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. Point mutations in the flanks can cause unwanted alterations in the genomic sequence, in particular if the flanks reach into the neighboring gene or the mutation modifies regulatory elements. This would affect all transformants and can cause phenotypes and side-effects unrelated to the desired gene deletion.
When planning the deletion strategy, the expected phenotype has to be considered to choose the genetic background. In the present case, AB33 23 and SG200 10 were chosen to allow for testing phenotypes in the morphological switch and plant infection. Similarly, many other read-outs may be of interest, e.g. fusions with cts1 are employed in export of heterologous proteins 14,26, and a strain background with fluorescently tagged rrm4 allows analysis of RNA transport on endosomes 27,28.
During strain verification, the high-throughput diagnostic PCRs allow a rapid reduction of the number of candidates to be tested in the rather laborious Southern blot. In particular, the first diagnostic PCR allows exclusion of candidates still containing the wildtype copy of the gene and thereby prevents the massive use of toxic phenol in gDNA preparations. In a standard case with a non-essential gene, approximately half of the colonies may contain the wildtype gene. This pre-screening can be of great interest for genes with potentially detrimental growth phenotypes. Hundreds of candidates can rapidly be screened.
If all candidates contain the wildtype copy, the gene deletion is most likely lethal. This can be confirmed by deleting one copy in a diploid strain background (e.g. d132) and analysis of the segregation after teliospore germination 29. Alternatively, a conditional deletion strategy using an inducible promoter can be chosen to follow the mutant phenotype. However, also contamination resulting from the crude NaOH-based method for gDNA preparation can interfere with the PCR and lead to false-negatives i.e. candidates do not show a band and therefore are kept even though they contain the gene. This can be excluded by testing a control gene with a comparable size product in an independent PCR reaction or by multiplex-PCR with the two gene-specific primers and primers for a control gene that result in a bigger fragment.
If a mutant is already available, e.g., cts1&#916;30 or rrm4&#916;, strain generation of tagged versions can be accelerated by replacing the gene deletion resistance cassette (e.g. hygR) in the genome with a novel construct containing, e.g., the fluorescently labelled rrm4 and a different resistance marker (e.g. natR). In this case, transformants can be screened by loss of the initial resistance marker 9. Correct candidates are resistant only against the new antibiotic (nourseothricin) and have lost their initial resistance (hygromycin). Additionally, complementation of the mutant phenotype in trans can be carried out to verify functionality of tagged constructs 30,31.
The here described gene deletion and modification strategy offers a reliable and exact tool in genetic analysis of U. maydis. For multiple deletions however, the strain generation can get time-consuming, since the alterations have to be carried out sequentially. Therefore, as a complementary tool, last year the CRISPR-Cas system has been established for U. maydis 32. It offers the possibility to disrupt several genes simultaneously and is an excellent addition to the genetic toolbox of U. maydis.
In summary, the protocol for genetic manipulation and U. maydis strain generation described here is a robust method that has been widely used in the community for years. Together with the comprehensive collection of respective resistance cassette modules it allows all sorts of genetic engineering in this fungus, addressing diverse questions ranging from basic to applied research.

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). Single and double mutants of cts1 with a second chitinolytic gene were generated sequentially with the same constructs in two different genetic backgrounds for analysis of the role of chitinases in virulence. Two different strain backgrounds were employed: AB33 allows induction of filamentous growth, the first step towards pathogenicity 23, SG200 is a solo-pathogenic strain that enables rapid disease scoring 10. After transformation of the cts3-deletion construct into the appropriate strain background, single colonies of the second selection plate were tested for the correct deletion by diagnostic PCRs (Table 1) and Southern blot (Figure 3). Interestingly, for cts3 the homologous recombination rate was much higher in SG200 than in AB33, but this difference was not consistently observed in deletions of the other chitinases.
The stringent strain verification ensures a single insertion of the deletion construct at the desired position. However, additional modification such as point mutations can remain undetected, which might mislead the interpretation of phenotypes. Therefore, at least two independent transformants are phenotyped.
<img alt="Figure 1" src="/files/ftp_upload/54522/54522fig1.jpg" /> 
Figure 1. Overview of the deletion strategy exemplified by cts3. This schematic overview includes the deletion construct with upstream flank (UF), downstream flank (DF), and the geneticin resistance cassette (G418R), the genomic locus of wildtype (WT) and cts3 deletion mutant and all primers as well as the restriction sites used in the strain verification. The first diagnostic PCR results in a product only if the wildtype gene copy is present, the two second diagnostic PCRs result in products if the resistance cassette has been correctly integrated. For the Southern blot the genomic DNA is digested with PstI, the probes span the UF and DF (red bars) leading to detection of a 6.7 kb band in wildtype&#160;and two bands of 5.6 kb and 2.0 kb in the mutant. In addition, the DF probe weakly recognizes a 2.6 kb product. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54522/54522fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54522/54522fig2.jpg" /> 
Figure 2. Microscopic verification of protoplasting reaction. (A) Untreated wildtype cells (FB2) appear cigar shaped. (B) Upon treatment with cell wall degrading enzymes (here for for 30 min), the appearance of round spheres at one end (s) and bar-bell like structures (b) indicates that the cell wall degradation has started. Finally the protoplasts are completely round (r), which is due to the lack of cell wall. Scale bar = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54522/54522fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54522/54522fig3.jpg" /> 
Figure 3. Southern blot for cts3 deletion. Genomic DNA was digested with PstI. Both flanks were labelled, mixed in equimolar ratios and used as a single probe. In the wildtype SG200 and deletions of the chitinases cts1 and cts2, a single band of 6.7 kb is detected. In cts3 deletions (g and k), two bands of 5.6 kb and 2.0 kb are visible indicative of the correct deletion of cts3. An additional band of 2.6 kb can be visualized by massive overexposure. This corresponds to a fragment that is recognized by an overlap of only 100 bp of the downstream flank probe. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54522/54522fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Some mutations lead to obvious growth defects that can be detected microscopically. In the chitinase deletions, single mutants did not display any obvious defect, while cts1/2 double mutants showed a pronounced cell separation defect 20. Staining of septa showed that cytokinesis was completed but the cells remained connected likely via residual chitin (Figure 4 from Langner et al. 2015). A similar microscopic growth phenotype is known for the deletion of khd4 24, a gene encoding an RNA-binding protein mediating post-transcriptional RNA turnover of genes involved in morphology and pathogenicity 25.
<img alt="Figure 4" src="/files/ftp_upload/54522/54522fig4.jpg" /> 
Figure 4. Microscopic analysis of deletion mutants. Cell morphology and septum formation of the chitinase deletion strains. cts1/2&#916; strains exhibit a cell separation defect and form large aggregates, which is not due to a lack of septum formation. Primary and secondary septa (see 5x&#160;enlargement in insets) were stained with Calcofluor White (CW) prior to microscopy. Scale bars = 10 &#181;m. This figure is reprinted with permission from Langner et al. 2015 Eukaryotic Cell 20. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54522/54522fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To test the contribution of chitinases to virulence, seedling infection assays were carried out using the mutants in the SG200 strain background. While mutants in khd4 were reduced in virulence, deletion of the chitinases did not affect infection of maize seedlings (Figure 5) 20,25.
<img alt="Figure 5" src="/files/ftp_upload/54522/54522fig5.jpg" /> 
Figure 5. Infection assay of deletion mutants. Disease rating of maize seedlings 9 days after infection with the respective U. maydis strains. (A) Macroscopic symptoms that are used for disease scoring. (B) Two independent transformants were tested for each strain (N &#62;100). All chitinase-deficient mutants (1/2/3&#916;: triple mutant endochitinases, 4&#916;: single mutant N-acetylglucosaminidase, 1/2/3/4&#916;: quadruple mutant) infected the host plant leading to heavy tumor formation as observed in wildtype infections with the solopathogenic strain SG200. By contrast, a strain carrying a deletion of the gene encoding the RNA-binding protein Khd4 was reduced in virulence as reported previously 24. Error bars indicate standard deviation of three independent experiments. This figure is modified with permission from Langner et al. 2015 Eukaryotic Cell 20. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54522/54522fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Genetic background</td>
			<td>1st diagn. PCR 
			(excluded/tested)</td>
			<td>2nd diagn. PCR 
			(confirmed/tested)</td>
			<td>Southern 
			(confirmed/tested)</td>
			<td>% recomb.</td>
		</tr>
		<tr>
			<td>AB33</td>
			<td>7/22</td>
			<td>-</td>
			<td>7/15</td>
			<td>32</td>
		</tr>
		<tr>
			<td>AB33 cts1&#916;</td>
			<td>19/24</td>
			<td>-</td>
			<td>5/5</td>
			<td>21</td>
		</tr>
		<tr>
			<td>SG200</td>
			<td>0/10</td>
			<td>8/10</td>
			<td>7/8</td>
			<td>70</td>
		</tr>
		<tr>
			<td>SG200 cts1&#916;</td>
			<td>0/20</td>
			<td>19/20</td>
			<td>14/19</td>
			<td>70</td>
		</tr>
	</tbody>
</table>
Table 1: Strain verification for cts3 deletions. The chitinase gene cts3 was deleted in four different genetic backgrounds to allow studies of filamentous growth (AB33) and infection (SG200) in single and multiple deletions.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Genetics

14.4K Views.  Heinrich-Heine University Düsseldorf.  We beschrijven een robuuste gen vervangende strategie om de vuiligheid schimmel Ustilago maydis genetisch te manipuleren. Dit protocol wordt uitgelegd hoe u deletiemutanten genereren om infectie fenotypes onderzoeken. Kan worden uitgebreid tot genen in elke gewenste wijze, bijvoorbeeld wijzigen door toevoeging van een sequentie die codeert voor een fluorescerend eiwit 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 ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

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Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

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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.

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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

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

Biology

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.

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

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