Name: genetic manipulation of the plant pathogen ustilago maydis to study fungal biology and plant microbe interactions
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Description: Watch this Scientific Journal Video about Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions at JoVE.com

Special thanks to Dr. Benedikt Steuten for critical reading of the manuscript. The original work on the chitinases was carried out by Dr. Thorsten Langner. The laboratory of VG is supported by the Cluster of Excellence in Plant Sciences (CEPLAS, DFG EXC 1028) and BioSC, the laboratory of KS is supported by BioSC. KB is supported by BioSC. The scientific activities of the Bioeconomy Science Center (BioSC) were financially supported by the Ministry of Innovation, Science and Research within the framework of the NRW Strategieprojekt BioSC (No. 313/323-400-00213). LF is supported by a doctoral fellowship of the DFG International Research Training Group 1525 iGRADplant.

1. Generation of Deletion Constructs
<ol>
	<li>Generate a plasmid containing the deletion construct (Figure 1) comprising a respective 1 kb upstream flank (UF) and downstream flank (DF) each and the appropriate resistance cassette flanked by blunt cutting restriction sites. 
	NOTE: Any cloning strategy can be used (for cloning see reference 22) we recommend Golden Gate cloning for such plasmids 9.</li>
	<li>Excise the deletion construct from the plasmid using a blunt cutter t...

<ol>
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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+Rapid+and+Efficient+Method+for+Assessing+Pathogenicity+of+Ustilago+maydis+on+Maize+and+Teosinte+Lines.">A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines.</a> Journal of Visualized Experiments. (83), e50712 (2014).</li><li>Khrunyk, Y., M&#252;nch, K., Schipper, K., Lupas, A. N., Kahmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+FLP-mediated+recombination+for+the+functional+analysis+of+an+effector+gene+family+in+the+biotrophic+smut+fungus+Ustilago+maydis.">The use of FLP-mediated recombination for the functional analysis of an effector gene family in the biotrophic smut fungus Ustilago maydis.</a> New Phytologist. 187, 957-968 (2010).</li><li>van Peer, A. F., de Bekker, C., Vinck, A., W&#246;sten, H. A. B., Lugones, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phleomycin+Increases+Transformation+Efficiency+and+Promotes+Single+Integrations+in+Schizophyllum+commune.">Phleomycin Increases Transformation Efficiency and Promotes Single Integrations in Schizophyllum commune.</a> Appl Environ Microb. 75, 1243-1247 (2009).</li><li>Sarkari, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+expression+of+single-chain+antibodies+in+Ustilago+maydis.">Improved expression of single-chain antibodies in Ustilago maydis.</a> Journal of Biotechnology. 191, 165-175 (2014).</li><li>Schirawski, J., Heinze, B., Wagenknecht, M., Kahmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mating+type+loci+of+Sporisorium+reilianum:+Novel+pattern+with+three+a+and+multiple+b+specificities.">Mating type loci of Sporisorium reilianum: Novel pattern with three a and multiple b specificities.</a> Eukaryotic Cell. 4, 1317-1327 (2005).</li><li>Cervantes-Chavez, J. 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F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloining: a laboratry manual. , (1989).</li><li>Brachmann, A., Weinzierl, G., K&#228;mper, J., Kahmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+genes+in+the+bW/bE+regulatory+cascade+in+Ustilago+maydis.">Identification of genes in the bW/bE regulatory cascade in Ustilago maydis.</a> Molecular Microbiology. 42, 1047-1063 (2001).</li><li>Becht, P., Vollmeister, E., Feldbr&#252;gge, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+RNA-binding+proteins+implicated+in+pathogenic+development+of+Ustilago+maydis.">Role for RNA-binding proteins implicated in pathogenic development of Ustilago maydis.</a> Eukaryotic Cell. 4, 121-133 (2005).</li><li>Vollmeister, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+KH+domains+of+Khd4+recognize+AUACCC+and+are+essential+for+regulation+of+morphology+as+well+as+pathogenicity+in+Ustilago+maydis.">Tandem KH domains of Khd4 recognize AUACCC and are essential for regulation of morphology as well as pathogenicity in Ustilago maydis.</a> RNA. 15, 2206-2218 (2009).</li><li>Stock, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+unconventional+secretion+of+the+endochitinase+Cts1+to+export+heterologous+proteins+in+Ustilago+maydis.">Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis.</a> Journal of Biotechnology. 161, 80-91 (2012).</li><li>Baumann, S., Pohlmann, T., Jungbluth, M., Brachmann, A., Feldbr&#252;gge, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin-3+and+dynein+mediate+microtubule-dependent+co-transport+of+mRNPs+and+endosomes.">Kinesin-3 and dynein mediate microtubule-dependent co-transport of mRNPs and endosomes.</a> Journal of Cell Science. 125, 2740-2752 (2012).</li><li>Pohlmann, T., Baumann, S., Haag, C., Albrecht, M., Feldbr&#252;gge, 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+FYVE+zinc+finger+domain+protein+specifically+links+mRNA+transport+to+endosome+trafficking.">A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking.</a> eLife. 4, (2015).</li><li>Kronstad, J. 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This protocol describes how to generate deletion mutants for reverse genetic studies in U. maydis. The starting point is a deletion construct that contains flanking sequences of the gene-of-interest containing sequences of about 1 kb upstream of the start and downstream of the stop-codon as well as an appropriate resistance cassette as it was previously optimized 7,9. The constructs have to be individually generated for each gene and carefully verified for sequence errors prior to deleting the gene. P...

Ustilago maydis is a phytopathogenic model fungus that has been studied extensively for decades 1,2. It exists in two morphologies, a yeast-like, non-pathogenic stage and a filamentous, infectious form 3. Universal breakthrough discoveries such as homologous recombination and DNA repair mechanisms were made in the yeast-like growth stage of this fungus 4. Furthermore, the morphological switch to the infectious filament and virulence factors important for infection are well-characterized 5,6. The increasing molecular knowledge about biology and virulence of this smut fungus relies on a straightforward gene replacement strategy 7-9 supported by an excellent genome annotation 10 and the ease of reverse genetics using, e.g., the well-organized plasmid collection at our institute (http://www.mikrobiologie.hhu.de/ustilago-community.html). Standardized, rapid infection assays in maize seedlings allow detailed studies of pathogenicity factors 11.
The genome of U. maydis contains about 6,900 genes 10. To study their function, they can be deleted individually or in combination due to an efficient homologous recombination system. Flanking regions of about 1 kb containing perfectly homologous ends are ideal for rates of homologous recombination greater than 50%, but already 250 bp with non-homologous ends allow some degree of correct integration of the construct 9. Currently, five different resistance cassettes, hygR, cbxR, natR, G418R, and phleoR mediating resistance against hygromycin, carboxin, nourseothricin, G418 and phleomycin, are employed to select for transformants 7,9. In addition, the hygromycin resistance has been developed into a recyclable cassette (FRT-hygR) that can be removed by the transient expression of a heterologous&#160;FLP recombinase 12. This allows removal of the resistance cassette and thereby in theory unlimited genetic modifications. Phleomycin is mutagenic 13, so that with the new cassettes, in particular the recyclable hygR cassette, the use of phleoR is decreasing. Quadruple mutants can thus be generated using the other four cassettes, but for quintuple mutants, the FRT-hygR system is recommended 14.
This general gene deletion strategy has been successfully transferred to other smut fungi such as Sporisorium reilianum 15, U. hordei 16, or U. esculenta 17 and therefore offers the potential for further applications in yet genetically intractable organisms with an efficient homologous recombination system. Moreover, organisms lacking homologous recombination can be modified to improve genetic engineering as exemplified by the deletion of genes involved in non-homologous-end-joining in Neurospora crassa 18,19.
Here we describe the published gene deletion strategy for U. maydis 7,9 in experimental detail with a focus on the rapid and accurate verification of the candidates. As an example, we use the fungal chitinases and depict the generation of single mutants as well as multiple deletion strains 20,21. Chitinases are interesting examples, because they act on chitin in the rigid cell wall. Cell wall remodeling is required for morphological changes during cell division, switch to filamentous growth, and spore formation. Hence, in deletion mutants phenotypes throughout the lifecycle can be expected.

<table border="0" cellpadding="0" cellspacing="0" width="1138">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Aminobenzoeic acid (Free Acid)</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">A-9878</td>
			<td style="width:485px;"></td>
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			<td height="21" style="height:21px;">Bacto Agar&#160;</td>
			<td style="width:183px;">BD</td>
			<td style="width:124px;">214010</td>
			<td>alternatively use local supplier</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Bacto Peptone</td>
			<td style="width:183px;">BD</td>
			<td style="width:124px;">211677</td>
			<td>alternatively use local supplier</td>
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			<td height="21" style="height:21px;">Bacto Yeast Extract&#160;</td>
			<td style="width:183px;">BD</td>
			<td style="width:124px;">212750</td>
			<td>alternatively use local supplier</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:347px;">CaCl2*2H2O</td>
			<td style="width:183px;">Gr&#252;ssing GmbH</td>
			<td style="width:124px;">10234</td>
			<td>alternatively use local supplier</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Ca-pantothenat (Hemi-Ca. salt)</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">P-2250</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Carboxin&#160;</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">45371</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Casamino acids&#160;</td>
			<td style="width:183px;">BD</td>
			<td style="width:124px;">223050</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Cholinchlorid</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td>C-1879</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Citric acid</td>
			<td style="width:183px;">ChemSolute</td>
			<td style="width:124px;">24,321,000</td>
			<td>alternatively use local supplier</td>
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			<td height="25" style="height:25px;width:347px;">CuSO4*5H2O</td>
			<td style="width:183px;">Fluka</td>
			<td style="width:124px;">61240</td>
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			<td height="21" style="height:21px;width:347px;">D(+)Sucrose&#160;</td>
			<td style="width:183px;">Roth</td>
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			<td>alternatively use local supplier</td>
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			<td height="21" style="height:21px;width:347px;">DNA degr. free acid&#160;</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:124px;">D-3159</td>
			<td></td>
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			<td height="21" style="height:21px;width:347px;">EDTA</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">E4378</td>
			<td></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:347px;">FeCl3*6H2O</td>
			<td style="width:183px;">Gr&#252;ssing GmbH</td>
			<td style="width:124px;">10288</td>
			<td>alternatively use local supplier</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Geneticin (G418) disulfate salt</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">A1720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichoderma lysing enzymes</td>
			<td>Sigma Aldrich</td>
			<td>L1412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Glucose</td>
			<td style="width:183px;">Caelo</td>
			<td style="width:124px;">2580</td>
			<td>alternatively use local supplier</td>
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			<td height="21" style="height:21px;width:347px;">Glycerin&#160;</td>
			<td style="width:183px;">Fisher Chemical</td>
			<td style="width:124px;">G065015</td>
			<td>alternatively use local supplier</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:347px;">H3BO3</td>
			<td style="width:183px;">AppliChem</td>
			<td style="width:124px;">A2940</td>
			<td>Dangerous substance. Please check manufacturer&#39;s safety instructions.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin sodium salt</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">H3393-50KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hygromycin B-solution</td>
			<td style="width:183px;">Roth</td>
			<td style="width:124px;">1287.2</td>
			<td>Dangerous substance.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">KCl</td>
			<td style="width:183px;">VWR</td>
			<td style="width:124px;">26764298</td>
			<td>alternatively use local supplier</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:347px;">KH2PO4</td>
			<td style="width:183px;">AppliChem</td>
			<td style="width:124px;">A3620</td>
			<td>alternatively use local supplier</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:347px;">MgSO4 waterfree</td>
			<td style="width:183px;">Merck</td>
			<td style="width:124px;">7487-88-9</td>
			<td>Water free is critical. Alternatively use local supplier</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">MnCl2*4H2O</td>
			<td style="width:183px;">AppliChem</td>
			<td style="width:124px;">A2087</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">myo-Inositol</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td>I-5125</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">Na2-EDTA*2H2O</td>
			<td style="width:183px;">AppliChem</td>
			<td style="width:124px;">A2937</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">Na2MoO4*2H2O</td>
			<td style="width:183px;">Roth</td>
			<td style="width:124px;">0274.2</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">Na2SO4</td>
			<td style="width:183px;">Gr&#252;ssing GmbH</td>
			<td style="width:124px;">12174</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">NaCl</td>
			<td style="width:183px;">Fisher Chemical</td>
			<td style="width:124px;">S316060</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">NaOH</td>
			<td style="width:183px;">ChemSolute</td>
			<td style="width:124px;">13,751,000</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">NH4NO3</td>
			<td style="width:183px;">Roth</td>
			<td style="width:124px;">K299.1</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nicotinic acid (Free Acid)</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">N-4126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nourseothricin dihydrogen sulfate&#160;&#160;</td>
			<td style="width:183px;">Werner BioAgents</td>
			<td style="width:124px;">5,001,000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Nutrient broth&#160;</td>
			<td style="width:183px;">Difco</td>
			<td style="width:124px;"></td>
			<td>local suppliers</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">Phenol:Chloroform:Isoamyl Alcohol (25:24:1) pH 6.7</td>
			<td>Sigma Aldrich</td>
			<td>P3803</td>
			<td>Dangerous substance. Please check manufacturer&#39;s safety instructions.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polyethylene glycol (PEG)</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">P-3640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium acetate</td>
			<td style="width:183px;">AppliChem</td>
			<td>121479</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pyridoxin (Monohydrochlorid)</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">P-9755</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Riboflavin&#160;</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">R4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">RNaseA</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">R5503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">SDS</td>
			<td style="width:183px;">Roth</td>
			<td style="width:124px;">Cn30.3</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">small syringe</td>
			<td>BD</td>
			<td>300300</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sterile filter, 22 &#181;m</td>
			<td>VWR</td>
			<td>28145-477</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Sorbitol</td>
			<td style="width:183px;">Roth</td>
			<td style="width:124px;">6213.1</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Thiamin-Hydrochloride</td>
			<td style="width:183px;">Serva</td>
			<td style="width:124px;">36020.02</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">tri-Na-Citrate</td>
			<td style="width:183px;">Fisher Chemical</td>
			<td style="width:124px;">S332060</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Tris- (hydroxymethyl) aminomethane</td>
			<td>VWR</td>
			<td style="width:124px;">103156X</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Tris hydrochloride</td>
			<td>Roth</td>
			<td style="width:124px;">9090.4</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100&#160;</td>
			<td style="width:183px;">Serva&#160;</td>
			<td style="width:124px;">37240</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">ZnCl2</td>
			<td style="width:183px;">Fluka</td>
			<td style="width:124px;">96470</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Name</td>
			<td style="width:183px;">Company</td>
			<td style="width:124px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Composition of solutions/preparation of material</td>
			<td></td>
			<td></td>
			<td>Composition of solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carboxin</td>
			<td></td>
			<td></td>
			<td>Stock: 5 mg/ml in methanol, final concentration: 2 &#181;g/ml</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CM plates</td>
			<td></td>
			<td></td>
			<td>0.25 % (w/v) Casamino acids, 0.1 % (w/v) Yeast Extract, 1.0 % (v/v) Holliday vitamin solution, 6.25 % (v/v); Holliday salt solution, 0.05 % (w/v) DNA degr. free acid, 0.15 % (w/v) NH4NO3, 2.0 % (w/v) Bacto Agar; adjust to pH 7.0 using 5 M NaOH; after autoclaving add 1 % glucose&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Geneticin (G418)</td>
			<td></td>
			<td></td>
			<td>Stock: 50 mg/ml in H2O, final concentration: 500 &#181;g/ml</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">HCl-washed glass beads (0,35-0,45 mm)</td>
			<td></td>
			<td></td>
			<td>Cover glass beads with concentrated HCl (25 %, 7.8 M) and incubate for 60 min. Sway several times. Decant HCl (keep decanted liquid) and wash glass beads with 3 M HCl (keep decanted liquid). Wash glass beads several times with double distilled H2O until the pH is 7 (the liquid should not be yellow-green anymore). Aliquot the glass beads and dry them at 180 &#176;C. The decanted HCl has to be neutralized before disposal.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin</td>
			<td></td>
			<td></td>
			<td>Stock: 15 mg/ml</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Holliday salt solution</td>
			<td></td>
			<td></td>
			<td>16.0 &#8240; (w/v) KH2PO4, 4.0 &#8240; (w/v) Na2SO4, 8.0 &#8240; (w/v) KCl, 1.32 &#8240; (w/v) CaCl2*2H2O, 8.0 &#8240; (v/v) trace elements, 2.0 &#8240; (w/v) MgSO4; sterile filtrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Holliday vitamin solution&#160;</td>
			<td></td>
			<td></td>
			<td>0.1&#8240; (w/v) Thiamin, 0.05&#8240; (w/v) Riboflavin, 0.05&#8240; (w/v) Pyridoxin, 0.2&#8240; (w/v) Ca-Pantothenat, (0.05&#8240; (w/v) Aminobenzoeic acid, 0.2&#8240; (w/v) Nicotinic acid, 0.2&#8240; (w/v) Cholinchlorid, 1.0&#8240; (w/v) myo-Inositol; may be stored at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hygromycin</td>
			<td></td>
			<td></td>
			<td>Stock: 50 mg/ml in PBS, final concentration: 200 &#181;g/ml</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Nourseothricin</td>
			<td></td>
			<td></td>
			<td>Stock: 200 mg/ml in H2O, final concentration: 150 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NSY-glycerol-medium</td>
			<td></td>
			<td></td>
			<td>0.8 % (w/v) Nutrient Broth, 0.1 % (w/v) Yeast Extract, 0.5 % (w/v) Sucrose,&#160; 80.0 % (v/v) 87% Glycerin (f.c. 69.6%)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RegLight</td>
			<td></td>
			<td></td>
			<td>1.0% (w/v) Yeast Extract&#160; 0.4 % (w/v) Bacto Peptone, 0.4 % (w/v) Sucrose, 18.22 % (w/v) Sorbitol, 1.5 % (w/v) Agar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SCS, pH 5.8</td>
			<td></td>
			<td></td>
			<td>Solution 1: 20 mM tri-Na-citrate, 1 M Sorbitol; colution 2: 20 mM Citric acid, 1 M Sorbitol, add solution 2 into solution 1 until pH 5.8 is reached; autoclave</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">STC, pH 8</td>
			<td></td>
			<td></td>
			<td>1 M Sorbitol, 10 mM Tris-HCl pH 7.5, 100 mM CaCl2; filter sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">STC/PEG</td>
			<td></td>
			<td></td>
			<td>40 % (v/v) PEG in STC-buffer&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">TE buffer, pH 8</td>
			<td></td>
			<td></td>
			<td>1.31 mM Tris-Base, 8.69 mM Tris-HCl, 10 mM Na2-EDTA*2H2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TE/RNase</td>
			<td></td>
			<td></td>
			<td>10 &#181;g/ml RNaseA in TE buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Trace elements</td>
			<td></td>
			<td></td>
			<td>0.06&#8240; (w/v) H3BO3, 0.14&#8240; (w/v) MnCl*4H2O, 0.4 &#8240; (w/v) ZnCl2, 0.4 &#8240; (w/v) Na2MoO4*2H2O, 0.1 &#8240; (w/v) FeCl3*6H2O, 0.04&#8240; (w/v) CuSO4*5H2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichoderma lysing enzymes solution</td>
			<td></td>
			<td></td>
			<td>12.5 mg/ml SCS; filter sterile; prepare shortly before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl pH 7.5</td>
			<td></td>
			<td></td>
			<td>806 mM Tris-HCl, 194 mM Tris-Base; check the pH and if necessary adjust with HCl; autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Usti-lysis buffer 1, pH 8</td>
			<td></td>
			<td></td>
			<td>10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 1 % (w/v) SDS, 2 % (v/v) TritonX-100, 1 mM EDTA. Do not measure pH using pH meter.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Usti-lysis buffer 2</td>
			<td></td>
			<td></td>
			<td>mix Usti lysis buffer 1 with 1 x TE in a 1:1 ratio</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">YEPS-Light medium</td>
			<td></td>
			<td></td>
			<td>1.0% (w/v) Yeast Extract, 0.4% (w/v) Bacto Peptone, 0.4% (w/v) Sucrose, for plates: 1.5% (w/v) Bacto Agar</td>
		</tr>
	</tbody>
</table>

genetic manipulation of the plant pathogen ustilago maydis to study fungal biology and plant microbe interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

The deletion constructs for all four chitinolytic genes encoded in the U. maydis genome were generated by Golden Gate cloning using the hygR cassette for deletion of cts1, the natR for deletion of cts2, the G418R cassette for deletion of cts3 and the cbxR for deletion of cts4 20. A general overview of the gene replacement strategy is exemplified by the deletion of cts3 (Figure 1</s...

Watch this Scientific Journal Video about Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions at JoVE.com

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Research

JoVE Journal

Genetics

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics ...

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

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We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms ...

Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula

Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula

Schrenkiella parvula is an extremophyte adapted to various abiotic stresses, including multiple ion toxicity stresses. Despite high-quality genomic ...

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast ...

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

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This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in ...