利用共聚焦显微镜对根结线虫取食部位的巨型细胞进行表型分析,突出了几丁质酶类似蛋白 1 在拟南芥中的作用。
A Phenotyping Method of Giant Cells from Root-Knot Nematode Feeding Sites by Confocal Microscopy Highlights a Role for CHITINASE-LIKE 1 in Arabidopsis.
机构信息
Facultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-La Mancha, Área de Fisiología Vegetal, Avda, Carlos III, s/n, 45071 Toledo, Spain.
Centro de Biotecnología y Genómica de Plantas (UPM-INIA), Pozuelo de Alarcón, 28223 Madrid, Spain.
出版信息
Int J Mol Sci. 2018 Feb 1;19(2):429. doi: 10.3390/ijms19020429.
Most effective nematicides for the control of root-knot nematodes are banned, which demands a better understanding of the plant-nematode interaction. Understanding how gene expression in the nematode-feeding sites relates to morphological features may assist a better characterization of the interaction. However, nematode-induced galls resulting from cell-proliferation and hypertrophy hinders such observation, which would require tissue sectioning or clearing. We demonstrate that a method based on the green auto-fluorescence produced by glutaraldehyde and the tissue-clearing properties of benzyl-alcohol/benzyl-benzoate preserves the structure of the nematode-feeding sites and the plant-nematode interface with unprecedented resolution quality. This allowed us to obtain detailed measurements of the giant cells' area in an line overexpressing () from optical sections by confocal microscopy, assigning a role for and adding essential data to the scarce information of the role of gene repression in giant cells. Furthermore, subcellular structures and features of the nematodes body and tissues from thick organs formed after different biotic interactions, i.e., galls, syncytia, and nodules, were clearly distinguished without embedding or sectioning in different plant species (, cucumber or ). The combination of this method with molecular studies will be valuable for a better understanding of the plant-biotic interactions.
大多数用于防治根结线虫的有效杀线虫剂已被禁用,这就要求我们更好地了解植物与线虫的相互作用。了解线虫取食部位的基因表达与形态特征的关系,可能有助于更好地描述这种相互作用。然而,线虫诱导的瘿瘤是由细胞增殖和肥大引起的,这阻碍了这种观察,需要进行组织切片或透明处理。我们证明了一种基于戊二醛产生的绿色自发荧光和苄醇/苯甲酸苄酯的组织透明特性的方法,可以在前所未有的分辨率质量下保存线虫取食部位和植物-线虫界面的结构。这使我们能够通过共聚焦显微镜从光学切片中获得过量表达 () 的 系中巨型细胞面积的详细测量值,为 基因的功能分配提供了依据,并为巨型细胞中基因抑制作用的作用提供了重要数据。此外,在不同的生物相互作用(如瘿瘤、合胞体和根瘤)后形成的厚器官(如黄瓜或拟南芥)中,无需包埋或切片即可清楚地区分线虫体和组织的亚细胞结构和特征。该方法与分子研究相结合,将有助于更好地了解植物与生物的相互作用。
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