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.
Highlights
The coordination of nuclear and chloroplast gene expression in both microalgae and higher plants is largely effected through nuclear-encoded proteins targeted to chloroplasts
Such factors act mainly at the post-transcriptional level by binding to chloroplast transcripts, often through the 50 UTR (Marın-Navarro et al, 2007; Bohne et al, 2009). These factors have been termed ‘nuclear-encoded regulators of organellar gene expression’ (ROGEs), and have been characterized in both higher plants and the unicellular green alga Chlamydomonas reinhardtii (Woodson and Chory, 2008). Examples from the latter include NAC2, which stabilizes the psbD mRNA encoding the core photosystem II (PSII) subunit D2 and has been used to create a psbD 50 UTR-linked system for inducible gene expression in the chloroplast (Nickelsen et al, 1999; Boudreau et al, 2000; Rochaix et al, 2014), MCA1, which is required for the stabilization of petA mRNA encoding the apo-cytochrome f subunit of cyt b6f and plays a central role in the regulation of cyt b6f biogenesis (Loiselay et al, 2008), and TCA1, which is required for translation of the same transcript (Wostrikoff et al, 2001; Raynaud et al, 2007)
We describe the development of a modified cytosine deaminase gene as a easy-to-use negative selectable marker for the C. reinhardtii chloroplast, and demonstrate its utility in the isolation of novel mutant alleles of three previously identified ROGEs that are required for expression of psaA or petA
Summary
The coordination of nuclear and chloroplast gene expression in both microalgae and higher plants is largely effected through nuclear-encoded proteins targeted to chloroplasts These trans-acting factors each regulate the expression of one or a few specific chloroplast genes, such as those encoding subunits of the photosynthetic complexes photosystem I (PSI), photosystem II (PSII), the cytochrome b6f complex (cyt b6f), ATP synthase and Rubisco. Such factors act mainly at the post-transcriptional level by binding to chloroplast transcripts, often through the 50 UTR (Marın-Navarro et al, 2007; Bohne et al, 2009) These factors have been termed ‘nuclear-encoded regulators of organellar gene expression’ (ROGEs), and have been characterized in both higher plants and the unicellular green alga Chlamydomonas reinhardtii (Woodson and Chory, 2008).
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