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>

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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,1...

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><tbody><tr><td>Confocal laser scanning microscope (CLSM)</td><td>Zeiss</td><td>LSM5</td><td>Any CLSM with similar capabilities is appropriate</td></tr><tr><td>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><td>Quickchange II site-directed mutagenesis kit&amp;nbsp;</td><td>Agilent</td><td>200523</td><td></td></tr><tr><td>Acetosyringone</td><td>Sigma-Aldrich</td><td>D134406</td><td></td></tr><tr><td>MES</td><td>Sigma-Aldrich</td><td>69892</td><td></td></tr><tr><td>Syringes without needles</td><td>BD</td><td>309659</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>FisherScientific</td><td>M33-500</td><td></td></tr><tr><td>Spectinomycin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S4014</td><td></td></tr><tr><td>Rifampicin</td><td>Sigma-Aldrich</td><td>R3501</td><td></td></tr><tr><td>Ampicillin&amp;nbsp;</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</...

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

Research

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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

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

使用吉布森大会和基因枪烟草中瞬时基因表达

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

科研

环境科学

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

一种高效的植物多嵌合体荧光融合蛋白的协同表达

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

化学

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

生物学

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

发育生物学

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

原位杂交技术在植物中的精确定位成绩单

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

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 ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

分离绿色荧光蛋白系统可视化细菌在感染过程中传递的效应

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

外周相关蛋白血浆膜分型的测定

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

<meta charset="utf8"></head><body>探索胞间连丝功能：靶向蛋白的共聚焦显微镜检查

Confocal Microscopy Analysis of Protein Sorting to Plasmodesmata in Nicotiana benthamiana

Plasmodesmata are membranous nanopores that connect the cytoplasm of adjacent plant cells and enable the cell-to-cell trafficking of nutrients, macromolecules, as well as invading viruses. Plasmodesmata play fundamental roles in the regulation of intercellular communication, contributing to plant development, environmental responses, and interactions with viral pathogens. Discovering plasmodesmal localization of plant or viral proteins could provide useful functional information about the protein and is important for understanding the mechanisms of plant-virus interactions. To facilitate these studies, we describe a protocol for confocal microscopy-based analysis of different plasmodesmal targeting proteins to select the best plasmodesmal marker for studying the virus-plasmodesmata interactions or plasmodesmal transport. Specifically, the analyses of these events are illustrated using the cell-to-cell movement protein (MP) of the Turnip vein-clearing virus (TVCV), the Arabidopsis Plasmodesmata-Localized Protein 5 (PDLP5) and Plasmodesmata Callose-Binding Protein 1 (PDCB1). The protein plasmodesmal localization data are analyzed in parallel with the global visualization of plasmodesmata using aniline blue staining of the sampled tissues. These approaches can be easily adapted to analyze the plasmodesmal localization of any cellular or pathogen proteins in planta.

<meta charset="utf8"></head><body>作者聚焦：植物胞间连丝蛋白质定位的显微分析

This protocol describes the selection of optimal plasmodesmal markers for confocal microscopy-based analyses of protein targeting to plasmodesmata during virus-plasmodesmata interactions or plasmodesmal transport.