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

Li, Xiaobo; Zhang, Ru; Patena, Weronika; Gang, Spencer S.; Blum, Sean R.; Ivanova, Nina; Yue, Rebecca; Robertson, Jacob M.; Lefebvre, Paul A.; Fitz-Gibbon, Sorel T.; Grossman, Arthur R.; Jonikas, Martin C.

doi:10.1105/tpc.15.00465

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

Journal Article · 12 January 2016 · The Plant Cell

DOI: https://doi.org/10.1105/tpc.15.00465 · OSTI ID:1418625

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Carnegie Inst. for Science, Stanford, CA (United States). Dept. of Plant Biology; US Dept. of Energy, Office of Scientific and Technical Information (OSTI)
Carnegie Inst. for Science, Stanford, CA (United States). Dept. of Plant Biology
Univ. of Minnesota, Minneapolis, MN (United States). Dept. of Plant Biology
Univ. of California, Los Angeles, CA (United States). Dept. of Chemistry and Biochemistry

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.

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Research Organization:: Carnegie Inst. for Science, Stanford, CA (United States); Univ. of California, Los Angeles, CA (United States); Univ. of Minnesota, Minneapolis, MN (United States)

Sponsoring Organization:: National Inst. of Health (NIH) (United States); National Science Foundation (NSF) (United States); USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)

Grant/Contract Number:: FC02-02ER63421

OSTI ID:: 1418625

Journal Information:: The Plant Cell, Journal Name: The Plant Cell Journal Issue: 2 Vol. 28; ISSN 1040-4651

Publisher:: American Society of Plant BiologistsCopyright Statement

Country of Publication:: United States

Language:: English

References (2)

Data from: An indexed, mapped mutant library enables reverse genetics studies of biological processes in Chlamydomonas reinhardtii Li, Xiaobo; Zhang, Ru; Patena, Weronika Dryad https://doi.org/10.5061/dryad.60mk5	dataset	January 2016
Data from: An indexed, mapped mutant library enables reverse genetics studies of biological processes in Chlamydomonas reinhardtii Li, Xiaobo; Zhang, Ru; Patena, Weronika Dryad https://doi.org/10.5061/dryad.60mk5	dataset	January 2016

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Additional file 10 of Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study Mao, Xuemei; Zhang, Yu; Wang, Xiaofei figshare https://doi.org/10.6084/m9.figshare.12144399	dataset	January 2020
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Additional file 12 of Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study Mao, Xuemei; Zhang, Yu; Wang, Xiaofei figshare https://doi.org/10.6084/m9.figshare.12144411	dataset	January 2020
Additional file 13 of Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study Mao, Xuemei; Zhang, Yu; Wang, Xiaofei figshare https://doi.org/10.6084/m9.figshare.12144414.v1	dataset	January 2020
Additional file 14 of Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study Mao, Xuemei; Zhang, Yu; Wang, Xiaofei figshare https://doi.org/10.6084/m9.figshare.12144423	dataset	January 2020
Additional file 15 of Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study Mao, Xuemei; Zhang, Yu; Wang, Xiaofei figshare https://doi.org/10.6084/m9.figshare.12144429.v1	dataset	January 2020
Additional file 16 of Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study Mao, Xuemei; Zhang, Yu; Wang, Xiaofei figshare https://doi.org/10.6084/m9.figshare.12144432.v1	dataset	January 2020
Additional file 17 of Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study Mao, Xuemei; Zhang, Yu; Wang, Xiaofei figshare https://doi.org/10.6084/m9.figshare.12144438	dataset	January 2020
Additional file 2 of Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study Mao, Xuemei; Zhang, Yu; Wang, Xiaofei figshare https://doi.org/10.6084/m9.figshare.12144447	dataset	January 2020
Additional file 4 of Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study Mao, Xuemei; Zhang, Yu; Wang, Xiaofei figshare https://doi.org/10.6084/m9.figshare.12144456.v1	dataset	January 2020
Additional file 5 of Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study Mao, Xuemei; Zhang, Yu; Wang, Xiaofei figshare https://doi.org/10.6084/m9.figshare.12144462	dataset	January 2020
Additional file 6 of Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study Mao, Xuemei; Zhang, Yu; Wang, Xiaofei figshare https://doi.org/10.6084/m9.figshare.12144465	dataset	January 2020
Additional file 7 of Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study Mao, Xuemei; Zhang, Yu; Wang, Xiaofei figshare https://doi.org/10.6084/m9.figshare.12144468.v1	dataset	January 2020
Additional file 8 of Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study Mao, Xuemei; Zhang, Yu; Wang, Xiaofei figshare https://doi.org/10.6084/m9.figshare.12144471	dataset	January 2020
Additional file 9 of Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study Mao, Xuemei; Zhang, Yu; Wang, Xiaofei figshare https://doi.org/10.6084/m9.figshare.12144477	dataset	January 2020
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Title: An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii

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