Comparative Phenotyping of Two Commonly Used Chlamydomonas reinhardtii Background Strains: CC-1690 (21gr) and CC-5325 (The CLiP Mutant Library Background)

Abstract

The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism to investigate many essential cellular processes in photosynthetic eukaryotes. Two commonly used background strains of Chlamydomonas are CC-1690 and CC-5325. CC-1690, also called 21gr, has been used for the Chlamydomonas genome project and several transcriptome analyses. CC-5325 is the background strain for the Chlamydomonas Library Project (CLiP). Photosynthetic performance in CC-5325 has not been evaluated in comparison with CC-1690. Additionally, CC-5325 is often considered to be cell-wall deficient, although detailed analysis is missing. The circadian rhythms in CC-5325 are also unclear. To fill these knowledge gaps and facilitate the use of the CLiP mutant library for various screens, we performed phenotypic comparisons between CC-1690 and CC-5325. Our results showed that CC-5325 grew faster heterotrophically in dark and equally well in mixotrophic liquid medium as compared to CC-1690. CC-5325 had lower photosynthetic efficiency and was more heat-sensitive than CC-1690. Furthermore, CC-5325 had an intact cell wall which had comparable integrity to that in CC-1690 but appeared to have reduced thickness. Additionally, CC-5325 could perform phototaxis, but could not maintain a sustained circadian rhythm of phototaxis as CC1690 did. Finally, in comparison to CC-1690, CC-5325 had longer cilia in the medium with acetate but slower swimming speed in the medium without nitrogen and acetate. Our results will be useful for researchers in the Chlamydomonas community to choose suitable background strains for mutant analysis and employ the CLiP mutant library for genome-wide mutant screens under appropriate conditions, especially in the areas of photosynthesis, thermotolerance, cell wall, and circadian rhythms.

Plant defensin MtDef4-derived antifungal peptide with multiple modes of action and potential as a bioinspired fungicide

“…For each captured cell, the cell surface binding intensity was quantified by free hand drawing a line intensity profile along the cell surface. The fluorescence intensity of each cell wall periphery was normalized by its perimeter length as described (47). The lower threshold was set to 7000 to subtract the signal in the cytoplasm.…”

Moderate High Temperature is Beneficial or Detrimental Depending on Carbon Availability in the Green AlgaChlamydomonas reinhardtii

“…Chlamydomonas reinhardtii wild-type strain CC-1690 (also called 21gr, mating type plus) (Pröschold et al, 2005;Zhang et al, 2022b;Sager, 1955-7) was purchased from the Chlamydomonas resource center and used in all experiments. Algal cultures were grown in Tris-acetate-phosphate (TAP, with acetate) or Tris-phosphate (TP, without acetate) medium with revised trace elements (Kropat et al, 2011) in 400 mL photobioreactors (PBRs) (Photon System Instruments, FMT 150/400-RB) as described before (Zhang et al, 2022a).…”

High-throughput Identification of Novel Heat Tolerance Genes via Genome-wide Pooled Mutant Screens in the Model Green AlgaChlamydomonas reinhardtii

“…However, their high temperature screens had several limitations (Supplemental Dataset 1a): (1) They emphasized 30°C heat which is a rather mild high temperature for CC-5325, the background wildtype (WT) strain of the CLiP mutant library (Zhang et al . 2014, 2022), and did not include heat at 40°C, which is acute high temperature for CC-5325. (2) They did not include heat at 35°C without carbon supply for parallel comparison with 35°C with carbon supply.…”

“…Additionally, several excellent molecular and genetic tools are available for Chlamydomonas, enabling efficient gene editing (Shimogawara, Fujiwara, Grossman & Usuda 1998;Greiner et al 2017;Wang et al 2019;Dhokane, Bhadra & Dasgupta 2020) and molecular engineering (Crozet et al 2018;Emrich-Mills et al 2021). Specifically, a mapped, indexed, genome-saturating Chlamydomonas insertional mutant library is available for both reverse and forward genetic screens (Chlamydomonas Library Project, CLiP, 62,389 mutants, covering 83% of nuclear protein-coding genes) (Zhang et al 2014(Zhang et al , 2022b.…”

“…Specifically, a mapped, indexed, genome-saturating Chlamydomonas insertional mutant library is available for both reverse and forward genetic screens (Chlamydomonas Library Project, CLiP, 62,389 mutants, covering 83% of nuclear protein-coding genes) (Zhang et al . 2014, 2022b).…”