Chapter 5 - Targeted Gene Silencing by RNA Interference in Chlamydomonas

Abstract

Small RNA-guided gene silencing is an evolutionarily conserved process that operates by a variety of molecular mechanisms and plays an essential role in developmental pathways and defense responses against genomic parasites in eukaryotes. Double-stranded RNA (dsRNA) triggered posttranscriptional gene silencing, termed RNA interference (RNAi), is also becoming a powerful tool for reverse genetics studies. Stable RNAi, induced by the expression of long dsRNAs or duplex small RNAs from genome-integrated transgenes, has been achieved in multiple organisms, including the green alga Chlamydomonas reinhardtii. However, the level of gene repression is often quite variable, depending on the type of construct, transgene copy number, site of integration, and target gene. Moreover, unintended transcripts partly complementary to a trigger dsRNA can also be silenced, making difficult the interpretation of observed phenotypes. To obviate some of these problems we have developed a tandem inverted repeat system that consistently induces cosilencing of a gene with a selectable RNAi-induced phenotype (encoding tryptophan synthase beta-subunit) and any other (nonessential) gene of interest. In addition, to circumvent off-target effects, for each tested gene, RNAi lines are generated with at least two transgenes, homologous to distinct and nonoverlapping sequences of the target transcript. A common phenotype among these independent RNAi strains is expected to result from suppression of expression of the gene of interest. We demonstrate this approach for the characterization of a gene of unknown function in Chlamydomonas, encoding a predicted exoribonuclease with weak similarity to 3'hExo/ERI-1.