由于篇幅不足, 我们主要引用了评论文章, 我们向我们的同事道歉, 他们的原创作品没有被引用。共实验室的工作得到 nih、NSF、美国农业部/NIFA、巴德和 BSF 共的资助, S.G.L. 实验室由国立卫生研究院和植物病理学和植物微生物生物学部门资助, S.G.L。

1. 植物材料
<ol><li>植物种类的选择</li>
	<li>使用的植物物种的利益蛋白,即, 其中一个编码这种蛋白质的内生蛋白或代表自然宿主的病原体的病毒蛋白。此外, 选定的植物种类必须服从选择的瞬态遗传转化方法。 注意: 这些研究通常使用烟草烟, 它代表了 TMV 的良好宿主, 并通过农杆菌介导的遗传转化技术 (即, agroinfiltration (参见步骤 3) 有效地转换。N. 烟植物也很容易生长并且有大叶子, 很容易被农杆菌接种并且通过共聚焦显微镜很容易分析。</li>
	<li>
植物生长
	<ol>
<li>在高密度的潮湿土壤中种植烟种子。让他们在一个受控环境室在20-25 ˚C 在 16 h 的光下在 ~ 75 µmol 光子 m-2 s-1和 8 h 黑暗。在 euphyll 直径达到0.5 厘米后, 小心地将幼苗转移到较大的盆中, 并在相同的条件下继续生长在同一个室内。</li>
		<li>在4周内, 当最大的叶子直径为4-6 厘米时, 保持植株生长到4-8 叶阶段;此时, 它们可用于 agroinfiltration (参见步骤 3)。 注意: 重要的是, 永远不要使用植物, 如果他们开始开花, 因为, 在这个发展阶段, 叶子建筑学经常变化24,25, 有时导致不一致的结果。</li>
	</ol>
</li>
</ol>2. 表达载体结构
<ol><li>
表达系统的选择
	<ol>
<li>选择表达系统,即, 向量和向量传递方法, 最适合在研究的植物物种中表达感兴趣的蛋白质。 注意: 有时, 这种特定的蛋白质/植物物种组合可能需要特定的载体和载体传递方法, 以获得最理想的蛋白质表达和功能。然而, 对于大多数双子叶植物植物, 选择二元质粒作为表达载体和 agroinfiltration 作为传递系统。</li>
	</ol>
</li>
	<li>
蛋白质荧光标记
	<ol>
<li>荧光标记每个表达的蛋白质用于分析其亚细胞分布22, 包括 Pd 定位。 注: 使用 autofluorescent 蛋白质, 如 CFP 和 DsRed2, 作为标签。标记被测试的蛋白质与 CFP 并且 coexpress 它与一个自由 DsRed2。CFP 功能作为标签和作为一个病毒无关的货物分子。DsRed2 函数来校准瞬态表达式的整体效率, 并因为它是细胞自主的, 来识别最初转化的细胞;这后一种功能是重要的评估细胞运动的潜在 non-cell 自主测试蛋白质。对于共与 Pd 标记 PDCB1, 这也是细胞自主, 标签的测试蛋白与 CFP 和 PDCB1 与 DsRed2。</li>
		<li>作为一个经验法则, 将 autofluorescent 标记融合到表达蛋白质的羧基末端。然而, 如果被标记的全长测试的蛋白质暴露了 Pd 靶向, 转移标记到蛋白质的氨基末端。</li>
		<li>克隆编码序列的蛋白质被测试成一个合适的植物表达载体。 注意: 有许多不同的植物表达载体, 允许荧光标记。我们使用一系列的 pSAT 质粒26或他们的一些衍生物27,28, 这是适合克隆的利益序列直接在帧与建议的 autofluorescent 标签 (见步骤 2.2.1) 在两个氨基和羧基末端方向。</li>
		<li>将每个表达式卡带转换为农杆菌二元向量。 注: 虽然 pSAT-based 构造适合直接枪法送到植物组织, agroinfiltration, 这是推荐的转换方法 (参见步骤 3), 要求表达式卡带位于 T-脱氧核糖核酸之间二进制向量29的边框。 注意: 所有 pSAT 向量都是为了允许这样的传输到 pPZP-RCS2 基因表达式二进制向量26,30由一步克隆使用罕见的切割核酸 AscI, 我-PpoI, i-SceI, 我-CeuI, pi 胶和 pi TliI26. 喜欢利用 recombinatorial 克隆的研究人员,例如, 使用外源液氧站点31, 可以采用 pSAT 矢量变体26,27。</li>
		<li>对于共, 传输两个表达式磁带,例如, 测试蛋白质-CFP 和 DsRed2 或被测试的蛋白质-CFP 和 PDCB1-DsRed2 (参见步骤 2.1), 对相同的 pPZP-RCS2 二进制向量。 注: 另一种方法是, 将单个二进制结构窝藏的农杆菌培养物混合在一起, 渗透到烟中 (参见步骤 3.1)。</li>
	</ol>
</li>
</ol>3. Agroinfiltration
<ol><li>
添加1µg 的 pPZP-RCS2-based 质粒从步2.2.5 到100µL 培养的农杆菌介导细胞菌株 EHA105 或 GV3101 准备的32, 在冰上孵育30分钟, 在液氮中闪光冷冻1分钟, 并孵育在28° c 为15分钟。
	<ol>
<li>然后, 添加1毫升的 LB 培养基 (10 克/升胰, 5 克/升酵母提取物, 10 克/升 NaCl) 到主管的细胞混合物和孵化在28° c 为2小时. 在3000分钟内将细胞旋转下来 1 x g, re-suspend 他们在0.2 毫升的 LB, 并将它们放在 LB 琼脂上, 辅以适当的抗生素 (例如, 100 毫克/升霉和50毫克/升利福平为 pPZP-RSC2-based 向量26,30)。生长在28° c 为 48 h, 直到各自的殖民地是可看见的。</li>
	</ol>
</li>
	<li>挑选和板几个单独的殖民地到一个新鲜的 LB 琼脂板 (见步骤 3.1), 生长在28° c 为48小时, 并采取少量的细菌进行分析的菌落 PCR33 , 以确认特定的二进制结构的存在。</li>
	<li>
在28° c 的5毫升培养基中, 用适当的抗生素补充一夜之间的利益结构, 增加每个农杆菌的菌落 (参见步骤 3.1.1)。离心的细胞在 3000 x g, 并 re-suspend 他们的 OD600= 0.5 在 agroinfiltration 缓冲区 (10 mm 氯化镁2, 10 mm MES (pH 5.6), 150 µM 丁香)。室温下孵育2小时。
	<ol>
<li>如果使用混合的二元质粒, 而不是一个单一的基因表达载体的蛋白质共, 混合相应的细胞培养在 1:1 v/v 比前渗透。然后在28° c 下孵育2小时的细胞。</li>
	</ol>
</li>
	<li>
将接种装入一个1毫升的塑料注射器中, 然后按下注射器 (无针) 的喷嘴, 对完全成熟的N. 烟叶的下部 (背面) 表皮进行按压。用戴着手套的手指在正面的脸上按住叶子34,35。
	<ol>
<li>采用慢注射法在渗透介质中引入农杆菌。为了统计意义, 每株植物都要渗透几片叶子。 注意: 请注意, 不使用最旧的叶, 因为它们不产生一致的表达式级别。</li>
		<li>接种所有的叶子原位。保持被渗透的植物直到观察如步骤1.2 所述。</li>
	</ol>
</li>
</ol>4. 共焦显微镜
<ol><li>用刀片将每片叶子切割成 24-48 h 之间的静脉浸润, (取决于特定的检测蛋白的瞬态表达效率);每个组织切片的大小应该是这样的, 切片是完全覆盖的盖子玻璃 (22 毫米 x 40 毫米) 用于显微镜。</li>
	<li>将叶片放在对象幻灯片上, 然后朝上, 将一滴水放在叶片上, 用盖玻璃覆盖。将盖玻璃固定在每边的小块胶带上。</li>
	<li>
在激光扫描共聚焦显微镜下观察 agroinfiltrated 组织, 记录产生的图像。
	<ol>
<li>首先, 使用10X 物镜来识别带有信号的细胞, 然后使用63x 物镜进行更详细的观察。</li>
		<li>使用氩离子激光器的 458 nm 波长激发 CFP, 543 nm 激发 DsRed2。保留图像采集的所有设置,即, 激光强度和光电倍增管 (PMT) 设置, 在实验之间。平均, 检查每一个实验条件的100-120 细胞。</li>
	</ol>
</li>
</ol>5. PLS 的识别
<ol><li>用共焦显微镜诊断检测蛋白的定位 (4 节)。检查测试的蛋白质是否有特征的外围点状图案25、36、37、38、39、40、41和 colocalizes使用 Pd 标记 PDCB123。这表明, 被测试的蛋白质最有可能包含 PLS 序列。</li>
	<li>经过测试的蛋白质 PLS 活性的确认, 逐步细分其开放阅读框架 (基因库) (BAF93925.1) 到更小的片段, autofluorescently, (例如, CFP,) 标记每一个, 并分析其如步骤4所述的亚细胞定位。最初, 建议每一个细分片段的100氨基酸残基的大小。</li>
	<li>进一步细分有 PLS 活动的被截断的片断和检查他们的亚细胞定位如描述在步4直到最小的片段仍然本地化到 Pd,即是足够运输 autofluorescent 标记货物到 Pd, 被确定。这个片断被推测代表 PLS 序列。</li>
	<li>从全长检测的蛋白质中删除假定 pls,即将测试蛋白的剩余部分与未假定的 autofluorescent 标记相融合, 并确定所产生的突变蛋白的亚细胞定位, 如步骤所述4. 如果这种突变蛋白缺乏假定的 pls 没有定位到 pd, 这将表明, 识别 pls 不仅是足够的 (见步骤 5.3), 但也有必要的 pd 运输的测试蛋白质。</li>
	<li>Agroinfiltrate 的叶子与农杆菌文化的混合物窝藏表达结构的 CFP 标签 PLS 和自由 DsRed2 (见步骤 3.3)。使用与步骤4.3 相同的设置, 使用10x 物镜的共聚焦显微镜观察浸润组织。</li>
	<li>将含有 CFP 和 DsRed2 信号的细胞分入最初渗透的细胞, 并将仅包含 CFP 信号的相邻细胞得分为表现运动的单元。 注: 此步骤检查已识别的 PLS 的潜在细胞运动能力;这是因为许多靶向 pd (包括大多数植物病毒 MPs) 的蛋白质也通过 pd 转移到相邻的细胞。土壤中的农杆菌浓度分别取决于不同结构的表达效率。对于 agroinfiltration, 确保使用细胞培养稀释, 确保 (i) 单细胞的转化, 使相邻的细胞未, (ii) 达到相似的表达效率。例如, 我们的实验使用 OD600的0.01 和0.005 分别表达 CFP 标记的 PLS 和自由 DsRed2。</li>
</ol>6. 使用丙氨酸扫描的关键 PLS 残留物的识别42
<ol><li>使用标准 PCR 协议放大 PLS 编码序列采用一对引物设计, 以包含所需的突变,即, 取代的目标氨基酸残留物与丙氨酸, 周围的中心区域的每一个底漆的描述21. 根据制造商的说明, 进行底漆设计并使用定点诱变试剂盒进行定点诱变。</li>
	<li>使用任何标准的 dna 纯化试剂盒纯化所产生的 PCR 产物, 并在37° c 的 DpnI 中将其消化, 以消除双亲 (即、non-mutated) 甲基化和 hemimethylated dna。在室温下冷却5分钟 (不在冰上)。然后将混合物直接转化为大肠杆菌主管细胞43, 并按照步骤3.2 中所述, 识别具有所需突变结构的菌落。</li>
	<li>如步骤2.2.4 所述, 将突变体构造引入二进制向量, 执行步骤3所述的 agroinfiltration, 并按照步骤4和5.1 所述评估 Pd 目标。</li>
</ol>

未声明任何利益冲突。

该协议有四核心成分: 确定一个序列的概念是必要的和足够的目标, 以 Pd, 系统划分的利益蛋白的片段, 逐步减少长度, 融合测试片段的 autofluorescent 蛋白, 既作为标签和高分子货物, 和功能测试的 Pd 靶向活植物组织后, 瞬态表达的检测融合蛋白。请注意, 农杆菌介导的瞬态表达式在相对较短的时间内生成数据,即, 与在类似研究中经常使用的转基因植物生产所需的月份相比, 是数天,15。
共的蛋白质的兴趣与亚细胞定位标记,例如, PDCB1 或自由 DsRed2, 通常是从基因表达载体执行。但是, 如果将多个表达式磁带传输到单个向量中是技术上的问题, 则共也可以通过将每个磁带传输到单独的二进制结构中, 并将等效的液体培养量结果农杆菌菌株为 agroinfiltration。由于该协议利用瞬态表达式, 二进制向量不需要进行稳定变换的选择标记, 尽管它们的存在不利于表达效率。
该协议针对烟中的蛋白质表达和目标进行了优化;然而, 它可以用于其他植物物种, 包括拟南芥45,46, 适于遗传转化的农杆菌。对于其他植物物种, 或如果其他需要, 此协议可适用于利用枪法传递41直接从 pSAT-based 向量的瞬态表达式, 避免了将表达式磁带传输到二进制向量的需要。
该协议的关键点是植物材料的生理和发育条件的一致性。具体地说, 所有的植物在整个实验中都必须是健康的, 而且, 对于 agroinfiltration, N. 烟植物必须小于4周, 并且在4-8 叶级, 最大的叶子直径为4-6 厘米。如果植株太小, agroinfiltration 的影响会太严重, 干扰标记蛋白的表达和定位。另一方面, 较老的植物通常表现出 pd24,25的可变结构, 也会影响 pd 目标模式的一致性。
尽管如此, 由于整个植物的生理条件的不同, 特别是其转化的细胞, 每个实验中 Pd 靶向的效率可能有所不同。因此, 重要的是要分析一个相对较大数量的细胞 (> 100) 每个实验, 并执行至少三生物复制每一个实验。通常, 我们认为一个测试片段, 以包含 pd 的目标活动, 如果它在至少70% 的转化细胞展示 pd 定位。每个片段的 pd 靶向效率的差异,例如, 显示 pd 特异的点状堆积模式的转化细胞百分比, 应由学生的 t 检验和 p 值 < 0.05-0. 01 对应于统计概率大于95-99%。
另一个重要的问题是荧光标记/货物在聚变分子中的位置。一些 Pd 靶向蛋白质也与内质网 (ER), 这可能需要无障碍的氨基末端序列。在这些情况下, 氨基末端融合, 蛋白质片段的羧基终点被熔化到荧光标记的氨基末端,例如, CFP 或 DsRed2, 应该使用。在以下情况下, 可以更改标记的此默认位置。(i) 如果氨基终端融合不可用--即, 它们不表现出有意义的亚细胞定位模式, 表达不充分, 或不产生足够可靠检测的荧光信号--如果感兴趣的蛋白质不包含氨基终端信号序列或羧基终端融合, 其中的氨基终点的测试片段被融合到的羧基终点的标签, 他们应该尝试。(ii) 如果感兴趣的蛋白质在其羧基末端含有信号序列, 则也应使用羧基端融合。(iii) 如果感兴趣的蛋白质具有氨基酸和羧基终端信号, 如在 GPI 锚定蛋白中发现的蛋白, 或者它含有氨基终端信号肽, 但其氨基终端融合不可用, 则应在内部放置标签 (无论是下游的氨基终端信号或上游的羧基终端信号)。我们的早期出版物47,48中描述了一些用于蛋白质内部荧光标记的有用程序。
目前, 还没有已知的协商一致的顺序。正如我们建议的那样 (在步骤5.2 中), 最初将蛋白质细分为连续的~100-200 残滓-长片段。这些可以根据观察到的 Pd 靶向活动或缺乏的情况逐步缩短。人们应该注意到在蛋白质中存在的其他潜在的靶向信号 (例如, 无源性, 信号肽,等)。然而, 由于这些序列与 PLS 序列的潜在重叠是未知的, 这些蛋白质区域应该包括在这个突变分析。
到目前为止, 这个协议被用来识别一个单 PLS 在蛋白质分子21。然而, 一个蛋白质可能包含不止一个信号序列;事实上, 许多核蛋白中已经描述了多个 NLSs, 包括 GATA 转录因子49。因此, 在分析被测蛋白质的不同片段用于 Pd 靶点时, 可能会发现不止一个这样的片段, 表明存在多个 PLSs, 它们可以独立或协同作用。
该协议的主要限制是, 在 agroinfiltration 后, 在表皮细胞和相对狭窄的生长阶段, 对瞬时表达的蛋白质的亚单位定位的可视化效果最好。在必要的时候, 可以通过生产转基因植物, 在大多数组织和细胞类型中稳定地表达每个被测试的蛋白质来克服这种限制。此外, 这项协议将不会有用的研究病毒 MPs 或其他 Pd 靶向蛋白质的功能只在植物物种, 不适合瞬态遗传转化。
虽然我们开发了这一技术, 以识别 TMV MP, 它可以用于发现 PLSs 在任何其他植物病毒 MPs 或植物内源性蛋白, 本地化为 Pd, 瞬时或永久性的。此外, 如果结合适当的亚细胞定位标记47, 此协议适用于在植物的感兴趣的蛋白质内的许多其他靶向信号的识别。

胞 (Pd) 作为植物发育和形态发生的关键调控器的细胞间转运的导管, 从转录因子到 mRNA 和小 RNA 分子不等。此外, 大多数植物病毒利用 Pd 的大分子转运能力在感染过程中进行细胞间传播;通过 pd, 植物病毒进化出专门的蛋白质, 称为运动蛋白 (MPs), 专门针对 pd1,2,3,45,7. Pd 转运的分子通路最有可能是与特定的序列紧密相连的, 靶向转移的蛋白质进入这些通路。因此, 识别这些 pd 定位信号 (PLSs) 可能是对相应的 pd 转运通路的诊断。这是通过类比的 Pd 传输8, 例如, 不同的核进口通路, 可以具体为不同的核定位信号 (不规则) 序列9,10。从概念上讲, NLSs 和 PLSs 都表示 non-cleavable 的亚细胞靶序列是必要和足够的目标。但是, 与 NLSs11不同的是, 有关 PLSs 的序列信息受到严重限制。具体来说, 只有四蛋白序列涉及 Pd 靶向已经报告, 所有这些都来源于内源植物蛋白。第一个是由 KN112的同源域表示的, 这是一个转录因子, 从内部细胞层到植物叶的表皮,13 , 以及它的诺克斯同系物14。第二个也是从转录因子, 自由度, 其中包含一个假定 PLS 描述为细胞间贩运 (IT) 主题15。第三个序列是从 PDLP1 胞的1型膜蛋白, 它由跨膜域16表示。最后, 第四 Pd 靶序列最近报告了 glycosylphosphatidylinositol (GPI) 锚定蛋白, 它由 glycosylphosphatidylinositol (GPI) 修饰信号17表示。
有趣的是, 直到最近, 没有 PLSs 的病毒的 MPs 报告。先前的研究表明, 假设 pls 序列在植物病毒 MPs18,19, 但没有真正 pls,即, 一个极小的氨基酸序列必要和充足为 Pd 靶向不相关的货物分子 (e. g, CFP) 已在病毒 MP 中被识别。然而, 其中的一种蛋白质, 即烟草花叶病毒的 MP (TMV), 是 Pd 局部化和传输被证明的第一个20。
为了解决这一差距, 我们制定了一个实验策略, 以确定 TMV MP PLS。该策略基于三概念。(i) 我们定义 PLS 作为一个极小的氨基酸序列是必要和充足的蛋白质靶向 Pd21。(ii) 由于 TMV MP 首先瞄准 pd, 然后通过这些通道22translocates, 我们的目的是拆下这两种活动并确定真正的 PLS, 它只对 pd 目标起作用, 而不适用于随后的传输。(iii) 我们分析了在结构上或功能上对其 Pd 靶向活性有重要意义的氨基酸残留物。利用这种方法, 我们在 TMV MP 的氨基末端划定了一个 50-氨基氨基酸残留序列, 作为善意的 PLS。这是通过产生一系列的 TMV MP 片段, 饱和整个蛋白质的长度, 标记他们的羧基总站与 CFP 和瞬时表达他们在植物组织。每个测试片段的 pd 定位是通过 coexpressing 他们与 pd 标记蛋白, PDCB1 (pd 胼胝结合蛋白 1)23。最小的片段, 仍然本地化到 pd, 但没有遍历 pd, 被认为是代表 PLS。最后, PLS 是丙氨酸扫描, 以确定其结构和/或功能所需的关键氨基酸残留物。
在这里, 我们通过描述 TMV MP 的识别来说明这种方法, 它可能被用来发现 PLSs 在任何其他 Pd 靶蛋白, 无论是由植物病原体或植物本身编码;这是因为我们的方法没有利用病毒 MPs 的任何独特的特点, 关于他们的能力目标 Pd。

<table border="0" cellpadding="0" cellspacing="0" width="1086">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:293px;">Confocal laser scanning microscope (CLSM)</td>
			<td style="width:176px;">Zeiss</td>
			<td style="width:268px;">LSM5</td>
			<td style="width:349px;">Any CLSM with similar capabilities is appropriate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zen software for confocal microscope imaging</td>
			<td>Zeiss</td>
			<td>2009 version</td>
			<td>The software should be compatible with the CLSM used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Quickchange II site-directed mutagenesis kit&#160;</td>
			<td>Agilent</td>
			<td>200523</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetosyringone</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td>D134406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MES</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td>69892</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringes without needles</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgCl2</td>
			<td>FisherScientific</td>
			<td>M33-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spectinomycin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S4014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rifampicin</td>
			<td>Sigma-Aldrich</td>
			<td>R3501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ampicillin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A0166</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of plasmodesmal localization sequences in proteins in planta

胞 (Pd) 是细胞连接, 它的作用是通过在植物细胞之间传输小分子和大分子的通路。而 Pd 运输的小分子, 如离子和水, 被推定为被动发生, 细胞运输的生物大分子, 这种蛋白质, 最有可能发生通过一个积极的机制, 涉及特定的靶向信号的运输分子。识别的胞 (Pd) 定位信号 (PLSs) 的稀缺性严重限制了对植物细胞大分子运输和通讯所涉及的蛋白质分选途径的理解。从大量的植物内源性和病毒蛋白已知的交通通过 Pd, 迄今只有三 PLSs 报告, 所有这些来自内源植物蛋白。因此, 重要的是要建立一个可靠的, 系统的实验策略, 以确定一个功能 PLS 序列, 这是必要的和足够的 Pd 靶向, 直接在活植物细胞。在这里, 我们描述一个这样的战略使用作为一个范例的细胞运动蛋白 (MP) 的烟草马赛克病毒(TMV)。这些实验, 确定和特点的第一植物病毒 pls, 可以适应发现 pls 序列在多数 Pd 靶向蛋白质。

代表数据, 忠实地说明预期的结果从被描述的协议和辨认 TMV MP PLS, 适应从元et al.21.图 1A首先总结了表达全长 TMV mp (1-268) 的主要构造, TMV mp PLS (包括蛋白质的前50氨基酸残滓, 1-50) 和它的丙氨酸扫描 V4A 衍生物熔化了 CFP (生成作为在步骤2.2、5.2 和6中描述), 而图 1B对这些标记蛋白质的亚细胞定位进行了总结和量化。图 1C说明了为 TMV mp-CFP 和 TMV mp PLS CFP 以及 coexpressed pd 标记 PDCB1-DsRed2 (参见步骤 5.1) 观察到的特征性外周点 Pd 定位模式。图 2A说明了 TMV mp-CFP 和 TMV mp CFP 和 nucleocytoplasmic coexpressed 自由 DeRed2 分布的 Pd 目标 (参见步骤 5.1)。最后,图 2B说明了 TMV mp 和 TMV mp pls 与单丙氨酸替代 V4A 的 Pd 靶向损失, 表明该残留物对 pls 结构或功能很重要;在同一个细胞 coexpressed pd 标记 PDCB1 仍然本地化对 pd (参见步骤 6)。
这些数据表明, 这一概念和技术上简单的过程, 系统生产的蛋白质片段, 他们的融合到一个 autofluorescent 标记货物的蛋白质, 并测试的能力, 本地化到 Pd 可以确定一个最低蛋白质序列必要和足够的 Pd 目标,即, PLS。为了正确解释这些本地化数据, 必须使用已知的 pd 蛋白进行定位实验,例如、PDLP 或 PDCB 家族成员16、23或一种对 pd44 进行排序的植物病毒 MPs。.显然, 定位不必须是完整的, 因为并非所有的 P 定位蛋白可能是针对相同的 pd, 但有统计学意义的定位与至少一个 pd 标记蛋白是必要的定义 PLS 活动。
<img alt="Figure 1" src="/files/ftp_upload/55301/55301fig1.jpg"> 图 1: TMV MP 的标识请(A) 用于识别 TMV mp 的主要 CFP 融合结构的摘要 TMV mp 序列, 绿色方框;CFP, 蓝色盒子;P, 启动子;T, 终结者左边的数字表示每个 TMV MP 片段中所含的氨基酸残留。(B) 综述了 (A) 中所示的融合蛋白的亚细胞定位。根据相应的亚细胞标记、PDCB1-DsRed2 和自由 DsRed2 的定位, 分别确定局部放电定位或 nucleocytoplasmic 定位。在三独立的实验中, 每一个结构的100个表达单元显示出每个定位模式的细胞百分比。数据是指±标准错误 (SE)。(C) pd 靶向 TMV mp-CFP, TMV mp PLS CFP, 和 pd 标记 PDCB1-DsRed2。CFP 信号是蓝色的;DsRed2 信号呈紫色;质的自发荧光被过滤掉了。图像是单个共焦剖面。缩放条形图 = 10 µm, 差分干涉对比显微图像。这个数字是改编自元et al.21.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55301/55301fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" src="/files/ftp_upload/55301/55301fig2.jpg"> 图 2: TMV mp、TMV mp 和他们的丙氨酸扫描突变体的 Pd 靶向性.(A) TMV mp-CFP、TMV mp CFP 和 nucleocytoplasmic 标记 DsRed2 的亚细胞定位。(B) TMV mp-CFP 和 TMV mp-CFP V4A 丙氨酸扫描突变体的 Pd 靶向能力损失。pd 标记的共 PDCB1-DsRed2。CFP 信号是蓝色的;DsRed2 信号呈紫色;质的自发荧光被过滤掉了。图像是单个共焦剖面。缩放条形图 = 10 µm, 差分干涉对比显微图像。这个数字是改编自元et al.21.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55301/55301fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Identification of Plasmodesmal Localization Sequences in Proteins In Planta at JoVE.com

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

植物细胞间连接, 胞 (Pd), 在植物生理学和植物-病毒相互作用中扮演中心角色。对 pd 传输的关键是分拣信号, 直接蛋白质的 pd。然而, 我们对这些序列的了解还处于起步阶段。我们描述了一种在 pd 靶蛋白中识别 pd 定位信号的策略。

蛋白质中 Plasmodesmal 定位序列的识别在底中

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

identification-plasmodesmal-localization-sequences-proteins

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

8.2K 次观看.  State University of New York. 植物细胞间连接, 胞 (Pd), 在植物生理学和植物-病毒相互作用中扮演中心角色。对 pd 传输的关键是分拣信号, 直接蛋白质的 pd。然而, 我们对这些序列的了解还处于起步阶段。我们描述了一种在 pd 靶蛋白中识别 pd 定位信号的策略。

视频: Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

科研

免疫与感染

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

可视登革病毒通过的Alexa Fluor标签

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms&#160;and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5&#39; or 3&#39; ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

We describe here a simple fluorescence in situ hybridization (FISH) method for the localization of viruses and bacteria in insect and plant tissues. This protocol can be extended for the visualization of mRNA in whole mount and microscopic sections.

荧光<em&gt;原位</em&gt;杂交（FISH）技术对病毒和植物和昆虫组织内共生细菌的本土化

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

利用荧光蛋白可视化和定量<em&gt;衣原体</em&gt;液泡生长动态的活细胞

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

病毒 DNA 的纯化及相关病毒和细胞蛋白的鉴定

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16&#8211;24 h), and higher incubation temperature (37 &#176;C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10&#8201;min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.