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

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.

Flavodiiron Proteins Promote Fast and Transient O2 Photoreduction in Chlamydomonas

“…To investigate the function of Flvs in the unicellular alga C. reinhardtii, we searched for insertion mutants in the Flvb (Cre16.g691800) gene region in the CLiP library (https://www.chlamylibrary.org; Li et al, 2016). We obtained four putative flvB mutants, three holding insertion of the paromomycin resistance cassette in introns and one with a possible large deletion ( Fig.…”

“…S1). We did not, however, succeed in mapping the insertion in the putative flvB-21 mutant, possibly due to atypical genomic rearrangement(s) that may occur with cassette insertion (Li et al, 2016). Immunodetection was then performed on total whole-cell protein extracts by using antibodies directed against recombinant FlvB and FlvA proteins (Fig.…”

“…The Chlamydomonas reinhardtii wilt-type strain CC-4533 and flvB mutants were obtained from the CLiP (Li et al, 2016). Upon reception, strains were plated on Tris-acetate-phosphate medium and streaked until exhaustion.…”

“…With the aim to better understand the function of Flvs in microalgal photosynthesis, and particularly to determine whether oxygen is the electron acceptor of Flv and what part of the electron flow is diverted toward oxygen photoreduction during photosynthesis, we study here C. reinhardtii flvB insertion mutants of the Chlamydomonas Library Project (CLiP) library (Li et al, 2016) devoid of both FlvB and FlvA proteins. By performing photosynthetic gas exchange measurements using […”

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in <em>Chlamydomonas reinhardtii</em>

“…However, targeted disruption of genes of interest is nearly impossible due to the lack of efficient homologous genomic integration. A random insertional disruption library is under construction, combined with identification of the disrupted site, so far yielding an arrayed set of 1,935 mapped disruptions representing 1,562 genes 3 . However, this approach (expected in general to produce null mutations) is not applicable to essential genes.…”