Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants

Forward genetics screens to isolate insertional mutants with specific phenotypes have been used successfully to identify genes involved in different metabolic and regulatory pathways in the eukaryotic green alga Chlamydomonas reinhardtii (Chlamydomonas throughout) [1-3]. However, these screens rely on selectable phenotype-based assays, making it impossible to identify lesions in genes that do not result in a clearly measurable phenotype. Recently, the sequencing of the entire Chlamydomonas nuclear genome [4] has revealed many putative genes encoding proteins with no known biochemical functions. To dissect the functions of such proteins, as well as the functions of individual members of multi-protein families, a robust approach for targeting mutations in specific gene is required. In contrast to forward genetics, reverse genetics approaches target specific gene mutations; the most facile methods for targeting gene lesions can be developed for organisms in which the nuclear DNA can be manipulated through homologous recombination [5,6]. However, homologous recombination does not occur at a high frequency, relative to non-homologous recombination, in most eukaryotic organism, including plants and algae.Within the last decade, some alternative approaches have been developed to generate specific mutations in organisms in which homologous recombination is a low frequency event. In Arabidopsis and rice, transformation with Agrobacterium tumefaciens T-DNA has led to the generation of hundreds of thousands of transformants with T-DNA insertions distributed with low bias in the genome [7,8]. Defining the insertion site for each transformant has allowed for the establishment of sequence-indexed libraries of mutant plants that can be stored as seed at low cost and for long periods of time. The availability of these libraries affords the scientific community the opportunity to characterize Arabidopsis and rice lines with a lesion in nearly any gene [7,8]. An indexed insertional m