Name: split green fluorescent protein system to visualize effectors delivered from bacteria during infection
Uploaded: 2018-05-24T14:30:00.000+00:00
Duration: 7 min 25 s
Description: Watch this Scientific Journal Video about Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection at JoVE.com

这项研究是通过由科学、信息和通信技术和未来规划部 (NRF-2018R1A2A1A05019892) 资助的韩国国家研究基金会 (NRF) 和植物分子育种中心的赠款来支持的。PMBC) 下一代 Biogreen 21 农村发展管理计划 (PJ013201) 向 EP。我们感谢国家环境管理仪器中心的成像中心, 为拍摄提供了共焦显微镜。

注: 所有步骤均在室温下执行, 除非另有说明。
1. 植物材料的制备 (4 周)
<ol><li>为benthamiana工厂准备<ol>
<li>在每个花盆的土面上播种benthamiana的2种子, 用塑料圆顶覆盖托盘, 并允许种子在25摄氏度、60% 湿度生长室中发芽, 其温度为16/8 小时/暗光周期。</li>
		<li>两周后, 挑选出并丢弃每壶中最小的幼苗, 并继续种植在相同的生长条件下, 在1.1.1 发芽的应用。每两天增加1升的水每托盘。 注意: 植物的生长条件可能在实验室中有所不同。因此, 遵循常规浇水协议, 种植植物在一个健康的条件。</li>
		<li>在一周内, 将植物转移到一个新的托盘, 并安排他们有足够的空间, 以进一步增长。在步骤1.1.1 中描述的条件下, 保持植物的生长, 直到它们准备在4周的年龄内渗入。 注: 植物生长可能会因不同实验室的生长情况而异。通常, 我们发现, 4 周大的benthamiana植物大约有六叶。</li>
	</ol>
</li>
	<li>对南芥转基因植物的制备<ol>
<li>请参阅材料表并订购转基因的拟南芥种子。</li>
		<li>在1毫升蒸馏水中浸泡 ~ 50-100 转基因的拟南芥种子, 并将它们贮存在4摄氏度, 在黑暗中保存3天, 以同步发芽的开始。</li>
		<li>在插头厂托盘的土壤表面播种 2-3 种子, 用塑料圆顶盖上托盘。允许种子发芽23摄氏度, 60% 湿度, 10/14 小时光/暗周期。 注: 种子应纯合体。然而, 我们建议重申的存在, 转基因在批。在这种情况下, 用70% 乙醇洗涤种子, 2 分钟, 50% 漂白剂 (约2% 次氯酸盐), 含 0.05% X-100 的5分钟. 跟随通过洗涤 5-6 次以无菌双重蒸馏水 (ddH2O)。灭菌后, 分层在4摄氏度3天, 并将其印在植物萌发培养基上, 含潮霉素 B 的25µg/升, 以选择转基因植物。</li>
		<li>一周后, 每个插头只留一株植物, 并继续生长在相同的生长条件下, 用于步1.2.3 的植物。 注: 四周大的植物用于野火感染。水植物每隔一天保持植物的健康。</li>
	</ol>
</li>
</ol>2. 准备假单胞菌文化 (~ 1 周)
<ol><li>铜绿假单胞菌转化质粒的构建<ol>
<li>请参阅材料表, 并订购 T3SS-based 效应传递系统向量25所需的向量。</li>
		<li>使用站点特定的重组克隆25将感兴趣的效应基因插入效应传递向量中。 注: 在监测全长效应蛋白的亚细胞定位时, 将全长基因 pBK-GW-1-2 或 pBG-GW-1-2。还可以选择含有 2x sfGFP11 的 pBK-GW-1-4 或 pBG-GW-1-4, 以增加荧光信号。对于缺乏信号肽的部分效应器, 使用 pBK-GW-2-2 或 pBK-GW-2-4。有关效应传递向量25的详细信息, 请参阅公园et .。</li>
	</ol>
</li>
	<li>将带有效应器的质粒转换为野火pv, 并将其与 sfGFP11 标记融合在一起。西红柿(Pst)CUCPB5500 使用标准电穿孔29。 注意: 如果需要, 可以使用其他铜绿假单胞菌。sfGFP11 标记系统是为范围很广的向量30构造的, 并且该效应器的基因表达式由 AvrRpm1 启动子来调节, 它与如假单胞菌荧光(伊桑)31之间的比较。</li>
	<li>将转化后的细菌细胞轻轻地散布在含有100µg/毫升利福平和25µg/毫升卡那霉素或25µg/毫升庆大霉素的王 B 琼脂板表面。孵育28°c 2 天。</li>
	<li>接种一个殖民地入国王的 B 液体媒介以适当为载体使用的抗生素, 并且在28°c 隔夜生长细胞与震动在200转每分钟。</li>
	<li>做甘油的存货。将蒸压甘油添加到最终浓度为 50%, 并贮存在-80 摄氏度。</li>
</ol>3. 在benthamiana (4 天) 中, 以细胞器为目标的 sfGFP1-10选择的瞬时表达式
<ol><li>农杆菌区域性的制备<ol>
<li>订购所需的细胞器目标 sfGFP1-10 可选质粒 (参见材料表)。</li>
		<li>将质粒转换成根癌农杆菌应变 GV3101 细胞32。在 Luria-Bertani (磅) 琼脂培养基上生长细胞, 辅以50µg/毫升卡那霉素和50µg/毫升利福平28摄氏度, 2 天。</li>
		<li>从单一菌落的 LB 琼脂培养基, 接种细胞成5毫升的液体 LB 培养基补充50µg/毫升卡那霉素和50µg/毫升利福平。在28摄氏度的时候以200转每分钟的抖动来生长细胞。</li>
		<li>以 3000 x g 为10分钟, 以离心的方法收割细胞, 然后从上清介质中并用重悬, 并在1毫升的新入渗缓冲液中注入颗粒。</li>
		<li>通过获取 600 nm (Abs 600 nm) 的吸收光密度 (OD) 值来测量农杆菌的数量。将细菌的 OD600调整为 0.5, 并使用浸润缓冲。 注: 1 毫升的悬浮液足以渗入两个斑点。</li>
		<li>在 1-5 小时的渗透前, 在室温下将培养基留在一个柔和的摇杆上。</li>
		<li>把一个洞戳进叶子的中心, 然后用10µL 的尖端渗入。使用1毫升无针注射器渗入农杆菌悬浮液。仔细和缓慢地注入约500µL 的悬浮液准备从步骤3.1.5 到叶正面通过注射器。重复对至少三种不同植物的渗透实验复制。 注意: 出于健康和安全的原因, 在渗透过程中应佩戴眼部防护。</li>
		<li>擦掉叶子上残留的细菌悬浮液, 标记浸润区域的边界。</li>
		<li>保持渗透植物在相同的生长条件下, 用于步骤 1.1.1 2 天。</li>
	</ol>
</li>
</ol>4. 接种假单胞菌(4 天)
<ol><li>将从步骤2.5 中的甘油库存中的已转换的铜绿假单胞菌与相应的抗生素在28摄氏度处连续2天进行条纹。 注意:铜绿假单胞菌的健康非常关键。如果殖民地不形成好, 再次条纹细胞或传播的细胞在国王的液体媒体之前, 继续进行。</li>
	<li>在28摄氏度的甘露醇-谷氨酸 (MG) 液体介质中接种一 loopful 的铜绿假单胞菌细胞, 一夜之间以200转每分钟的抖动。</li>
	<li>以 3000 x g 为10分钟, 以离心的方法收割细胞. 倒入上清介质, 在10毫米氯化镁2中并用重悬颗粒, 并将 N . benthamiana 叶和 1 (10 x7 0.002) 的OD 600 调整为 0.02 (1 x10 6 cfu/毫升);cfu/mL) 为拟南芥留下.
</li>
	<li>对于benthamiana, 将铜绿假单胞内的悬浮液渗透到叶子的区域, 其中农杆菌携带 sfGFP1-10选择构造被渗透2天前 (如步骤 3.1.7)。对于 sfGFP1-10选择转基因拟南芥, 将假单胞菌的悬浮液渗透到两个4周长的短日生长的叶子中。 注意: 实验复制需要至少三株植物。</li>
</ol>5. 通过共聚焦显微镜观察 sfGFP 信号 (1 天)
<ol><li>从假单胞菌接种的叶子中剪下叶盘。在铜绿假单渗入后的特定时间点, 图像两个2厘米2叶盘从单一植物使用激光扫描共焦系统与 40 x/1.2 NA c 复消色差水浸泡目标或 63 x/0.8 na c-复消色差油浸泡目的。为了避免被伤致死的细胞, 观察细胞远离浸润孔。</li>
	<li>开始观察在低功率设置的 488 nm 氩激光。增加激光功率以检测 sfGFP。 注: 我们通常使用 2-15% 的激光强度来检测荧光信号。但是, 激光功率和检测设置应根据用户的显微系统进行调整。在这里, 发射过滤器被设置为 520-550 nm。死细胞通常在488纳米激光激发下发出自动荧光。因此, 作为一个负控制, 在相同的观测条件下, 没有 sfGFP11 标记的同样的效应器应该被渗透和观察。此外, 高激光激发能诱发叶绿素自发荧光。因此, 用控制植物细胞调节激光强度, 不诱导叶绿素自发荧光。<ol>
<li>对于细胞壁的 counterstaining, 在观察前 5-10 分钟内浸润20毫米碘碘化物 (PI) 到叶盘中。</li>
		<li>对于细胞核染色, 将叶盘浸入0.1% 多聚甲醛5分钟, 然后用水冲洗。然后, 在显微镜观察之前, 将10毫米 PI 渗透到叶盘中 5-10 分钟。重复实验至少三次。 注意: 这一步并不重要, 但有助于定义在等离子膜或原子核上的效应器定位。如果感兴趣的效应器显示他们在特定的细胞器中的定位, 使用一个标记为给定的细胞, 以确认该效应器的本地化。</li>
	</ol>
</li>
</ol>

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作者没有披露的利益冲突。

这里描述的协议用于监测细菌 T3SS 注射的效应蛋白在感染时对宿主植物细胞的准确定位。以前, 分离 GFP 系统被用来作为一个工具, 研究哺乳动物蛋白的亚细胞定位23,36,沙门氏菌T3E 本地化, 和农杆菌VirE2 交付通过 T4SS 到植物细胞37。为了在植物细胞中应用这个系统, 以前的研究使用了一种转基因玉米植株, 组成性表达 GFP1-10 ?...

植物是一种无梗的有机体, 在整个生命周期中遇到许多入侵的病原体, 包括细菌、真菌、病毒、昆虫和线虫。在 phytopathogens 中, 革兰阴性细菌病原体, 如铜绿假单胞菌和青, 通过进入伤口或自然开口, 如气孔和具1, 感染寄主植物。为了成功地将寄主植物殖民化, 细菌病原体已经进化形成了各种致病因子2。当细菌侵入寄主植物时, 它们会注射一系列的毒力蛋白--被称为效应者--直接进入植物细胞, 以促进其致病性。这些效应抑制或调节植物先天免疫, 并操纵宿主细胞过程, 导致细菌生存3。
致病细菌主要使用 T3SS 将效应蛋白直接传递到宿主细胞中4。T3SS 类似于一个分子注射器, 其针状通道连接从支架蛋白结构横跨内部和外部细菌膜到宿主细胞的注射点5。这种 T3SS-mediated 效应器 (T3E) 分泌机制在植物的各种革兰氏阴性细菌病原体以及人类中都保存良好。一个代表性植物病原体,野火pv。西红柿DC3000 hrcC变种通常有缺陷的 T3SS, 限制了植物的生长, 可能是由于这个突变体无法完全抑制植物免疫 (通过注射效应蛋白)6。在转移到宿主细胞后, 效应靶针对宿主细胞系统中重要的各种宿主蛋白, 包括植物防御反应、基因转录、细胞死亡、蛋白酶体、囊泡贩运和激素通路7,8,9,10. 因此, 跟踪宿主细胞中效应蛋白的细胞定位是了解它们在调节植物免疫力方面的作用的一个很有吸引力的目标。
大多数 T3Es 的定位研究都采用了农杆菌-介导的大荧光蛋白在寄主植物9中的过度表达。然而, 其他物种引入的基因的异种表达方法已被证明是错误本地化的, 或者偶尔不起作用的11,12,13。此外, 一些研究显示, 细菌效应在宿主细胞中进行了适当的靶向定位14,15,16,17。因此, 瞬时表达的效应在植物细胞的细胞质可能不是功能上或数量相同的效应者, 由 T3SS 提供的病原体感染18。此外, 大荧光标记与效应蛋白的融合可能会扰乱适当的效应传递和可视化18,19。因此, 这些检测 T3E 功能的方法可能无法充分反映 T3SS-secreted 效应的本土化。
绿色荧光蛋白 (GFP) 由一个11股的β桶组成, 其中包含一个包括生色20的中心链。金都et报道了一个新的分裂-GFP 系统, 由一个小组成部分 (gfp β链 11;GFP11) 和一个大的互补片段 (GFP β链 1-10;GFP1-10)21。片段不荧光自己, 但荧光后, 他们的自我联想, 当两个片断是在密切接近。对于蛋白质折叠效率的优化, gfp 的鲁棒折叠变体,即, sfGFP 和 sfGFP选择, 随后为分割的 gfp 系统20,21,22而开发。最近, sfGFP1-10 可选-sfYFP1-10选择和 sfCFP1-10选择的单个氨基酸突变变种, 可以用 sfGFP11 片段进行重建, 并分别显示黄色和青色荧光, 生成23.此外, sfCherry 是 mCherry 的导数, 可以以与 sfGFP23相同的方式拆分为 sfCherry1-10 和 sfCherry11 片段。
该系统已经适应了标签和跟踪 T3SS 在 HeLa 细胞感染期间使用的效应器从沙门氏菌24.然而, 它以前未被优化为寄主植物细菌病原体系统。最近, 我们优化了基于改进的 sfGFP1-10 可选的分割 GFP 系统, 以监视从野火发送到植物单元25的 T3Es 的亚细胞定位。为了促进 T3Es 对不同亚细胞间的定位研究, 一组转基因的拟南芥植物在不同的亚细胞间隔内生成 sfGFP1-10 可选25. 此外, 携带各种细胞器靶向的 sfGFP1-10 的质粒选择用于农杆菌介导的瞬态过度表达和 T3SS-based 效应传递的 sfGFP11-tagged 向量也产生。各种转基因的拟南芥线和质粒来表达 T3Es 的种子, 可以从材料表26,27中提到的来源获得。
在下面的协议中, 我们描述了一个优化系统, 以监测由细菌在宿主细胞中使用分裂 sfGFP 系统传递的效应器的动力学。表达 sfGFP1-10选择的植物感染转基因假单胞菌携带重组 sfGFP11 质粒导致 sfGFP11-tagged 效应器从假单胞菌交付宿主细胞。因此, 这些蛋白质被重组, 并植物常常将到特定的效应靶室 (s)。假单胞野火pv。西红柿CUCPB5500 菌株, 其中18个效应被删除, 使用, 因为这种菌株显示低或没有细胞死亡, 在A. 南芥和N. benthamianas28。然而, 这里描述的所有材料和步骤可以被替换或修改, 以适应分裂 sfGFP 系统的调查其他生物问题或优化在给定的实验室条件。

<table><tbody><tr><td>&lt;strong&gt;Arabidopsis transgenic lines&lt;/strong&gt;</td><td></td><td></td><td>Park, E., Lee, H. Y., Woo, J., Choi, D. &amp;amp; Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)</td></tr><tr><td>CYTO-sfGFP1-10</td><td>ABRC</td><td>CS69831</td><td></td></tr><tr><td>NU-sfGFP1-10</td><td>ABRC</td><td>CS69832</td><td></td></tr><tr><td>PT-sfGFP1-10</td><td>ABRC</td><td>CS69833</td><td></td></tr><tr><td>MT-sfGFP1-10</td><td>ABRC</td><td>CS69834</td><td></td></tr><tr><td>PX-sfGFP1-10</td><td>ABRC</td><td>CS69835</td><td></td></tr><tr><td>ER-sfGFP1-10</td><td>ABRC</td><td>CS69836</td><td></td></tr><tr><td>GO-sfGFP1-10</td><td>ABRC</td><td>CS69837</td><td></td></tr><tr><td>PM-sfGFP1-10</td><td>ABRC</td><td>CS69838</td><td></td></tr><tr><td>&lt;strong&gt;Organelle-targeted sfGFP1-10OPT plasmid&lt;/strong&gt;</td><td></td><td></td><td>Park, E., Lee, H. Y., Woo, J., Choi, D. &amp;amp; Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)</td></tr><tr><td>CYTO-sfGFP1-10</td><td>Addgene</td><td>97387</td><td></td></tr><tr><td>NU-sfGFP1-10</td><td>Addgene</td><td>97388</td><td></td></tr><tr><td>PT-sfGFP1-10</td><td>Addgene</td><td>97389</td><td></td></tr><tr><td>MT-sfGFP1-10</td><td>Addgene</td><td>97390</td><td></td></tr><tr><td>PX-sfGFP1-10</td><td>Addgene</td><td>97391</td><td></td></tr><tr><td>ER-sfGFP1-10</td><td>Addgene</td><td>97392</td><td></td></tr><tr><td>GO-sfGFP1-10</td><td>Addgene</td><td>97393</td><td></td></tr><tr><td>PM-sfGFP1-10</td><td>Addgene</td><td>97394</td><td></td></tr><tr><td>ER-sfCherry1-10</td><td>Addgene</td><td>97403</td><td></td></tr><tr><td>ER-sfYFP1-10</td><td>Addgene</td><td>97404</td><td></td></tr><tr><td>CYTO-sfCFP1-10</td><td>Addgene</td><td>97405</td><td></td></tr><tr><td>&lt;strong&gt;sfGFP11-tagged Gateway compatible vector for T3SS-based effector delivery system&lt;/strong&gt;</td><td>Park, E., Lee, H. Y., Woo, J., Choi, D. &amp;amp; Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)</td><td></td><td></td></tr><tr><td>pBK-GW-1-2</td><td>Addgene</td><td>98250</td><td>pAvrRpm1:GW:HA-sfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td></tr><tr><td>pBK-GW-1-4</td><td>Addgene</td><td>98251</td><td>pAvrRpm1:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td></tr><tr><td>pBK-GW-2-2</td><td>Addgene</td><td>98252</td><td>pAvrRpm1:AvrRPM1sp:GW:HA-sfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td></tr><tr><td>pBK-GW-2-4</td><td>Addgene</td><td>98253</td><td>pAvrRpm1:AvrRPM1sp:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td></tr><tr><td>pBG-GW-1-2</td><td>Addgene</td><td>98254</td><td>pAvrRpm1:GW:HA-sfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td></tr><tr><td>pBG-GW-1-4</td><td>Addgene</td><td>98255</td><td>pAvrRpm1:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td></tr><tr><td>pBG-GW-2-2</td><td>Addgene</td><td>98256</td><td>pAvrRpm1:AvrRPM1sp:GW:HA-sfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td></tr><tr><td>pBG-GW-2-4</td><td>Addgene</td><td>98257</td><td>pAvrRpm1:AvrRPM1sp:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td></tr><tr><td>&lt;strong&gt;Bacterial strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Agrobacterium tumefaciens &lt;/em&gt;GV3101</td><td></td><td></td><td>Csaba Koncz and Jeff Schell, The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet. 204,383-396 (1986); Resistant to gentamycin (50 ug/ml) and rifampicin (50 ug/ml)</td></tr><tr><td>&lt;em&gt;Pseudomonas syringae&lt;/em&gt; pv. &lt;em&gt;Tomato&lt;/em&gt; CUCPB5500</td><td></td><td></td><td>Kvitko, B. H. et al. Deletions in the repertoire of Pseudomonas syringae pv. tomato DC3000 type III secretion effector genes reveal functional overlap among effectors. PLoS Pathog. 5 (4) (2009).; Resistant to rifampicin (100 ug/ml)</td></tr><tr><td>&lt;strong&gt;Media components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Plant germination media&lt;/strong&gt;</td><td></td><td></td><td>Add 2.165g/L Murashige &amp;amp; Skoog powder, 10 g/L sucrose to water. Adjust to pH 5.8 and add 2.2 g/L phytagel. Autocalve.</td></tr><tr><td>Murashige &amp;amp; Skoog medium including vitamins</td><td>Duchefa Biochemie</td><td>M0222</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>Sucrose</td><td>Duchefa Biochemie</td><td>S0809</td><td></td></tr><tr><td>Phytagel</td><td>Sigma-Aldrich</td><td>P8169</td><td></td></tr><tr><td>&lt;strong&gt;LB media&lt;/strong&gt;</td><td></td><td></td><td>Add 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl to water. For solid media, add 15 g/L micro agar. Autoclave.&amp;nbsp; Allow solution to cool to 55 &amp;deg;C, and add antibiotic if needed.</td></tr><tr><td>Tryptone</td><td>BD Bioscience</td><td>211705</td><td></td></tr><tr><td>Yeast extract</td><td>BD Bioscience</td><td>212750</td><td></td></tr><tr><td>NaCl</td><td>Duchefa Biochemie</td><td>S0520</td><td></td></tr><tr><td>Micro agar</td><td>Duchefa Biochemie</td><td>M1002</td><td></td></tr><tr><td>&lt;strong&gt;King&#039;s B media&lt;/strong&gt;</td><td></td><td></td><td>10 g/L protease peptone #2, 1.5 g/L anhydrous K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 15 g/L of agar to water. Autoclave. Cool down to 55 &amp;deg;C and add sterile 15 ml/L glycerol, 5 ml/L MgSO&lt;sub&gt;4&lt;/sub&gt; to the medium. Add antibiotics if needed.</td></tr><tr><td>Proteose peptone</td><td>BD Bioscience</td><td>212120</td><td></td></tr><tr><td>Anhydrous K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>1551128 USP</td><td></td></tr><tr><td>Glycerol</td><td>Duchefa Biochemie</td><td>G1345</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M7506</td><td></td></tr><tr><td>Bacto Agar</td><td>BD Bioscience</td><td>214010</td><td></td></tr><tr><td>&lt;strong&gt;Mannitol-Glutamate (MG) liquid media &lt;/strong&gt;</td><td></td><td></td><td>Add 10 g/L of mannitol, 2 g/L of L-glutamic acid, 0.5 g/L of KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 0.2 g/L of NaCl, and 0.2 g/L of MgSO&lt;sub&gt;4&lt;/sub&gt; to water. Adjust to pH 7</td></tr><tr><td>Mannitol</td><td>Duchefa Biochemie</td><td>M0803</td><td></td></tr><tr><td>L-glutamic acid</td><td>Duchefa Biochemie</td><td>G0707</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>NIST200B</td><td></td></tr><tr><td>Infiltration buffer</td><td></td><td></td><td>10 mM MES (2-(N-morpholino)-ethane sulfonic acid), 10 mM MgCl2, 150 &amp;micro;M acetosyringone. pH 5.6; Prepare a fresh buffer before use.</td></tr><tr><td>MES</td><td>Duchefa Biochemie</td><td>M1503</td><td>Prepare 100 mM (pH 5.6) stock in water. Filter sterilize.</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M8266</td><td>Prepare 100 mM stock in water. Autoclave.</td></tr><tr><td>Acetosyringone</td><td>Sigma-Aldrich</td><td>D134406</td><td>Prepare 150 mM stock in DMSO.</td></tr><tr><td>Confocal microscope equipments/materials</td><td></td><td></td><td></td></tr><tr><td>710 laser scanning confocal system</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Axio observer Z1 inverted microscope</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Propidium iodide</td><td>ThermoFisher</td><td>P1304MP</td><td></td></tr></tbody></table>

split green fluorescent protein system to visualize effectors delivered from bacteria during infection

细菌是各种植物病害中最重要的致病因子之一, 它将一组效应蛋白分泌到宿主植物细胞中, 破坏植物的免疫系统。在感染期间细胞质效应通过 III. 型分泌系统 (T3SS) 传递给宿主细胞质。传递到植物细胞后, 效应器 (s) 靶向特定的隔间, 调节宿主细胞的过程以生存和复制病原体。虽然对宿主细胞中效应蛋白的亚型定位进行了研究, 以了解其在致病性中的作用, 利用荧光蛋白, 直接从细菌中注射的效应研究由于 T3SS 和荧光蛋白之间的不相容性而受到了挑战。
在这里, 我们描述了我们最近的方法, 优化分裂 superfolder 绿色荧光蛋白系统 (sfGFP选择), 以可视化的效应传递通过细菌 T3SS 在宿主细胞。通过 T3SS 分泌的 sfGFP 的 sfGFP11 (11th β链) 可以与特定的细胞器 (sfGFP1-10 可选(1-10 β链 sfGFP) 一起组装, 从而导致现场的荧光发射.该协议提供了一个程序, 以可视化重组 sfGFP 荧光信号的效应蛋白从假单胞菌野火在一个特定的细胞器在拟南芥和烟草 benthamiana植物。

GFP 的β桶结构由十一β链组成, 可分为两个片段, 1-10th链 (GFP1-10 可选) 和 11th (GFP11) 链。尽管两个碎片本身都不是荧光的, 但自组装 sfGFP 可以在接近接近度 (图 1A) 存在时发出荧光。在该系统中, sfGFP1-10 "选择"-表示拟南芥或N. benthamiana植物接种了假单胞菌携带一个 sfGFP11 标记的效应器。由铜绿假单?...

Watch this Scientific Journal Video about Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection at JoVE.com

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

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

以荧光蛋白为基础的方法来监测细菌分泌到宿主细胞中的效应, 这是很有挑战性的。这是由于荧光蛋白与 III. 型分泌系统之间的不相容。在这里, 一个优化的分裂 superfolder GFP 系统是用来可视化的效应, 细菌分泌的宿主植物细胞。

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 ...

split-green-fluorescent-protein-system-to-visualize-effectors

Research

JoVE Journal

Biology

10.4K 次观看.  Seoul National University. 以荧光蛋白为基础的方法来监测细菌分泌到宿主细胞中的效应, 这是很有挑战性的。这是由于荧光蛋白与 III. 型分泌系统之间的不相容。在这里, 一个优化的分裂 superfolder GFP 系统是用来可视化的效应, 细菌分泌的宿主植物细胞。

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

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

基于荧光的感染组织细菌基因调控方法

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

科研

遗传学

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

电功能性细菌效应器到哺乳动物细胞

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

免疫与感染

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

分析<em&gt;小肠结肠炎耶尔森菌</em&gt;效应易位到宿主细胞中使用β-内酰胺酶效应融合体

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

应用荧光共振能量转移（FRET）审查效应转运效率由<em&gt;贝氏柯克斯体</em&gt; siRNA沉默中

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

增强型绿色荧光蛋白表达大肠杆菌评价小鼠腹腔巨噬细胞吞噬细胞增多

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

应用活细胞成像和低温电子断层扫描解决军团菌肺瘤点/Icm分泌系统的时空特征

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

单细胞物种间细菌相互作用的动力学可视化

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

通过电穿孔内在和生物分子荧光观察生活中的微生物

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

生物学

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host cells. Upon entry, the injected effector proteins cooperatively modulate eukaryotic signaling pathways and reprogram cellular functions, enabling bacterial entry and survival. Monitoring and localizing these secreted effector proteins in the context of infections provides a footprint for defining the dynamic interface of host-pathogen interactions. However, labeling and imaging bacterial proteins in host cells without disrupting their structure/function is technically challenging. 
Constructing fluorescent fusion proteins does not resolve this problem, because the fusion proteins jam the secretory apparatus and thus are not secreted. To overcome these obstacles, we recently employed a method for site-specific fluorescent labeling of bacterial secreted effectors, as well as other difficult-to-label proteins, using genetic code expansion (GCE). This paper provides a complete step-by-step protocol to label Salmonella secreted effectors using GCE site-specifically, followed by directions for imaging the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM) 
Recent findings suggest that the incorporation of non-canonical amino acids (ncAAs) via GCE, followed by bio-orthogonal labeling with tetrazine-containing dyes, is a viable technique for selective labeling and visualization of bacterial secreted proteins and subsequent image analysis in the host. The goal of this article is to provide a straightforward and clear protocol that can be employed by investigators interested in conducting super-resolution imaging using GCE to study various biological processes in bacteria and viruses, as well as host-pathogen interactions.

This article provides a straightforward and clear protocol to label Salmonella secreted effectors using genetic code expansion (GCE) site-specifically and image the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM)

使用遗传密码扩增对细菌分泌蛋白进行超分辨率成像

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host ...

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

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