Name: genetic manipulation of the plant pathogen ustilago maydis to study fungal biology and plant microbe interactions
Uploaded: 2016-09-30T15:00:00.000+00:00
Duration: 11 min 42 s
Description: 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

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。

1.删​​除生成的构造<ol><li>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。 </li><li>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，并通过使用商购试剂缓冲器22 例如并按照制造商的说明。 注:载体骨架可以保留在转化混合物。 </li></ol> 2.原生质体的制备注:在电子商务时刻保持无菌条件xperiment。细胞壁是限制分子的访问到质膜很强的保护屏障。以允许含有缺失构建体的DNA的摄取，细胞壁需要通过细胞壁的原生质体反应降解酶除去。在原生质体的制备关键步骤是:1）介质的渗透稳定和2）避免对原生质体的机械应力。 <ol><li>标记2ml管与圆底冻结原生质体并冷却下来在-20℃。从具有期望遗传背景的菌株的新鲜板开始在3ml YEPS光强的预培养。孵育在28℃的旋转轮24小时。 注:该菌株可以是solopathogenic应变SG200 10，野生型株，测试菌株如AB33 23，或用于产生双重突变体中的任何突变体的背景。确保应变是自由的所需阻力。 </li><li>稀释在50毫升YEPS光强的文化在挡板FLASk和在28℃下用200rpm的定轨振荡器上孵育。 注:倍增时间是野生型菌株约2小时。允许3重复最小。 </li><li>生长，直到指数期。确保光密度，在600nm（OD 600）的波长测量，大约是0.8（0.6至1.0是可接受的）。的OD 1.0 600对应于大约1至2×10 7个U.每玉米须毫升细胞。 </li><li>检查污染的细胞在显微镜下。使用放大40倍。沉淀完全50ml培养的细胞5分钟，1500 xg离心并弃去上清液。重悬在25毫升的柠檬酸钠，山梨糖醇溶液（SCS），离心5分钟，1500 xg离心沉淀，并弃去上清液。 注:细菌污染物可确定为小，经常移动的细胞。真菌污染物可以基于不同的细胞的形状或大小相比U的雪茄形细胞被识别玉米小斑病 。 </li><li>在岑trifugation制备原生质体溶液（12.5毫克/毫升木霉裂解酶，在SCS;制备每个细胞沉淀3毫升;过滤消毒通过一个22微米的过滤器;溶液是新鲜的最佳的酶活性）。 SCS是渗透稳定剂，以防止在除去细胞壁的原生质体的破裂。 </li><li>悬浮颗粒在2毫升原生质体的解决方案。孵育5到20分钟，在RT和在显微镜下检查，直至细胞的30至40％是圆形或像猪头（ 图2）。 注意:请在冰上所有步骤从现在开始，并小心处理原生质体！ </li><li>洗3次在10毫升冷（4℃）SCS，离心机在1000×g离心5分钟。 </li><li>重悬在10ml冷山梨醇，TrisHCl， 氯化钙溶液（STC），旋粒料以1000×g离心5分钟，弃上清。重悬在1ml冷的STC沉淀，使在冷却管道100微升等分试样。在-80°C，直到进一步利用冻结等分。</li></ol> 3. U.改造玉米小斑病 注意:U的转化小斑病菌原生质依赖于聚乙二醇（PEG）介导的转化方法，该方法在技术上是简单和相对电穿孔或基因枪转化不需要专门的设备。 <ol><li>制备双层选择平板（每个转化反应2块板）。底层包含双倍浓度的抗生素。吸管12毫升RegLight的含有400微克/毫升潮霉素（或300微克/毫升诺尔丝菌素或4微克/毫升萎或1mg / ml的G418）成培养皿。避免气泡。 注:底层板可以存储在几天，在4℃，但转化过程中的新鲜制备的顶层（见下文）。 </li><li>在冰上解冻原生质体（1管/转换）。加入1μl肝素（15毫克/毫升）。加入1微克DNA（线性结构）或10微升H 2 </子&gt; O（水控机）。孵育在冰上10分钟。 </li><li>在此期间，为上层蔓延12毫升液化RegLight（煮沸，冷却至60°C）到RegLight底板。 注:此层不含有抗生素。 </li><li>加入500微升STC / PEG（聚乙二醇）到变换管（3 2），并通过反相管小心混合。在冰上孵育15分钟。 </li><li>分发两个双层RegLight板的各转化反应。用玻璃吸管小心缓慢地扩散。在28°C直立孵育板5〜7天，直到转化成长为殖民地。获取每盘约100-200菌落。 注:下数据可能通过"坏"原生质体，要么含有太多细胞壁，因此可以不占用DNA或不能再生而引起。前者可以通过转化具有自我复制的质粒，后者由平板上测试再生没有抗生素进行测试。 </li> &lt;利&gt;准备含有适当抗生素YEPS-光板，在200微克/毫升潮霉素（或150微克/毫升诺尔丝菌素或2微克/毫升萎或500微克/毫升G418的;这些浓度用于底板的一个等于一半）。再净化部对这些板块至少24假定转化体菌落。单一菌落来获得。在非选择性培养基同时条纹出原始菌株。 注:将板可以储存在4℃下1-2周。 </li></ol> 4.正确删除事件的验证注:突变的三步骤过程中被验证（ 图1）。首先，含有该基因的野生型拷贝的候选人通过PCR排除。第二，电阻磁带插入基因座的整合通过PCR验证。第三，整合的抗性盒只成所需轨迹由Southern印迹分析证实。细心的应变验证ESSE微分方程边值问题的，使突变表型真正与缺失相关。 <ol><li>首先PCR诊断20 <ol><li>在基因设计的引物配对要删除该结果在250-600碱基（ 图1）的产物。这种小产品很容易在菌落PCR扩增，和足够长的检测标准凝胶电泳。 </li><li>每个候选和原始菌株作为阳性对照，悬浮单个菌落的细胞材料一点点（针头量）与在20μl的0.02M的NaOH牙签。孵育在室温下进行30分钟。 注意:使用新的殖民地。如果这些板块都超过1周再连胜老年，获取新的殖民地。 </li><li>立即使用1微升的细胞悬液用于PCR。运行标准PCR反应，分析产生的碎片。 注意:这些样品不能存储。 注:野生型基因的存在这个PCR试验，从而允许负C和快速鉴定在其中的感兴趣的基因没有被替换idates。产生在该PCR反应条带的克隆将被丢弃。一个这样的负面克隆无论如何都应该被一起作为一个额外的阴性对照进行。没有PCR产物考生必须进一步分析。对于基因无不利影响大约一半的候选应包含野生型基因，而另一半不应该在的频带造成的。这将对应于50％的同源重组率。 </li></ol></li><li>第二PCR诊断<ol><li>基因组DNA的制备。 注:基因组DNA（gDNA的）可以用两种不同的协议来制备。第一个涉及毒性试剂像苯酚和氯仿，因此，应仅由有经验的研究人员（在4.2.1.1至4.2.1.5中描述），而第二也可以由经验较少的学生（4.2.1.6至4.2中所述进行处理来处理。 1.11）。这两个协议都可靠工作。步骤4.2.1.1是为protoc相同醇。 <ol><li>接种5毫升YEPS光强与每个候选和在28℃在旋转轮孵育24小时。掉落在CM板2微升的文化。保持无菌的文化。 注:以下是一个标准协议（注意:有毒的步骤）。 </li><li>把1勺（约200微升）的盐酸洗玻璃珠的2ml管。加入2毫升的U.玉米小斑病文化。自旋12000 xg离心5分钟。弃上清（颗粒可以在此阶段被冻结）。加入500μl苯酚/氯仿/异戊醇（25:24:1）进行沉淀。警告！苯酚是有毒的！ </li><li>加入500μl拉贝裂解缓冲液1.摇为6至10分钟，在一个vibrax以1,000rpm。验证该细胞沉淀完全重悬，但避免延长摇动防止的gDNA的剪切。旋在12,000 xg离心15分钟。转移400微升的水相到新的反应管（请勿触摸相间）。 </li><li>加入1ml 100％（体积/体积）乙醇。反转管3X混合，观察的C大声说前不久出现的DNA。旋在12,000 xg离心5分钟。除去上清并用200μl70％乙醇洗涤。不重悬在该阶段沉淀。 </li><li>完全取出上清液（不要让小球完全干燥）。加入50μlTE / RNA酶。通过在50℃下以400rpm振摇10分钟溶解的gDNA。在0.8％琼脂糖凝胶上20分析2微升的gDNA的。 注:下面的步骤包括另一种无毒的协议。 </li></ol></li><li>基因组DNA的候补制备<ol><li>把1勺（约200微升）的盐酸洗玻璃珠的2ml管。加入2毫升的U.小斑病菌培养和自旋在12,000 xg离心5分钟。弃上清（粒料可以在此阶段被冻结）。 </li><li>加入500微升拉贝裂解缓冲液2到沉淀。以1000rpm摇动5至15分钟，在一个vibrax。验证该细胞沉淀完全重悬。 </li><li>在65℃下进行15至20分钟孵育的管子。 Appro公司使用旨在承载2ml管priate孵化器是至关重要的。然后，将其放置在冰上5分钟。 </li><li>加入100微升8M的乙酸钾，涡旋或反转8至10倍。自旋12000×g离心在RT 15分钟。 </li><li>转移500μl的上清液至新的1.5毫升管，并添加400μl的异丙醇。涡流或倒置8到10倍。自旋12000×g离心15分钟。除去上清并用500μl70％乙醇洗涤。自旋12000 xg离心5分钟。 </li><li>完全浸泡过上清！最终，添加短的自旋（10秒），以去除残留的液体。让为3至5分钟，将DNA沉淀干燥。加入50μl的TE / RNA酶，并在50℃下孵育10至15分钟，在400rpm。 </li></ol></li><li> PCR <ol><li>设计2种引物对，在侧翼外其中之一的结合和中相应的一个的抗性盒内结合（UF-F，RC-R; RC-F，DF-R， 图1）9。 </li><li>使用1微升1:10稀释每一个C的基因组DNA的andidate如2 PCR反应的模板验证正确的插入上游和下游9。 </li><li>使用每个候选标准PCR反应，并分析产生的碎片22。 注意:这些的PCR测试对于抗性盒在正确的基因组基因座的插入。如果产品是双方获得，候选人可能是一个正确的插入。然而，考生也只有一个侧（上游或下游），可以确认应沿的情况下，进一步的分析，认为PCR反应根本没有一个侧面进行。 </li></ol></li></ol></li><li>通过Southern印迹分析22基因正确插入的验证<ol><li>消化突变体候选的gDNA的15微升（4.2.1）和相应的野生型/祖应变与轨迹外切割/构造，另外，切割无论是在基因或抗性盒导致清楚地区的适当的酶inguishable图案（ 图1）8。 注:预期的条带中没有应大于10kb的，以使它们从未消化的DNA有明显的区别。 </li><li>使用上游和下游侧翼作为探针。要贴上标签，例如使用PCR DIG标记试剂盒或任何类似的标准方法。在一个1混合上述标记探针:1摩尔比，并进行Southern印迹。保持在至少两个独立的正确转化体。 注:野生型图案应在原始菌株清楚地检测和正确的克隆只包含预期带。只存在于候选额外条带表明在一个错误的轨迹构建体的其他插入。这种克隆应该被丢弃。在第一次诊断PCR鉴定负候选人应包含集成错误删除结构的野生型带和条带（尺寸不可预测）。 </li></ol></li><li>甘油股票 注:甘油股起到维持克隆多年来在应变集合。至少两个独立的转化体应当保持对于每个突变体。这允许比较表型应是相同的。 <ol><li>生长5毫升培养物在YEPS-光确认的突变株上的旋转轮24小时，在28℃混合500微升每种培养物用500μlNSY-甘油的培养基。反转管，直到文化和中充分混合。甘油培养基中防止冰晶的形成。 </li><li>冻结在-80℃。重新连胜冷冻文化的一部分来测试，如果股票是功能。 </li></ol></li></ol> 5.显微表型<ol><li>生长5毫升培养野生型和突变株在YEPS-光对旋转轮12小时在28℃。 </li><li>第二天，稀释培养至约0.1的OD 600和成长它，直到0.5和0.7之间的OD 600。 1.0 corresp的OD 600哔声至约1至2×10 7个U.每玉米须毫升细胞。 </li><li>显微镜检查细胞形态学。 注:健康的细胞出现雪茄形（ 图4，WT）。发音的空泡或丝的形成表明，所述细胞强调。 注意:根据预期的突变表型，分析可以适于， 例如 ，如果使用的话记者菌株的合适的测定可以在此阶段进行。如果细胞形态改变的统计分析应进行， 例如 ，以确定平均细胞长度。 </li></ol> 6.感染测定注:苗感染试验已经在本杂志11先前可视化。它可以是使用一个单倍体，solopathogenic菌株背景如SG200 10或通过混合兼容交配伙伴其中两个携带突变进行。 <ol><li>每增长至少40玉米幼苗应变7天16小时，200μE，28℃/ 22℃（昼/夜）的光周期。它们应在3叶阶段和10-15厘米高在感染一天。 </li><li>感染前两天，在28°C开始菌株的预培养5毫升YEPS光强在一个旋转的车轮24小时。种植主培养在50毫升YEPS光强可达600 OD = 1.0（细胞分裂率= 2小时，允许3师最小，可以做到O / N）。 </li><li>降速50ml培养的细胞在1,500×g离心5分钟并弃上清。洗3次，用无菌H 2 O悬浮细胞在无菌H 2 O操作的外径为3.0 600。如果需要的话，混合交配伙伴。 </li><li>注入250 - 500微升细胞悬浮液成采用一个小注射器7天老玉米幼苗的茎。使用无菌H 2 O作为对照。保持感染幼苗在相同光照和温度条件下的额外7-14天。分数每个工厂的表型ccording健康，萎黄，花青素的形成，小的肿瘤，肿瘤大，植株枯死10。 注:评分可以早7天完成，14天重复遵循感染随着时间的推移。 </li></ol>

<ol>
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The authors have nothing to disclose.

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?...

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。

<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

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。

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...

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

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。

植物病原体的遗传操作<em&gt;玉米黑粉菌</em&gt;研究真菌生物学和植物微生物相互作用

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

Research

JoVE Journal

Genetics

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

14.4K 次观看.  Heinrich-Heine University Düsseldorf. 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。 

视频: 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.

评估 乌西拉戈 病原性的快速高效方法，可在玉米和特奥辛特线上使用

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

科研

环境科学

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.

真菌快速缺失生产<em&gt;农杆菌</em&gt;介入OSCAR删除构造的转换

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

遗传学

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.

使用乌斯蒂拉戈梅迪斯作为特洛伊木马在现场交付玉米蛋白

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast ...

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

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

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

Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.

crispr 介导的人类真菌病原体白色念珠菌基因组编辑

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

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

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

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

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

转基因玉米中黄曲霉生长和黄曲霉毒素的产生表达表达.

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.

遗传操纵<em&gt;Δku80</em&gt;株功能基因组分析<em&gt;弓形虫</em

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

免疫与感染

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.

CRISPR-Cas9-中切基因组编辑在纤维阿莫西特 亨蒂埃拉阿曼尼西斯

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.

用于在细胞水平上探测真菌 - 微生物相互作用的微流体工具

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

生物工程

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.

用于植物/微生物分子相互作用和信号的同时系统分析的水培共培养系统

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

生物学

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

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

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

利用 Microrespiration 分析和显微解剖研究 Teliospore 萌发

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

植物病原体的遗传操作

摘要

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此视频中的章节

评估乌西拉戈病原性的快速高效方法，可在玉米和特奥辛特线上使用

真菌快速缺失生产

使用乌斯蒂拉戈梅迪斯作为特洛伊木马在现场交付玉米蛋白

crispr 介导的人类真菌病原体白色念珠菌基因组编辑

转基因玉米中黄曲霉生长和黄曲霉毒素的产生表达表达.

遗传操纵

CRISPR-Cas9-中切基因组编辑在纤维阿莫西特亨蒂埃拉阿曼尼西斯

用于在细胞水平上探测真菌 - 微生物相互作用的微流体工具

用于植物/微生物分子相互作用和信号的同时系统分析的水培共培养系统

利用 Microrespiration 分析和显微解剖研究 Teliospore 萌发

植物病原体的遗传操作

摘要

探索更多视频

此视频中的章节

评估 乌西拉戈 病原性的快速高效方法，可在玉米和特奥辛特线上使用

真菌快速缺失生产

使用乌斯蒂拉戈梅迪斯作为特洛伊木马在现场交付玉米蛋白

crispr 介导的人类真菌病原体白色念珠菌基因组编辑

转基因玉米中黄曲霉生长和黄曲霉毒素的产生表达表达.

遗传操纵

CRISPR-Cas9-中切基因组编辑在纤维阿莫西特 亨蒂埃拉阿曼尼西斯

用于在细胞水平上探测真菌 - 微生物相互作用的微流体工具

用于植物/微生物分子相互作用和信号的同时系统分析的水培共培养系统

利用 Microrespiration 分析和显微解剖研究 Teliospore 萌发

评估乌西拉戈病原性的快速高效方法，可在玉米和特奥辛特线上使用

CRISPR-Cas9-中切基因组编辑在纤维阿莫西特亨蒂埃拉阿曼尼西斯