Genetisk Manipulation af afgroedesygdomme<em> Ustilago maydis</em> At studere Fungal Biology og Plant Microbe Interactions
Summary
Vi beskriver en robust generstatning strategi til genetisk manipulere smut svampen Ustilago maydis. Denne protokol forklarer, hvordan at generere deletionsmutanter at undersøge infektion fænotyper. Det kan udvides til at modificere gener i enhver ønsket måde, fx ved tilsætning af en sekvens, der koder et fluorescerende protein-tag.
Abstract
Gendeletion spiller en vigtig rolle i analysen af genfunktion. En af de mest effektive metoder til at forstyrre gener i målrettet er udskiftningen af hele genet med en selekterbar markør via homolog rekombination. Under homolog rekombination, udveksling af DNA finder sted mellem sekvenser med høj lighed. Derfor kan lineære genomiske sekvenser, der flankerer et target-gen anvendes til specifikt dirigere en selekterbar markør til den ønskede integration site. Stumpe ender af deletionskonstruktion aktivere cellens DNA reparationssystemer og dermed fremme integration af konstruktionen enten via homolog rekombination eller ved ikke-homolog-endesammenbinding. I organismer med effektiv homolog rekombination, kan hastigheden af succesfulde gendeletion nå mere end 50% at gøre denne strategi et værdifuldt gen afbrydelse system. Den smut svamp Ustilago maydis er en eukaryot model mikroorganisme viser sådan effektiv homolog recombination. Ud af de omkring 6900 gener, har mange været funktionelt karakteriseret ved hjælp af deletionsmutanter, og gentagne overtrædelser af gen udskiftning forsøg peger på væsentlige funktion af genet. Efterfølgende karakterisering af genet funktion ved tagging med fluorescerende markører eller mutationer af forudsagte domæner også afhængig DNA udveksling via homolog rekombination. Her præsenterer vi U. maydis stamme generation strategi i detaljer ved hjælp af simpleste eksempel genet deletion.
Introduction
Ustilago maydis er en fytopatogene model svamp, der er blevet omfattende undersøgt i årtier 1,2. Den findes i to morfologier, en gær-lignende, ikke-patogene fase og en filamentøs, smitsom formular 3. Universelle gennembrud opdagelser såsom homolog rekombination og DNA-reparationsmekanismer blev foretaget i gær-lignende vækst fase af denne svamp 4. Endvidere den morfologiske kontakten til de infektiøse filament og virulensfaktorer vigtige for infektion velkarakteriseret 5,6. Den stigende molekylær viden om biologi og virulens af denne smut svamp bygger på en simpel gen udskiftning strategi 7-9 understøttet af en fremragende genom annotation 10 og lethed af revers genetik hjælp, f.eks den velorganiserede plasmid samling på vores institut (http : //www.mikrobiologie.hhu.de/ustilago-community.html). Standardiserede, hurtige infektion analyser i majs seedlings tillader detaljerede undersøgelser af patogenicitet faktorer 11.
Genomet i U. maydis indeholder omkring 6900 gener 10. For at undersøge deres funktion, kan de slettes enkeltvis eller i kombination på grund af en effektiv homolog rekombination system. Flankerende regioner på ca. 1 kb indeholdende perfekt homologe ender er ideelle til satserne for homolog rekombination større end 50%, men allerede 250 bp med ikke-homologe ender tillader en vis grad af korrekt integration af konstruktionen 9. I øjeblikket er fem forskellige modstandsværdier kassetter, hygR, cbxR, J.Clin.Natr, G418R, og phleoR mediere resistens mod hygromycin, carboxin, nourseothricin, G418 og phleomycin, anvendes til at selektere for transformanter 7,9. Derudover har hygromycinresistens blevet udviklet til en genanvendelig kassette (FRT-hygR), der kan fjernes ved den transiente ekspression af et heterologt FLP rekombinase 12. Dette tillader fjernelse af resistente kassette og dermed i teorien ubegrænset genetiske modifikationer. Phleomycin er mutagent 13, således at de nye kassetter, navnlig genanvendeligt hygR kassette, brug af phleoR er faldende. Quadruple mutanter kan således dannes ved hjælp af de øvrige fire kassetter, men for femdobbelt mutanter er FRT-hygR systemet anbefales 14.
Denne generelle gendeletion strategi er blevet overført til andre smut svampe såsom Sporisorium reilianum 15, U. hordei 16, eller U. esculenta 17 og tilbyder derfor potentialet for yderligere ansøgninger i endnu genetisk umedgørlige organismer med et effektivt homolog rekombination system. Desuden kan organismer mangler homolog rekombination modificeres for at forbedre gensplejsning som eksemplificeret ved deletion af gener involveret i ikke-homolog-end-sammenføjning i Neurospora crassa 18,19.
Her beskriver vi den offentliggjorte gendeletion strategi for U. maydis 7,9 i eksperimentel detaljer med fokus på hurtig og præcis kontrol af kandidaterne. Som et eksempel, bruger vi de svampeangreb chitinaser og skildrer generation af enkelte mutanter samt flere sletning stammer 20,21. Chitinaser er interessante eksempler, fordi de handler om kitin i stiv cellevæg. Cellevæg remodeling er påkrævet for morfologiske forandringer under celledeling, skifte til filamentøs vækst, og sporedannelse. Derfor, i deletionsmutanter kan forventes fænotyper hele livscyklussen.
Protocol
Representative Results
Discussion
Denne protokol beskriver, hvordan at generere deletionsmutanter for reverse genetiske undersøgelser i U. maydis. Udgangspunktet er en deletion konstrukt, der indeholder flankerende sekvenser af genet af interesse, der indeholder sekvenser på ca. 1 kb opstrøms for starten og nedstrøms stop-codon samt et passende resistent kassette, som det tidligere blev optimeret 7,9 . Konstruktionerne har at være individuelt genereret for hvert gen og omhyggeligt kontrolleres for sekvensfejl før sletning genet…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Særlig tak til Dr. Benedikt Steuten for kritisk læsning af manuskriptet. Den oprindelige arbejde på chitinaser blev udført af Dr. Thorsten Langner. Laboratoriet for VG er støttet af Cluster of Excellence i Plant Sciences (CEPLAS, DFG EXC 1028) og BioSC er laboratorium KS støttet af BioSC. KB er støttet af BioSC. De videnskabelige aktiviteter Bioeconomy Science Center (BioSC) blev støttet af Ministeriet for Innovation, Videnskab og forskning inden for rammerne af NRW Strategieprojekt BioSC (nr 313 / 323-400-00213). LF er understøttet af en ph.d.-stipendium af DFG International Research Training Group 1525 iGRADplant.
Materials
| 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 | |
| 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 |
| Name | Company | Catalog Number | Comments |
| 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*2H2O, 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 1 x 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 |
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