Molecular analysis of the eggshell and cuticle surface of the potato cyst nematode, Globodera rostochiensis (dataset)

Plant-parasitic nematodes regulate their interactions with the external environment and their host. The surface of the nematodes, whether the cuticle or the eggshell, plays a key role in this process. Work undertaken in this project aimed to exploit new genome resources for potato cyst nematodes (PCN) to allow characterisation of these structures. The eggshell is a key survival structure for PCN. Hatching of juvenile potato cyst nematodes occurs in response to host derived hatching factors. Little is known about the molecular mechanisms that underpin hatching in response to these host diffusates and nothing is known about the molecular composition of the PCN eggshell. Methods that allow extraction of proteins and lipids from large numbers of isolated PCN eggshells were developed. These extraction techniques permitted identification of a range of proteins present in the eggshell, including chondroitin proteoglycans and a calcium dependent phospholipid-binding annexin. The annexin was focused on due to the apparent major role of calcium in PCN hatching. This annexin was localised to the eggshells of Globodera rostochiensis by immunofluorescence. To our knowledge, this makes the annexin the first eggshell protein to be identified and localised in any plant-parasitic nematode. The annexin was shown to bind to phospholipids and RNAi of the annexin showed that reducing expression of the eggshell annexin alters nematode hatching patterns in response to host hatching factors. The eggshell permeability barrier is essential for the development of a juvenile within the embryo. Eggshell lipid analysis has given insight into components of the permeability barrier. Identified lipid species highlight potential function within the nematode hatching cascade through calcium interaction or down stream signalling. Attempts to identify the presence of nematode specific glycolipids, ascarosides, within the PCN eggshell showed that ascarosides or ascarylose were not detectable within PCN eggs, cysts or juveniles. It has previously been shown that the infective juveniles are able to alter their surface composition to avoid damage from host defence mechanisms. Here, methods that allow labelling of proteins present on the cuticle surface of PCN were developed. Three proteins; Galectin-5, Cri-2 and a nematode specific hypothetical protein that are present on the surface of PCN second stage juveniles were further investigated. These proteins were tested for their potential to suppress host defences through a variety of protein-small molecule interactions. This analysis showed that surface coat associated galectins could offer protection to an invasive juvenile through sequestering cell wall breakdown products, silencing detection of the pathogen.

植物寄生线虫可调控自身与外界环境及宿主之间的相互作用。线虫体表（无论是角质层还是卵壳）在此过程中发挥关键作用。本项目旨在利用马铃薯胞囊线虫（potato cyst nematodes, PCN）的新型基因组资源，对上述结构进行表征分析。卵壳是PCN的关键存活结构。马铃薯胞囊线虫幼虫的孵化受宿主产生的孵化因子诱导。目前，关于这些宿主扩散物诱导孵化的分子机制知之甚少，且PCN卵壳的分子组成尚属未知。本研究建立了从大量分离的PCN卵壳中提取蛋白质和脂质的方法。这些提取技术成功鉴定出卵壳中的多种蛋白质，包括软骨素蛋白聚糖（chondroitin proteoglycans）和一种钙依赖性磷脂结合膜联蛋白（annexin）。鉴于钙在PCN孵化中明显的重要作用，本研究重点关注该膜联蛋白。通过免疫荧光技术，该膜联蛋白被定位到罗氏球胞囊线虫（Globodera rostochiensis）的卵壳上。据我们所知，这是首个在植物寄生线虫中被鉴定并定位的卵壳蛋白。研究表明，该膜联蛋白可与磷脂结合；通过RNA干扰（RNA interference, RNAi）抑制该膜联蛋白表达后，线虫对宿主孵化因子的响应模式发生改变。卵壳渗透屏障对胚胎内幼虫的发育至关重要。卵壳脂质分析为渗透屏障的成分研究提供了线索。已鉴定的脂质种类揭示了其通过钙相互作用或下游信号传导在线虫孵化级联反应中的潜在功能。本研究尝试鉴定PCN卵壳中线虫特异性糖脂（即ascarosides）的存在，但结果显示在PCN卵、胞囊或幼虫中均未检测到ascarosides或ascarylose。已有研究表明，侵染性幼虫可通过改变体表成分来规避宿主防御机制的损伤。本研究建立了PCN角质层表面蛋白质的标记方法。研究人员进一步分析了PCN二龄幼虫体表的三种蛋白质：半乳糖凝集素-5（Galectin-5）、Cri-2以及一种线虫特异性假设蛋白（hypothetical protein）。研究人员通过多种蛋白质-小分子相互作用实验，检测了这些蛋白质抑制宿主防御的潜力。分析结果表明，体表被膜相关的半乳糖凝集素可通过结合细胞壁降解产物，抑制宿主对病原体的识别，从而为侵染性幼虫提供保护。