A genome-wide algal mutant library and functional screen identifies genes required for eukaryotic photosynthesis

Abstract

Photosynthetic organisms provide food and energy for nearly all life on Earth, yet half of their protein-coding genes remain uncharacterized^1,2. Characterization of these genes could be greatly accelerated by new genetic resources for unicellular organisms. Here we generated a genome-wide, indexed library of mapped insertion mutants for the unicellular alga Chlamydomonas reinhardtii. The 62,389 mutants in the library, covering 83% of nuclear protein-coding genes, are available to the community. Each mutant contains unique DNA barcodes, allowing the collection to be screened as a pool. We performed a genome-wide survey of genes required for photosynthesis, which identified 303 candidate genes. Characterization of one of these genes, the conserved predicted phosphatase-encoding gene CPL3, showed that it is important for accumulation of multiple photosynthetic protein complexes. Notably, 21 of the 43 higher-confidence genes are novel, opening new opportunities for advances in understanding of this biogeochemically fundamental process. This library will accelerate the characterization of thousands of genes in algae, plants, and animals.

Fig. 1 ∣. A genome-wide library of Chlamydomonas mutants was generated by random insertion of barcoded cassettes and mapping of insertion sites.

a, Chlamydomonas reinhardtii is used for studies of various cellular processes and organism-environment interactions. b, Our library contains 62,389 insertional mutants maintained as 245 plates of 384-colony arrays. Each mutant contains at least one insertion cassette at a random site in its genome; each insertion cassette contains one unique barcode at each end (Supplementary Fig. 1a-c). c, The insertion density is largely random over the majority of the genome. This panel compares the observed insertion density over the genome (the left column above each chromosome number) to three simulations with insertions randomly distributed over all mappable positions in the genome (the three narrow columns to the right for each chromosome). Areas that are white throughout all columns represent regions where insertions cannot be mapped to a unique genomic position due to highly repetitive sequence. See also Supplementary Fig. 4.

Fig. 2 ∣. The library covers 83% of Chlamydomonas genes.

a, 83% of all Chlamydomonas genes have one or more insertions in the library. b, In various functional groups, more than 75% of genes are represented by insertions in the library. c, The number of insertions per gene is roughly correlated with gene length. Box heights represent quartiles, whiskers represent 1^st and 99^th percentiles, and outliers are plotted as crosses. Box widths are proportional to the number of genes in each bin. d, Insertion density varies among different gene features, with the lowest density in exons. e, More than 1,800 mutants were distributed to approximately 200 laboratories around the world during the first 18 months of its availability. f, Distributed mutants are being used to study a variety of biological processes. Only genes with some functional annotation are shown.

Fig. 3 ∣. A high-throughput screen using the library identifies many genes with known roles in photosynthesis and many novel components.

a, Unique barcodes allow screening mutants in a pool. Mutants deficient in photosynthesis can be identified because their barcodes will be less abundant after photosynthetic growth relative to after heterotrophic growth. b, Biological replicates were highly reproducible, with a Spearman’s correlation of 0.982. Each dot represents one barcode. See also Supplementary Fig. 5. c, The phenotype of each insertion was determined by comparing its read count under photosynthetic and heterotrophic conditions. Insertions that fell below the phenotype cutoff were considered to show a defect in photosynthesis. cpl3 alleles are highlighted. d, Exon and intron insertions are most likely to show strong phenotypes, while 3’UTR insertions rarely do. The plot is based on all insertions for the 43 higher-confidence genes. e, The photosynthetic/heterotrophic ratio of all the alleles are shown for hit and control genes. Each column is a gene; each horizontal bar is an allele. f, The 303 candidate genes were categorized based on statistical confidence in this screen and based on whether the genes had a previously known function in photosynthesis (see Supplementary Note). g, Known higher-confidence genes, novel higher-confidence genes, and lower-confidence genes are all enriched in predicted chloroplast-targeted proteins (P < 0.011). h, A schematic summary illustrates the numbers of candidate genes in each category (panel f) and the specific functions of the genes with a known role in processes related to photosynthesis.

Fig. 4 ∣. CPL3 is required for photosynthetic growth and accumulation of photosynthetic protein complexes in the thylakoid membranes.

a, The cpl3 mutant contains cassettes inserted in the first exon of CPL3. The locations of conserved protein phosphatase motifs are indicated (see Supplementary Fig. 6e). b, cpl3 is deficient in growth under photosynthetic conditions and can be rescued upon complementation with the wild-type CPL3 gene (comp1–3 represent three independent complemented lines). c, cpl3 has a lower relative photosynthetic electron transport rate than the wild-type strain (WT) and comp1. Error bars indicate standard deviations (n = 3 for WT and comp1; n = 7 for cpl3). d, Whole-cell proteomics (Supplementary Table 14) indicate that cpl3 is deficient in accumulation of PSII, PSI, and the chloroplast ATP synthase. Each gray dot represents one Chlamydomonas protein. PSII, PSI and ATP synthase subunits are highlighted as black or red symbols. e, Western blots show that CPL3 is required for normal accumulation of the PSII subunit CP43, the PSI subunit PsaA, and the chloroplast ATP synthase subunit ATPC. α-tubulin was used as a loading control. See also Supplementary Fig. 7c. f, A heatmap of the protein abundance of subunits in the light reactions protein complexes or enzymes in the CBB cycle in cpl3 relative to the wild type based on proteomics data. Depicted subunits that were not detected by proteomics are filled with gray. Nuclear- and chloroplast-encoded proteins are labeled in black and red fonts respectively. A stack of horizontal ovals indicates different isoforms for the same enzyme, such as FBA1, FBA2, and FBA3.