Name: rapid deletion production in fungi via agrobacterium mediated transformation of oscar deletion constructs
Uploaded: 2017-06-12T13:00:00
Description: Watch this Scientific Journal Video about Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs at JoVE.com

The authors thank the following undergraduate and high school students for their work to generate OSCAR mutants in Fusarium verticillioiodes: Anjellica Miller, Athar Naseer, Xiu Lin, Katelyn Woodburry, Chelsea Patterson, Kathleen Robertson, Krystina Bradley, Ashton Rogers, Alexis McKensie, Manny Hernandez, Ashli Crepsac, Jeff Delong, Christian King, Gi Jeong, Maria Belding, Christy Burre, Daniel O&#39;Meara, Lauren (Victoria) Cook, Jake Goodman, Sampriti De, Oge Okoye, Alyssa Beckstead, Garrett Hibbs, Nick Goldstein, Caroline Twum, Chris Benson, Louis Stokes, Hannah Itell, Jane Hulse, Jasim Mohammed, James Loggins, Kelli Russell, Gre&#39;Nisha Jones, Kristin Sheaffer, Mariam Hammady, Ava Wilson, Katrina Bazemore, Toney Harper, Karlin McGhee, Mohmed Momin, Rima Momin, Thi Ngoc Le and Angel Pham.

1. Primer Design for PCR Amplification of Gene Flanks
<ol>
	<li>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.</li>
	<li>Highlight the ORF intended for deletion and label start and stop codons.</li>
	<li>Identify and highlight adjacent ORFs within the downloaded sequence.</li>
	<li>Use the 2 kb 5&#39; end of the GOI ORF and the primer design to...

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-prep+in+ten+minutes.">Mini-prep in ten minutes.</a> Biotechniques. 8 (2), 172 (1990).</li><li>Sanger, F., Nicklen, S., Coulson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequencing+with+chain-terminating+inhibitors.">DNA sequencing with chain-terminating inhibitors.</a> P Natl Acad SciUSA. 74 (12), 5463-5467 (1977).</li><li>Khang, C. H., Park, S. Y., Rho, H. S., Lee, Y. H., Kang, S., Wang, K. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamentous+fungi+(Magnaporthe+grisea+and+Fusarium+oxysporum).">Filamentous fungi (Magnaporthe grisea and Fusarium oxysporum).</a> Agrobacterium Protocols. 2, 403-420 (2007).</li><li>Zhang, Y. J., Zhang, S., Liu, X. Z., Wang Wen, H. A., M, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+genomic+DNA+extraction+suitable+for+analysis+of+bulk+fungal+strains.">A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains.</a> Lett Appl Microbiol. 51 (1), 114-118 (2010).</li><li>Pluthero, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+purification+of+high-activity+Taq+DNA+polymerase.">Rapid purification of high-activity Taq DNA polymerase.</a> Nucleic Acids Res. 21 (20), 4850-4851 (1993).</li><li>McCluskey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boosting+Research+and+Industry+by+Providing+Extensive+Resources+for+Fungal+Research.">Boosting Research and Industry by Providing Extensive Resources for Fungal Research.</a> Gene Expression Systems in Fungi: Advancements and Applications. , 361-384 (2016).</li></ol>

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...

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&#39;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. Figure 1 depicts the assembly process in the OSCAR approach. The method described here has the advantage of combining rapid construction of individual gene deletion vectors in a single multipart reaction in combination with subsequent Agrobacterium tumefaciens mediated transformation (ATMT). OSCAR is very rapid and compares well with other strategies such as use of Gibson assembly in yeast2. The OSCAR method has been used successfully with several Ascomycota species of fungi. These species include: Fusarium verticillioides (unpublished), Verticillium dahliae3, Setosphaeria turcica4, Metarhizium robertsii5, Fusarium oxysporum f. sp. vasinfectum6, Pestalotiopsis microspora7, Colletotrichum higginsianum8, and Dothistroma septosporum9 and Sarocladium zeae (unpublished).
This protocol provides step-by-step instruction for the method including primer design, flank PCR amplification, the OSCAR BP reaction, deletion construct structure confirmation, transformation of Agrobacterium with the construct followed by ATMT based transfer of the deletion construct into the fungal cells, and finally differentiating fungal deletion mutants from those with ectopically integrated deletion constructs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>FungiDB</td>
			<td></td>
			<td></td>
			<td>Database/ http://fungidb.org/fungidb/</td>
		</tr>
		<tr>
			<td>IDT PrimerQuest</td>
			<td>IDT</td>
			<td></td>
			<td>Primer design online software/ http://www.idtdna.com/Primerquest/Home/Index</td>
		</tr>
		<tr>
			<td>Microsoft Word</td>
			<td></td>
			<td></td>
			<td>Sequence file manipulation</td>
		</tr>
		<tr>
			<td>Low Na LB Spec 100 medium</td>
			<td></td>
			<td></td>
			<td>E. coli transformant selection, composition: 1% tryptone, 0.05% NaCl, 0.5% yeast extract, 1.5 % agar if for solid medium</td>
		</tr>
		<tr>
			<td>Co-cultivation medium</td>
			<td></td>
			<td></td>
			<td>ATMT transformation induction (Reference 12)</td>
		</tr>
		<tr>
			<td>Aspergillus minimal medium with Hygromycin</td>
			<td></td>
			<td></td>
			<td>Fungal transformant selection</td>
		</tr>
		<tr>
			<td>PDA medium</td>
			<td>Acumedia</td>
			<td>7149A</td>
			<td>Single spore slant tubes</td>
		</tr>
		<tr>
			<td>PDA-Hyg-Kan medium</td>
			<td></td>
			<td></td>
			<td>Fungal ransformant isolation, PDA containing 150 &#956;g/ml hygromycin B and 100 &#956;g/ml Kanamycin;</td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>Genlantis</td>
			<td>C400100</td>
			<td>Plate spreading</td>
		</tr>
		<tr>
			<td>Nitrocellulose filters (47mm)</td>
			<td>Fisher</td>
			<td>09-719-555</td>
			<td>Co-culturing for ATMT</td>
		</tr>
		<tr>
			<td>Various centifuge tubes</td>
			<td></td>
			<td></td>
			<td>multiple preps</td>
		</tr>
		<tr>
			<td>Petri plates (various)</td>
			<td></td>
			<td></td>
			<td>Culturing of bacteria and Fungi</td>
		</tr>
		<tr>
			<td>pA-Hyg OSCAR</td>
			<td>Addgene</td>
			<td>29640</td>
			<td>Selectable marker vector</td>
		</tr>
		<tr>
			<td>pOSCAR</td>
			<td>Addgene</td>
			<td>29639</td>
			<td>Assembly vector</td>
		</tr>
		<tr>
			<td>DH5a One Shot Competent E. coli cells</td>
			<td>Life Technologies&#160;</td>
			<td>12297-016</td>
			<td>BP reaction transformation</td>
		</tr>
		<tr>
			<td>ccdB survival E. coli cells</td>
			<td>Life Technologies&#160;</td>
			<td>A10460</td>
			<td>Maintenance of pOSCAR</td>
		</tr>
		<tr>
			<td>Wooden transfer sticks</td>
			<td></td>
			<td></td>
			<td>Colony streaking</td>
		</tr>
		<tr>
			<td>Toothpicks</td>
			<td></td>
			<td></td>
			<td>Colony picking</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td></td>
			<td></td>
			<td>Pelleting Bacteria etc</td>
		</tr>
		<tr>
			<td>Preparative centrifuge</td>
			<td></td>
			<td></td>
			<td>Fungal spore collection</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td></td>
			<td></td>
			<td>Single spore isolation</td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td></td>
			<td></td>
			<td>Spore suspension calculation</td>
		</tr>
		<tr>
			<td>Compound microscope</td>
			<td></td>
			<td></td>
			<td>Hemocytometer cell counting</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit&#160;</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>PCR gene flank produict purification</td>
		</tr>
		<tr>
			<td>TaKaRa LA Taq&#160;</td>
			<td>Takara Bio USA</td>
			<td>RR002A</td>
			<td>Hi Fidelity taq polymerase for OSCAR flank generation</td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>InvivoGen</td>
			<td>ant-hg-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>Sigma</td>
			<td>22189-32-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Cefotaxim&#160;</td>
			<td>TCI America</td>
			<td>C2224</td>
			<td></td>
		</tr>
		<tr>
			<td>Moxalactam&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>43963</td>
			<td></td>
		</tr>
		<tr>
			<td>GelRed</td>
			<td>&#160;Phenix Research Products</td>
			<td>RGB-4103</td>
			<td>Post staining agarose gels</td>
		</tr>
		<tr>
			<td>Qiagen QIAquick PCR Purification Kit (Cat. No. 28104)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(OneShot_ Mach1TM T1R or One Shot_ OmniMAX&#8482; 2 T1R from Invitrogen)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>C862003</td>
			<td></td>
		</tr>
		<tr>
			<td>Gateway BP Clonase II Enzyme mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>11789020</td>
			<td>Used to assemble deletion construct in pOSAR</td>
		</tr>
		<tr>
			<td>PrimerQuest tool</td>
			<td>IDT&#160;</td>
			<td></td>
			<td>Used in step 1.4; available on http://www.idtdna.com/Primerquest/Home/Index</td>
		</tr>
	</tbody>
</table>

rapid deletion production in fungi via agrobacterium mediated transformation of oscar deletion constructs

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular gene&#39;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&#39; and 3&#39; 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.

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...

Watch this Scientific Journal Video about Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs at JoVE.com

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

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.

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

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular ...

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