Whole mount multiplexed visualization of DNA, mRNA, and protein in plant-parasitic nematodes
Whole mount multiplexed visualization of DNA, mRNA, and protein in plant-parasitic nematodes
复制标题
植物寄生线虫 DNA、mRNA 和蛋白质的整体多重可视化
DOI：
10.1101/2023.09.23.559107
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Sperling A
中科院分区：
文献类型：
--
作者：
Sperling A
背景植物-寄生线虫危害世界范围内最常见的各种作物的农业。利用小分子染色、抗体或原位杂交观察完整线虫的能力，限制了获取这些生物的基本生物学以及它们如何感染植物的信息。因此，关于线虫的内部组成或它们用来重新编程宿主植物的效应分子的生物学信息有限。结果我们提出了Sperling Prep-一种完整的线虫制备方法，能够用小分子、抗体或原位杂交链式反应进行染色。这种方法不需要专门的仪器，使用的是典型的实验室设备和材料。通过分离坚固的角质层和内部肌肉层，我们能够使小分子染料进入组织。渗透后，小分子染色可用DNA染色DAPI显示细胞核，用丝状肌动蛋白染色显示消化道和纵向肌肉的内部结构。这种通透性甚至允许更大的抗体进入，尽管效率较低。最后，这种方法在原位HCR中工作得特别好。利用这种方法，我们可以可视化甜菜孢囊线虫的背部腺体和腹下腺体的特异效应转录本。结论我们能够可视化甜菜孢囊线虫的内部结构以及在植物侵染和寄生过程中使用的关键效应物转录本。因此，该方法为植物寄生线虫生物学研究提供了一个重要的工具包。
BackgroundPlant-parasitic nematodes compromise the agriculture of a wide variety of the most common crops worldwide. Obtaining information on the fundamental biology of these organisms and how they infect the plant has been restricted by the ability to visualize intact nematodes using small molecule stains, antibodies, or in situ hybridization. Consequently, there is limited information available about the internal composition of the nematodes or the biology of the effector molecules they use to reprogram their host plant.ResultsWe present the Sperling prep - a whole mount method for nematode preparation that enables staining with small molecules, antibodies, or in situ hybridization chain reaction. This method does not require specialized apparatus and utilizes typical laboratory equipment and materials. By dissociating the strong cuticle and interior muscle layers, we enabled entry of the small molecule stains into the tissue. After permeabilization, small molecule stains can be used to visualize the nuclei with the DNA stain DAPI and the internal structures of the digestive tract and longitudinal musculature with the filamentous actin stain phalloidin. The permeabilization even allows entry of larger antibodies, albeit with lower efficiency. Finally, this method works exceptionally well with in situ HCR. Using this method, we have visualized effector transcripts specific to the dorsal gland and the subventral grand of the sugar beet cyst nematode,Heterodera schachtii, multiplexed in the same nematode.ConclusionWe were able to visualize the internal structures of the nematode as well as key effector transcripts that are used during plant infection and parasitism. Therefore, this method provides an important toolkit for studying the biology of plant-parasitic nematodes.
影响因子：
4.5
作者：
Lilley CJ;Maqbool A;Wu D;Yusup HB;Jones LM;Birch PRJ;Banfield MJ;Urwin PE;Eves-van den Akker S
通讯作者：
Eves-van den Akker S
影响因子：
48
作者：
Schindelin, Johannes;Arganda-Carreras, Ignacio;Frise, Erwin;Kaynig, Verena;Longair, Mark;Pietzsch, Tobias;Preibisch, Stephan;Rueden, Curtis;Saalfeld, Stephan;Schmid, Benjamin;Tinevez, Jean-Yves;White, Daniel James;Hartenstein, Volker;Eliceiri, Kevin;Tomancak, Pavel;Cardona, Albert
通讯作者：
Cardona, Albert