Resistance in crop roots: functional analysis of the H2-mediated response to nematodes

植物寄生线虫是粮食安全的重大威胁，造成重大但往往被忽视的产量损失。如果不加以解决，到2040年，马铃薯胞囊线虫(PCN)可能导致仅苏格兰马铃薯种植业就损失1.25亿GB。近年来，我们对支撑植物病原菌抗性的分子机制的理解有了很大的进步。然而，这在很大程度上是利用叶片病原体在叶片中建立的，而对支撑对侵根线虫的抗性的机制知之甚少。抗性基因H2最近被定位在马铃薯基因组的0.8MB区域，该基因对PCN种的PA1致病型具有主要抗性，对Pa2/3致病型具有部分抗性。对含H2的马铃薯品系P55/7进行RenSeq富集测序和PacBio测序，发现了多个候选基因，KASP标记分析表明其中两个基因与抗性表型高度相关。这些候选基因将在一个敏感的马铃薯品系中表达，作为鉴定真正的H2基因的初步努力。同时，已经对P55/7的BAC文库进行了筛选，以寻找携带这些候选的抗性等位基因的克隆，并将对其进行测序，以揭示可能的邻近候选。如有必要，序列数据还可用于进一步精细定位H2。已鉴定了多个衍生H2的马铃薯品种的种质，并获得了它们的真实种子。将对这些菌株进行梅毒螺旋体抗药性筛选，并通过RenSeq浓缩测序获得抗药性基因序列。这将被用来识别H2基因的变异，从中SNPs可以与线虫抗性相关，作为对H2抗性进行功能分析的第一步。最近，英联邦马铃薯种质资源中的多个材料被进行了PacBio和RenSeq测序，以鉴定抗性基因。这些材料将对可能产生新的PCN抗性基因的多个PCN物种进行抗性筛选，并将通过产生荧光标记进行进一步的功能鉴定。抗性基因的重新定位是防御信号的关键组成部分，这将在H2中进行探索。参与H2防御信号的相互作用蛋白将通过标记的H2的下拉和质谱分析来鉴定。H2的信号网络将进一步通过产生具有结构性活性的H2突变体来表征。这将通过随机诱变和瞬时表达在烟草叶片中产生，用于筛选抗性基因诱导的细胞死亡。在多裂链霉菌种质测序中发现的抗性相关多态也可用于定向突变方法。H2突变体将在缺乏多种防御信号成分的番茄品系中表达，以表征H2介导的信号转导途径。突变体还可以通过瞬时共表达在烟草中筛选可能的细胞死亡抑制因子。H2的鉴定和鉴定将揭示支撑线虫抗性的分子机制。这将有助于培育抗线虫作物品系。开发转基因抗性品系是减轻作物产量损失的关键战略，符合当前减少作物生产中农药和化肥使用量的目标。

Plant parasitic nematodes are a significant threat to food security, causing major but often overlooked yield loss. If unaddressed, the potato cyst nematode (PCN) could result in £125m losses to the potato growing industry in Scotland alone by 2040. Recently, there have been rapid advancements in our understanding of the molecular mechanisms that underpin plant-pathogen resistance. However, much of this has been established in the leaf using foliar pathogens and less is known on the mechanisms underpinning resistance against root-attacking nematodes. The resistance gene H2 which provides major resistance to the Pa1 pathotype of the PCN species Globodera pallida and partial resistance to the Pa2/3 pathotype has recently been mapped to a 0.8MB region of the potato genome. RenSeq enrichment sequencing and PacBio sequencing of the H2-containg potato line P55/7 revealed multiple candidate genes, and a KASP marker assay indicated that two of these are highly associated with the resistant phenotype. These candidates will be expressed in a susceptible potato line as an initial effort to identify the true H2 gene. Simultaneously, a BAC library of P55/7 has been screened for clones carrying the resistant allele of these candidates which will be sequenced to reveal possible neighbouring candidates. If necessary, the sequence data can also be used for further fine mapping of H2.Multiple accessions of the potato species Solanum multidissectum from which H2 is derived have been identified and their true seed obtained. These will be screened for G. pallida resistance, and resistance gene sequences will be obtained via RenSeq enrichment sequencing. This will be used to identify variants of the H2 gene, from which SNPs can be correlated to nematode resistance as a first step towards functional analysis of H2 resistance. Recently, multiple accessions in the Commonwealth Potato Collection have been PacBio and RenSeq sequenced to identify resistance genes. These accessions will be screened for resistance to multiple PCN species which could yield novel PCN resistance genes.Further functional characterisation will be carried out by producing fluorescently tagged. Re-localisation of resistance genes is a key component of defence signalling, and this will be explored for H2. Interacting proteins involved in H2 defence signalling will be identified through a pull-down and mass spectrometry of tagged H2. The signalling network of H2 will be further characterised by the production of a constitutively active H2 mutant. This will be produced by random mutagenesis and transient expression in tobacco leaves for screening of resistance gene induced cell death. Resistance associated polymorphisms identified in the sequencing of S. multidissectum accessions may also be used in a targeted mutagenesis approach. The constitutively active H2 mutant will be expressed in tomato lines deficient in various defence signalling components to characterise the H2-mediated signalling pathway.The mutant can also be used to screen for putative cell-death suppressors in the G. pallida Pa2/3 pathotype by transient co-expression in tobacco.The identification and characterisation of H2 will reveal the molecular mechanisms that underpin nematode resistance. This will contribute to the development of nematode resistant crop lines. Developing genetically resistant lines is a key strategy to mitigate crop yield loss and agrees with current goals to reduce pesticide and fertiliser usage in crop production.