In planta observation of live fluorescent plant endoparasitic nematodes during early stages of infection
In planta observation of live fluorescent plant endoparasitic nematodes during early stages of infection
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感染早期活荧光植物内寄生线虫的植物观察
DOI：
10.3725/jjn.40.15
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发表时间：
2010
期刊：
影响因子：
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通讯作者：
Michael G. K. Jones
中科院分区：
文献类型：
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作者：
Derek B. Goto;J. Fosu‐Nyarko;F. Sakuma;J. Sadler;Melita Flottman;T. Uehara;N. Kondo;J. Yamaguchi;Michael G. K. Jones
植物内寄生线虫的大部分生命周期都隐藏在植物根部内，每年都会给世界各地的农业造成重大损失。根结线虫（‘RKNs’，Meloidogyne spp.）是定居的内寄生线虫，它们在第二阶段幼虫 (J2) 时在植物根部建立永久的取食位点，然后在此位点度过其剩余的生命周期。相比之下，根病线虫（短体线虫属）是一种迁徙性内寄生虫，它们在根部移动并以不同的根细胞为食，引起棕色病斑的典型症状。人们对研究静止型和迁徙性内寄生线虫的初始感染阶段非常感兴趣。就 RKN 而言，已知宿主细胞在感染和宿主细胞选择的前 48 小时内会经历细胞周期激活、DNA 合成和核分裂的关键初始变化（de Almeida Engler 等，1999；Jones，1981；Jones 和 Payne，1978；Niebel 等，1996）。就根部病变线虫而言，跟踪根组织中的侵入运动和行为也有可能揭示限制生命周期完成的抗性反应的本质。一个主要挑战是，目前在感染早期识别植物根内线虫的方法要么具有破坏性，要么无法提供足够的时间或空间分辨率。荧光化合物已被用来追踪寄生线虫感染期间宿主植物组织中的溶质运动（Böckenhoff 等，1996；Hofmann 等，2007；Hutangura，1999）。荧光蛋白的表达也被用于研究摄食细胞生物学（Hofmann 和 Grundler，2006；Hoth 等，2005；Hoth 等，2008），并且是识别植物根部特定细胞类型的常用方法（Birnbaum 等，2003；Brady 等，2007；Lee 等，2006）。因此，线虫感染初始阶段的研究将极大地受益于基于荧光的方法，该方法还能够以高空间分辨率识别植物根部中的入侵线虫。最近使用异硫氰酸荧光素 (FITC) 对活胞囊线虫进行荧光标记（Schroeder 和 MacGuidwin，2007）。 FITC 是一种荧光素偶联物，也已用于显示 J2 RKN 从溶液中的摄取（Rosso 等人，2005）。早期的一项研究利用不同的荧光素结合物荧光素二乙酸酯 (FDA) 来标记 RKN 幼虫并区分活线虫和死线虫 (Bird, 1979)。 FDA 是一种非荧光结合物，进入细胞并被内源性酯酶水解后会发出荧光。除了促进活线虫的识别之外，背景荧光信号和大量清洗的要求也保持在最低限度，因为 FDA 不发出荧光，并且游离荧光素保留在活细胞中。在这项研究中，我们测试了 FDA 是否可以用于荧光标记单个线虫，以便直接无损观察它们在植物根部内的运动。
Plant endoparasitic nematodes spend most of their life cycle hidden within plant roots and worldwide cause major losses to agriculture every year. Root-knot nematodes (‘RKNs’, Meloidogyne spp.) are sedentary endoparasitic nematodes that establish permanent feeding sites within plant roots as 2nd-stage juveniles (J2), and then spend the rest of their life-cycle at this site. In contrast, root-lesion nematodes (Pratylenchus spp.) are migratory endoparasites that move in roots and feed on different root cells, causing typical symptoms of brown lesions. There is great interest in studying the initial infection stages of both sedentary and migratory endoparasitic nematodes. In the case of RKNs, it is known that host cells undergo key initial changes of cell-cycle activation, DNA synthesis and nuclear division during the first 48 hr of infection and host cell selection (de Almeida Engler et al., 1999; Jones, 1981; Jones and Payne, 1978; Niebel et al., 1996). In the case of root-lesion nematodes, tracking invasive movement and behavior in root tissues also has potential to reveal the nature of resistant responses limiting the completion of the life-cycle. A major challenge is that current methods to identify nematodes inside plant roots during early stages of infection are either destructive or do not provide sufficient temporal or spatial resolution. Fluorescent compounds have been used to track solute movement in host plant tissues during parasitic nematode infection (Böckenhoff et al., 1996; Hofmann et al., 2007; Hutangura, 1999). Expression of fluorescent proteins has also been used to study feeding cell biology (Hofmann and Grundler, 2006; Hoth et al., 2005; Hoth et al., 2008) and is a common approach to identify specific cell types in plant roots (Birnbaum et al., 2003; Brady et al., 2007; Lee et al., 2006). Research on initial stages of nematode infection would thus greatly benefit from a fluorescence-based approach that would also enable invading nematodes to be identified in plant roots with high spatial resolution. Live cyst nematodes have recently been marked fluorescently using fluorescein isothiocyanate (FITC) (Schroeder and MacGuidwin, 2007). FITC is a fluorescent fluorescein conjugate that has also been used to show uptake from solution by J2 RKNs (Rosso et al., 2005). An earlier study made use of a different fluorescein conjugate, fluorescein diacetate (FDA), to label RKN juveniles and to distinguish between living and dead nematodes (Bird, 1979). FDA is a non-fluorescent conjugate that becomes fluorescent after entering a cell and being hydrolyzed by endogenous esterases. In addition to facilitating identification of live nematodes, background fluorescent signals and requirement for extensive washing are kept to a minimum because FDA does not fluoresce, and free fluorescein remains in live cells. In this study, we tested whether FDA can be applied to label individual nematodes fluorescently for direct non-destructive observation of their movement inside plant roots.