Golden Gate vectors for efficient gene fusion and gene deletion in diverse filamentous fungi

Abstract

The cloning of plasmids can be time-consuming or expensive. Yet, cloning is a prerequisite for many standard experiments for the functional analysis of genes, including the generation of deletion mutants and the localization of gene products. Here, we provide Golden Gate vectors for fast and easy cloning of gene fusion as well as gene deletion vectors applicable to diverse fungi. In Golden Gate cloning, restriction and ligation occur simultaneously in a one-pot reaction. Our vector set contains recognition sites for the commonly used type IIS restriction endonuclease BsaI. We generated plasmids for C- as well as N-terminal tagging with GFP, mRFP and 3xFLAG. For gene deletion, we provide five different donor vectors for selection marker cassettes. These include standard cassettes for hygromycin B, nourseothricin and phleomycin resistance genes as well as FLP/FRT-based marker recycling cassettes for hygromycin B and nourseothricin resistance genes. To make cloning most feasible, we provide robust protocols, namely (1) an overview of cloning procedures described in this paper, (2) specific Golden Gate reaction protocols and (3) standard primers for cloning and sequencing of plasmids and generation of deletion cassettes by PCR and split-marker PCR. We show that our vector set is applicable for the biotechnologically relevant Penicillium chrysogenum and the developmental model system Sordaria macrospora. We thus expect these vectors to be beneficial for other fungi as well. Finally, the vectors can easily be adapted to organisms beyond the kingdom fungi.

Tracking Fungal Growth: Establishment of Arp1 as a Marker for Polarity Establishment and Active Hyphal Growth in Filamentous Ascomycetes

“…For the construction of the pArp1-KO knockout plasmid, the Golden Gate cloning system according to Dahlmann et al, 2020, was used [ 57 ]. The 5′-(1311 bp) and 3′-(1791 bp) flanking regions of the Smarp1 gene were amplified from S. macrospora wt gDNA with the Arp1-ko-5f/Arp1-ko-5r and Arp1-ko-3f/Arp1-ko-3r primer pairs, respectively.…”

“…The 5′-(1311 bp) and 3′-(1791 bp) flanking regions of the Smarp1 gene were amplified from S. macrospora wt gDNA with the Arp1-ko-5f/Arp1-ko-5r and Arp1-ko-3f/Arp1-ko-3r primer pairs, respectively. Together with the donor vector pGG-hph and the destination vector pDest-Amp, the fragments were cloned via the Golden Gate procedure [ 57 ].…”

Analysis of the Putative Nucleoporin POM33 in the Filamentous Fungus Sordaria macrospora

“…All plasmids and primers (Sigma-Aldrich Chemie GmbH Taufkirchen, Germany) used in this study are shown in Tables S1 and S2 in Supplementary Materials . Plasmids were constructed via HR in S. cerevisiae [ 34 ] or the Golden Gate (GG) cloning system [ 45 ]. For p5′sci1-egfp_hyg, the S. macrospora sci1 native promotor and ORF and the trpC terminator ( TtrpC ) of Aspergillus nidulans were amplified from plasmid p5′sci1gfp_nat [ 14 ] with the primer pair 5′sci1-f/pRS426GFPrev.…”

“…To generate the knockout-plasmid pPom33-KO, the Golden Gate cloning system according to [ 45 ] was used. The 5′ (726 bp) and 3′ (1021 bp) flanking regions of the pom33 gene were amplified from S. macrospora wt gDNA with primer pairs Pom33-GG-ko-5f/Pom33-GG-ko-5r and Pom33-GG-ko-3f/Pom33-GG-ko-3r, respectively.…”

“…The 5′ (726 bp) and 3′ (1021 bp) flanking regions of the pom33 gene were amplified from S. macrospora wt gDNA with primer pairs Pom33-GG-ko-5f/Pom33-GG-ko-5r and Pom33-GG-ko-3f/Pom33-GG-ko-3r, respectively. Together with donor vector pGG-nat1 and the destination vector pDest-Amp, the fragments were cloned via the Golden Gate procedure [ 45 ].…”