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，马铃薯孢囊线虫（PCN）可能导致苏格兰马铃薯种植业损失1.25亿英镑。最近，我们对植物病原体抗性的分子机制的理解取得了快速进展。然而，这已经建立了在叶片中使用叶面病原体和鲜为人知的机制，对根攻击线虫的抗性基础。H2基因是一个主要抗性基因，对马铃薯的Pa 1致病型有较强的抗性，对Pa 2/3致病型有较强的抗性，它位于马铃薯基因组的0.8MB区域。含H2的马铃薯品系P55/7的RenSeq富集测序和PacBio测序揭示了多个候选基因，并且KASP标记测定表明其中两个与抗性表型高度相关。这些候选基因将在易感马铃薯品系中表达，作为鉴定真正H2基因的初步努力。同时，已经筛选了P55/7的BAC文库以获得携带这些候选者的抗性等位基因的克隆，这些克隆将被测序以揭示可能的邻近候选者。如果有必要，序列数据也可以用于进一步精细定位H2。已经鉴定了多个马铃薯物种Solanum multisdisctum的种质，并获得了它们的真实种子。这些将被筛选为G。pallida抗性，并且抗性基因序列将通过RenSeq富集测序获得。这将用于鉴定H2基因的变体，其中SNP可以与线虫抗性相关，作为H2抗性功能分析的第一步。最近，联邦马铃薯保藏中心的多个品种已被PacBio和RenSeq测序，以确定抗性基因。这些材料将被筛选出对多个PCN物种的抗性，从而产生新的PCN抗性基因。抗性基因的重新定位是防御信号传导的关键组成部分，这将在H2中进行探索。参与H2防御信号传导的相互作用蛋白将通过标记H2的下拉和质谱法来鉴定。H2的信号传导网络将进一步通过产生组成型活性H2突变体来表征。这将通过随机诱变产生并在烟草叶片中瞬时表达，用于筛选诱导细胞死亡的抗性基因。耐药相关的多态性在S.多裂属种质也可用于靶向诱变方法。组成型活性的H2突变体将在各种防御信号组分缺陷的番茄品系中表达，以阻断H2介导的信号传导途径。通过在烟草中的瞬时共表达，将H2转化到pallida Pa 2/3致病型中。H2的鉴定和表征将揭示支撑线虫抗性的分子机制。这将有助于抗线虫作物品系的开发。开发遗传抗性品系是减轻作物产量损失的关键策略，也符合目前减少作物生产中农药和化肥使用的目标。

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.