Supplementary MaterialsSupplementary Tables and Figures. photosystem II, resulting in a much smaller PSI pool size and diminished chlorophyll antenna size. The lack of CrLTD did not prevent photoautotrophic growth of the cells. These results are substantially different from those for Arabidopsis null mutant, indicating LTD function in LHCP delivery and PSI assembly may not be as stringent in BIBW2992 inhibitor as it is in higher plants. gene family is encoded in the nuclear genome (Jansson, 1999; Stauber null mutant showed a drastic lowering of total chlorophyll (Chl) and LHCP and no growth under photoautotrophic conditions. These severe phenotypes suggested a critical role of the LTD protein in LHCP trafficking (Cui ((Baek mutant, total Chl content was decreased to 33% of the wild-type level, but the Chl ratio was not changed. The level of the BIBW2992 inhibitor PSI-LHCI complex in the mutant was severely reduced, to a far greater extent than that of the PSII-LHCII complex. Concomitantly, the ultrastructure of the thylakoid membranes was drastically altered. These results suggest that the CrLTD is specifically involved in the transport and assembly of the PSI-LHCI complexes, and that the associated CpSRP pathway may act differently in microalgae than in higher plants. Materials and methods Cell growth conditions wild-type strain CC-4349 (Baek (1993). CRISPRCCas9 driven mutagenesis All procedures were performed according to Baek (2016) and Yu (2017). Recombinant Cas9 protein (200 g) and transcribed single guide RNA (sgRNA; 140 g) were mixed and preincubated for 10 BIBW2992 inhibitor min at room temperature. Then, cells (5 105 cells) were transformed with the ribonucleoprotein (RNP) complex in a Gene Pulser Xcell Electroporation System (Bio-Rad). After transformation, cells were diluted and plated on TAP medium containing 1.5% agar to obtain single colonies for further investigation. For genotype characterization, genomic DNA was extracted as described in Jeong (2017) and DNA segments were analysed as given by Baek (2016) using Cas-Analyzer (Park (2017) with the modification of illumination with incandescent light ranging from 25 to 1200 mol photons m?2 s?1. Spectrophotometric and kinetic analysis Thylakoid membranes were isolated as described in Kirst (2012PSI and PSII was measured from the first-order rate constants of P700 photo-oxidation and QA photoreduction, conducted upon weak continuous green actinic illumination of isolated thylakoid membranes (Polle for 10 min to remove unsolubilized material. Thylakoid membrane proteins (25 g Chl per lane) were separated by gradient Deriphat-PAGE; the running gel had an acrylamide concentration gradient from 3.5 to 10.5% (w/v) (29:1 acrylamideCbisacrylamide) containing 12 mM TrisCHCl pH 8.5, 48 mM glycine, and a glycerol gradient from 10 to 14% (w/v). The stacking gel had 3.5% (w/v) acrylamide, 12 mM TrisCHCl pH 8.5, 48 mM glycine, and 10% (w/v) glycerol. The electrophoresis Mouse monoclonal to p53 anode buffer was 12 mM TrisCHCl pH 8.3, 96 mM glycine. The cathode buffer had the same components as the anode buffer except for the addition of 0.1% (w/v) Deripat-160 (Cognis). The gel was electrophoresed at 50 V constant voltage overnight. For two-dimensional electrophoresis analysis, proteins were extracted from one-dimensional native Deriphat-PAGE strips by soaking them in SDS-PAGE stacking buffer containing 5 M urea twice for 25 min each and resolved by denaturing in a 12% SDS-polyacrylamide gel containing 2 M urea (second dimension). Acrylamide gels were stained with Coomassie Blue. Results Targeted gene knockout using CRISPRCCas9 technology The gene (Cre12.g551950) contains one exon, which is 504 bp in length, and encodes a protein of 167 amino acids including a 38-amino-acid- long chloroplast transit peptide predicted by Predalgo software (Tardif LTD knockout strains (online). Then, preassembled sgRNA and the Cas9 protein forming a CRISPRCCas9 ribonucleoprotein (RNP) complex were transfected into the by electroporation. Because the Arabidopsis knockout mutant has yellow leaves and lower Chl content than the wild-type (Ouyang deletion mutants were initially selected on the basis of pale green coloration of transformant colonies (Supplementary Fig. S1). In a second screening step, the gene knockout was confirmed by Sanger sequencing. Out of 388 colonies, four knockout mutants were isolated, which was calculated to represent a 1.12% transformation efficiency. All knockout mutants were isolated by using sgRNA 1 (see Supplementary Table S2). At the same cell density in liquid culture, the coloration of these mutants was lighter green compared with the wild-type and was similar to that of the.