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 to obtain 1 &#181;g DNA of the deletion construct 9. Purify the deletion construct to eliminate enzyme and buffer 22 e.g. by using commercial reagents and follow the manufacturer&#39;s instructions. 
	NOTE: The vector backbone can remain in the transformation mixture.</li>
</ol>
2. Preparation of Protoplasts
NOTE: Keep sterile conditions during all times of the experiment. The cell wall is a strong protective barrier that limits access of molecules to the plasma membrane. To allow uptake of the DNA containing the deletion construct, the cell wall needs to be removed by cell wall degrading enzymes in a protoplasting reaction. Critical steps in protoplast preparation are 1) osmotic stabilization of the medium and 2) avoiding mechanical stress on the protoplasts.
<ol>
	<li>Mark 2 ml tubes with a round bottom to freeze the protoplasts and cool them down at -20 &#176;C. Start a pre-culture in 3 ml YEPS-Light from a fresh plate of the strain with the desired genetic background. Incubate on a rotating wheel for 24 hr at 28 &#176;C. 
	NOTE: The strain can be the solopathogenic strain SG200 10, wildtype strains, tester strains such as AB33 23, or any mutant background for generation of double mutants. Make sure the strain is free of the desired resistance.</li>
	<li>Dilute the culture in 50 ml YEPS-Light in a baffled flask and incubate on an orbital shaker at 28 &#176;C with 200 rpm. 
	NOTE: The doubling time is about 2 hr for wildtype strains. Allow three duplications minimum.</li>
	<li>Grow until exponential phase. Ensure that the optical density, measured at a wavelength of 600 nm (OD600), is around 0.8 (0.6 to 1.0 is acceptable). An OD600 of 1.0 corresponds to about 1 to 2 x 107U. maydis cells per ml.</li>
	<li>Check the cells for contamination under a microscope. Use 40x&#160;magnification. Pellet the cells of the complete 50 ml culture for 5 min, 1,500 x g and discard the supernatant. Resuspend the pellet in 25 ml sodium citrate, sorbitol solution (SCS), centrifuge 5 min, 1,500 x g, and discard supernatant. 
	NOTE: Bacterial contaminations can be identified as small and often moving cells. Fungal contaminations can be identified based on a different cell shape or size compared to the cigar shaped cells of U. maydis.</li>
	<li>During centrifugation prepare protoplasting solution (12.5 mg/ml Trichoderma lysing enzymes, in SCS; prepare 3 ml per cell pellet; filter sterilize through a 22 &#181;m filter; solution has to be fresh for optimal enzymatic activity). SCS is an osmotic stabilizer to prevent rupture of protoplast upon removal of the cell wall.</li>
	<li>Resuspend the pellet in 2 ml protoplasting solution. Incubate for 5 to 20 min at RT and check under the microscope until 30 to 40% of the cells are round or like pinheads (Figure 2). 
	NOTE: Perform all steps on ice from now on and handle protoplasts with care!</li>
	<li>Wash 3x in 10 ml cold (4 &#176;C) SCS, centrifuge at 1,000 x g for 5 min.</li>
	<li>Resuspend the pellet in 10 ml cold sorbitol, TrisHCl, CaCl2 solution (STC), spin at 1,000 x g for 5 min, discard supernatant. Resuspend the pellet in 1 ml cold STC, make 100 &#181;l aliquots in the cooled tubes. Freeze the aliquots at -80 &#176;C until further use.</li>
</ol>
3. Transformation of U. maydis
NOTE: The transformation of U. maydis protoplasts relies on the polyethylene glycol (PEG)-mediated transformation method, which is technically simple and opposed to electroporation or biolistic transformation does not require specialized equipment.
<ol>
	<li>Prepare the two-layered selection plates (2 plates per transformation reaction). The bottom layer contains the antibiotic in a doubled concentration. Pipet 12 ml of RegLight containing 400 &#181;g/ml hygromycin (or 300 &#181;g/ ml nourseothricin or 4 &#181;g/ml carboxin or 1 mg/ml G418) into a Petri dish. Avoid bubbles. 
	NOTE: Bottom layer plates may be stored a couple of days at 4 &#176;C, but prepare the top layer freshly during transformation (see below).</li>
	<li>Thaw protoplasts on ice (1 tube/transformation). Add 1 &#181;l heparin (15 mg/ml). Add 1 &#181;g DNA (linearized construct) or 10 &#181;l H2O (water control). Incubate for 10 min on ice.</li>
	<li>During this time, for the upper layer spread 12 ml liquefied RegLight (boil and cool to 60 &#176;C) onto the RegLight bottom plate. 
	NOTE: This layer does not contain antibiotics.</li>
	<li>Add 500 &#181;l STC/PEG (polyethylene glycol) to the transformation tube (3. 2) and mix carefully by inverting the tube. Incubate 15 min on ice.</li>
	<li>Distribute each transformation reaction on two of the two-layered RegLight plates. Spread carefully and slowly with a glass pipet. Incubate plates upright at 28 &#176;C for 5 to 7 days until transformants grow as colonies. Obtain about 100-200 colonies per plate. 
	NOTE: Lower numbers might be caused by &#34;bad&#34; protoplasts that either contain too much cell wall and therefore cannot take up DNA or cannot regenerate. The former can be tested by transformation with a self-replicating plasmid, the latter by testing regeneration on plates without antibiotics.</li>
	<li>Prepare YEPS-Light plates containing the appropriate antibiotic at 200 &#181;g/ml hygromycin (or 150 &#181;g/ ml nourseothricin or 2 &#181;g/ml carboxin or 500 &#181;g/ml G418; these concentrations equal half of the one used for bottom plates). Re-purify at least 24 putative transformant colonies on these plates. Single colonies should be obtained. At the same time streak out the original strain on non-selective media. 
	NOTE: Plates can be stored at 4 &#176;C for 1-2 weeks.</li>
</ol>
4. Verification of Correct Deletion Events
NOTE: The mutations are verified in a three-step procedure (Figure 1). First, candidates that contain the wildtype copy of the gene are excluded by PCR. Second, the integration of the resistance cassette into the locus is verified by PCR. Third, the integration of the resistance cassette only into the desired locus is verified by Southern blot analysis. Careful strain verification is essential, so that mutant phenotypes truly correlate with the deletion.
<ol>
	<li>First Diagnostic PCR 20
	<ol>
		<li>Design primers pairing in the gene to be deleted that result in a product of 250-600 bp (Figure 1). Such small products are easy to amplify in a colony PCR, and long enough for detection in standard gel electrophoresis.</li>
		<li>For each candidate and the original strain as a positive control, resuspend a little bit (amount of a pinhead) of cell material of a single colony with a toothpick in 20 &#181;l 0.02 M NaOH. Incubate at RT for 30 min. 
		NOTE: Use fresh colonies. If the plates are older than 1 week re-streak to obtain a fresh colony.</li>
		<li>Use 1 &#181;l of the cell suspension for the PCR immediately. Run a standard PCR reaction and analyze resulting fragments. 
		NOTE: These samples cannot be stored. 
		NOTE: This PCR tests for presence of the wildtype gene and thereby allows rapid identification of negative candidates in which the gene of interest has not been replaced. Clones yielding bands in this PCR reaction will be discarded. One such negative clone should anyway be carried along as an additional negative control. Candidates without a PCR product have to be analyzed further. For genes without detrimental effects approximately half of the candidates should contain the wildtype gene, while the other half should not result in a band. This would correspond to a homologous recombination rate of 50%.</li>
	</ol>
	</li>
	<li>Second Diagnostic PCR
	<ol>
		<li>Preparation of genomic DNA. 
		NOTE: Genomic DNA (gDNA) can be prepared using two different protocols. The first involves toxic reagents like phenol and chloroform and hence, should be handled only by experienced researchers (described in 4.2.1.1 to 4.2.1.5) while the second can also be handled by less experienced students (described in 4.2.1.6 to 4.2.1.11). Both protocols are working reliably. Step 4.2.1.1 is identical for both protocols.
		<ol>
			<li>Inoculate 5 ml YEPS-Light with each candidate and incubate on a rotating wheel for 24 hr at 28 &#176;C. Drop 2 &#181;l of the culture on a CM plate. Keep the culture sterile. 
			NOTE: Following is a standard protocol (CAUTION: toxic steps).</li>
			<li>Put 1 scoop (~200 &#181;l) of HCl-washed glass beads to 2 ml tubes. Add 2 ml of the U. maydis culture. Spin 12,000 x g for 5 min. Discard supernatant (pellet can be frozen at this stage). Add 500 &#181;l Phenol/Chloroform/Isoamylalcohol (25:24:1) to the pellet. CAUTION! PHENOL IS TOXIC!</li>
			<li>Add 500 &#181;l Usti-lysis-buffer 1. Shake for 6 to 10 min on a vibrax at 1,000 rpm. Verify that the cell pellet is completely resuspended, but avoid extended shaking to prevent shearing of the gDNA. Spin at 12,000 x g for 15 min. Transfer 400 &#181;l of the aqueous phase into the new reaction tube (do not touch the interphase).</li>
			<li>Add 1 ml 100 % (v/v) ethanol. Invert the tube 3x to mix, observe the cloud of DNA that shortly appears. Spin at 12,000 x g for 5 min. Remove supernatant and wash with 200 &#181;l 70% EtOH. Do not resuspend the pellet in this stage.</li>
			<li>Remove supernatant completely (do not let the pellet dry completely). Add 50 &#181;l TE/RNase. Dissolve gDNA by shaking at 400 rpm at 50&#176;C for 10 min. Analyze 2 &#181;l of the gDNA on a 0.8 % agarose gel 20. 
			NOTE: The steps below comprise an alternative non-toxic protocol.</li>
		</ol>
		</li>
		<li>Alternate preparation of genomic DNA
		<ol>
			<li>Put 1 scoop (~200 &#181;l) of HCl washed glass beads to 2 ml tubes. Add 2 ml of the U. maydis culture and spin at 12,000 x g for 5 min. Discard supernatant&#160;(pellets can be frozen at this stage).</li>
			<li>Add 500 &#181;l Usti Lysis buffer 2 to the pellet. Shake for 5 to 15 min on a vibrax at 1,000 rpm. Verify that the cell pellet is completely resuspended.</li>
			<li>Incubate the tubes at 65 &#176;C for 15 to 20 min. Using appropriate incubators designed to host 2 ml tubes is critical. Then, place it on ice for 5 min.</li>
			<li>Add 100 &#181;l 8 M potassium acetate, vortex or invert 8 to 10 times. Spin 12,000 x g for 15 min at RT.</li>
			<li>Transfer 500 &#181;l of the supernatant to a fresh 1.5 ml tube and add 400 &#181;l isopropanol. Vortex or invert 8 to 10 times. Spin 12,000 x g for 15 min. Remove supernatant and wash with 500 &#181;l 70% EtOH. Spin 12,000 x g for 5 min.</li>
			<li>Soak off supernatant completely! Eventually, add a short spin (10 sec) to remove residual liquid. Let the DNA pellet dry for 3 to 5 min. Add 50 &#181;l TE/RNAase and incubate at 50 &#176;C for 10 to 15 min at 400 rpm.</li>
		</ol>
		</li>
		<li>PCR
		<ol>
			<li>Design two primer pairs with one of them binding outside the flanks and the corresponding one binding within the resistance cassette (UF-f, RC-r; RC-f, DF-r, Figure 1) 9.</li>
			<li>Use 1 &#181;l of a 1:10 dilution of the gDNA of every candidate as a template in 2 PCR reactions verifying the correct insertion upstream and downstream 9.</li>
			<li>Use standard PCR reactions for each candidate and analyze resulting fragments 22. 
			NOTE: These PCRs test for the insertion of the resistance cassette in the correct genomic locus. If products are obtained for both sides, the candidate is likely a correct insertion. However, also candidates where only one side (upstream or downstream) could be confirmed should be carried along for further analysis in case that PCR reactions simply failed for one flank.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Verification of Correct Genomic Insertion by Southern Blot Analysis 22
	<ol>
		<li>Digest 15 &#181;l of gDNA (4.2.1) of the mutant candidates and the corresponding wildtype/progenitor strain with an appropriate enzyme that cuts outside the locus/construct and in addition, cuts either in the gene or in the resistance cassette leading to clearly distinguishable patterns (Figure 1) 8. 
		NOTE: None of the expected bands should be larger than 10 kb, so that they are clearly distinguishable from undigested DNA.</li>
		<li>Use the upstream and the downstream flank as probes. To label them for example use the PCR DIG labelling Kit or any similar standard method. Mix the labelled probes in a 1:1 molecular ratio and carry out the Southern blot. Keep at least two independent correct transformants. 
		NOTE: The wildtype pattern should be clearly detectable in the original strain and the correct clones should contain only the expected bands. Additional bands only present in the candidates indicate other insertions of the construct in a wrong locus. Such clones should be discarded. The negative candidate identified in the first diagnostic PCR should contain the wildtype bands and the bands (unpredictable in size) of the wrongly integrated deletion construct.</li>
	</ol>
	</li>
	<li>Glycerol Stocks 
	NOTE: Glycerol stocks serve to maintain the clones for years in strain collections. At least two independent transformants should be kept for each mutant. This allows comparing phenotypes that should be identical.
	<ol>
		<li>Grow 5 ml cultures of the confirmed mutant strains in YEPS-Light on a rotating wheel for 24 hr at 28 &#176;C Mix 500 &#181;l of each culture with 500 &#181;l NSY-Glycerol-medium. Invert tube until culture and medium are well mixed. Glycerol in the medium prevents the formation of ice crystals.</li>
		<li>Freeze at -80 &#176;C. Re-streak a part of the frozen culture to test if the stock is functional.</li>
	</ol>
	</li>
</ol>
5. Microscopic Phenotypes
<ol>
	<li>Grow 5 ml cultures of wildtype and mutant strains in YEPS-Light on a rotating wheel for 12 hr at 28 &#176;C.</li>
	<li>Next day, dilute the culture to an OD600 of about 0.1 and grow it till an OD600&#160; between 0.5 and 0.7. An OD600 of 1.0 corresponds to about 1 to 2 x 107 U. maydis cells per ml.</li>
	<li>Inspect the cell morphology microscopically. 
	NOTE: Healthy cells appear cigar shaped (Figure 4, WT). Pronounced vacuoles or filament formation indicate that the cells are stressed. 
	NOTE: Depending on the expected mutant phenotype, the analysis can be adapted, e.g., if reporter strains are used the appropriate assay can be carried out at this stage. If cell morphology is changed statistical analysis should be conducted, e.g., to determine the mean cell length.</li>
</ol>
6. Infection Assay
NOTE: The seedling infection assay has been visualized previously in this journal 11. It can either be carried out using a haploid, solopathogenic strain background such as SG200 10 or by mixing compatible mating partners which both carry the mutation.
<ol>
	<li>Grow at least 40 maize seedlings per strain for 7 days at a light cycle of 16 hr, 200 &#181;E, 28 &#176;C/22 &#176;C (day/night). They should be at the 3-leaf stage and 10-15 cm tall at the day of infection.</li>
	<li>Two days before infection, start a pre-culture of the strains in 5 ml YEPS-Light on a rotating wheel for 24 hr at 28 &#176;C. Grow a main culture in 50 ml YEPS-Light up to OD600 = 1.0 (cell division rate = 2 hr, allow 3 divisions minimum, can be done O/N).</li>
	<li>Spin down the cells of 50 ml culture at 1,500 x g for 5 min and discard the supernatant. Wash 3x with sterile H2O. Resuspend the cells in sterile H2O to an OD600 of 3.0. If necessary, mix the mating partners.</li>
	<li>Inject 250 - 500 &#181;l of the cell suspensions into the stem of 7 day old maize seedlings using a small syringe. Use sterile H2O as a control. Keep infected seedlings for an additional 7-14 days in the same light and temperature conditions. Score the phenotype of each plant according to healthy, chlorosis, anthocyanin formation, small tumors, large tumors, dead plants 10. 
	NOTE: Scoring can be done as early as 7 days and repeated at 14 days to follow infection over time.</li>
</ol>

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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. W., Leong, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+two+Alleles+of+the+b-Locus+of+Ustilago.">Isolation of two Alleles of the b-Locus of Ustilago.</a> P Natl Acad Sci USA. 86, 978-982 (1989).</li><li>Koepke, 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=The+RNA-binding+protein+Rrm4+is+essential+for+efficient+secretion+of+endochitinase+Cts1.">The RNA-binding protein Rrm4 is essential for efficient secretion of endochitinase Cts1.</a> Molecular &amp; cellular proteomics. 10, (2011).</li><li>Schuler, D., 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=Hxt1,+a+monosaccharide+transporter+and+sensor+required+for+virulence+of+the+maize+pathogen+Ustilago+maydis.">Hxt1, a monosaccharide transporter and sensor required for virulence of the maize pathogen Ustilago maydis.</a> New Phytologist 206. , 1086-1100 (2015).</li><li>Schuster, M., Schweizer, G., Reissmann, S., 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=Genome+editing+in+Ustilago+maydis+using+the+CRISPR-Cas+system.">Genome editing in Ustilago maydis using the CRISPR-Cas system.</a> Fungal Genetics and Biology. , (2015).</li></ol>

The authors have nothing to disclose.

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><tbody><tr><td>Aminobenzoeic acid (Free Acid)</td><td>Sigma Aldrich</td><td>A-9878</td><td></td></tr><tr><td>Bacto agar&amp;nbsp;</td><td>BD</td><td>214010</td><td>alternatively use local supplier</td></tr><tr><td>Bacto peptone</td><td>BD</td><td>211677</td><td>alternatively use local supplier</td></tr><tr><td>Bacto yeast extract&amp;nbsp;</td><td>BD</td><td>212750</td><td>alternatively use local supplier</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Gr&amp;uuml;ssing GmbH</td><td>10234</td><td>alternatively use local supplier</td></tr><tr><td>Ca-Pantothenat (Hemi-Ca. salt)</td><td>Sigma Aldrich</td><td>P-2250</td><td></td></tr><tr><td>Carboxin&amp;nbsp;</td><td>Sigma Aldrich</td><td>45371</td><td></td></tr><tr><td>Casamino acids&amp;nbsp;</td><td>BD</td><td>223050</td><td></td></tr><tr><td>Cholinchlorid</td><td>Sigma Aldrich</td><td>C-1879</td><td></td></tr><tr><td>Citric acid</td><td>ChemSolute</td><td>24,321,000</td><td>alternatively use local supplier</td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;*5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fluka</td><td>61240</td><td>alternatively use local supplier</td></tr><tr><td>D(+)Sucrose&amp;nbsp;</td><td>Roth</td><td>4621.1</td><td>alternatively use local supplier</td></tr><tr><td>DNA degr. free acid&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D-3159</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E4378</td><td></td></tr><tr><td>FeCl&lt;sub&gt;3&lt;/sub&gt;*6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Gr&amp;uuml;ssing GmbH</td><td>10288</td><td>alternatively use local supplier</td></tr><tr><td>Geneticin (G418) disulfate salt</td><td>Sigma Aldrich</td><td>A1720</td><td></td></tr><tr><td>Trichoderma lysing enzymes</td><td>Sigma Aldrich</td><td>L1412</td><td></td></tr><tr><td>D(+) Glucose</td><td>Caelo</td><td>2580</td><td>alternatively use local supplier</td></tr><tr><td>Glycerin&amp;nbsp;</td><td>Fisher Chemical</td><td>G065015</td><td>alternatively use local supplier</td></tr><tr><td>H&lt;sub&gt;3&lt;/sub&gt;BO&lt;sub&gt;3&lt;/sub&gt;</td><td>AppliChem</td><td>A2940</td><td>Dangerous substance. Please check manufacturer&#039;s safety instructions.</td></tr><tr><td>Heparin sodium salt</td><td>Sigma Aldrich</td><td>H3393-50KU</td><td></td></tr><tr><td>Hygromycin B-solution</td><td>Roth</td><td>1287.2</td><td>Dangerous substance.&amp;nbsp;</td></tr><tr><td>KCl</td><td>VWR</td><td>26764298</td><td>alternatively use local supplier</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>AppliChem</td><td>A3620</td><td>alternatively use local supplier</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt; waterfree</td><td>Merck</td><td>7487-88-9</td><td>Water free is critical. Alternatively use local supplier</td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;*4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>AppliChem</td><td>A2087</td><td>alternatively use local supplier</td></tr><tr><td>myo-Inositol</td><td>Sigma Aldrich</td><td>I-5125</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;-EDTA*2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>AppliChem</td><td>A2937</td><td>alternatively use local supplier</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;MoO&lt;sub&gt;4&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Roth</td><td>0274.2</td><td>alternatively use local supplier</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Gr&amp;uuml;ssing GmbH</td><td>12174</td><td>alternatively use local supplier</td></tr><tr><td>NaCl</td><td>Fisher Chemical</td><td>S316060</td><td>alternatively use local supplier</td></tr><tr><td>NaOH</td><td>ChemSolute</td><td>13,751,000</td><td>alternatively use local supplier</td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt;</td><td>Roth</td><td>K299.1</td><td>alternatively use local supplier</td></tr><tr><td>Nicotinic acid (Free acid)</td><td>Sigma Aldrich</td><td>N-4126</td><td></td></tr><tr><td>Nourseothricin dihydrogen sulfate&amp;nbsp;&amp;nbsp;</td><td>Werner BioAgents</td><td>5,001,000</td><td></td></tr><tr><td>Nutrient broth&amp;nbsp;</td><td>Difco</td><td></td><td>local suppliers</td></tr><tr><td>Phenol:Chloroform:Isoamyl alcohol (25:24:1) pH 6.7</td><td>Sigma Aldrich</td><td>P3803</td><td>Dangerous substance. Please check manufacturer&#039;s safety instructions.</td></tr><tr><td>polyethylene glycol (PEG)</td><td>Sigma Aldrich</td><td>P-3640</td><td></td></tr><tr><td>Potassium acetate</td><td>AppliChem</td><td>121479</td><td>alternatively use local supplier</td></tr><tr><td>Pyridoxin (monohydrochlorid)</td><td>Sigma Aldrich</td><td>P-9755</td><td></td></tr><tr><td>Riboflavin&amp;nbsp;</td><td>Sigma Aldrich</td><td>R4500</td><td></td></tr><tr><td>RNaseA</td><td>Sigma Aldrich</td><td>R5503</td><td></td></tr><tr><td>SDS</td><td>Roth</td><td>Cn30.3</td><td>alternatively use local supplier</td></tr><tr><td>small syringe</td><td>BD</td><td>300300</td><td>alternatively use local supplier</td></tr><tr><td>sterile filter, 22 &amp;micro;m</td><td>VWR</td><td>28145-477</td><td>alternatively use local supplier</td></tr><tr><td>Sorbitol</td><td>Roth</td><td>6213.1</td><td>alternatively use local supplier</td></tr><tr><td>Thiamin-hydrochloride</td><td>Serva</td><td>36020.02</td><td>alternatively use local supplier</td></tr><tr><td>tri-Na-Citrate</td><td>Fisher Chemical</td><td>S332060</td><td>alternatively use local supplier</td></tr><tr><td>Tris- (hydroxymethyl) aminomethane</td><td>VWR</td><td>103156X</td><td>alternatively use local supplier</td></tr><tr><td>Tris hydrochloride</td><td>Roth</td><td>9090.4</td><td>alternatively use local supplier</td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Serva&amp;nbsp;</td><td>37240</td><td>alternatively use local supplier</td></tr><tr><td>ZnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fluka</td><td>96470</td><td>alternatively use local supplier</td></tr><tr><td>Composition of solutions/preparation of material</td><td></td><td></td><td>Composition of solutions</td></tr><tr><td>Carboxin</td><td></td><td></td><td>Stock: 5 mg/ml in methanol, final concentration: 2 &amp;micro;g/ml</td></tr><tr><td>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) NH&lt;sub&gt;4&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt;, 2.0 % (w/v) Bacto Agar; adjust to pH 7.0 using 5 M NaOH; after autoclaving add 1 % glucose&amp;nbsp;</td></tr><tr><td>Geneticin (G418)</td><td></td><td></td><td>Stock: 50 mg/ml in H&lt;sub&gt;2&lt;/sub&gt;O, final concentration: 500 &amp;micro;g/ml</td></tr><tr><td>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 H&lt;sub&gt;2&lt;/sub&gt;O until the pH is 7 (the liquid should not be yellow-green anymore). Aliquot the glass beads and dry them at 180 &amp;deg;C. The decanted HCl has to be neutralized before disposal.&amp;nbsp;</td></tr><tr><td>Heparin</td><td></td><td></td><td>Stock: 15 mg/ml</td></tr><tr><td>Holliday salt solution</td><td></td><td></td><td>16.0 &amp;permil; (w/v) KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 4.0 &amp;permil; (w/v) Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;, 8.0 &amp;permil; (w/v) KCl, 1.32 &amp;permil; (w/v) CaCl&lt;sub&gt;2&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O, 8.0 &amp;permil; (v/v) trace elements, 2.0 &amp;permil; (w/v) MgSO&lt;sub&gt;4&lt;/sub&gt;; sterile filtrate</td></tr><tr><td>Holliday vitamin solution&amp;nbsp;</td><td></td><td></td><td>0.1&amp;permil; (w/v) Thiamin, 0.05&amp;permil; (w/v) Riboflavin, 0.05&amp;permil; (w/v) Pyridoxin, 0.2&amp;permil; (w/v) Ca-Pantothenat, (0.05&amp;permil; (w/v) Aminobenzoeic acid, 0.2&amp;permil; (w/v) Nicotinic acid, 0.2&amp;permil; (w/v) Cholinchlorid, 1.0&amp;permil; (w/v) myo-Inositol; may be stored at -20 &amp;deg;C</td></tr><tr><td>Hygromycin</td><td></td><td></td><td>Stock: 50 mg/ml in PBS, final concentration: 200 &amp;micro;g/ml</td></tr><tr><td>Nourseothricin</td><td></td><td></td><td>Stock: 200 mg/ml in H&lt;sub&gt;2&lt;/sub&gt;O, final concentration: 150 &amp;micro;g/ml</td></tr><tr><td>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,&amp;nbsp; 80.0 % (v/v) 87% Glycerin (f.c. 69.6%)&amp;nbsp;</td></tr><tr><td>RegLight</td><td></td><td></td><td>1.0% (w/v) Yeast Extract&amp;nbsp; 0.4 % (w/v) Bacto Peptone, 0.4 % (w/v) Sucrose, 18.22 % (w/v) Sorbitol, 1.5 % (w/v) Agar</td></tr><tr><td>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><td>STC, pH 8</td><td></td><td></td><td>1 M Sorbitol, 10 mM Tris-HCl pH 7.5, 100 mM CaCl&lt;sub&gt;2&lt;/sub&gt;; filter sterile</td></tr><tr><td>STC/PEG</td><td></td><td></td><td>40 % (v/v) PEG in STC-buffer&amp;nbsp;</td></tr><tr><td>TE buffer, pH 8</td><td></td><td></td><td>1.31 mM Tris-Base, 8.69 mM Tris-HCl, 10 mM Na&lt;sub&gt;2&lt;/sub&gt;-EDTA*2H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>TE/RNase</td><td></td><td></td><td>10 &amp;micro;g/ml RNaseA in TE buffer</td></tr><tr><td>Trace elements</td><td></td><td></td><td>0.06&amp;permil; (w/v) H&lt;sub&gt;3&lt;/sub&gt;BO&lt;sub&gt;3&lt;/sub&gt;, 0.14&amp;permil; (w/v) MnCl*4H&lt;sub&gt;2&lt;/sub&gt;O, 0.4 &amp;permil; (w/v) ZnCl&lt;sub&gt;2&lt;/sub&gt;, 0.4 &amp;permil; (w/v) Na&lt;sub&gt;2&lt;/sub&gt;MoO&lt;sub&gt;4&lt;/sub&gt;*H2O:2, 0.1 &amp;permil; (w/v) FeCl&lt;sub&gt;3&lt;/sub&gt;*6H&lt;sub&gt;2&lt;/sub&gt;O, 0.04&amp;permil; (w/v) CuSO&lt;sub&gt;4&lt;/sub&gt;*5H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Trichoderma lysing enzymes solution</td><td></td><td></td><td>12.5 mg/ml SCS; filter sterile; prepare shortly before use</td></tr><tr><td>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><td>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.&amp;nbsp;</td></tr><tr><td>Usti-lysis buffer 2</td><td></td><td></td><td>mix Usti lysis buffer 1 with 1x TE in a 1:1 ratio</td></tr><tr><td>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...

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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 ...

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Research

JoVE Journal

Genetics

14.4K Views.  Heinrich-Heine University Düsseldorf. 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.

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

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.1 Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut2. However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.
Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

Environment

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

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular gene&#39;s function in the living organism. In this protocol the OSCAR method of precise and rapid deletion plasmid construction is described. OSCAR relies on the cloning system in which a single recombinase reaction is carried out containing the purified PCR-amplified 5&#39; and 3&#39; flanks of the gene of interest and two plasmids, pA-Hyg OSCAR (the marker vector) and pOSCAR (the assembly vector). Confirmation of the correctly assembled deletion vector is carried out by restriction digestion mapping followed by sequencing. Agrobacterium tumefaciens is then used to mediate introduction of the deletion construct into fungal spores (referred to as ATMT). Finally, a PCR assay is described to determine if the deletion construct integrated by homologous or non-homologous recombination, indicating gene deletion or ectopic integration, respectively. This approach has been successfully used for deletion of numerous genes in Verticillium dahliae and in Fusarium verticillioides among other species.

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

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

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

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 ...

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are potent carcinogens that substantially reduce crop value in maize and other oil rich crops like peanut besides posing serious threat to human and animal health. Different approaches, including traditional breeding, transgenic expression of resistance associated proteins, and RNA interference (RNAi)-based host-induced gene silencing of critical A. flavus gene targets, are being evaluated to increase aflatoxin resistance in susceptible crops. Past studies have shown an important role of &#945;-amylase in A. flavus pathogenesis and aflatoxin production, suggesting this gene/enzyme is a potential target to reduce both A. flavus growth and aflatoxin production. In this regard, the current study was undertaken to evaluate heterologous expression (under control of the constitutive CaMV 35S promoter) of a Lablab purpureus L. &#945;-amylase inhibitor-like protein (AILP) in maize against A. flavus. AILP is a 36-kDa protein, which is a competitive inhibitor of A. flavus &#945;-amylase enzyme and belongs to the lectin&#8211;arcelin&#8211;&#945;-amylase inhibitor protein family in common bean. In vitro studies prior to the current work had demonstrated the role of AILP in inhibition of A. flavus &#945;-amylase activity and fungal growth. Fungal growth and aflatoxin production in mature kernels were monitored in real time using a GFP-expressing A. flavus strain. This kernel screening assay (KSA) is very simple to set up and provides reliable and reproducible data on infection and the extent of spread that could be quantified for evaluation of germplasm and transgenic lines. The fluorescence from the GFP strain is closely correlated to fungal growth and, by extension, it is well-correlated to aflatoxin values.&#160; The goal of the current work was to implement this previous knowledge in a commercially important crop like maize to increase aflatoxin resistance. Our results show a 35%&#8211;72% reduction in A. flavus growth in AILP-expressing transgenic maize kernels which, in turn, translated into a 62%&#8211;88% reduction in aflatoxin levels.

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.

Here we present a protocol to analyze Aspergillus flavus growth and aflatoxin production in maize kernels expressing an antifungal protein.&#160; Using a GFP-expressing A. flavus strain we monitored the infection and spread of the fungus in mature kernels in real time. The assay is rapid, reliable, and reproducible.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Immunology and Infection

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species alike. This technology can be used for a variety of genome editing approaches, from gene knockouts and knockins to more specific changes like the introduction of a few nucleotides at a targeted location. Genome editing can be used for a multitude of applications, including the partial functional characterization of genes, the production of transgenic organisms and the development of diagnostic tools. Compared to previously available gene editing strategies, the CRISPR-Cas9 system has been shown to be easy to establish in new species and boasts high efficiency and specificity. The primary reason for this is that the editing tool uses an RNA molecule to target the gene or sequence of interest, making target molecule design straightforward, given that standard base pairing rules can be exploited. Similar to other genome editing systems, CRISPR-Cas9-based methods also require efficient and effective transformation protocols as well as access to good quality sequence data for the design of the targeting RNA and DNA molecules. Since the introduction of this system in 2013, it has been used to genetically engineer a variety of model species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Mus musculus. Subsequently, researchers working on non-model species have taken advantage of the system and used it for the study of genes involved in processes as diverse as secondary metabolism in fungi, nematode growth and disease resistance in plants, among many others. This protocol detailed below describes the use of the CRISPR-Cas9 genome editing protocol for the truncation of a gene involved in the sexual cycle of Huntiella omanensis, a filamentous ascomycete fungus belonging to the Ceratocystidaceae family.

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is an easy-to-use genome editor that has been used in model and non-model species. Here we present a protein-based version of this system that was used to introduce a premature stop codon into a mating gene of a non-model filamentous ascomycete fungus.

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species ...

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

Bioengineering

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Biology

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are ...

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

Summary

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Chapters in this video

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

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

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

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

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the α-amylase Inhibitor from Lablab purpureus L.

Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection