Background

Cyanidioschizon merolae 10D is a small single-cell eukaryotic red algae, that inhabits acidic hot springs. Lately, all three of its genomes were sequenced: nucleus (Matsuzaki et al., 2004), mitochondrion (Ohta et al., 1998), and plastid (Ohta et al., 2003). Its simple cell anatomy, the presence of a single mitochondrion and chloroplast, a highly reduced spliceosome (Stark et al., 2015), almost deprived of introns nuclear genome make this organism a suitable candidate for a model organism in broad range of plant research, from cell and plastid anatomy, through physiology and up to genetic engineering (Kuroiwa et al., 1998; Nozaki et al., 2003). Its simplicity makes it particularly useful for genetic manipulations. Thus, many genetic techniques have been applied and optimized, for this organism, including antisense suppression, transient expression, gene disruption, and stable insertion into the nuclear genome (Newell, 2000; Walker et al., 2005; Hallmann, 2007; Minoda et al., 2004; Ohnuma et al., 2008; Imamura et al., 2009; Ohnuma et al., 2009; Imamura et al., 2010; Watanabe et al., 2011, Zienkiewicz et al., 2017a). Recently, also stable transformation of the chloroplast genome has been achieved (Zienkiewicz et al., 2017b; Zienkiewicz et al., 2018) in our laboratory. The developed method takes advantage of double homologs recombination in C. merolae, allowing for integration of foreign DNA into the genome in specific regions. A gene knock-out mutation replaces or disrupts the gene of interest with the cat (chloramphenicol acetyltransferase; Zienkiewicz et al., 2017a) gene under the original promoter or any familiar nuclear or chloroplast promoter (Krupnik et al., 2018). Promoters driving the transcription of the cat gene should exhibit at least moderate to significant constitutive activity; therefore application of promoters already tested is advised (Krupnik et al., 2018). By introducing Diphtheria toxin (fragment A-DTA) under a constitutive promoter into the transformation vector we managed to virtually exclude the possibility of random, illegitimate recombination events (Zienkiewicz et al., 2018). A single recombination event, random integration or otherwise unspecific incorporation of the transformation vector into the nuclear genome would likely cause expression of the A-DTA protein and in effect results in cell lysis. However, the heterogeneity of chloroplast transformation still remains a challenge. We have shown that continued growth of chloroplast mutants in increasing concentration of chloramphenicol allows in principal for reduction of heterogeneity and selection of homogenous mutants. Chloramphenicol is the only antibiotic that is toxic to C. merolae (out of c.a. 10 tested, Zienkiewicz et al., 2017a). This method is just as simple as application of lipofectamine for DNA delivery and is as efficient as the gene-gun method but without its drawbacks and complexity (Zienkiewicz et al., 2017b).