3.3 Efficient creation of marker-free transplastomic lines using CpPosNeg
To validate the CpPosNeg system (Figure 1 ), plasmids were designed in which the dual marker was placed downstream of lucCP , a codon-optimized reporter encoding firefly luciferase.[42] For both gene cassettes, the same 3’ UTR element from rbcL was used in order to create a 258 bp direct repeat (see: Supplementary Figure 1) . This size of repeat was chosen since it is significantly smaller than the 462 bp needed for high rates of intramolecular recombination in the absence of selection in the C. reinhardtiichloroplast,[24] but larger than the minimum size (~210 bp) reported for such recombination to occur.[26] The dual marker was therefore predicted to be stably maintained in the intermediate transformant lines (R1) in the absence of Spc selection, but efficiently lost in the R2 lines following counter-selection on 5-FC (Figure 1b ). Left and right homology arms of ~1000 bp were placed upstream and downstream of the reporter and marker cassettes in order to target the genes to two different neutral insertion sites: either downstream ofpsbA [39] (plasmid pLuc1) or downstream ofpsbH [43] (plasmid pLuc2).
WT C. reinhardtii was transformed using pLuc1 or pLuc2 with selection based on Spc resistance conferred by the dual marker. For each transformation, six colonies were re-streaked on Spc over three generations to drive the cells to the R1 homoplasmic state (Figure 1d ). As illustrated in Figure 4a , a four-primer multiplex PCR analysis of either the psbA locus or the psbH locus was employed to confirm the genotype with diagnostic band sizes for the WT, R1 and R2 loci. All six transformant lines showed the R1 genotype and appeared to be homoplasmic following three rounds of Spc selection, with no detection of the WT band. Importantly, two further rounds of replating on medium lacking Spc demonstrated that the codA-aadA marker DNA was stably maintained in the plastome despite being flanked by the rbcL direct repeat, with the PCR analysis showing maintenance of the R1 band and no appearance of the R2 band (Figure 4c ). Conversely, two rounds of plating on 5-FC medium led to the rapid loss of the marker, with the R2 plastome appearing to be homoplasmic since only the PCR product from primers P2 and P4 was detected (Figure 4d ). Sequencing of this PCR product confirmed the loop-out of the marker at both the psbAand psbH loci via recombination between the rbcL copies.
An assay of luciferase activity in the R1 and R2 lines confirmed that the introduced lucCP cassette was expressed and that the level of expression was not affected by the subsequent loop-out of the cassette with both pairs of R1 and R2 lines showing similar activities (Figure 5e ). There was a small but significant difference in expression in Luc1.R2 relative to Luc1.R1 in the conditions tested (3.6%; p = 0.03; Student’s t -test). While this could be due to the removal of the selection cassette, it is more likely an artefact due to subtle differences in the culture history and incubation conditions of the samples. There was no significant difference between expression in Luc2.R1 and Luc2.R2 (p = 0.13; Student’st -test). Interestingly, the targeting of lucCP into the plastome’s large inverted repeat region downstream of psbA(transformant Luc1) such that two copies are present per plastome molecule rather than one as in the case of the psbH transformants (Luc2) gave more than twice the luciferase activity. This suggests that the activities of the rrnS promoter and psaA 5’UTR elements used to drive lucCP expression are not limiting, and that the level of recombinant protein is directly related to copy number. This is a surprising finding given that copy number is assumed not to be a key factor in chloroplast expression[44] and that transgene expression is mainly controlled at the translational level.[45]However, we cannot rule out genomic context and the influence of upstream/downstream transcriptional units as an alternative explanation for the different expression levels.
After removal of the codA-aadA cassette, the R2 phenotype should be the same as the WT with respect to sensitivity to Spc and resistance to 5-FC. To confirm this, spot tests were carried out on WT, Luc1.R2 and Luc2.R2 (Figure 5f ). C-A.f (Spc resistant; 5-FC sensitive) was included as a positive control. Luc1.R2 and Luc2.R2 showed the same phenotype as WT: full dieback on Spc and similar levels of growth in the presence or absence of 5-FC. This further confirmed that thecodA-aadA marker had been completely lost in the R2 cell lines.