An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in <i>Chlamydomonas reinhardtii</i>

TLDR

Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes, yet its utility has been limited by difficulties in obtaining mutants in specific genes of interest. The study aimed to enable generation of large numbers of mapped mutants by developing high‑throughput methods for strain maintenance, insertion site identification, and validation via flanking sequence acquisition. Using these methods, the authors built a stably maintained library of 1,935 mapped mutants disrupting 1,562 genes, and applied it to analyze lipid content in mutants affecting algal lipid droplet proteome genes. Characterization of random mutants confirmed 75 % of insertion sites by PCR and 74 % had a single insertion, and the library revealed that the long‑chain acyl‑CoA synthetase LCS2 is central to triacylglycerol production from de novo fatty acids.

Abstract

The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids.