Testing of Auxotrophic Selection Markers for Use in the Moss Physcomitrella Provides New Insights into the Mechanisms of Targeted Recombination
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 thir...
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Frontiers Media S.A.
2017-11-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | http://journal.frontiersin.org/article/10.3389/fpls.2017.01850/full |
| _version_ | 1819151835388182528 |
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| author | Mikael Ulfstedt Guo-Zhen Hu Monika Johansson Hans Ronne |
| author_facet | Mikael Ulfstedt Guo-Zhen Hu Monika Johansson Hans Ronne |
| author_sort | Mikael Ulfstedt |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-22T14:39:43Z |
| format | Article |
| id | doaj.art-989f280598964a35ab251348c4055729 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-22T14:39:43Z |
| publishDate | 2017-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-989f280598964a35ab251348c40557292022-12-21T18:22:35ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2017-11-01810.3389/fpls.2017.01850291775Testing of Auxotrophic Selection Markers for Use in the Moss Physcomitrella Provides New Insights into the Mechanisms of Targeted RecombinationMikael Ulfstedt0Guo-Zhen Hu1Monika Johansson2Hans Ronne3Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Uppsala, SwedenDepartment of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Uppsala, SwedenDepartment of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala, SwedenDepartment of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Uppsala, SwedenThe 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.http://journal.frontiersin.org/article/10.3389/fpls.2017.01850/fullauxotrophic selection markerscomplementationimidazoleglycerol-phosphate dehydratasephosphoribosylanthranilate isomerasePhyscomitrella patensplasmid rescue |
| spellingShingle | Mikael Ulfstedt Guo-Zhen Hu Monika Johansson Hans Ronne Testing of Auxotrophic Selection Markers for Use in the Moss Physcomitrella Provides New Insights into the Mechanisms of Targeted Recombination Frontiers in Plant Science auxotrophic selection markers complementation imidazoleglycerol-phosphate dehydratase phosphoribosylanthranilate isomerase Physcomitrella patens plasmid rescue |
| title | Testing of Auxotrophic Selection Markers for Use in the Moss Physcomitrella Provides New Insights into the Mechanisms of Targeted Recombination |
| title_full | Testing of Auxotrophic Selection Markers for Use in the Moss Physcomitrella Provides New Insights into the Mechanisms of Targeted Recombination |
| title_fullStr | Testing of Auxotrophic Selection Markers for Use in the Moss Physcomitrella Provides New Insights into the Mechanisms of Targeted Recombination |
| title_full_unstemmed | Testing of Auxotrophic Selection Markers for Use in the Moss Physcomitrella Provides New Insights into the Mechanisms of Targeted Recombination |
| title_short | Testing of Auxotrophic Selection Markers for Use in the Moss Physcomitrella Provides New Insights into the Mechanisms of Targeted Recombination |
| title_sort | testing of auxotrophic selection markers for use in the moss physcomitrella provides new insights into the mechanisms of targeted recombination |
| topic | auxotrophic selection markers complementation imidazoleglycerol-phosphate dehydratase phosphoribosylanthranilate isomerase Physcomitrella patens plasmid rescue |
| url | http://journal.frontiersin.org/article/10.3389/fpls.2017.01850/full |
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