Characterization of Chlamydomonas RSP6 Mutant

The parental strain CC-5325 and an insertional mutant in RSP6 (CLiP ID: LMJ.RY0402.248886) from the Chlamydomonas Library Project (CLiP; https://www.chlamylibrary.org)^{32 (link)} were obtained from the Chlamydomonas resource center and maintained on Tris-Acetate-Phosphate (TAP) solid media (1.6% agar, USP grade, Thermo Fisher Scientific) with Hutner’s trace elements⁶⁸ and 20 mg/mL paromomycin (Sigma). After isolation of single colonies and confirmation of the insertion site in the RSP6 mutant by PCR (Extended Data Fig. 9b), antibiotics was eliminated from the media. The resemblance in swimming phenotype of the CLiP RSP6 mutant and other reported RSP6 mutants^{33 (link)}, as well as the ability to restore motility in the CLiP RSP6 by complementation with wild-type RSP6 (Fig. 6a–b and Extended Data Fig. 9d), were considered as indications that the examined swimming phenotype of the CLiP RSP6 mutant was largely due to disruption of the RSP6 gene. Prior to motility analysis cells were grown in liquid TAP under constant illumination.