Core effectors and host targets of plant parasitic nematodes

植物寄生线虫（PPN）对世界上所有主要作物造成危害。最具破坏性的PPN是定居的内寄生物种。PPN的主要分支之一包括囊肿线虫（Globodera/Heterodera）、肾形线虫（Rotylenchulus reniformis）和假根结线虫（Nacobbus aberrans）。这些线虫在寄主植物的根部诱导一个大的多核、代谢活跃的取食部位。虽然它们的形态不同，但它们都是合胞体的一种，由线虫选择的初始细胞和邻近细胞之间的细胞壁破裂形成，然后是原生质体的融合。线虫一生中只能诱导一个合胞体。因此，合胞体必须保持活力并免受宿主防御反应的保护，以提供线虫所需的营养。PPN与宿主的相互作用，包括诱导和维持摄食部位，是由效应物介导的；线虫分泌的操纵宿主生物化学以使寄生虫受益的蛋白质。效应物已经在一系列ppn中被发现，并且通常以大基因家族的形式存在。了解效应物的功能作用和诱导/保护合胞体的分子基础，为开发新的寄生虫防治策略提供了前景。本项目的主管参与了苍白球虫和罗斯托奇球虫、肾形球虫和畸变球虫基因组/转录组数据的生成。我们已经开发了生物信息学管道，允许从这些数据中识别候选效应物。该项目的主要目的是利用这些数据来识别“核心”宿主蛋白质或过程，这些蛋白质或过程由所有物种操纵，它们的本质可能是线虫与其宿主植物之间相互作用的关键参与者。为了实现这一目标，将确定在所有三种线虫群之间保守的“核心”效应物的目标。项目前期可预见的主要工作领域如下：“核心”效应物的鉴定：鉴定的三个属跨越门的两个关键分支，并拥有大型序列数据库，包括基因组和转录组信息。来自三个线虫类群的所有基因将被聚类，并且那些包含所有类群中包含先前确定的效应物的序列的基因将被突出显示。此外，假定的新的保守效应簇将通过用于区分效应与管家基因的特征（用于分泌的信号肽和与寄生作用一致的转录谱）的存在来识别。在这两种情况下，这些分析将通过原位杂交验证，以确认这些序列在线虫组织中表达，并具有向植物分泌蛋白质的能力。保守宿主靶点/过程的鉴定：这三个属都能感染一些常见的宿主，如番茄和马铃薯，并产生具有相似特征的取食点。因此，很可能必须操纵普通宿主蛋白或过程。最初将重点放在物种之间的那些保守效应上，可以最大限度地确定这些“关键参与者”。将采用两种互补的策略。首先，在植物中标记过表达的效应物将被拉下来，并通过质谱法鉴定与效应物结合的任何伴随的宿主蛋白。其次，利用效应剂筛选酵母-2杂交文库（马铃薯和番茄）。相互作用将通过共免疫沉淀和/或FRET分析来确认。工作的后期阶段将取决于在第一阶段获得的数据。关键实验可能包括通过过表达、沉默或基因组编辑对保守靶点在线虫感染中的作用进行功能分析。它们也可能包括效应靶相互作用的结构分析。

Plant-parasitic nematodes (PPN) cause damage to all major crops across the world. The most damaging PPN are the sedentary endoparasitic species. One of the major clades of PPN includes the cyst nematodes (Globodera/Heterodera spp.), the reniform nematode Rotylenchulus reniformis and the false root-knot nematode Nacobbus aberrans. These nematodes induce a large multinucleate, metabolically active feeding site in the root of their host plants. Although different in their morphology, each is a type of syncytium, formed by the breakdown of the cell wall between the initial cell selected by the nematode and its neighbours followed by fusion of the protoplasts. The nematodes can only induce one syncytium in their life cycle. The syncytium must therefore be kept alive and protected from host defence responses in order to provide the nutrients required by the nematode. The interactions of PPN with their hosts, including the induction and upkeep of the feeding site, are mediated by effectors; proteins secreted by the nematode that manipulate host biochemistry to benefit the parasite. Effectors have been identified in a range of PPNs and are often present as large gene families. Understanding the functional roles of effectors and the molecular basis by which syncytia are induced/protected offers the prospects of new control strategies against these parasites. The supervisors of this project have been involved in the generation of genome/transcriptome data from Globodera pallida and G. rostochiensis, R. reniformis and N. aberrans. We have developed bioinformatic pipelines allowing for candidate effectors to be identified from these data. The main aim of this project is to use the data to allow the identification of "core" host proteins or processes, manipulated by all species, which by their very nature are likely to be key players in the interaction between the nematode and its host plant. To achieve this, the targets of "core" effectors, conserved between all three nematode groups, will be identified. The main work areas that are foreseen for the early part of the project are as follows; Identification of "core" effectors: The three genera identified span two key bifurcations in the phylum, and have large sequence databases, including genome and transcriptome information. All genes from the three nematode groups will be clustered, and those containing sequences from all groups which contain previously identified effectors will be highlighted. In addition, putative novel conserved effector clusters will be identified by the presence of signatures used to distinguish effectors from house-keeping genes (signal peptide for secretion and transcriptional profile consistent with a role in parasitism). In both cases, these analyses will be validated by in situ hybridisation to confirm that these sequences are expressed in nematode tissues with the capacity to secrete proteins into plants. Identification of conserved host targets/processes: All three genera can infect some common hosts, e.g. tomato and potato, and produce feeding sites with similar features. It is therefore likely that common host proteins or process must be manipulated. Initially focusing on those conserved effectors between species maximises the likelihood of identifying such 'key players'. Two complementary strategies will be used. Firstly, tagged effectors over expressed in planta will be pulled down, and any accompanying host proteins bound to the effectors will be identified by mass spectrometry. Secondly, yeast-2-hybrid libraries (potato and tomato) will be screened with effectors. Interactions will be confirmed by co-immunoprecipitation and/or FRET analysis. The later stages of the work will depend on the data obtained in the first phases. Key experiments may include functional analysis of the roles of conserved targets in nematode infection through over-expression, silencing, or genome editing. They may also include structural analysis of effector-target interactions.