我们要感谢保罗. 弗罗斯特医生使用他的 microrespirometer, 妮可瓦格纳和亚历克斯贝尔的技术援助。这项工作由 NSERC 赠款资助 B.J.S。

1. 玉米芯感染
<ol><li>种植玉米玉蜀黍(cv 金矮脚鸡), 直到棒子形成, 并开始丝绸 (约60天)。</li>
	<li>与以前描述的7使用标准协议的区域性兼容的单倍体U. 玉米菌株。</li>
	<li>使用以前描述的7标准协议感染玉米棒子。</li>
</ol>2. Teliospore 收获
<ol><li>蒸压设备 (Büchner 漏斗, Büchner 烧瓶, 搅拌器, 250 毫升离心瓶, 扁平铲子, 和水) 使用标准的干燥循环, 至少30分钟的灭菌121摄氏度 (水的标准液体循环)。</li>
	<li>使用剃刀将受感染的棒子从植物中取出 (大约28–35天后感染), 并将棒子放在工作台保护罩上的托盘上。</li>
	<li>用剃刀刀片从棒子上取出肿瘤, 然后在烧杯中采集。</li>
	<li>填充250毫升实验室搅拌器杯与肿瘤, 直到大约1/3 满, 并添加蒸压 dH2O, 直到搅拌机杯约3/4 满。通过在低的搅拌机中脉冲来扰乱肿瘤, 直到均匀。</li>
	<li>将真空泵连接到一个疏水器, 然后再用1升 Büchner 瓶。</li>
	<li>将大型 Büchner 漏斗插入瓶中, 并在 Büchner 漏斗底部排上四层棉布。</li>
	<li>打开真空泵。</li>
	<li>将匀质肿瘤的一部分倒入棉布, 用刮刀刮。</li>
	<li>将一些 dH2O 倒入棉布中, 以刷新冬孢子。</li>
	<li>重复直到通过棉布的 dH2O 是清楚的。</li>
	<li>将含有匀质肿瘤材料的棉布拧出过滤器, 以确保最大 teliospore 回收率。</li>
	<li>当1升 Büchner 瓶接近满, 清空它成一个大的锥形瓶, 并把它放在一边。</li>
	<li>将新的棉布放入过滤器中, 重复步骤 (2.7–2.12), 直到所有肿瘤被破坏和过滤。</li>
	<li>将过滤后的冬孢子倒入蒸压250毫升离心机瓶和离心机, 1000 x 克5分钟, 醒酒上清。</li>
	<li>重复步骤 2.14, 直到所有的过滤冬孢子被离心颗粒化。</li>
	<li>将小球悬浮在少量的水中, 转移到50毫升离心管。</li>
	<li>离心管在 1000 x g 5 分钟, 醒酒上清。</li>
	<li>将颗粒悬浮在大约50毫升的 dH 中2O, 离心管在 1000 x g 处为5分钟, 醒酒上清。用刮刀轻轻刮掉灰色的顶层, 然后把它扔掉。重复, 直到上面不再有灰色图层。</li>
	<li>用真空干燥在一夜之间干燥样品。</li>
	<li>将干燥的冬孢子在4摄氏度, 直到使用。</li>
	<li>
如果需要, 在诱导发芽前用硫酸铜8治疗冬孢子。如果不治疗, 则对冬孢子进行彻底的显微分析, 以确认样品代表纯冬孢子, 缺乏细菌或其他污染物。
	<ol>
<li>在1.5 毫升离心管中称量约50毫克的冬孢子。</li>
		<li>将大约1.0 毫升0.75% 丘索4添加到包含冬孢子的1.5 毫升离心管中。吸管向上和向下悬浮在丘索4解决方案中的冬孢子, 然后将该样本搅拌3小时。</li>
		<li>将样品离心 2500 x 克5分钟, 取出上清液。用无菌水并用重悬 teliospore 颗粒, 重复离心, 取出上清液。</li>
		<li>重复步骤2.21.3 两次。</li>
	</ol>
</li>
</ol>3. Teliospore 活力和发芽试验
<ol><li>在1.5 毫升离心管中称量出大约10毫克的玉米冬孢子, 以评估它们的生存能力和发芽时间。</li>
	<li>在生物安全柜中, 制备马铃薯葡萄糖汤 (PDB, 24 克/升), 辅以硫酸链霉素 (160 µg/毫升)。</li>
	<li>在 PDB 的500µL 中暂停冬孢子。轻轻的吸管, 混合和打破所有的冬孢子团簇。</li>
	<li>将 teliospore 悬浮液转移到含10毫升 PDB 的蒸压250毫升锥形烧瓶中。</li>
	<li>孵化的烧瓶在28°c 颤抖在 90 rpm 为 12–16 h。</li>
	<li>在生物安全柜中, 取出20µL 样品, 冬孢子诱导发芽并准备显微镜滑动。</li>
	<li>
使用显微镜, 直观地评估存在的发芽阶段和细菌污染的存在。
	<ol>
<li>使用 hemocytometer 计算第一阶段冬孢子的数量, 并确定发芽的百分比。</li>
		<li>如果只有一级冬孢子存在, 在评估 teliospore 发芽之前, 继续孵化24小时的烧瓶。在认定样品不可行之前, 继续孵化48小时。</li>
		<li>如果细菌污染存在, 补充 PDB 硫酸卡那霉素 (50 µg/毫升) 以及硫酸链霉素 (160 µg/毫升), 然后重复步骤3.1 到3.7。如果细菌污染持续, 用硫酸铜治疗冬孢子, 重复步骤3.1 至3.7。</li>
	</ol>
</li>
</ol>4. 呼吸监测的发芽诱导
<ol><li>对每种呼吸实验的冬孢子量相等 (e.g, 50 毫克)。</li>
	<li>在生物安全柜中, 将冬孢子添加到蒸压呼吸室。</li>
	<li>用 PDB (24 克/升) 补充硫酸链霉素 (160 µg/毫升) 和硫酸卡那霉素 (50 µg/毫升) 填充腔室。</li>
	<li>吸管向上和向下创建 teliospore 悬浮。</li>
	<li>将室盖放在会议厅内, 以制造空气密封。</li>
</ol>5. 获得氧气消耗率 (OCR) 测量
<ol><li>安置房间在房间架子在水浴之内 (预热到28°c)。</li>
	<li>将 O2探头置于会议厅的开口内。</li>
	<li>监视实时显示在 "SensorTrace 速率" 程序上的数据点, 并让探头稳定 (在探头放置在腔室中3分钟)。</li>
	<li>单击 "度量值" 以连续6小时测量 O2级别, 并以2秒为间隔记录测量值。</li>
	<li>停止测量, 并对每个要分析的示例重复步骤4.1–5.4。</li>
	<li>通过单击 "文件 |" 将数据导出到 Microsoft Excel 中出口 |另存为. xls "。</li>
</ol>6. 数据分析
<ol><li>
计算 OCR
	<ol>
<li>在 "Within_Rates" 选项卡下的导出的 Excel 文件中, 记录每个分庭测量的 "速率" (nmol/小时)。</li>
		<li>对于每个实验样本, 从实验样品室的 "速率" 中减去空白腔的 "速率" 以获得修正的 OCR 值, 并取该数字的绝对值。</li>
		<li>计算每毫克冬孢子的总 ocr, 方法是用所用的细胞质量除以校正后的绝对 ocr 值。</li>
		<li>每个应变的平均复制 "每毫克冬孢子的 OCR" 值。</li>
	</ol>
</li>
	<li>使用其他统计软件的 Microsoft Excel, 使用适当的统计方法 (e. g、学生的t测试、方差分析) 分析数据。</li>
	<li>若要绘制原始数据, 请计算要绘制的每个时间点的剩余氧气百分比。先将自己的读数除以 100 (剩余的100% 氧气), 然后除以一读后的每一次读数, 乘以 100, 以获得腔内剩余氧气的百分比。</li>
</ol>7. 诱导 Teliospore 萌发分离冬孢子在萌发的不同阶段
<ol><li>准备 PDB (24 克/升) 在生物安全柜中辅以硫酸链霉素 (160 µg/毫升)。</li>
	<li>将大约10毫克的U 玉米冬孢子放入1.5 毫升离心管中。</li>
	<li>在 PDB 的500µL 中暂停冬孢子。轻轻吸管混合, 直到没有在培养基中的冬孢子团簇。</li>
	<li>将 teliospore 悬浮液转移到含 PDB 的蒸压250毫升锥形烧瓶中, 并用硫酸链霉素补充。</li>
	<li>在90转每分钟28摄氏度的抖动中孵化烧瓶。</li>
</ol>8. 培养皿和机器人的制备
<ol><li>
通过吹打排的水滴进行 microcapillary 准备和样品收集, 准备一个培养皿 (57 厘米2)。
	<ol>
<li>吸管5µL (x4) dH2O 滴横跨培养皿的顶部。</li>
		<li>吸管2µL (x3) 的 RNA 稳定溶液在培养皿用于标本收集。</li>
		<li>吸管5µL (x30) 在培养皿上萌发冬孢子的水滴。</li>
	</ol>
</li>
	<li>在培养皿中加入15毫升矿物油。在继续之前, 确保所有的水滴都被油覆盖。</li>
	<li>准备一个具有15µm 内径, 1 毫米法兰, 55 毫米长度的 microcapillary, 并把它放在 microcapillary 持有者, 并将其浸入矿物油中, 毛细管作用将允许矿物油进入 microcapillary。在把 microcapillary 的压力放到水滴之前, 先释放它。吸水准备 microcapillary。</li>
</ol>9. 特定阶段萌发冬孢子的分离
<ol><li>使用机器人的控制, 将准备好的 microcapillary 移动到其中一个发芽水滴。穿透液滴, 降低 microcapillary, 并使 microcapillary 的嘴在萌发的过程中发芽 teliospore。</li>
	<li>慢慢地吸入捕捉发芽的 teliospore。一旦 teliospore 进入 microcapillary, 停止吸气。重复, 直到有大约五冬孢子在 microcapillary。</li>
	<li>用机器人提高 microcapillary, 并将其带到 RNA 稳定溶液的收集液滴上。穿透液滴并将冬孢子注入液滴中。</li>
	<li>重复步骤8.1 到 8.3, 直到捕获了大约1000冬孢子。</li>
</ol>10. 回收液滴
<ol><li>将收集液滴吸干并将其转移到 RNase/DNase 2.0 毫升离心管的盖子上。小心地用吸管除去矿物油而不干扰收集液滴。</li>
	<li>将冬孢子用于下游应用程序, 如 RNA 隔离。</li>
</ol>

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作者没有竞争的金融利益或其他利益冲突来披露。

担子 biotrophic 植物病原体每年造成数以亿计的农作物损失。绝大多数这些病原体产生冬孢子, 是真菌散布和性繁殖不可分割的组成部分。了解冬孢子的发展和发芽对理解这些真菌造成的破坏性疾病的传播至关重要。为了识别关键控制点的分子变化, 我们设计了一种方法来确定生理移位的时间, 另一种是在萌发的不同阶段分离冬孢子。et 等。(未发表) 注意到 teliospore 萌发的五个阶段的光显微术 (图 2)。为了研究 i. 期的生理活化, 并对发芽过程中呼吸速率进行评估, 我们用克拉克型 microrespirometer 精确测量了氧消耗的变化。我们的样本数据表明, 我们的方法是精确和高度重现性。我们的发现证实, 发芽的U. 玉米冬孢子与未诱导的休眠冬孢子相比, 细胞呼吸的急剧增加。我们首次发现, 被诱导发芽的U. 玉米冬孢子在吸氧量上的延迟为45分钟。这表明,玉米冬孢子可能需要一些时间来处理发芽信号 (e.g), 在反应之前, 增加的吸氧量不是对发芽信号的立即反应, 也不是我们的化验不够灵敏, 无法检测初始吸氧量的最小变化。
以前的研究检测黑穗病的呼吸率冬孢子3依赖于华宝烧瓶设备测量氧水平 manometrically 5. 简单地, 该方法通过直接观察压力表臂中的液位变化来检测密封瓶中的压强变化, 测量耗氧量和 CO2的产量。实验可能很难建立, 测量可能是不精确的。该仪器必须在整个测量期间进行, 并且需要进行大量的计算来估计 OCR。我们的协议利用技术进步, 消除了用户在实验期间留在仪器上的要求, 用眼睛测量, 并使用广泛的数学公式。其他人使用了早期的克拉克类型 respirometers 来测量粗糙霉9和Botryodpilodia theobromae10孢子的 OCR, 但是这些早期仪器允许连续测量最多20分钟。这种限制将不会允许识别45分钟的氧气摄取延迟, 我们观察到新的模型呼吸。我们的协议使数据解释更加简单, 因为读数是在腔室中剩余的氧气浓度, 可以直接绘制, 无需任何计算或数据操作。此外, 有可能连续测量 (每2秒) 的时间无限期, 直到可用氧气完全耗尽。这允许发现在呼吸的小变化在长时间期间。因此, 我们已经改进了早期的技术, 并制定了一个简单的, 精确的, 可重复的方法来测量真菌孢子的耗氧量。根据我们的知识, 这是第一个使用现代克拉克型呼吸的研究, 研究休眠与发芽冬孢子的呼吸。
尽管该协议简单明了, 但需要进行优化, 并且存在一些限制分析的生物现实。首先, 必须确定适当的样本大小, 以达到合理的 OCRs。过多的样品会导致氧气水平过早的崩溃, 样本太少会导致无法观察到氧气消耗的有意义的变化。其次, 有必要让探测时间稳定 (约3分钟), 以提供准确的初始数据。最后, 用抗菌剂 (如硫酸链霉素) 补充发芽培养基是很重要的, 以确保细菌污染不会改变 OCR 读数。我们所面临的生物限制是在测量时间过程中的低发芽率, (~ 1%) 通过观察视觉形态学变化来确定。确定孢子的生存能力将允许这种速率的决心转化为每孢子数的速率和隔离冬孢子与较高的发芽率将导致更高的 OCRs。U. 玉米冬孢子11的异步发芽是一个必须考虑的现实, 可能导致了在发芽早期无法检测到氧气消耗的情况.
为了提高测量 teliospore 呼吸变化的准确性和精确度, 今后对该方法的适应可能包括在单个细胞基础上测量 OCR。微操作系统技术可用于分离单个 teliospore, 然后诱导其发芽, 并可监测其呼吸速率。这可以改善分辨率, 提供有关 OCR 的信息, 在休眠-发芽转移每 teliospore, 而不是每毫克的冬孢子。另外, 这也解决了异步发芽的混杂问题。
在发芽的后期, 我们开发了一种微操作系统的方法来分离冬孢子在发芽的共同阶段。这使得建立相对同步的 teliospore 种群进行分析。对分离单微生物的各种方法进行了描述, 并在过去的几年中改进了12。这些方法包括对孢子悬浮液进行稀释, 以获得单一微生物, 半机械方法, 利用 microcapillaries 获得转移到培养基进行培养的孢子, 以及使用并联的机械方法。.以前的方法, 我们用来获得冬孢子在同一阶段的发芽包括逆流离心淘洗和过滤发芽冬孢子通过一个特定的孔大小尼龙膜。使用这些方法, 我们可以丰富的发芽冬孢子, 但是, 我们的样品仍然包含冬孢子在不同的阶段发芽13。现有的单微生物微操作系统技术已得到改进, 引进了更高的放大倍数和仪器, 精细控制毛细管针, 吸入和转移微生物。以前的微操作系统技术侧重于隔离单细胞进行培养或用于单细胞 PCR 应用 14.使用并联分离单一真菌孢子以前并没有建立。以前一种分离单一真菌孢子的方法涉及使用细钳或针头来挑选含有发芽孢子的小块固体培养基15。微操作系统与并联的使用广泛应用于酵母研究中, 子囊孢子的聚类可以在培养基上孢子培养基中分离, 用于减数分裂遗传分析16。我们已经开发了一种方法, 结合微操作系统技术的细菌细胞14和 体外施肥方法, 以隔离发芽冬孢子。我们已经表明, 数以百计的共同萌发阶段冬孢子可以得到这种技术。这些样品可用于下游表达研究使用的技术, 如 RT-qPCR 或 RNA, 以获得一个冬孢子的种群, 发芽是同步允许分析基因表达的具体变化发生在teliospore 萌发的早期、中期和后期阶段。
阶段特异性发芽冬孢子的显微解剖可能需要建立和认识不同萌发阶段的经验, 但是, 这种经验可以通过实践快速获得。有几个步骤, 必须遵循成功的显微解剖后, RNA 分离。首先, 发芽培养基必须辅以抗生素 (e. g, 硫酸链霉素), 以抑制细菌污染的增长, 在发芽开始时, 以及在收集发芽冬孢子。其次, 使用稳定的解决方案来稳定和保护 RNA 的隔离是很重要的。RNA 稳定的解决方案也防止收集冬孢子的进展到下一个发芽阶段, 同时收集额外的冬孢子。第三, 一旦回收液滴, 以确保 RNA 提取成功, 就必须去除矿物油。最后, 我们注意到, 如果分离的冬孢子在 rna 稳定溶液中储存了较长的时间, rna 的质量就会下降;因此, 建议在采集萌发期特定冬孢子后立即分离 RNA。该方法的一个局限性是, i 冬孢子收集的阶段可能包含休眠, 死亡, 并诱导发芽冬孢子, 因为这三阶段是形态上无法区分的。另外, 在收集 III. 冬孢子时, 可以获得冬孢子减数分裂 i 或减数分裂 II 的混合物。帮助区分真正休眠和死冬孢子的一种方法是确定样本的生存能力。利用活/死细胞活性测定法评估真菌孢子活力的方法可能能够评估可行的冬孢子的百分比, 从中可以确定更多的信息萌发率 17.此外, DAPI 的细胞核染色, 例如, 可以用来可视化的事件发生在第三阶段和过渡到第四阶段, 以便进一步表征冬孢子形态上第三阶段。在使用我们的显微解剖方法时, 这将有助于冬孢子在发芽的一个阶段的收集。
最后, 我们开发了一种简单、精确和可重现的方法来测量在黑穗病玉米冬孢子休眠-萌发转移过程中发生的细胞呼吸变化。此外, 我们还开发了一种方法来收集特定阶段的发芽冬孢子, 可用于下游应用, 如 RNA-我们的方法可以适应各种细胞类型和种类。我们预计, 改进我们的技术将有助于检测单一孢子水平的呼吸道变化, 并进一步确定发生在后期发芽的事件。

黑穗病真菌 (Ustilaginales) 由1600多个物种组成, 其中包括玉米、大麦和小麦等重要谷物作物, 每年造成的农作物损失数以亿计.1。这些真菌的特点是冬孢子的生产, 其中有黑暗的色素细胞壁和分散剂。冬孢子在寄主植物间分散的压力下保护遗传物质的作用, 并且可以在休眠状态持续多年 2.因此, 冬孢子是疾病传播的一个重要组成部分。
为了研究 teliospore 生物学, 我们的实验室利用了黑穗病真菌黑穗病玉米(U 玉米), 这是该病 "玉米常见黑穗病" 的因果动因。成熟的U. 玉米冬孢子的特点是生长骤停, 细胞代谢减少, 细胞呼吸的水平低 3.在有利的环境条件下 (e. g., 特定糖的存在), U. 玉米冬孢子发芽和完全减数分裂, 产生 basidiospores, 可引发新一轮的感染。发芽的特点是增加的呼吸, 恢复代谢活动, 并通过可见的形态学阶段的发芽4的进展。
发芽的初始阶段包括增加的呼吸和新陈代谢功能, 但是, 没有形态学的变化迹象。最初测量的呼吸变化在U. 玉米是50年前进行的, 测量耗氧量 manometrically 与华宝瓶设备5。我们开发了一种新的, 简单的方法来研究在 teliospore 发芽过程中的呼吸的精确变化, 用克拉克型 microrespirometer 测量在发芽的时间过程中的耗氧量。我们以前使用这种方法来研究野生型U. 玉米单倍体细胞和突变体与缺陷线粒体6之间呼吸速率的变化, 并在这里修改了该协议来研究 teliospore 呼吸过程中的变化。萌发。这为准确确定呼吸变化的时间提供了一种方法, 以便我们能够在发芽开始后适当的时间针对冬孢子进行研究, 以调查早期的分子事件。一旦 promycelia 从 teliospore 出现, 就可以微观地观察萌发过程, 但异步性质抑制了在特定阶段对足够冬孢子的隔离, 以供调查。我们开发了一种类似于体外受精的显微切割技术, 用于在不同形态的萌发阶段物理地收集冬孢子。

<table><tbody><tr><td>Streptomycin Sulfate</td><td>BioShop</td><td>STP101</td><td></td></tr><tr><td>Kanamycin Sulfate</td><td>BioShop</td><td>KAN201</td><td></td></tr><tr><td>Potato Dextrose Broth</td><td>BD Difco</td><td>254920</td><td></td></tr><tr><td>1 L Waring Laboratory blender</td><td>Waring</td><td>7011S</td><td></td></tr><tr><td>Cheesecloth</td><td>VWR</td><td>470150-438</td><td></td></tr><tr><td>Nalgene Polypropylene Desiccator with Stopcock</td><td>ThermoFisher Scientific</td><td>5310-0250</td><td></td></tr><tr><td>&lt;em&gt;Unisense MicroRespiration system&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>MicroRespiration Sensor (O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Unisense</td><td>OX10</td><td></td></tr><tr><td>MicroOptode Meter Amplifier</td><td>Unisense</td><td>N/A</td><td></td></tr><tr><td>MR-Ch Small</td><td>Unisense</td><td>MR-Ch</td><td></td></tr><tr><td>SensorTrace Rate Software</td><td>Unisense</td><td>N/A</td><td></td></tr><tr><td>MicroRespiration Rack</td><td>Unisense</td><td>MR2-Rack</td><td></td></tr><tr><td>MicroRespiration Stirrer</td><td>Unisense</td><td>MR2-Co</td><td></td></tr><tr><td>&lt;em&gt;Microdissection system&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Axio Vert.A1 Inverted Light Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Coarse Manipulator</td><td>Narishige</td><td>MMN-1</td><td></td></tr><tr><td>Three-axis Hanging Joystick Oil Hydraulic Micromanipulator</td><td>Narishige</td><td>MMO-202ND</td><td></td></tr><tr><td>Pneumatic Microinjector</td><td>Narishige</td><td>IM-11-2</td><td></td></tr><tr><td>TransferTip (ES)</td><td>Eppendorf</td><td>5175107004</td><td></td></tr></tbody></table>

investigating teliospore germination using microrespiration analysis and microdissection

黑穗病真菌是几种破坏性农业病害的致病因子。它们的特点是冬孢子的生产, 这是厚壁分散剂。冬孢子可以保持休眠数十年。休眠的特点是低代谢率, 暂停大分子生物合成和大大降低呼吸水平。在接收到所需的环境信号后, 冬孢子发芽产生单倍体细胞, 这会引发新一轮的感染。Teliospore 萌发的特点是恢复大分子生物合成, 增加呼吸和戏剧性的形态学变化。为了精确测量发芽初期细胞呼吸的变化, 我们开发了一种使用克拉克型呼吸的简单协议。发芽的后期阶段是由特定的形态学变化来区分的, 但发芽是异步的。我们开发了一种显微切割技术, 使我们能够收集冬孢子在不同的发芽阶段。

利用克拉克型 microrespirometer 测量 teliospore 休眠和萌发过程中呼吸变化的方法, 我们证实休眠冬孢子与发芽相比具有低呼吸水平 (约1075µmol/小时/毫克)冬孢子 (2614 µmol/小时/毫克;图 1A)。这表示休眠冬孢子和冬孢子诱导发芽的平均呼吸速率有2.4 倍的变化。此外, 我们还发现, 诱导发芽的冬孢子在吸氧量上有45分钟的延迟 (图1B)。这是由30分钟的氧摄取延迟 (图 1B) 所示, 除了诱导发芽和氧气测量开始之间的 ~ 15 分钟的延迟。这确定了一个时间点开始评估的冬孢子发芽的分子变化, 没有明显变化。
在发芽过程中的后续变化可以显微镜观察。确定了发芽的五阶段。萌芽期的第一阶段代表冬孢子诱导发芽, 但仍与休眠冬孢子无法区分。第二阶段冬孢子有一个新兴的 promycelium, 长度小于或等于 teliospore 的直径。第三阶段冬孢子的 promycelia 大于 teliospore 直径。发芽阶段 IV 是 basidiospores 从 promycelia 的初始萌芽, 而 V 期是由芽分化而成的单倍体 basidiospores (图 2)。利用我们开发的显微切割技术, 我们成功地分离出500到1000发芽冬孢子, 用于下游应用, 如 rna 分离用于 RT PCR 或 rna 序列 (表1)。图 3显示了用于显微切割的培养皿的一般设置以及使用机器人进行显微切割的步骤。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57628/57628fig1.jpg"> 图 1: teliospore 发芽期间氧气消耗的时间过程.休眠冬孢子诱导发芽, 氧水平连续记录6小时使用克拉克型 microrespirometer。采用非诱导休眠冬孢子作为对照, 所有测量均归为空白样本。(A)数据表示为平均 OCR。(B)为获取呼吸曲线而绘制的原始数据, 允许在时间过程中检测 OCR 的变化。冬孢子诱导的发芽消耗氧的平均速率比未诱导的休眠冬孢子快2.4 倍( p < 0.01;学生的t测试)。猪: 发芽后诱导。<a href="/files/ftp_upload/57628/57628fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57628/57628fig2.jpg"> 图 2: teliospore 萌发的阶段.阶段 I 通过 V 的 teliospore 发芽被说明(A-E)。缩放条 = 20 µm.<a href="/files/ftp_upload/57628/57628fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57628/57628fig3.jpg"> 图 3: 显微解剖以分离出萌发冬孢子的不同形态阶段。介绍了一种用于显微切割的培养皿的一般设置, 以及在发芽的第三阶段分离 teliospore 的步骤。(A)图示一种培养皿, 其设置有一排含有无菌水、RNA 稳定溶液或发芽冬孢子的水滴。萌发诱导后, 冬孢子在萌发的特定阶段被分离出来, 采用显微解剖。(B)在 I. 期内诱导冬孢子发芽的萌发液滴。(C)已准备好的 microcapillary 被提交给第三阶段的 teliospore, 以便通过愿望收集。(D)将玻璃 microcapillary 中的第三阶段 teliospore 从发芽液滴中移除, 并移到收集液滴上。(E)将 microcapillary 插入包含 RNA 稳定溶液的收集液滴中, 并将 III. 级 teliospore 注入液滴中。(F)在 RNA 稳定化解决方案中第三阶段冬孢子的集合。刻度条 = 20 µm (A D, F) 和50µm (E)。<a href="/files/ftp_upload/57628/57628fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" table=""><tbody>
<tr>
<td>萌发阶段</td>
			<td>冬孢子萌发数</td>
		</tr>
<tr>
<td>第一阶段</td>
			<td>1000</td>
		</tr>
<tr>
<td>第二阶段</td>
			<td>500</td>
		</tr>
<tr>
<td>第三阶段</td>
			<td>650</td>
		</tr>
</tbody></table>表 1: 标准隔离实验中每个发芽阶段成功分离发芽冬孢子的数量。该表说明了在收集下游应用程序之前, 通过对 I 阶段 I 至 III 级的显微解剖分离出的冬孢子的平均数量。

Watch this Scientific Journal Video about Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection at JoVE.com

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

黑穗病真菌引起许多破坏性的农业病害。它们被分散为休眠冬孢子, 在响应环境提示时发芽。我们概述了两种方法来研究发芽过程中的分子变化: 测量呼吸增加, 以检测代谢活化和评估变化的分子事件通过隔离冬孢子在不同的形态学阶段。

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 ...

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JoVE Journal

Biology

7.2K 次观看.  Trent University. 黑穗病真菌引起许多破坏性的农业病害。它们被分散为休眠冬孢子, 在响应环境提示时发芽。我们概述了两种方法来研究发芽过程中的分子变化: 测量呼吸增加, 以检测代谢活化和评估变化的分子事件通过隔离冬孢子在不同的形态学阶段。

视频: Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

剖析酿酒酵母 ASCI

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

科研

生物学

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and genetics because of its simple life cycle, ease of growth and manipulation for genetic analysis, genomic resources, and transformability of the nucleus and both organelles. Mating strains is a common practice when genetic approaches are used in Chlamydomonass, to create vegetative diploids for analysis of dominance, or following tetrad dissection to ascertain nuclear vs. organellar inheritance, to test allelism, to analyze epistasy, or to generate populations for the purpose of map-based cloning. Additionally, genetic crosses are routinely used to combine organellar genotypes with particular nuclear genotypes. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad separation. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas. Key parameters and trouble spots are explained. Finally, resources for further information and alternative methods are provided.

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.

交配和四分体分离衣藻遗传分析

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Detecting Cortex Fragments During Bacterial Spore Germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

检测皮质片段在细菌孢子萌发

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

免疫与感染

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

微流控装置在极小空间中研究尖端生长植物细胞的伸长能力

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

生物工程

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

枯草芽孢杆菌中细菌体和内膜的可视化

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

从长期来看，高解析度激光共聚焦时间流逝成像<em&gt;拟南芥子叶</em&gt;表皮萌发过程中

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The establishment of an auxin gradient is a key event leading to phototropism and gravitropism. Previously, polar auxin transport (PAT) was shown to establish an auxin gradient in different cellular domains of plants. However, Han et al. recently demonstrated that for proper auxin gradient formation, plasmodesmal callose-mediated symplasmic connectivity between the adjacent cells is also a critical factor. In this manuscript, the strategy to elucidate the role of particular genes, which can affect phototropism and gravitropism by altering the symplasmic connectivity through modulating plasmodesmal callose synthesis, is discussed. The first step is to screen aberrant tropic responses from 3-day-old etiolated seedlings of mutants or over-expression lines of a gene along with the wild type. This preliminary screening can lead to the identification of a range of genes functioning in PAT or controlling symplasmic connectivity. The second screening involves the sorting of candidates that show altered tropic responses by affecting symplasmic connectivity. To address such candidates, the movement of a symplasmic tracer and the deposition of plasmodesmal callose were examined. This strategy would be useful to explore new candidate genes that can regulate symplasmic connectivity directly or indirectly during tropic responses and other developmental processes.

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

的策略，以验证胼胝质介导的Plasmodesmal浇注在热带反应中的作用

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

用于解释霉菌异养植物组织中真菌定植和种子共生萌发的显微镜技术

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...