Abstract
The moss Physcomitrella patens is unique among plants in that homologous recombination can be used to knock out genes, just like in yeast. Furthermore, transformed plasmids can be rescued from Physcomitrella back into Escherichia coli, similar to yeast. In the present study, we have tested if a third important tool from yeast molecular genetics, auxotrophic selection markers, can be used in Physcomitrella. Two auxotrophic moss strains were made by knocking out the PpHIS3 gene encoding imidazoleglycerol-phosphate dehydratase, and the PpTRP1 gene encoding phosphoribosylanthranilate isomerase, disrupting the biosynthesis of histidine and tryptophan, respectively. The resulting PpHIS3Δ and PpTRP1Δ knockout strains were unable to grow on medium lacking histidine or tryptophan. The PpHIS3Δ strain was used to test selection of transformants by complementation of an auxotrophic marker. We found that the PpHIS3Δ strain could be complemented by transformation with a plasmid expressing the PpHIS3 gene from the CaMV 35S promoter, allowing the strain to grow on medium lacking histidine. Both linearized plasmids and circular supercoiled plasmids could complement the auxotrophic marker, and plasmids from both types of transformants could be rescued back into E. coli. Plasmids rescued from circular transformants were identical to the original plasmid, whereas plasmids rescued from linearized transformants had deletions generated by recombination between micro-homologies in the plasmids. Our results show that cloning by complementation of an auxotrophic marker works in Physcomitrella, which opens the door for using auxotrophic selection markers in moss molecular genetics. This will facilitate the adaptation of shuttle plasmid dependent methods from yeast molecular genetics for use in Physcomitrella.
Highlights
Much of our knowledge about basic functions of the eukaryotic cell such as the cell cycle, intracellular transport, and gene expression was obtained by research in the budding yeast Saccharomyces cerevisiae
Our results show that cloning by complementation of an auxotrophic marker works in Physcomitrella, which opens the door for using auxotrophic selection markers in moss molecular genetics
Many moss genes are duplicated, but we found that the moss orthologs of the yeast TRP1 (Pp3s17_23550) and HIS3 genes (Pp3c13_21930) are present in only one copy in the moss genome, making them suitable knockout targets
Summary
Much of our knowledge about basic functions of the eukaryotic cell such as the cell cycle, intracellular transport, and gene expression was obtained by research in the budding yeast Saccharomyces cerevisiae. A second important method in yeast molecular genetics is the use of shuttle plasmids that are able to replicate both in yeast and in bacteria (Beggs, 1978; Stinchcomb et al, 1979). Shuttle plasmids make it possible to clone genes from plasmid libraries by complementation of yeast mutants, and enable more advanced methods such as dosage suppressor screens (Rine, 1991). Gene targeting and dosage suppression complement each other in that gene targeting looks at what happens when a gene function is lost, whereas dosage suppressor screens look at what happens when a gene is overexpressed By using both methods much can be elucidated about the function of a given gene. It is possible to identify all proteins in a given signaling or metabolic pathway in one single dosage suppressor screen
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