Rescue and characterization of episomally replicating DNA from the moss Physcomitrella

Abstract

The moss Physcomitrella is unique among plants in that it permits efficient gene targeting by homologous recombination. Furthermore, transformed DNA can replicate episomally in Physcomitrella. Here we show that episomally replicating DNA can be rescued back into Escherichia coli, and we use such rescue to study the fate of the transformed DNA. Significantly, plasmids rescued from moss transformed with circular DNA are identical to the original plasmid, whereas plasmids rescued from moss transformed with linearized DNA frequently have deletions created by direct repeat recombination. These events are highly predictable in that they target the longest direct repeat on the plasmid if this repeat is at least 12 bp. Episomal transformants obtained with linearized DNA show a more than 1,000-fold amplification of the DNA whereas transformants obtained with circular DNA have much lower copy numbers. Most episomal transformants quickly lose the plasmid in the absence of selection, but a semistable type of transformant that loses the plasmid at a much lower frequency was also observed. The consistent rescue of the original plasmid, or of predictable derivatives thereof, suggests that molecular genetics methods which rely on shuttle plasmids are feasible in Physcomitrella.episomal replication ͉ plasmid rescue ͉ recombination ͉ shuttle plasmid T he discovery of gene targeting by homologous recombination (1) and the development of yeast shuttle plasmids (2-4) were key advances in eukaryotic molecular genetics. Gene targeting made reverse genetics possible, and shuttle plasmids made it easy to clone both yeast genes and genes from other eukaryotes by complementation of yeast mutants (5). Furthermore, the effects of overexpressing a gene could be studied by using plasmids that replicate in high copy number; by screening genomic libraries made in such plasmids, dosage suppressors of yeast mutants could be cloned, one of the most powerful methods available in molecular genetics (6).The finding that gene targeting is feasible in the moss Physcomitrella (7) transformed this small plant into an important model organism (8-11). Interestingly, early studies also showed that foreign DNA can replicate episomally in Physcomitrella (12-15). Three types of transformants were described by Schaefer (14). Class I have the DNA integrated into the moss genome and further studies of these transformants led to the discovery of gene targeting in moss (7). Class II, which comprise most transformants, quickly lose the marker under nonselective growth. Class III transformants also lose the marker in the absence of selection, but the rate of loss is lower (14). Further studies by Ashton et al. (15) showed that foreign DNA can replicate episomally in moss for at least 10 years if selection is maintained. Southern blots and PCR experiments suggested that the episomal DNA forms concatemers with 3-40 copies of the plasmid (14, 15). A notable finding in both studies was that moss DNA does not seem to be required for replication, unlike the case in yeast....

Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 in Physcomitrella patens

“…For this purpose, we took advantage of the possibility in Physcomitrella of episomal replication of the transformed DNA (Murén et al 2009), and used the pBNRF plasmid containing a 35S::neoR cassette for transient resistance of the transformed protoplasts to geneticin. This approach relies on the fact that (1) circular plasmids do not integrate stably into Physcomitrella chromosomes; and (2) due to the high copy number of plasmids, transformed protoplasts take up all constructs present.…”

“…The pBNRF plasmid was used here for transient selection. The rationale for this assay was the capacity, in P. patens , of transformed DNA to replicate episomally allowing antibiotic selection to be maintained during multiple cycles of divisions (Ashton et al 2000; Murén et al 2009; Schaefer et al 1991). Plants were put onto a new PpNH 4 plate for 1 wk, and then individualized; 1 or 2 wk later, a sample of each clone was taken for high-throughput DNA extraction.…”

Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes control auxin biosynthesis and affect growth and development in the moss Physcomitrella patens

“…Southern blots confirmed this, but also revealed the inclusion of vector DNA in some of the repeats. Concatenation can be the result of either recombination between microhomologies, preferentially those longer than 12 bp, or of ligation of compatible sticky ends (Murén et al, 2009). None of our targeting fragments possesses direct repeats longer than 12 bp, and as we mainly used different restriction enzymes for their release from the vector, the vector was most likely used as a linker to produce the tandem repeats.…”

“…DNA extractions, blotting and hybridisation were performed as described (Murén et al, 2009) using 0.4 g protonemata and subsequently 10 g of DNA per restriction digest. Probe templates were PCR amplified with primers PpSHI1-5Јprobe and PpSHI1-3Јprobe and with PpSHI2-5Јprobe and PpSHI1-3Јprobe, respectively (see Table S1 in the supplementary material), and cloned into pCR-BluntII-TOPO, from which they were excised with EcoRI and isolated by gel purification prior to DIG labelling using the DIG High Prime DNA Labelling and Detection Kit (Roche).…”

Physcomitrella patens mutants affected on heat dissipation clarify the evolution of photoprotection mechanisms upon land colonization

“…At least four independent clones with the insertion in the correct position were isolated and retained for further characterization. During transformation in Physcomitrella, multiple insertions at the same site might occur (24,26,27). To select single copy insertions, we used a PCR assay with primers annealing to the genomic sequences at either side of the insertion region.…”