An efficient gene deletion procedure for the mushroom-forming basidiomycete Schizophyllum commune
- First Online:
- Cite this article as:
- Ohm, R.A., de Jong, J.F., Berends, E. et al. World J Microbiol Biotechnol (2010) 26: 1919. doi:10.1007/s11274-010-0356-0
- 539 Downloads
Gene deletion in Schizophyllum commune is hampered by a low incidence of homologous integration. As a consequence, extensive screening is required to identify a transformant with the desired genotype. To alleviate this and to facilitate the assembly of deletion plasmids, vector pDelcas was constructed. This construct has a set of restriction sites, which allows for directional cloning of the flanking sequences at both sides of a nourseothricin resistance cassette. Moreover, it contains a phleomycin resistance cassette elsewhere in the plasmid, which is used to screen for transformants with an ectopic integration of the pDelcas derivative. The use of pDelcas derivatives in combination with an improved PCR screening protocol permitted the efficient identification of S. commune deletion strains. This procedure may also function in other basidiomycetes.
KeywordsBasidiomyceteSchizophyllum communeHomologous integrationTransformation
Gene inactivation is an important tool in functional genetics and the most reliable way of implementing it is gene deletion through homologous recombination. The efficiency of this process depends on the pathway that is used by the organism to repair double stranded DNA breaks (Ninomiya et al. 2004). Homologous integration occurs with a high frequency in organisms such as Saccharomyces cerevisae that primarily use the homologous recombination pathway. Plants, animals and filamentous fungi use mainly non homologous end joining for DNA repair. As a result, the most frequent outcome of a DNA transformation is ectopic integration. Ten gene deletions have been reported in the basidiomycete Schizophyllum commune. Gene X of the MATAα mating type locus and the mtd1 gene were inactivated with a frequency of 33% (Marion et al. 1996) and 50% (Lengeler and Kothe 1999b), respectively. The other genes were deleted with an average frequency of only 3% (van Wetter et al. 1996, 2000; Robertson et al. 1996; Horton et al. 1999; Lugones et al. 2004; Schubert et al. 2006; Lengeler and Kothe 1999a). The low incidence of homologous integration in most of the cases where a gene was deleted prompted us to set up a screening system for transformants with such an integration event. To this end, pDelcas (accession number GQ184463) was constructed. This vector allows easy directional cloning of flanking sequences at either side of a nourseothricin resistance cassette. The presence of a phleomycin resistance cassette elsewhere in the construct enables elimination of transformants with an ectopic integration due to their resistance to the antibiotic phleomycin. In the second step, phleomycin sensitive colonies are screened using a fast colony PCR protocol to confirm gene inactivation by homologous integration.
Materials and methods
Construction of pDelcas
Transformation and first screening
Circular DNA of derivatives of pDelcas was introduced in S. commune strain 4–8 (MATA43 MATB41, Fowler et al. 1999) according to van Peer et al. (2009). Selection was done on minimal medium (Dons et al. 1979) with 8 μg ml−1 nourseothricin (Jena Biosciences, Jena, Germany). Transformants were transferred to a second selection plate with nourseothricin and then screened on minimal medium plates with 25 μg ml−1 phleomycin (Cayla S.A.R.L., Toulouse, France). Transformants that failed to grow on phleomycin were candidates to have a gene deletion due to homologous integration resulting from a double cross-over.
Derivatives of strain 4–8 with a gene inactivation were crossed with the compatible isogenic strain 4–8b (MATA41 MATB43). Segregation of nourseothricin resistance in the F1 was assessed to exclude multiple integrations (integration in a single locus should give a 1:1 ratio of resistant and sensitive colonies). Single integrations were confirmed by Southern analysis. Crossing was also used to clean heterokaryons that harbor transformed and untransformed nuclei. Such heterokaryons can result from fusion of transformed and non-transformed (regenerating) protoplasts and give PCR products in both the positive and negative controls.
Results and discussion
Primers used in this study to delete myn6
Results of the screening of transformants from four different gene deletion experiments
Nour(+) Phleo(−) colonies
Nour(+) Phleo(−) colonies (% of Nour(+) colonies)
Transformants with a gene deletion
Transformants with a gene deletion (% of Nour(+) Phleo(−) colonies)
The use of simultaneous selection for transformation and homologous integration would simplify the screening even more. Kothe et al. (1993) suggested the use of trp1 (involved in tryptophan synthesis) as a marker for ectopic integration. In their proposed setup the deletion construct is transformed to a trp1−frl1− strain that shows fluorescence under UV exposure as a consequence of anthranilic acid accumulation. Colonies with an ectopic integration are not fluorescent because the product of trp1 processes anthranilic acid to tryptophan. Unfortunately, this strategy implies the use of a strain with two mutations. In contrast, pDelcas can be used in any strain.
Recently, we described that the number of S. commune transformants can be increased tenfold by adding a low non-selective concentration of phleomycin in the regeneration medium (van Peer et al. 2009). This increase can be explained by the fact that this antibiotic introduces double strand breaks in the DNA. We have also observed that the low concentration of phleomycin promotes single integrations. However, addition of a low concentration of phleomycin in the regeneration medium did not improve efficiency of gene deletion using pDelcas. In fact, no gene deletions were observed in nearly a thousand transformants that had been transformed with pDelcas derivatives. These data and the fact that phleomycin increases expression of genes involved in non homologous end joining (van Peer et al. 2009) indicates that this antibiotic promotes ectopic integrations.
Efficient gene deletion protocols are essential for functional gene analysis. The genomes of the mushroom forming fungi Phanerochaete chrysosporium, Coprinopsis cinerea, Laccaria bicolor and S. commune have been sequenced and those of Pleurotus ostreatus and Agaricus bisporus will be ready soon. Vector pDelcas and the described PCR procedure allow rapid screening of transformants in S. commune. Possibly, the same procedure can also be used for deletion of genes in other basidiomycetes. This is indicated by the fact that expression cassettes of S. commune are functional in other basidiomycetes like Pycnoporus cinnabarinus (Alves et al. 2004), Agaricus bisporus and Pleurotus ostreatus (L.G. Lugones, unpublished).
This research is supported by the Dutch Technology Foundation STW, applied science division of NWO and the Technology Program of the Ministry of Economic Affairs.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.