植物病原体的遗传操作<em>玉米黑粉菌</em>研究真菌生物学和植物微生物相互作用
Summary
我们描述了一个强大的基因替代策略，以基因操纵黑粉菌玉米黑粉菌 。该协议说明了如何生成缺失突变体研究感染表型。它可以扩展到修改的基因以任何期望的方式， 例如 ，通过添加编码荧光蛋白标记的序列。
Abstract
基因缺失在基因功能的分析的重要作用。其中最有效的方法来破坏有针对性地基因是通过同源重组的选择性标记的更换整个基因。在同源重组，DNA的交换以高相似度序列之间的地方。因此，侧翼的靶基因的线性基因组序列可用于特异性指示一个可选择的标记到所需的整合位点。缺失构建体的钝端激活细胞的DNA修复系统，从而或者通过同源重组或通过非同源末端接合促进构建体整合。在高效的同源重组微生物，成功的基因缺失的速率可以达到50％以上，使此策略的有价值的基因破坏系统。该黑粉菌玉米黑粉菌是表示该有效的同源recombinat真核微生物模式离子。其约6900个基因，许多已在功能上表征与缺失突变体的帮助下，在该基因的基本功能重复的基因置换的尝试点故障。通过与荧光标记或预测域的突变标记基因功能的后续特性也依赖于通过同源重组DNA交换。在这里，我们提出了U.用最简单的例子中，基因缺失详细小斑病菌应变生成策略。
Introduction
玉蜀黍黑粉菌是已经数十年1,2-广泛研究一个植物致病模型真菌。它存在于两个形态，酵母样的，非致病阶段和丝状，感染形式3。万向突破性发现诸如同源重组和DNA修复机制在这种真菌4的酵母样生长阶段进行的。此外，该形态切换到传染性长丝和毒力因子感染重要被充分表征5,6。这个黑粉菌生物学和毒力的日益分子知识依赖于一个优秀的基因组注释10和反向遗传学的使用简便， 例如 ，组织严密的质粒收集在我院（HTTP支持一个简单的基因替换策略7-9 ：//www.mikrobiologie.hhu.de/ustilago-community.html）。玉米组织规范化，快速感染试验eedlings允许致病性的详细研究因素11。
美国的基因组玉米小斑病包含约6900个基因10。研究它们的功能，它们可以单独或组合由于一个高效的同源重组系统中删除。含完全同源末端的约1kb的侧翼区是理想的同源重组大于50％的比率，但已经250bp的非同源末端允许某种程度的正确整合的构建体9组成。目前，五个不同的抗性盒，hygR，cbxR，NATR，G418R，和phleoR介导针对潮霉素，萎，诺尔丝菌素，G418和腐草霉素抗性，被用来选择转化7,9。此外，潮霉素抗性已被开发成可回收盒（FRT-hygR），可以通过异源FLP重组酶12的瞬时表达被移除。这样做允许移除，从而在理论上无限的遗传修饰的抗性盒和。腐草霉素是诱变13，使之与新盒带，特别是可回收hygR盒，使用phleoR的正在减少。四倍突变体因此可利用其他四个盒产生，但对于五元组突变体中，FRT-hygR系统建议14。
这是一般的基因缺失策略已成功转移到其他黑穗病菌如丝黑穗病的 15 U.大麦 16，或U.星虫 17岁 ，因此提供了一个高效的同源重组系统，但转基因生物顽固进一步应用的潜力。此外，缺乏同源重组生物体可以被修饰以提高基因工程所涉及的基因的缺失例举非同源烯D-加盟链孢霉 18,19。
在这里，我们描述了U.公布的基因缺失策略实验细节7,9 玉米须 ，重点对考生的快速和准确的验证。作为一个例子，我们使用真菌几丁质酶和描绘单突变体的产生以及多个缺失菌株20,21。几丁质酶是有趣的例子，因为它们作用于刚性细胞壁甲壳素。细胞壁重塑需要细胞分裂过程中的形态变化，切换到丝状生长，孢子形成。因此，在缺失突变体在整个生命周期中的表型可以预期。
Protocol
1.删除生成的构造产生含有缺失构建体（ 图1），包括一个相应的1kb的上游侧翼（UF）和下游侧翼（DF），每个与由钝切割型限制性位点侧接的适当抗性盒的质粒。 注意：任何克隆策略可以使用（用于克隆见参考22），建议金门克隆这种质粒9。 切除删除使用钝刀具，以获得缺失1微克DNA构建从9质粒构建。净化的删除构建体，以消除酶，并…
Representative Results
缺失构建在U.编码的所有四个基因几丁质通过使用hygR盒为CTS1缺失金门克隆生成小斑病菌的基因组中，NATR为CTS2的删除，则G418R盒为CTS3的删除和cbxR为CTS4 20的删除。该基因替换策略的概述由缺失CTS3的（ 图1）举例说明。与第二个几丁质基因CTS1的单，双突变体在几丁质酶的毒…
Discussion
本协议描述了如何为在美国反向遗传学研究的缺失突变体玉米小斑病 。起点是缺失构建体，其中包含基因的感兴趣含有的约1kb的开始的上游和终止密码子的下游，以及适当的抗性盒序列的侧翼序列，因为它先前优化7,9- 。该构建体已经为每个基因单独生成并认真核实为之前删除该基因序列的错误。在侧翼点突变可以导致在基因组序列不需要的改变，特别是如果侧面伸入相邻?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
特别感谢本尼迪克特Steuten博士的手稿的批判性阅读。在几丁质酶的原创作品是由和Thorsten兰纳博士进行。 VG的实验室是由卓越的植物科学（CEPLAS，DFG EXC 1028）和BioSC的群集支持，堪萨斯州的实验室是由BioSC支持。 KB由BioSC支持。生物经济科学中心（BioSC）的科学活动是由财政创新，科学和研究部的NRW Strategieprojekt BioSC（313号/ 323-400-00213）的框架内支持。 LF是由东风集团国际研究培训集团1525 iGRADplant的博士生奖学金支持。
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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Cite This Article
Bösch, K., Frantzeskakis, L., Vraneš, M., Kämper, J., Schipper, K., Göhre, V. Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions. J. Vis. Exp. (115), e54522, doi:10.3791/54522 (2016).