The juxtaposition of variability and stability in the HYP effectors of globally important plant-parasites.

Plant-parasitic nematodes threaten current and future global food security, and are estimated to cost world agriculture over $100 billion per year. In the UK, the most economically important species are the "potato cyst nematodes", which cause over £50 million of damages to the potato industry each year. Given that potato cyst nematodes are already present in every major potato growing region of the world, including emerging markets, a strong case can be made for exploring any area of parasite biology that may lead to new control measures. One area of parasite biology of increasing interest is "effectors" - nematode-derived molecules injected into the plant to cause disease.


In this proposal we will focus our efforts on an unusual group of effectors called "HYPs", for two main reasons: Firstly, the genetics underlying HYPs are academically fascinating; Secondly, disrupting HYPs could lead to robust nematode control.

Understanding the unusual genetics of HYP effectors:
All of the 75 unique HYP genes identified to date share two continuous strings of coding sequence that are near identical between genes: together, these "conserved regions" make up approximately half of the total gene length. What is remarkable about HYPs is that between these two near-identical conserved regions lies a "hyper-variable domain". This hyper-variable domain can encode several "motifs" of variable sequence and organisation with almost no observable patterns. To add to the complexity, no one individual nematode genome encodes all HYPs, and no two individuals tested encode the same repertoire of HYPs. To the best of our knowledge, there is no known mechanism that can account for both the hyper-variable domain organisations and the gene number variation between sisters of the same population.

To understand the combination of HYP genetic variability and stability, we need to know where HYPs are in the genomes of individuals, and how they are arranged. To do this we will use recent advances in technology to sequence HYP-containing ultra-long DNA molecules from individual nematodes. While we will not be able to determine whether any two molecules came from the same individual (even if they are identical), we can be absolutely certain that each read came from an individual. We can use these ultra-long DNA sequences, in combination with a high-quality consensus genome of the population, to explore the genetic basis of HYP genetic variability and stability.

Disrupting HYP function:
We know that the sequence of HYP genes are "evolutionarily constrained". The species which contain HYP effectors diverged from one another approximately 30 million years ago, and yet, since that time, the conserved regions of their HYP genes have remained almost unchanged. It stands to reason that there must be some explanation why the nucleotide sequence is so highly conserved, in spite of the tremendous diversity of the variable domain. Given the evolutionary constraint on HYPs, targeting the conserved regions in all HYP genes, in all potato cyst nematode species, may produce strong, durable, and broad spectrum resistance.

To disrupt HYP function we propose to use a targeted approach called RNA interference. RNA interference can disrupt the function of a gene based on its sequence. By targeting RNA interference to the near-identical conserved regions, we can disrupt the function of all HYP genes, in all potato cyst nematodes. To deliver RNA interference to nematodes during infection, we will create transgenic potato plants that encode an RNA interference gene which targets the nematode HYPs. When the nematode feeds on the plant, they eat the interfering RNA, and their corresponding HYP genes are disrupted.


This proposal thus combines interesting how/what/why pure science questions (about the genomic evolution of this system) with real potential for an agricultural impact.

This proposal is designed to understand the HYP effectors of globally economically important potato cyst nematodes in sufficient detail to develop disease resistance.

Aims
1) Reconstruct the local genomic neighbourhood of HYP effectors in individuals to understand the unprecedented genomic variability.
Based on unique non-HYP surrounding sequence, we will assign ultra-long HYP containing single DNA molecules from individuals (generated using Oxford Nanopore sequencing), onto a highly contiguous consensus genome assembly of the population (generated using PacBio sequencing).


2) Determine the full extent of HYP diversity, including in successful (female) and non-successful (male) individuals, to accurately target multiple HYP members and multiple nematode species.
We will exploit the genetic structure of HYPs by using homology-dependent hybridisation to biotin labelled oligonucleotide "probes" to selectively capture, and ultimately sequence, the HYP complement of different species, populations, and life stages of potato cyst nematode.


3) Generate a HYP loss-of-function phenotype in planta to understand how HYPs contribute to parasitism.
All HYP genes will be aligned, and a number of chimeric consensus sequences synthesised that share at least 50 contiguous bp at 100% identity, and 100 contiguous bp >95% identity, with every individual HYP gene. RNA interference hairpin constructs, generated from these chimeric sequences, will be cloned under the expression of the strong CaMV35S promoter, transformed into Agrobacterium tumefaciencs, and ultimately used to generate transgenic potato plants (cv Desirée). Transgenics will be used to determine the HYP loss-of-function phenotype, and the efficacy of targeting HYPs to control potato cyst nematodes, through nematode infection trials.

The beneficiaries of this proposal are detailed in the relevant sections of the JeS form and the pathways to impact. To summarise, my proposal has substantial potential to impact the academic, societal, and industrial sectors. A series of measures are detailed to realise that impact over the course of the grant, and these timescales are detailed in the diagrammatic work plan.

Academic impact:
The major academic impact of the proposal is an improved understanding of the genomic basis of genetic stability and variability. One of the central hypotheses of this proposal is that the juxtaposition of variability and stability in the HYP effectors is the result of as yet undescribed mechanisms. Such mechanisms have the potential for broad academic impact outside plant-pathology. In order to address this hypothesis, we need to generate sufficiently contiguous genomic resources for the most important UK plant-parasitic nematodes. While not the aim of this proposal, these resources will nevertheless also positively impact the academic community well beyond the lifetime of the proposal.

Societal impact:
Informing the public, and training the next generation of scientists, is our responsibility. During the grant, we will continue to train young plant scientists to address the major global issue of food security. The engagement infrastructure at the University of Cambridge will help appropriately deliver the messages of the outcomes to a broad spectrum of audiences.

Industrial impact:
Although a challenging aim, the long-term goal of the research is to develop approaches with the potential to increase agricultural stability by combatting plant-parasitic nematodes. There are a number of potential routes to realise industrial impact in the UK and globally. Globally, potato is the most important non-cereal food crop and represented an economic value of over $30 billion (USD) in 2008. The British Potato Council estimated the UK potato production, processing, and retail markets to be worth around £3 billion per annum.