is to use long hairpin RNA (lhRNA) constructs containing inverted repeats (IRs), which are processed into small interfering RNAi (siRNA) that silence target genes based on sequence homology. We developed a novel restriction enzyme-mediated method, Phi29-Amplified RNAi Construct (PARC), which converts a cDNA library into a lhRNA expression library by only three steps, i.e., ligation, amplification and digestion. We generated a suppression subtractive hybridization (SSH) cDNA library in which cold-responsive genes were enriched. We subsequently converted the SSH library into a RNAi library which contains lhRNA constructs for the majority of the enriched genes. Seventeen randomly selected lhRNA constructs from the cold-stress RNAi library were delivered en masse into Brachypodium by Agrobacterium-mediated transformation. This forward genetics-based approach would allow for rapid gene discovery in plant species in which limited genomic resources are available.