Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

Summary

Gene deletion mutants generated through homologous recombination are the gold standard for gene function studies. The OSCAR (One Step Construction of Agrobacterium-Recombination-ready-plasmids) method for rapid generation of deletion constructs is described. Agrobacterium mediated fungal transformation follows. Finally, a PCR based confirmation method of gene deletions in fungal transformants is presented.

Abstract

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular gene's function in the living organism. In this protocol the OSCAR method of precise and rapid deletion plasmid construction is described. OSCAR relies on the cloning system in which a single recombinase reaction is carried out containing the purified PCR-amplified 5' and 3' flanks of the gene of interest and two plasmids, pA-Hyg OSCAR (the marker vector) and pOSCAR (the assembly vector). Confirmation of the correctly assembled deletion vector is carried out by restriction digestion mapping followed by sequencing. Agrobacterium tumefaciens is then used to mediate introduction of the deletion construct into fungal spores (referred to as ATMT). Finally, a PCR assay is described to determine if the deletion construct integrated by homologous or non-homologous recombination, indicating gene deletion or ectopic integration, respectively. This approach has been successfully used for deletion of numerous genes in Verticillium dahliae and in Fusarium verticillioides among other species.

Introduction

Genetic dissection is a powerful methodology for determining the functional importance of individual or combinations of genes. A standard approach to understand the role of specific genes is production of single gene mutants unaltered in any other gene. The most powerful and least potentially confounding approach is complete and precise deletion of a gene of interest's open reading frame (GOI ORF) without damage to any other gene function.

Because standard ligation approaches for deletion plasmid generation require multiple steps, the rational for OSCAR¹ was to produce a more rapid in vitro approach.

Protocol

1. Primer Design for PCR Amplification of Gene Flanks

1. Download to a word processing file the genomic region of the gene of interest (GOI) including the open reading frame (ORF) and at least 2 kb flanking the gene on each side from FungiDB or other genomic data resource.
2. Highlight the ORF intended for deletion and label start and stop codons.
3. Identify and highlight adjacent ORFs within the downloaded sequence.
4. Use the 2 kb 5' end of the GOI ORF and the primer design tool (see Materials List) to design PCR primer pair O1 and O2 to generate a minimum size product of 1 kb. Take care to not impact adjacent ORFs.

Results

The OSCAR method, in a single reaction, generates a plasmid containing the flanks of the target gene to be deleted surrounding the selectable marker cassette. The production of deletion constructs using OSCAR is very efficient. The system can, however, produce partial constructs containing some but not all three fragments (the two gene flanks and the selectable marker). Generally, the majority of E. coli transformants contain the correct OSCAR construct. For example, Figu.......

Discussion

One Step Construction of Agrobacterium-Recombination-ready-plasmids (OSCAR) has been successfully employed with an ever-increasing number of Ascomycota fungi. The method should also easily be applicable to the Basidiomycota and species from other fungal phyla (with appropriate promoters driving selectable marker genes), assuming Agrobacterium mediated transformation and homologous recombination are possible. Additional marker vectors have been generated to diversify choices of anti-fungal compound as we.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
FungiDB			Database/ http://fungidb.org/fungidb/
IDT PrimerQuest	IDT		Primer design online software/ http://www.idtdna.com/Primerquest/Home/Index
Microsoft Word			Sequence file manipulation
Low Na LB Spec 100 medium			E. coli transformant selection, composition: 1% tryptone, 0.05% NaCl, 0.5% yeast extract, 1.5% agar if for solid medium
Co-cultivation medium			ATMT transformation induction (Reference 12)
Aspergillus minimal medium with Hygromycin			Fungal transformant selection
PDA medium	Acumedia	7149A	Single spore slant tubes
PDA-Hyg-Kan medium			Fungal ransformant isolation, PDA containing 150 μg/mL hygromycin B and 100 μg/mL Kanamycin
Glass beads	Genlantis	C400100	Plate spreading
Nitrocellulose filters (47 mm)	Fisher	09-719-555	Co-culturing for ATMT
Various centifuge tubes			multiple preps
Petri plates (various)			Culturing of bacteria and Fungi
pA-Hyg OSCAR	Addgene	29640	Selectable marker vector
pOSCAR	Addgene	29639	Assembly vector
DH5α One Shot Competent E. coli cells	Life Technologies	12297-016	BP reaction transformation
ccdB survival E. coli cells	Life Technologies	A10460	Maintenance of pOSCAR
Wooden transfer sticks			Colony streaking
Toothpicks			Colony picking
Microcentrifuge			Pelleting Bacteria, etc.
Preparative centrifuge			Fungal spore collection
Dissecting microscope			Single spore isolation
Automated Cell Counter			Spore suspension calculation
Compound microscope			Hemocytometer cell counting
QIAquick PCR Purification Kit	Qiagen	28104	PCR gene flank produict purification
TaKaRa LA Taq	Takara Bio USA	RR002A	Hi Fidelity taq polymerase for OSCAR flank generation
Hygromycin B	InvivoGen	ant-hg-5
Spectinomycin	Sigma	22189-32-8
Cefotaxime	TCI America	C2224
Kanamycin		11815032
Moxalactam	Sigma-Aldrich	43963
GelRed	Phenix Research Products	RGB-4103	Post staining agarose gels
Qiagen QIAquick PCR Purification Kit (Cat. No. 28104)
(OneShot_ Mach1TM T1R or One Shot_ OmniMAX™ 2 T1R from Invitrogen)	Thermo Fisher Scientific	C862003
Gateway BP Clonase II Enzyme mix	Thermo Fisher Scientific	11789020	Used to assemble deletion construct in pOSAR