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

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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.