Identification and functional analysis of LCI15, a suppressor of the air dier phenotype of LCIB mutants in Chlamydomonas reinhardtii

The eukaryotic alga, Chlamydomonas reinhardtii, acclimates to limiting CO2 conditions by the induction of the CO2-concentrating mechanism (CCM) – a complex system of changes in its metabolism, gene expression patterns, and physiology – to compensate for the reduction in the amount of available CO2 and counter the hindrance to its ability to photosynthesize and grow. LCIB, a gene upregulated in such conditions, encodes a protein potentially involved in uptake of CO2 into the cell and in preventing the leakage of CO2 out of the cell. This protein is indispensable for growth in air-level CO2 (~350-400 ppm), since mutants in this gene are unable to grow (hence they are called air dier mutants). Several mutants that have second-site alterations that restore growth in air-level CO2 (i.e., suppress the air dier phenotype) have been isolated. Identifying the genes that are mutated in these suppressors and the functions of the encoded proteins will help us better discern the role of LCIB and comprehend the workings of the entire mechanism. To identify the locus of the mutated gene in one suppressor mutant, crosses were performed between the mutant strain and a non-mutant, polymorphic wild-type strain, and a large population of recombinant progeny that segregated against the mutation of interest was amassed. Using a strain that has unique single nucleotide polymorphisms (SNPs) as the non-mutant parent allowed us to seek a particular characteristic (the polymorphisms) in the region of interest in the genome of the progeny. With a sequenced genome, a library of SNPs in the polymorphic strain, and a pool of the genomic DNA from the entire population, we mapped the mutation to a specific region of the genome and narrowed potential candidates down to a small number of genes. By cosegregation analysis, we were able to confirm one of the candidates, LCI15, as the implicated gene. Preliminary functional analyses with semi-quantitative RT-PCR and Western immunoblots reveal the LCI15 protein as possibly playing an overarching role in regulation in the CCM and offer terms for discussing potential methods by which the lack of LCI15 might potentially mask the deleterious effects of the absence of LCIB

Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

Programmable site-specific nucleases, such as the clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas (Chlamydomonas reinhardtii). However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous doublestranded DNA (dsDNA) encoding a selectable marker gene. Repair of the dsDNA breaks induced by the RNPs is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. Here, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker)— in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precise edits in the homolog of bacterial ftsY or the WD and TetratriCopeptide repeats protein 1 genes in ~1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonasacetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways, or structures

Identification and functional analysis of LCI15, a suppressor of the air dier phenotype of LCIB mutants in Chlamydomonas reinhardtii

The eukaryotic alga, Chlamydomonas reinhardtii, acclimates to limiting CO2 conditions by the induction of the CO2-concentrating mechanism (CCM) – a complex system of changes in its metabolism, gene expression patterns, and physiology – to compensate for the reduction in the amount of available CO2 and counter the hindrance to its ability to photosynthesize and grow. LCIB, a gene upregulated in such conditions, encodes a protein potentially involved in uptake of CO2 into the cell and in preventing the leakage of CO2 out of the cell. This protein is indispensable for growth in air-level CO2 (~350-400 ppm), since mutants in this gene are unable to grow (hence they are called air dier mutants). Several mutants that have second-site alterations that restore growth in air-level CO2 (i.e., suppress the air dier phenotype) have been isolated. Identifying the genes that are mutated in these suppressors and the functions of the encoded proteins will help us better discern the role of LCIB and comprehend the workings of the entire mechanism. To identify the locus of the mutated gene in one suppressor mutant, crosses were performed between the mutant strain and a non-mutant, polymorphic wild-type strain, and a large population of recombinant progeny that segregated against the mutation of interest was amassed. Using a strain that has unique single nucleotide polymorphisms (SNPs) as the non-mutant parent allowed us to seek a particular characteristic (the polymorphisms) in the region of interest in the genome of the progeny. With a sequenced genome, a library of SNPs in the polymorphic strain, and a pool of the genomic DNA from the entire population, we mapped the mutation to a specific region of the genome and narrowed potential candidates down to a small number of genes. By cosegregation analysis, we were able to confirm one of the candidates, LCI15, as the implicated gene. Preliminary functional analyses with semi-quantitative RT-PCR and Western immunoblots reveal the LCI15 protein as possibly playing an overarching role in regulation in the CCM and offer terms for discussing potential methods by which the lack of LCI15 might potentially mask the deleterious effects of the absence of LCIB.</p

Recurrent evolutionary switches of mitochondrial cytochrome c maturation systems in Archaeplastida

Abstract Mitochondrial cytochrome c maturation (CCM) requires heme attachment via distinct pathways termed systems I and III. The mosaic distribution of these systems in Archaeplastida raises questions about the genetic mechanisms and evolutionary forces promoting repeated evolution. Here, we show a recurrent shift from ancestral system I to the eukaryotic-specific holocytochrome c synthase (HCCS) of system III in 11 archaeplastid lineages. Archaeplastid HCCS is sufficient to rescue mutants of yeast system III and Arabidopsis system I. Algal HCCS mutants exhibit impaired growth and respiration, and altered biochemical and metabolic profiles, likely resulting from deficient CCM and reduced cytochrome c-dependent respiratory activity. Our findings demonstrate that archaeplastid HCCS homologs function as system III components in the absence of system I. These results elucidate the evolutionary trajectory and functional divergence of CCM pathways in Archaeplastida, providing insight into the causes, mechanisms, and consequences of repeated cooption of an entire biological pathway