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

Summary

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.

Abstract

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'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.

Introduction

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 replacemen....

Protocol

1. Generation of Deletion Constructs

1. 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.
2. Excise the deletion construct from the plasmid using a blunt cutter to obtain 1 µg DNA of the deletion construct ⁹. Purify the deletion construct to eliminate enzyme and buffer ²² e.g.

Results

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

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Aminobenzoeic acid (Free Acid)	Sigma Aldrich	A-9878
Bacto agar	BD	214010	alternatively use local supplier
Bacto peptone	BD	211677	alternatively use local supplier
Bacto yeast extract	BD	212750	alternatively use local supplier
CaCl2*2H2O	Grüssing GmbH	10234	alternatively use local supplier
Ca-Pantothenat (Hemi-Ca. salt)	Sigma Aldrich	P-2250
Carboxin	Sigma Aldrich	45371
Casamino acids	BD	223050
Cholinchlorid	Sigma Aldrich	C-1879
Citric acid	ChemSolute	24,321,000	alternatively use local supplier
CuSO4*5H2O	Fluka	61240	alternatively use local supplier
D(+)Sucrose	Roth	4621.1	alternatively use local supplier
DNA degr. free acid	Sigma-Aldrich	D-3159
EDTA	Sigma Aldrich	E4378
FeCl3*6H2O	Grüssing GmbH	10288	alternatively use local supplier
Geneticin (G418) disulfate salt	Sigma Aldrich	A1720
Trichoderma lysing enzymes	Sigma Aldrich	L1412
D(+) Glucose	Caelo	2580	alternatively use local supplier
Glycerin	Fisher Chemical	G065015	alternatively use local supplier
H3BO3	AppliChem	A2940	Dangerous substance. Please check manufacturer's safety instructions.
Heparin sodium salt	Sigma Aldrich	H3393-50KU
Hygromycin B-solution	Roth	1287.2	Dangerous substance.
KCl	VWR	26764298	alternatively use local supplier
KH2PO4	AppliChem	A3620	alternatively use local supplier
MgSO4 waterfree	Merck	7487-88-9	Water free is critical. Alternatively use local supplier
MnCl2*4H2O	AppliChem	A2087	alternatively use local supplier
myo-Inositol	Sigma Aldrich	I-5125
Na2-EDTA*2H2O	AppliChem	A2937	alternatively use local supplier
Na2MoO4*2H2O	Roth	0274.2	alternatively use local supplier
Na2SO4	Grüssing GmbH	12174	alternatively use local supplier
NaCl	Fisher Chemical	S316060	alternatively use local supplier
NaOH	ChemSolute	13,751,000	alternatively use local supplier
NH4NO3	Roth	K299.1	alternatively use local supplier
Nicotinic acid (Free acid)	Sigma Aldrich	N-4126
Nourseothricin dihydrogen sulfate	Werner BioAgents	5,001,000
Nutrient broth	Difco		local suppliers
Phenol:Chloroform:Isoamyl alcohol (25:24:1) pH 6.7	Sigma Aldrich	P3803	Dangerous substance. Please check manufacturer's safety instructions.
polyethylene glycol (PEG)	Sigma Aldrich	P-3640
Potassium acetate	AppliChem	121479	alternatively use local supplier
Pyridoxin (monohydrochlorid)	Sigma Aldrich	P-9755
Riboflavin	Sigma Aldrich	R4500
RNaseA	Sigma Aldrich	R5503
SDS	Roth	Cn30.3	alternatively use local supplier
small syringe	BD	300300	alternatively use local supplier
sterile filter, 22 µm	VWR	28145-477	alternatively use local supplier
Sorbitol	Roth	6213.1	alternatively use local supplier
Thiamin-hydrochloride	Serva	36020.02	alternatively use local supplier
tri-Na-Citrate	Fisher Chemical	S332060	alternatively use local supplier
Tris- (hydroxymethyl) aminomethane	VWR	103156X	alternatively use local supplier
Tris hydrochloride	Roth	9090.4	alternatively use local supplier
Triton X-100	Serva	37240	alternatively use local supplier
ZnCl2	Fluka	96470	alternatively use local supplier
Composition of solutions/preparation of material			Composition of solutions
Carboxin			Stock: 5 mg/ml in methanol, final concentration: 2 µg/ml
CM plates			0.25 % (w/v) Casamino acids, 0.1 % (w/v) Yeast Extract, 1.0 % (v/v) Holliday vitamin solution, 6.25 % (v/v); Holliday salt solution, 0.05 % (w/v) DNA degr. free acid, 0.15 % (w/v) NH4NO3, 2.0 % (w/v) Bacto Agar; adjust to pH 7.0 using 5 M NaOH; after autoclaving add 1 % glucose
Geneticin (G418)			Stock: 50 mg/ml in H2O, final concentration: 500 µg/ml
HCl-washed glass beads (0.35-0.45 mm)			Cover glass beads with concentrated HCl (25 %, 7.8 M) and incubate for 60 min. Sway several times. Decant HCl (keep decanted liquid) and wash glass beads with 3 M HCl (keep decanted liquid). Wash glass beads several times with double distilled H2O until the pH is 7 (the liquid should not be yellow-green anymore). Aliquot the glass beads and dry them at 180 °C. The decanted HCl has to be neutralized before disposal.
Heparin			Stock: 15 mg/ml
Holliday salt solution			16.0 ‰ (w/v) KH2PO4, 4.0 ‰ (w/v) Na2SO4, 8.0 ‰ (w/v) KCl, 1.32 ‰ (w/v) CaCl2*2H2O, 8.0 ‰ (v/v) trace elements, 2.0 ‰ (w/v) MgSO4; sterile filtrate
Holliday vitamin solution			0.1‰ (w/v) Thiamin, 0.05‰ (w/v) Riboflavin, 0.05‰ (w/v) Pyridoxin, 0.2‰ (w/v) Ca-Pantothenat, (0.05‰ (w/v) Aminobenzoeic acid, 0.2‰ (w/v) Nicotinic acid, 0.2‰ (w/v) Cholinchlorid, 1.0‰ (w/v) myo-Inositol; may be stored at -20 °C
Hygromycin			Stock: 50 mg/ml in PBS, final concentration: 200 µg/ml
Nourseothricin			Stock: 200 mg/ml in H2O, final concentration: 150 µg/ml
NSY-glycerol-medium			0.8 % (w/v) Nutrient Broth, 0.1 % (w/v) Yeast Extract, 0.5 % (w/v) Sucrose,  80.0 % (v/v) 87% Glycerin (f.c. 69.6%)
RegLight			1.0% (w/v) Yeast Extract  0.4 % (w/v) Bacto Peptone, 0.4 % (w/v) Sucrose, 18.22 % (w/v) Sorbitol, 1.5 % (w/v) Agar
SCS, pH 5.8			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
STC, pH 8			1 M Sorbitol, 10 mM Tris-HCl pH 7.5, 100 mM CaCl2; filter sterile
STC/PEG			40 % (v/v) PEG in STC-buffer
TE buffer, pH 8			1.31 mM Tris-Base, 8.69 mM Tris-HCl, 10 mM Na2-EDTA*2H2O
TE/RNase			10 µg/ml RNaseA in TE buffer
Trace elements			0.06‰ (w/v) H3BO3, 0.14‰ (w/v) MnCl*4H2O, 0.4 ‰ (w/v) ZnCl2, 0.4 ‰ (w/v) Na2MoO4*H2O:2, 0.1 ‰ (w/v) FeCl3*6H2O, 0.04‰ (w/v) CuSO4*5H2O
Trichoderma lysing enzymes solution			12.5 mg/ml SCS; filter sterile; prepare shortly before use
Tris-HCl pH 7.5			806 mM Tris-HCl, 194 mM Tris-Base; check the pH and if necessary adjust with HCl; autoclave
Usti-lysis buffer 1, pH 8			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.
Usti-lysis buffer 2			mix Usti lysis buffer 1 with 1x TE in a 1:1 ratio
YEPS-Light medium			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