Name: live-cell imaging of fungal cells to investigate modes of entry and subcellular localization of antifungal plant defensins
Uploaded: 2017-12-24T12:30:00
Description: Watch this Scientific Journal Video about Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins at JoVE.com

我们感谢 Dr. r. 伯格, 在唐纳德丹佛斯植物科学中心的综合显微镜设施主任, 为他的指导和帮助与共焦显微镜。作者没有利益冲突的声明。

1. 用荧光标记防御
<ol>
	<li>选择一种对抗菌性能影响最小的荧光, 以及对活细胞内防御的吸收和定位。 注意: 选择最佳荧光取决于特定的实验目标。还应考虑荧光的光谱和化学性质、光、大小和电荷。</li>
	<li>标签防御与选定的荧光使用适当的肽标签套件可从商业供应商。另外, 化学合成荧光标记的防御。在这项研究中, 标签 MtDef5 防御与 DyLight550 使用商业标签套件根据制造商的协议和四甲基罗?...

<ol>
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A., Thevissen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modes+of+antifungal+action+and+in+planta+functions+of+plant+defensins+and+defensin-like+peptides.">Modes of antifungal action and in planta functions of plant defensins and defensin-like peptides.</a> Fungal Biol Rev. 26 (4), 109-120 (2013).</li><li>Kaur, J., Sagaram, U. S., Shah, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+plant+defensins+be+used+to+engineer+durable+commercially+useful+fungal+resistance+in+crop+plants?.">Can plant defensins be used to engineer durable commercially useful fungal resistance in crop plants?.</a> Fungal Biol. Rev. 25 (3), 128-135 (2011).</li><li>Vriens, K., Cammue, B. P. A., Thevissen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+plant+defensins:+Mechanisms+of+action+and+production.">Antifungal plant defensins: Mechanisms of action and production.</a> Molecules. 19 (8), 12280-12303 (2014).</li><li>Sagaram, U. S., Kaur, J., Shah, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+plant+defensins:+Structure-activity+relationships,+modes+of+action,+and+biotech+applications.">Antifungal plant defensins: Structure-activity relationships, modes of action, and biotech applications.</a> ACS Symp. Ser. 1095, 317-336 (2012).</li><li>Thevissen, K., Francois, I. E. J. A., Aerts, A. M., Cammue, B. P. 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A., Thevissen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mode+of+antifungal+action+of+plant,+insect+and+human+defensins.">The mode of antifungal action of plant, insect and human defensins.</a> Cell. Mol. Life Sci. 65 (13), 2069-2079 (2008).</li><li>Van Der Weerden, N. L., Lay, F. T., Anderson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plant+defensin,+NaD1,+enters+the+cytoplasm+of+Fusarium+oxysporum+hyphae.">The plant defensin, NaD1, enters the cytoplasm of Fusarium oxysporum hyphae.</a> J. Biol. Chem. 283 (21), 14445-14452 (2008).</li><li>Lobo, D. S., Pereira, I. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+Pisum+sativum+Defensin+1+Interacts+with+Neurospora+crassa+Cyclin+F+Related+to+the+Cell+Cycle.">Antifungal Pisum sativum Defensin 1 Interacts with Neurospora crassa Cyclin F Related to the Cell Cycle.</a> Biochemistry. 46 (4), 987-996 (2007).</li><li>El-Mounadi, K., Islam, K. T., Hernandez-Ortiz, P., Read, N. D., Shah, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+mechanisms+of+a+plant+defensin+MtDef4+are+not+conserved+between+the+ascomycete+fungi+Neurospora+crassa+and+Fusarium+graminearum.">Antifungal mechanisms of a plant defensin MtDef4 are not conserved between the ascomycete fungi Neurospora crassa and Fusarium graminearum.</a> Mol. Microbiol. 100 (3), 542-559 (2016).</li><li>Baxter, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tomato+Defensin+TPP3+Binds+Phosphatidylinositol+(4,5)-Bisphosphate+via+a+Conserved+Dimeric+Cationic+Grip+Conformation+To+Mediate+Cell+Lysis.">The Tomato Defensin TPP3 Binds Phosphatidylinositol (4,5)-Bisphosphate via a Conserved Dimeric Cationic Grip Conformation To Mediate Cell Lysis.</a> Mol. and Cell. Biol. 35 (11), 1964-1978 (2015).</li><li>Kvansakul, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+phosphatidic+acid+by+NsD7+mediates+the+formation+of+helical+defensin-lipid+oligomeric+assemblies+and+membrane+permeabilization.">Binding of phosphatidic acid by NsD7 mediates the formation of helical defensin-lipid oligomeric assemblies and membrane permeabilization.</a> Proc. Natl. Acad. Sci. 113, 11202-11207 (2016).</li><li>Poon, I. K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoinositide-mediated+oligomerization+of+a+defensin+induces+cell+lysis.">Phosphoinositide-mediated oligomerization of a defensin induces cell lysis.</a> eLife. 3, e01808 (2014).</li><li>Mu&#241;oz, A., Read, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+and+analysis+shed+light+on+the+complexity+and+dynamics+of+antimicrobial+Peptide+action.">Live-cell imaging and analysis shed light on the complexity and dynamics of antimicrobial Peptide action.</a> Front. Immunol. 3, 248 (2012).</li><li>Hayes, B. M. 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在这项研究中, 一个可靠的活细胞成像方法, 使用荧光标记的抗真菌防御被描述为研究的动力学, 这些肽内化的真菌细胞和确定其亚型靶点。这种方法是一个强大的工具, 以可视化的动态的互动之间的防御和真菌细胞的时空。
采用多种方法研究了真菌细胞内植物防御的内部化和细胞定位。在这些方法中, 防御处理的细胞通常是固定的, 然后处理的 immunolocalization, 电子显微镜, 或 X ...

植物进化了一个复杂的先天免疫系统, 以抵御微生物植物病原体1。他们表达了许多基因编码的宿主防御肽与假定的抗菌活性2。事实上, 这些肽中的许多都是在体外3中显示抗菌活性的。防御包括植物王国中最大的寄主防御肽组之一4。这些富含半胱氨酸的阳离子肽在 micromolar 浓度下表现出对真菌和卵病原体的有效生长抑制作用, 并且是抵御这些病原体的第一条防线5,6。由于其强大的抗真菌活性, 防御可以被利用在 agribiotechnological 的应用, 以产生抗病作物。几种植物防御的本构表达提高了温室的抗病性和转基因作物的田间试验6。重要的是要解开这些抗真菌肽的作用机制 (MOA), 以便充分利用其潜力作为有效的作物保护工具。然而, 对这些植物防御的 MOA 了解得很差。目前的证据表明, 他们表现出不同的 MOA5,6,7,8。一些防御行为 extracellularly 真菌和靶向特定细胞壁/细胞膜驻留鞘, 扰乱膜完整性和激活细胞毒性通路9,10,11。最近, 然而, 抗真菌防御, 转移到真菌细胞已被发现12,13,14。这些防御结合到膜驻留生物活性磷脂, 形成聚复合物和 permeabilize 等离子膜15,16,17。从而阐明了植物防御的某些方面。然而, 植物防御的 MOA 可能涉及一系列复杂的事件, 它们尚未被识别并集成到一个综合模型中。特别是, 在我们对这些肽的细胞靶点的理解上仍然存在很大的差距。
近年来, 随着显微镜技术的发展和新的荧光探针的开发, 活体细胞成像技术已被广泛应用于抗菌肽 (安培) 的研究。这些技术补充了广泛使用的方法, 如 immunolocalization, 电子显微镜, 原子力显微镜或 X 射线断层摄影术18, 主要用于分析抗真菌肽对形态学的影响和真菌细胞的生长, 包括细胞壁完整性的研究, 细胞生长/分支模式的改变, 以及血浆膜性和杀戮。然而, 这些研究仅限于在治疗后的某个时间点的影像细胞, 而不是在同一个细胞上进行延时成像, 以监测它们对防御挑战的动态变化。近年来, 使用荧光标记多肽与活细胞成像结合使用共焦显微镜, 使 real-time 可视化动力学的 AMP–microbe 相互作用。天然纯化和化学合成的抗真菌肽可以标记为荧光标签 (如, DyLight, 罗丹明, BODIPY, 或 Alexa 的染料), 并直接观察在他们与细胞的互动时, 延时活细胞成像。这些标记多肽的使用大大增加了我们对其 MOA 的不同方面的理解, 包括进入模式、亚细胞定位、胞内贩运以及活真菌细胞中的抗真菌作用部位18。
最近, 有几项研究表明, 包括植物防御在内的各种抗真菌肽都是由活真菌细胞内化的12,14,19,20。这些防御的 MOA 可能涉及与细胞内目标的相互作用。我们最近报告了植物防御 MtDef4 在两个囊真菌的抗真菌作用,孢菌和镰刀菌菌。MtDef4 被证明使用不同的途径真菌细胞进入和亚细胞定位这些真菌14。本研究采用化学合成的四甲基罗丹明 (TAMRA) 标记 MtDef4 与生命荧光染料 (膜选择染料, FM4-64; 膜 permeant 染料, SYTOX 绿色; 细胞死亡记者染料, 碘碘化物) 和代谢抑制剂。这些分析显示了 MtDef4 内化的动力学, 其胞内转运机制和亚细胞靶向14。
本文提出了一种利用共焦显微镜进行活细胞成像的协议。该协议利用荧光标记多肽与生命荧光染料结合, 研究植物防御真菌相互作用, 特别是真菌细胞的易位途径和胞内防御的细胞靶点。

<table>
	<tbody>
		<tr>
			<td>FM4-64 Dye</td>
			<td>Life Technologies</td>
			<td>T13320</td>
			<td></td>
		</tr>
		<tr>
			<td>DyLight 550 Antibody Labeling Kit</td>
			<td>Thermo Scientific</td>
			<td>84530</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Microwell Dishes</td>
			<td>Mat TeK</td>
			<td>P35G-1.5-10-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Mira cloth</td>
			<td>EMD Millipore Corp</td>
			<td>475855-1R</td>
			<td></td>
		</tr>
		<tr>
			<td>SP8-X confocal microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>FiJi</td>
			<td></td>
			<td>For Image analysis</td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane</td>
			<td></td>
			<td>For Image analysis</td>
		</tr>
	</tbody>
</table>

live-cell imaging of fungal cells to investigate modes of entry and subcellular localization of antifungal plant defensins

小的半胱氨酸丰富的防御是在所有植物中存在的最大的寄主防御肽群之一。许多植物防御展示了强有力的在体外抗真菌活性的 broad-spectrum 霉菌病原体, 因此有潜力被用作抗真菌剂在转基因作物。为了充分发挥植物防御对疾病控制的潜力, 阐明其作用机制是至关重要的。随着先进的显微技术的问世, 活细胞成像已经成为了解植物防御抗真菌的动力学的有力工具。在这里, 用两个荧光标记植物防御 (MtDef4 和 MtDef5) 结合生命荧光染料描述了一种基于共聚焦显微镜的活体细胞成像方法。这一技术使 real-time 可视化和分析的动态事件的 MtDef4 和 MtDef5 内化为真菌细胞。重要的是, 这种分析产生了丰富的信息, 包括内化动力学, 进入方式和亚细胞定位这些肽。这些方法与其他细胞生物工具一起, 为这些肽的动态和复杂性提供了重要的洞察力。这些工具也可以用来比较这些肽对不同真菌的 MOA。

从苜蓿蒺藜中进行了活细胞成像, 以追踪和比较两个防御、MtDef4 和 MtDef5 的内化和亚单位定位;在真菌细胞中。TMR-MtDef4 是化学合成的, 而 MtDef5 被标记为 Dylight550 (Dylight550-MtDef5)。用防御与膜选择性染料 FM4-64 联合培养孢子。图 1显示, 与F. 菌相比, TMR-MtDef4 在N. 菌中具有不同的贩运路径。在N. 菌中, FM4-64 不 co-localize 与防御, 而?...

Watch this Scientific Journal Video about Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins at JoVE.com

Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins

植物防御在植物防御病原体中起着重要的作用。为了有效地使用这些抗真菌肽作为抗真菌剂, 了解其作用方式 (MOA) 是至关重要的。在这里, 描述了一个活细胞成像方法, 以研究这些肽的 MOA 的关键方面。

真菌细胞的活细胞成像研究抗真菌植物防御的进入和亚单位定位

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro ...

Research

JoVE Journal

Biology

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

科研

生物学

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

活细胞成像早期自噬活动：Omegasomes与超越

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

扫描电子显微镜（SEM）协议有问题的工厂，卵菌和真菌样本

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

利用pHluorin标记的受体监视亚细胞定位和贩运

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

活细胞成像血小板脱水和分泌流量

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

Super-Resolution Live Cell Imaging of Subcellular Structures

亚细胞结构的超分辨率活细胞成像

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

从土壤中分离可培养酵母和霉菌以研究真菌种群结构

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular ...

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

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

Visualization of Cellular Interactions Between the Maize Sheaths and Fungi

Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens

Investigating Fungal Pathogenicity Mechanisms in Maize (Video) | JoVE

用于真菌叶面玉米病原体感染的活细胞成像的分离玉米鞘

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one ...