Centre for Plant Sciences, University of Leeds, Leeds, United Kingdom ; Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee, United Kingdom.
Centre for Plant Sciences, University of Leeds, Leeds, United Kingdom.
PLoS One. 2014 Jan 28;9(1):e87289. doi: 10.1371/journal.pone.0087289. eCollection 2014.
Plant parasitic nematodes comprise several groups; the most economically damaging of these are the sedentary endoparasites. Sedentary endoparasitic nematodes are obligate biotrophs and modify host root tissue, using a suite of effector proteins, to create a feeding site that is their sole source of nutrition. They feed by withdrawing host cell assimilate from the feeding site though a structure known as the feeding tube. The function, composition and molecular characteristics of feeding tubes are poorly characterised. It is hypothesised that the feeding tube facilitates uptake of host cell assimilate by acting as a molecular sieve. Several studies, using molecular mass as the sole indicator of protein size, have given contradictory results about the exclusion limits of the cyst nematode feeding tube. In this study we propose a method to predict protein size, based on protein database coordinates in silico. We tested the validity of these predictions using travelling wave ion mobility spectrometry--mass spectrometry, where predictions and measured values were within approximately 6%. We used the predictions, coupled with mass spectrometry, analytical ultracentrifugation and protein electrophoresis, to resolve previous conflicts and define the exclusion characteristics of the cyst nematode feeding tube. Heterogeneity was tested in the liquid, solid and gas phase to provide a comprehensive evaluation of three proteins of particular interest to feeding tube size exclusion, GFP, mRFP and Dual PI. The data and procedures described here could be applied to the design of plant expressed defence compounds intended for uptake into cyst nematodes. We also highlight the need to assess protein heterogeneity when creating novel fusion proteins.
植物寄生线虫包括几个类群；其中最具经济破坏性的是固着内寄生线虫。固着内寄生线虫是专性生物营养体，它们利用一系列效应蛋白来修饰宿主根组织，从而创建一个仅作为其营养来源的取食位点。它们通过称为取食管的结构从取食位点撤回宿主细胞同化产物来进行取食。取食管的功能、组成和分子特征尚未得到充分描述。据推测，取食管通过充当分子筛来促进宿主细胞同化产物的吸收。有几项研究仅使用分子质量作为蛋白质大小的唯一指标，这些研究对胞囊线虫取食管的排除限制得出了相互矛盾的结果。在这项研究中，我们提出了一种基于蛋白质数据库坐标的预测蛋白质大小的方法。我们使用行波离子淌度 - 质谱法测试了这些预测的有效性，预测值和测量值之间的差异约为 6%。我们使用这些预测值结合质谱、分析超速离心和蛋白质电泳来解决先前的冲突，并定义胞囊线虫取食管的排除特征。我们在液体、固体和气相中测试了异质性，为特别关注取食管大小排除的三种蛋白质 GFP、mRFP 和 Dual PI 的异质性提供了全面评估。这里描述的数据和程序可应用于设计旨在被胞囊线虫吸收的植物表达防御化合物。我们还强调在创建新型融合蛋白时需要评估蛋白质异质性。
