Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: a strategy for the isolation of nuclear mutations that affect chloroplast gene expression

Abstract

Negative selectable markers are useful tools for forward-genetic screens aimed at identifying trans-acting factors that are required for expression of specific genes. Transgenic lines harbouring the marker fused to a gene element, such as a promoter, may be mutagenized to isolate loss-of-function mutants able to survive under selection. Such a strategy allows the molecular dissection of factors that are essential for expression of the gene. Expression of individual chloroplast genes in plants and algae typically requires one or more nuclear-encoded factors that act at the post-transcriptional level, often through interaction with the 5′ UTR of the mRNA. To study such nuclear control further, we have developed the Escherichia coli cytosine deaminase gene codA as a conditional negative selectable marker for use in the model green alga Chlamydomonas reinhardtii. We show that a codon-optimized variant of codA with three amino acid substitutions confers sensitivity to 5-fluorocytosine (5-FC) when expressed in the chloroplast under the control of endogenous promoter/5′ UTR elements from the photosynthetic genes psaA or petA. UV mutagenesis of the psaA transgenic line allowed recovery of 5-FC-resistant, photosynthetically deficient lines harbouring mutations in the nuclear gene for the factor TAA1 that is required for psaA translation. Similarly, the petA line was used to isolate mutants of the petA mRNA stability factor MCA1 and the translation factor TCA1. The codA marker may be used to identify critical residues in known nuclear factors and to aid the discovery of additional factors required for expression of chloroplast genes.

A Nucleus-Encoded Chloroplast Protein Regulated by Iron Availability Governs Expression of the Photosystem I Subunit PsaA inChlamydomonas reinhardtii

“…The mapping of this mutation confirms that the TAA1 gene was properly identified. Using a different genetic screen, Young and Purton (2014) identified several PSI-deficient mutants that could be rescued by transformation with TAA1 genomic DNA (cosmid 64B4). In one of these mutants, allelism to taa1 was confirmed by the presence of a mutation creating a stop codon in exon 5 of TAA1.…”

Genetic tools and techniques for Chlamydomonas reinhardtii

“…In contrast to positive selectable markers, negative selectable markers represent factors that confer sensitivity towards a certain compound once expressed in the host cell and can be useful e.g., to identify trans-acting factors (Young and Purton 2014) or as markers for gene silencing approaches (Jiang et al 2014;Rohr et al 2004). Three established negative selectable marker for C. reinhardtii are the endogenous MAA7 gene (encoding the tryptophan synthase ß subunit; presence leads to sensitivity to 5-fluoroindole) (Rohr et al 2004), the endogenous FKBP12 gene (encoding the 12-kD FK506-binding protein; presence leads to sensitivity to rapamycin) (Crespo et al 2005), and the E. coli codA gene (encoding cytosine deaminase; presence leads to sensitivity to 5-fluorocytosine) (Young and Purton 2014).…”

“…Three established negative selectable marker for C. reinhardtii are the endogenous MAA7 gene (encoding the tryptophan synthase ß subunit; presence leads to sensitivity to 5-fluoroindole) (Rohr et al 2004), the endogenous FKBP12 gene (encoding the 12-kD FK506-binding protein; presence leads to sensitivity to rapamycin) (Crespo et al 2005), and the E. coli codA gene (encoding cytosine deaminase; presence leads to sensitivity to 5-fluorocytosine) (Young and Purton 2014).…”

Multigenic engineering of the chloroplast genome in the green alga Chlamydomonas reinhardtii

“…In this study we have explored multigenic engineering by combining two approaches: namely, the integration of three gene cassettes into a single neutral locus, followed by a further round of transformation to integrate a fourth gene cassette plus the aadA marker cassette at a second locus. The four genes were selected from previous studies in our group where each coding sequence was codon-optimised for expression in the chloroplast, and each was modified to encode the haemagglutinin epitope tag (HA-tag), YPYDVPDYA at the C terminus [25]. The gene products are unrelated, and were chosen based on their different sizes and the different levels of accumulation observed previously in chloroplast transformants expressing just the single gene.…”