2.2. Construction of Deletion Cassettes by PCR Fusion

The orthologue of the yeast Snf1 gene was identified in B. cinerea by bidirectional blast hit (BBH) and named Bcsnf1, corresponding to the Bcin14g03370 reference gene (strain B05.10). For targeted gene replacement, the split-marker technique was used [44]. With this approach, two constructs are required per transformation, each containing a flanking region of the target gene and a truncated selectable marker cassette (Figure 1). Homologous recombination between the overlapping regions of the selectable marker gene and between the flank regions and their genome counterparts results in a targeted gene deletion and replacement with an intact marker gene. Genomic DNA from B. cinerea B05.10 strain was extracted with a DNeasy Plant Mini Kit (Qiagen, Germany) and used as a template. In the first PCR round, the 5′ and 3′ flanking regions were amplified with the primer pairs Snf1-5′-For and Snf1-5′-Rev, and the Snf1-3′-For and Snf1-3′-Rev (Table S1). The hygromycin resistance gene was amplified with the primers Hyg-For and Hyg-Rev, using a hygromycin cassette as a template [45]. Double-joint PCR [46] was performed with iProofTM High-Fidelity DNA Polymerase (BIO-RAD, Hercules, CA, USA), following the manufacturer’s instructions, and the DNA fragments were purified from agarose gel using GFX PCR and a Gel Band Purification Kit (GE Healthcare, Chicago, IL, USA). Equimolar amounts of the purified fragments were mixed and fused in a second round of linear PCR. The joint fragments were amplified with nested primers in a third round of PCR. The primer pair Snf1-5′-For and Hyg-Nest-Rev was used to amplify the 5′ flanking region and the 5′ fragment of the hygromycin gene, and the Hyg-Nest-For and Snf1-3′-Rev primers to amplify the 3′ fragment of the hygromycin gene and 3′ flanking region (Table S1). The DNA fragments were purified from agarose gel using GFX PCR and a Gel Band Purification Kit.

Construction and verification of B. cinerea Δsnf1 deletion mutants. (A) Illustration of the wild type B05.10 and mutant alleles with the indication of the restriction sites and the probes used in the Southern blot analysis. (B) To verify the gene replacement, genomic DNA from the wild type and the mutant strains was digested with SnaBI and hybridized with a 5′ flanking region-specific probe. Wild type, as a control, shows a 5.57 Kb band, while the two mutant strains Δsnf1.1 and Δsnf1.4 show a 11.3 Kb band, confirming the homologous integration of the hygromycin gene at the 5′ flanking region of the snf1 gene. (C) To verify that no other ectopic integration had occurred in the mutants, genomic DNA from the wild type and the mutant strains were digested with SpeI and hybridized with a hygromycin-specific probe. The two mutants displayed only a unique band of the 6.35 Kb expected size.