Phytopathology. 2014 May;104(5):497-506. doi: 10.1094/PHYTO-08-13-0240-R.
The secluded lifestyle of endoparasitic plant nematodes hampers progress toward a comprehensive understanding of plant-nematode interactions. A novel technique that enables nondestructive, long-term observations of a wide range of live nematodes in planta is presented here. As proof of principle, Pratylenchus penetrans, Heterodera schachtii, and Meloidogyne chitwoodi were labeled fluorescently with PKH26 and used to infect Arabidopsis thaliana grown in microscopy rhizosphere chambers. Nematode behavior, development, and morphology were observed for the full duration of each parasite's life cycle by confocal microscopy for up to 27 days after inoculation. PKH26 accumulated in intestinal lipid droplets and had no negative effect on nematode infectivity. This technique enabled visualization of Meloidogyne gall formation, nematode oogenesis, and nematode morphological features, such as the metacorpus, vulva, spicules, and cuticle. Additionally, microscopy rhizosphere chambers were used to characterize plant organelle dynamics during M. chitwoodi infection. Peroxisome abundance strongly increased in early giant cells but showed a marked decrease at later stages of feeding site development, which suggests a modulation of plant peroxisomes by root-knot nematodes during the infection process. Taken together, this technique facilitates studies aimed at deciphering plant-nematode interactions at the cellular and subcellular level and enables unprecedented insights into nematode behavior in planta.
内寄生植物线虫的隔离生活方式阻碍了对植物-线虫相互作用的全面理解。本文提出了一种新的技术，可以对植物中广泛的活体线虫进行非破坏性、长期观察。作为原理验证，用 PKH26 荧光标记根结线虫、大豆胞囊线虫和南方根结线虫，并将其用于感染在显微镜根际室中生长的拟南芥。通过共聚焦显微镜，在接种后长达 27 天的时间内，观察到线虫的行为、发育和形态，直到每个寄生虫生命周期的全部时间。PKH26 积累在肠道脂滴中，对线虫的感染力没有负面影响。该技术能够可视化南方根结线虫的虫瘿形成、线虫卵母细胞发生以及线虫形态特征，如后体、阴道、刺和角质层。此外，还使用显微镜根际室来描述南方根结线虫感染过程中植物细胞器的动态变化。过氧化物酶体在早期巨型细胞中大量增加，但在食窦发育的后期阶段明显减少，这表明根结线虫在感染过程中对植物过氧化物酶体进行了调节。总之，该技术有助于研究植物-线虫相互作用的细胞和亚细胞水平，并使人们对线虫在植物体内的行为有了前所未有的了解。
