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 ...

The overall goal of this protocol is to genetically modify the smut fungus, ustilago maydis. This strain generation method allows functional characterization of genes in a variety of ways. For example, we can delete the genes, we can exchange the promotus, or we can generate fusion constructs.The main advantage of this method is that we can precisely verify the modification, so that any phenotype can be directly linked to the change in the gene of interest. Visual demonstration of this method is essential, because knowing when to stop the protoplasting reaction is critical for successful transformation. In preparation, cool down two milliliter round bottom tubes at minus 20 degrees Celsius.Next, inoculate a three milliliter preculture in YEPS-Light using a fresh plate. Incubate it for 24 hours on a rotating wheel at 28 degrees Celsius. The next day, add 25 microliters of the preculture to 50 milliliters of YEPS-Light in a baffled flask.Then, grow up the culture on an orbital shaker until it reaches the exponential phase. An optical density of 1.0 corresponds to one to two times 10 to the seventh cells in ustilago maydis. Check the optical density at 600 nanometers.A value of 0.6 to 1.0 is acceptable. Next, check the cells for contamination under 40x magnification. Bacterial contaminations can be identified as small and often moving cells.Fungal contaminations can be identified based on their shape or size. Always discard contaminated cultures and only work from clean ones. After pelleting the cells for five minutes at 1, 500 G, discard the supernantant.Then, re-suspend the pellet in 25 milliliters of SCS. Repeat the centrifugation. And meanwhile, prepare three milliliters of protoplasting solution per cell pellet.Pass this solution through a 22-micron filter. Now discard the supernatant and re-suspend the pellet in two milliliters of protoplasting solution. Then let the cells rest at room temperature until 30 to 40%of them are round or pinhead shaped.The reaction can take five to 20 minutes. Start observing it under the microscope after five minutes. To obtain the maximum transformation efficiency, the protoplastic reaction has to be stopped at the right moment.And then, the protoplasts have to be handled very carefully as if they were soap bubbles. Proceed with the protocol, always keeping the protoplasts on ice. The next step is to wash them with 10 milliliters of cold SCS, and then collect them with a five minute spin at 1, 000 G.Repeat this wash step a total of three times.After the washes, re-suspend the pellet in 10 milliliters of cold STC. Then collect the cells again with another five minute spin at 1, 000 G, followed by re-suspending them in 1 milliliter of cold STC. Now make 100 microliter aliquots in the cold tubes and store these aliquots at minus 80 degrees Celsius.To transform ustilago maydis protoplasts, prepare two selections per transformation. First, melt RegLight agar and cool it to 60 degrees Celsius. Then, to some of the melted agar, add the selective antibiotic at double the working concentration, and add 12 milliliters to each plate to form the bottom layer.The sample should be mixed carefully. Avoid making bubbles. Keep the remaining RegLight without antibiotics at 60 degrees Celsius.Next, thaw one tube of protoplasts on ice for each transformation. Once thawed, add one microliter of heparin solution and one microgram of linearized construct DNA. Then keep them on ice for 10 minutes.While waiting, spread 12 milliliters of RegLight without antibiotics over the RegLight with antibiotics. Next, add 500 microliters of STC/PEG to each transformation tube and carefully mix them with inversions. Then continue the incubation on ice for 15 more minutes.Now distribute each transformation reaction onto two separate selective RegLight plates. Make sure all the liquid has soaked into the plates before proceeding. Then, incubate the plates upright at 28 degrees Celsius for five to seven days to get 100 to 200 colonies.If only a few colonies grow, the protoplasts might contain too much cell wall, and this can be tested by transforming them with a cell-fabricating plasma or they might not be able to regenerate and this can be tested by planting them on plates without antibiotics. Later, select 24 transformants for testing. Re-purify them on selective YEPS-Light to obtain single colonies.On each plate, also streak out some of the original strain to make sure the antibiotics are selecting against non-transformants. It is critical to verify the mutations using a three step process that involves two PCRs and a Southern Blot analysis. These are detailed in the text protocol.This section explains how to prepare the genomic DNA needed for these analyses. First, inoculate five milliliters of YEPS-Light with the candidate transformant, and incubate it on a rotating wheel for 24 hours at 28 degrees Celsius. The next day, drop two microliters of the culture on a CM plate.Keep the culture sterile. Then prepare a two milliliter tube with about 200 microliters of hydrochloric acid-washed glass beads and two milliliters of the 24-hour culture. After spinning the tube at 12, 000 G for five minutes, aspirate and discard the supernatant.To the pellet, which may be frozen for storage, add 500 microliters of usti-lysis buffer two. Then shake the tube at 1, 000 RPM on a Vibrax for five to 15 minutes. Make sure that the cells are completely re-suspended, then incubate the tube at 65 degrees Celsius for 15 to 20 minutes.Next, place the tube on ice for five minutes. Once cooled down, add 100 microliters of 8 molar potassium acetate, and apply a brief vortex, or ten inversions. Next, spin the tube at 12, 000 G for 15 minutes at room temperature.Then, aliquot 500 microliters of the supernatant to a 1.5 milliliter tube, and add 400 microliters of isopropanol to the aliquot. After mixing, spin down the tube for five minutes at 12, 000 G.Discard the supernatant and wash the pelleted DNA with 500 microliters of 70%ethanol. Now apply a five minute spin and completely remove the supernatant.After another brief spin, let the DNA pellet air dry for a few minutes, and then add 50 microliters of RNase NTE. After adding the RNase, incubate with shaking, and then proceed to use the DNA for the analyses. Single and double mutants of the chintinases cts3 delta and multiple mutants with up to the three other chitinase genes were generated sequentially with the same constructs in two different genetic backgrounds.Transformants were confirmed by PCR and then by Southern Blot analysis. For the cts3 gene, the homologous recombination rate was 70%in the SG200 strain, suggesting cts3 has a non-essential function. Some of the mutations lead to obvious growth defects.For instance, the cts1/2 double mutant had a pronounced cell separation defect. Staining the septa showed that cytokinesis would complete, but the cells would remain connected. To test the contribution of chitinases to virulence, seedling infection assays were carried out using the SG200 strain mutants.While mutation of a controlled gene, khd4, reduced virulence, deletion of all the chitinases did not affect the infection of maize seedlings. After watching this video, you should have a good understanding of how to genetically modify smut fungi like ustilago maydis. Once mastered, strains can easily be generated in about four weeks.When using this procedure, it is really important to plan the strain verification detail by using future cloning programs. Do this before you start to work with fungal cultures. With slight modifications to this procedure, other smut fungi like sporsorium reilianum or ustilago esculenta or even filamentous fungi, like piriformospora indica, can be genetically modified.Don't forget that working with genetically modified organisms need to be carried out according to regulations in your country.

genetic-manipulation-plant-pathogen-ustilago-maydis-to-study-fungal

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 PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species.
Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family,&#160;it could be easily used to other prokaryote and eukaryote species

Here, we present a protocol for tracing genomic DNA (gDNA) contamination in RNA samples. The presented method utilizes primers specific for the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) genes. The method is suited for reliable and sensitive detection of DNA contamination in most eukaryotes and prokaryotes.

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 of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 &#181;g DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., &#62; 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.

Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome 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

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

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

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

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 a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

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 abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

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

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms to questions surrounding insect's interactions with their host plants. Genomic resources are available for several aphid species, and with advances in the next-generation sequencing, transcriptomic studies are being extended to non-model organisms that lack genomes. Furthermore, aphid cultures can be collected from the field and reared in the laboratory for the use in organismal and molecular experiments to bridge the gap between ecological and genetic studies. Last, many aphids can be maintained in the laboratory on their preferred host plants in perpetual, parthenogenic life cycles allowing for comparisons of asexually reproducing genotypes. Aphis nerii, the milkweed-oleander aphid, provides one such model to study insect interactions with toxic plants using both organismal and molecular experiments. Methods for the generation and maintenance of the plant and aphid cultures in the greenhouse and laboratory, DNA and RNA extractions, microsatellite analysis, de novo transcriptome assembly and annotation, transcriptome differential expression analysis, and qPCR verification of differentially expressed genes are outlined and discussed here.

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

The aphid Aphis nerii colonizes on highly-defended plants in the dogbane family (Apocyanaceae) and provides numerous opportunities to study plant-insect interactions. Here, we present a series of protocols for the maintenance of plant and aphid cultures, and the generation and analysis of molecular and -omic data for A. nerii.

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

Schrenkiella parvula is an extremophyte adapted to various abiotic stresses, including multiple ion toxicity stresses. Despite high-quality genomic resources available to study how plants adapt to environmental stresses, its value as a functional genomics model and tool has been limited by the lack of a feasible transformation system. In this protocol, we report how to generate stable transgenic S. parvula lines using an Agrobacterium-mediated floral-dip method. We modified the transformation protocol used for A. thaliana to account for unique traits of S. parvula, such as an indeterminate flowering habit and a high epicuticular wax content on leaves. Briefly, S. parvula seeds were stratified at 4 &#176;C for five days before planting. Plants were grown at a photoperiod of a 14 h light and 10 h dark and a 130 &#181;mol m-2s-1 light intensity, at 22 &#176;C to 24 &#176;C. Eight to nine week-old plants with multiple inflorescences were selected for transformation. These inflorescences were dipped in an infiltration solution of Agrobacterium tumefaciens GV3101 carrying the pMP90RK plasmid. We performed two rounds of flower dipping with an interval of three to four weeks to increase the transformation efficiency. The T1 seeds were collected and dried for four weeks in a container with desiccants before germination to screen for candidate transformed lines. Resistance to BASTA was used to screen T1 plants. We sprayed the BASTA solution three times with an interval of three days starting at two week-old plants to reduce false positives. A BASTA drop test was performed on surviving individual plants to identify true positive transformants. The transformation efficiency was 0.033%, yielding 3&#8211;4 transgenic plants per 10,000 T1 seeds propagated.

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

Agrobacterium-mediated transformation using a floral-dip method can be successfully employed to create stable transgenic lines of the extremophyte model Schrenkiella parvula. We present a protocol modified from that for Arabidopsis thaliana, considering different growth habits and physiological characteristics of the extremophyte.

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

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast permitting in vivo phenotypic analysis. This method does not rely on maize transformation but exploits microbial genetics and secretory capabilities of pathogens. Herein, it allows inspection of in vivo delivered secreted proteins with high spatiotemporal resolution at different kinds of infection sites and tissues. The Trojan horse strategy can be utilized to transiently complement maize loss-of-function phenotypes, to functionally characterize protein domains, to analyze off-target protein effects, or to study onside protein overdosage, making it a powerful tool for protein studies in the maize crop system. This work contains a precise protocol on how to generate a Trojan horse strain followed by standardized infection protocols to apply this method to three different maize tissue types.

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

This work describes the cloning of an Ustilago maydis Trojan horse strain for the in situ delivery of secreted maize proteins into three different types of maize tissues.

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

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

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

Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.

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 regulating gene expression at the post-transcriptional level. Sequencing sRNA libraries by Next Generation Sequencing (NGS) methods has been widely employed to identify and analyze miRNA transcriptomes in the last decade, resulting in a rapid increase of miRNA discovery. However, two major challenges arise in plant miRNA annotation due to increasing depth of sequenced sRNA libraries as well as the size and complexity of plant genomes. First, many other types of sRNAs, in particular, short interfering RNAs (siRNAs) from sRNA libraries, are erroneously annotated as miRNAs by many computational tools. Second, it becomes an extremely time-consuming process for analyzing miRNA transcriptomes in plant species with large and complex genomes. To overcome these challenges, we recently upgraded miRDeep-P (a popular tool for miRNA transcriptome analyses) to miRDeep-P2 (miRDP2 for short) by employing a new filtering strategy, overhauling the scoring algorithm and incorporating newly updated plant miRNA annotation criteria. We tested miRDP2 against sequenced sRNA populations in five representative plants with increasing genomic complexity, including Arabidopsis, rice, tomato, maize and wheat. The results indicate that miRDP2 processed these tasks with very high efficiency. In addition, miRDP2 outperformed other prediction tools regarding sensitivity and accuracy. Taken together, our results demonstrate miRDP2 as a fast and accurate tool for analyzing plant miRNA transcriptomes, therefore a useful tool in helping the community better annotate miRNAs in plants.

A bioinformatics pipeline, namely miRDeep-P2 (miRDP2 for short), with updated plant miRNA criteria and an overhauled algorithm, could accurately and efficiently analyze microRNA transcriptomes in plants, especially for species with complex and large genomes.

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