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>

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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 border="0" cellpadding="0" cellspacing="0" width="1138">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Aminobenzoeic acid (Free Acid)</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">A-9878</td>
			<td style="width:485px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Agar&#160;</td>
			<td style="width:183px;">BD</td>
			<td style="width:124px;">214010</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Peptone</td>
			<td style="width:183px;">BD</td>
			<td style="width:124px;">211677</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Yeast Extract&#160;</td>
			<td style="width:183px;">BD</td>
			<td style="width:124px;">212750</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">CaCl2*2H2O</td>
			<td style="width:183px;">Gr&#252;ssing GmbH</td>
			<td style="width:124px;">10234</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Ca-pantothenat (Hemi-Ca. salt)</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">P-2250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carboxin&#160;</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">45371</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Casamino acids&#160;</td>
			<td style="width:183px;">BD</td>
			<td style="width:124px;">223050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Cholinchlorid</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td>C-1879</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Citric acid</td>
			<td style="width:183px;">ChemSolute</td>
			<td style="width:124px;">24,321,000</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">CuSO4*5H2O</td>
			<td style="width:183px;">Fluka</td>
			<td style="width:124px;">61240</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">D(+)Sucrose&#160;</td>
			<td style="width:183px;">Roth</td>
			<td style="width:124px;">4621.1</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">DNA degr. free acid&#160;</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:124px;">D-3159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">EDTA</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">E4378</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">FeCl3*6H2O</td>
			<td style="width:183px;">Gr&#252;ssing GmbH</td>
			<td style="width:124px;">10288</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Geneticin (G418) disulfate salt</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">A1720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichoderma lysing enzymes</td>
			<td>Sigma Aldrich</td>
			<td>L1412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Glucose</td>
			<td style="width:183px;">Caelo</td>
			<td style="width:124px;">2580</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Glycerin&#160;</td>
			<td style="width:183px;">Fisher Chemical</td>
			<td style="width:124px;">G065015</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">H3BO3</td>
			<td style="width:183px;">AppliChem</td>
			<td style="width:124px;">A2940</td>
			<td>Dangerous substance. Please check manufacturer&#39;s safety instructions.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin sodium salt</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">H3393-50KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hygromycin B-solution</td>
			<td style="width:183px;">Roth</td>
			<td style="width:124px;">1287.2</td>
			<td>Dangerous substance.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">KCl</td>
			<td style="width:183px;">VWR</td>
			<td style="width:124px;">26764298</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">KH2PO4</td>
			<td style="width:183px;">AppliChem</td>
			<td style="width:124px;">A3620</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">MgSO4 waterfree</td>
			<td style="width:183px;">Merck</td>
			<td style="width:124px;">7487-88-9</td>
			<td>Water free is critical. Alternatively use local supplier</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">MnCl2*4H2O</td>
			<td style="width:183px;">AppliChem</td>
			<td style="width:124px;">A2087</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">myo-Inositol</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td>I-5125</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">Na2-EDTA*2H2O</td>
			<td style="width:183px;">AppliChem</td>
			<td style="width:124px;">A2937</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">Na2MoO4*2H2O</td>
			<td style="width:183px;">Roth</td>
			<td style="width:124px;">0274.2</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">Na2SO4</td>
			<td style="width:183px;">Gr&#252;ssing GmbH</td>
			<td style="width:124px;">12174</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">NaCl</td>
			<td style="width:183px;">Fisher Chemical</td>
			<td style="width:124px;">S316060</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">NaOH</td>
			<td style="width:183px;">ChemSolute</td>
			<td style="width:124px;">13,751,000</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">NH4NO3</td>
			<td style="width:183px;">Roth</td>
			<td style="width:124px;">K299.1</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nicotinic acid (Free Acid)</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">N-4126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nourseothricin dihydrogen sulfate&#160;&#160;</td>
			<td style="width:183px;">Werner BioAgents</td>
			<td style="width:124px;">5,001,000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Nutrient broth&#160;</td>
			<td style="width:183px;">Difco</td>
			<td style="width:124px;"></td>
			<td>local suppliers</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">Phenol:Chloroform:Isoamyl Alcohol (25:24:1) pH 6.7</td>
			<td>Sigma Aldrich</td>
			<td>P3803</td>
			<td>Dangerous substance. Please check manufacturer&#39;s safety instructions.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polyethylene glycol (PEG)</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">P-3640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium acetate</td>
			<td style="width:183px;">AppliChem</td>
			<td>121479</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pyridoxin (Monohydrochlorid)</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">P-9755</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Riboflavin&#160;</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">R4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">RNaseA</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:124px;">R5503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">SDS</td>
			<td style="width:183px;">Roth</td>
			<td style="width:124px;">Cn30.3</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">small syringe</td>
			<td>BD</td>
			<td>300300</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sterile filter, 22 &#181;m</td>
			<td>VWR</td>
			<td>28145-477</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Sorbitol</td>
			<td style="width:183px;">Roth</td>
			<td style="width:124px;">6213.1</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Thiamin-Hydrochloride</td>
			<td style="width:183px;">Serva</td>
			<td style="width:124px;">36020.02</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">tri-Na-Citrate</td>
			<td style="width:183px;">Fisher Chemical</td>
			<td style="width:124px;">S332060</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Tris- (hydroxymethyl) aminomethane</td>
			<td>VWR</td>
			<td style="width:124px;">103156X</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Tris hydrochloride</td>
			<td>Roth</td>
			<td style="width:124px;">9090.4</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100&#160;</td>
			<td style="width:183px;">Serva&#160;</td>
			<td style="width:124px;">37240</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:347px;">ZnCl2</td>
			<td style="width:183px;">Fluka</td>
			<td style="width:124px;">96470</td>
			<td>alternatively use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Name</td>
			<td style="width:183px;">Company</td>
			<td style="width:124px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Composition of solutions/preparation of material</td>
			<td></td>
			<td></td>
			<td>Composition of solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carboxin</td>
			<td></td>
			<td></td>
			<td>Stock: 5 mg/ml in methanol, final concentration: 2 &#181;g/ml</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">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) NH4NO3, 2.0 % (w/v) Bacto Agar; adjust to pH 7.0 using 5 M NaOH; after autoclaving add 1 % glucose&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Geneticin (G418)</td>
			<td></td>
			<td></td>
			<td>Stock: 50 mg/ml in H2O, final concentration: 500 &#181;g/ml</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">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 H2O until the pH is 7 (the liquid should not be yellow-green anymore). Aliquot the glass beads and dry them at 180 &#176;C. The decanted HCl has to be neutralized before disposal.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin</td>
			<td></td>
			<td></td>
			<td>Stock: 15 mg/ml</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Holliday salt solution</td>
			<td></td>
			<td></td>
			<td>16.0 &#8240; (w/v) KH2PO4, 4.0 &#8240; (w/v) Na2SO4, 8.0 &#8240; (w/v) KCl, 1.32 &#8240; (w/v) CaCl2*2H2O, 8.0 &#8240; (v/v) trace elements, 2.0 &#8240; (w/v) MgSO4; sterile filtrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Holliday vitamin solution&#160;</td>
			<td></td>
			<td></td>
			<td>0.1&#8240; (w/v) Thiamin, 0.05&#8240; (w/v) Riboflavin, 0.05&#8240; (w/v) Pyridoxin, 0.2&#8240; (w/v) Ca-Pantothenat, (0.05&#8240; (w/v) Aminobenzoeic acid, 0.2&#8240; (w/v) Nicotinic acid, 0.2&#8240; (w/v) Cholinchlorid, 1.0&#8240; (w/v) myo-Inositol; may be stored at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hygromycin</td>
			<td></td>
			<td></td>
			<td>Stock: 50 mg/ml in PBS, final concentration: 200 &#181;g/ml</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Nourseothricin</td>
			<td></td>
			<td></td>
			<td>Stock: 200 mg/ml in H2O, final concentration: 150 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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,&#160; 80.0 % (v/v) 87% Glycerin (f.c. 69.6%)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RegLight</td>
			<td></td>
			<td></td>
			<td>1.0% (w/v) Yeast Extract&#160; 0.4 % (w/v) Bacto Peptone, 0.4 % (w/v) Sucrose, 18.22 % (w/v) Sorbitol, 1.5 % (w/v) Agar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="25">
			<td height="25" style="height:25px;">STC, pH 8</td>
			<td></td>
			<td></td>
			<td>1 M Sorbitol, 10 mM Tris-HCl pH 7.5, 100 mM CaCl2; filter sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">STC/PEG</td>
			<td></td>
			<td></td>
			<td>40 % (v/v) PEG in STC-buffer&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">TE buffer, pH 8</td>
			<td></td>
			<td></td>
			<td>1.31 mM Tris-Base, 8.69 mM Tris-HCl, 10 mM Na2-EDTA*2H2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TE/RNase</td>
			<td></td>
			<td></td>
			<td>10 &#181;g/ml RNaseA in TE buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Trace elements</td>
			<td></td>
			<td></td>
			<td>0.06&#8240; (w/v) H3BO3, 0.14&#8240; (w/v) MnCl*4H2O, 0.4 &#8240; (w/v) ZnCl2, 0.4 &#8240; (w/v) Na2MoO4*2H2O, 0.1 &#8240; (w/v) FeCl3*6H2O, 0.04&#8240; (w/v) CuSO4*5H2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichoderma lysing enzymes solution</td>
			<td></td>
			<td></td>
			<td>12.5 mg/ml SCS; filter sterile; prepare shortly before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Usti-lysis buffer 2</td>
			<td></td>
			<td></td>
			<td>mix Usti lysis buffer 1 with 1 x TE in a 1:1 ratio</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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

14.3K 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

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species.
Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family,&#160;it could be easily used to other prokaryote and eukaryote species

Here, we present a protocol for tracing genomic DNA (gDNA) contamination in RNA samples. The presented method utilizes primers specific for the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) genes. The method is suited for reliable and sensitive detection of DNA contamination in most eukaryotes and prokaryotes.

基于核糖体 dna 的 RNA 样本 dna 污染评价

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

科研

遗传学

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 &#181;g DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., &#62; 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.

Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

超高分子量基因组DNA的分离纯化白粉病对长读测序

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics ...

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms to questions surrounding insect's interactions with their host plants. Genomic resources are available for several aphid species, and with advances in the next-generation sequencing, transcriptomic studies are being extended to non-model organisms that lack genomes. Furthermore, aphid cultures can be collected from the field and reared in the laboratory for the use in organismal and molecular experiments to bridge the gap between ecological and genetic studies. Last, many aphids can be maintained in the laboratory on their preferred host plants in perpetual, parthenogenic life cycles allowing for comparisons of asexually reproducing genotypes. Aphis nerii, the milkweed-oleander aphid, provides one such model to study insect interactions with toxic plants using both organismal and molecular experiments. Methods for the generation and maintenance of the plant and aphid cultures in the greenhouse and laboratory, DNA and RNA extractions, microsatellite analysis, de novo transcriptome assembly and annotation, transcriptome differential expression analysis, and qPCR verification of differentially expressed genes are outlined and discussed here.

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

The aphid Aphis nerii colonizes on highly-defended plants in the dogbane family (Apocyanaceae) and provides numerous opportunities to study plant-insect interactions. Here, we present a series of protocols for the maintenance of plant and aphid cultures, and the generation and analysis of molecular and -omic data for A. nerii.

nerii 蚜生物培养和基因表达的维持--植物-昆虫相互作用的非模型系统

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms ...

Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula

Schrenkiella parvula is an extremophyte adapted to various abiotic stresses, including multiple ion toxicity stresses. Despite high-quality genomic resources available to study how plants adapt to environmental stresses, its value as a functional genomics model and tool has been limited by the lack of a feasible transformation system. In this protocol, we report how to generate stable transgenic S. parvula lines using an Agrobacterium-mediated floral-dip method. We modified the transformation protocol used for A. thaliana to account for unique traits of S. parvula, such as an indeterminate flowering habit and a high epicuticular wax content on leaves. Briefly, S. parvula seeds were stratified at 4 &#176;C for five days before planting. Plants were grown at a photoperiod of a 14 h light and 10 h dark and a 130 &#181;mol m-2s-1 light intensity, at 22 &#176;C to 24 &#176;C. Eight to nine week-old plants with multiple inflorescences were selected for transformation. These inflorescences were dipped in an infiltration solution of Agrobacterium tumefaciens GV3101 carrying the pMP90RK plasmid. We performed two rounds of flower dipping with an interval of three to four weeks to increase the transformation efficiency. The T1 seeds were collected and dried for four weeks in a container with desiccants before germination to screen for candidate transformed lines. Resistance to BASTA was used to screen T1 plants. We sprayed the BASTA solution three times with an interval of three days starting at two week-old plants to reduce false positives. A BASTA drop test was performed on surviving individual plants to identify true positive transformants. The transformation efficiency was 0.033%, yielding 3&#8211;4 transgenic plants per 10,000 T1 seeds propagated.

Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula

Agrobacterium-mediated transformation using a floral-dip method can be successfully employed to create stable transgenic lines of the extremophyte model Schrenkiella parvula. We present a protocol modified from that for Arabidopsis thaliana, considering different growth habits and physiological characteristics of the extremophyte.

植物生长与农杆菌介导的植物浸水转化

Schrenkiella parvula is an extremophyte adapted to various abiotic stresses, including multiple ion toxicity stresses. Despite high-quality genomic ...

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 ...

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in regulating gene expression at the post-transcriptional level. Sequencing sRNA libraries by Next Generation Sequencing (NGS) methods has been widely employed to identify and analyze miRNA transcriptomes in the last decade, resulting in a rapid increase of miRNA discovery. However, two major challenges arise in plant miRNA annotation due to increasing depth of sequenced sRNA libraries as well as the size and complexity of plant genomes. First, many other types of sRNAs, in particular, short interfering RNAs (siRNAs) from sRNA libraries, are erroneously annotated as miRNAs by many computational tools. Second, it becomes an extremely time-consuming process for analyzing miRNA transcriptomes in plant species with large and complex genomes. To overcome these challenges, we recently upgraded miRDeep-P (a popular tool for miRNA transcriptome analyses) to miRDeep-P2 (miRDP2 for short) by employing a new filtering strategy, overhauling the scoring algorithm and incorporating newly updated plant miRNA annotation criteria. We tested miRDP2 against sequenced sRNA populations in five representative plants with increasing genomic complexity, including Arabidopsis, rice, tomato, maize and wheat. The results indicate that miRDP2 processed these tasks with very high efficiency. In addition, miRDP2 outperformed other prediction tools regarding sensitivity and accuracy. Taken together, our results demonstrate miRDP2 as a fast and accurate tool for analyzing plant miRNA transcriptomes, therefore a useful tool in helping the community better annotate miRNAs in plants.

A bioinformatics pipeline, namely miRDeep-P2 (miRDP2 for short), with updated plant miRNA criteria and an overhauled algorithm, could accurately and efficiently analyze microRNA transcriptomes in plants, especially for species with complex and large genomes.

生物信息学管道，用于准确、高效地分析植物中的微RNA转录

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in ...

植物病原体的遗传操作

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