Controlling important diseases in potato by cloning functional NB-LRR-type resistance genes

Lead Research Organisation: Earlham Institute
Department Name: Research Faculty

Abstract

Potato is one of the world's most important food crops, and production is threatened by several pests and pathogens that severely reduce crop yield and quality. These pathogens include late blight, the organism that caused the infamous Irish potato famine, soil-dwelling cyst nematodes that attack the roots, and a number of bacteria and viruses. Current control methods for many of these pathogens in most parts of the world are based mainly on the use of chemical sprays which are both environmentally hazardous and often ineffective. Moreover, some of these chemicals, for example nematicides, are being removed from use by EU legislation, severely limiting the ability of growers to control cyst nematodes. Modelling suggests that global potato production could increase by up to a third if the diseases that reduce yields could be controlled.

It is widely thought that the most promising and environmentally benign route for combating crop pests and diseases is by deployment of naturally-occurring plant resistance genes. In plants, including potato, disease resistance is typically controlled by resistance genes, known as 'R genes' that have a recognizable DNA 'domain' structure. In the plant innate immune system, the R proteins encoded by R genes provide resistance by recognising 'effector' molecules produced by the pathogen and then activating various defence processes. Hence R genes are key players in plant disease resistance, and they are effective towards diverse pathogens of all major types (oomycetes, fungi, viruses, nematodes and bacteria). We recently analysed the published potato genome and identified ~760 R genes using a novel technique we have developed for rapid isolation of all R genes from potato varieties or wild species. This new method, called RenSeq (R gene enrichment sequencing), takes advantage of recent developments in genome sequencing and in 'capturing' targeted DNA sequences from complex mixtures. Using this technique we can now sequence the R genes, which represent only about 0.24% of the DNA in the potato genome, from any potato or closely related plant in an efficient and cost effective manner. Moreover, given the appropriate sequence information, this method can be easily applied to other crop species.

This project makes use of the wide diversity of R genes present in wild potatoes and other Solanaceae. The main aims of this project are to combine the RenSeq method with the latest sequencing technologies to determine the R gene complements of selected Solanum species harbouring functionally effective resistances against one or more potato pathogen. We will use the sequence data generated to identify markers and gene candidates for functional resistances using an efficient genetic pooling approach. This will make use of existing crosses between resistant and susceptible plants to map the functional resistances to a chromosomal position in the plant genome. RenSeq will be deployed to sequence all R genes in the parents of crosses as well as pools of offspring plants that are resistant and susceptible. Comparing these sequence data using new bioinformatics approaches we will develop genetic markers that are within or immediately flanking the genes conferring resistance. Markers will be deployed via conventional plant breeding to select for resistance and candidate genes will be subjected to functional studies for potential future biotechnological deployment. A key aspect of this project is the partnership between the JHI, UoD and TSL, the plant genome sequencing expertise of TGAC as well as the link with Simplot, who will test resistance genes in field trials in the USA.

Technical Summary

We have developed RenSeq, an NB-LRR-specific enrichment and sequencing workflow that enables a detailed annotation of R genes in genome sequences and rapid mapping of resistances in segregating populations. The underlying principle for this project is RenSeq-based comparative bulked segregant analysis to reveal candidate R genes among closely-related paralogous and non-functional alleles.

RenSeq will be used to generate error corrected, long, R gene-specific sequences from parental plants (Objective 1). Up to 1.5 kb, highly accurate sequencing reads obtained by combining PacBio with MiSeq paired-end reads will be assembled to generate high-quality contigs that best describe the NB-LRRs in the resistant and susceptible parents, respectively, to enable discrimination between alleles and paralogs.

In Objective 2, shorter but highly accurate MiSeq-based RenSeq reads will be obtained from bulked resistant and bulked susceptible plants and compared to the parental NB-LRRs established in Objective 1. Candidate NB-LRRs are unique to the resistant parent as well as the resistant bulks and absent in the corresponding susceptible plants.

In Objective 3, transgenic potato lines expressing candidate R genes will be used to verify resistance to corresponding pathogens. In parallel, we will take advantage of on-going complementary research to identify and clone effectors from P. infestans, G. pallida and PVY. From these we will identify avirulence (Avr) genes matching the R genes in this study by co-infiltration with R gene candidates in the model Solanaceae N. benthamiana. Identification of avirulences will facilitate prediction of R genes durability. Markers will be developed from functional R genes to aid marker assistant selection in the JHI/MRS breeding programme.

In Objective 4, together with the industrial partner Simplot, we will test and combine R genes effective against late blight, PCN and PVY in selected US/UK potato cultivars using Golden Gate cloning.

Planned Impact

Who might benefit from this research?
The immediate beneficiaries of this research will include other academic researchers, as described in detail above. This project has been developed by the academic partners in collaboration with our commercial sponsor Simplot, who have a major investment in potato through ownership of various processed potato products. One of the main outputs of this project will be technical 'know-how' of how to efficiently isolate resistance gene repertoires from potato, which will be applicable to any plant. A second major output is a large amount of information about potato resistance genes, including some genes of known function that can be exploited via potato breeding as well as by biotechnological approaches. This project therefore will provide information, namely markers and functional resistances that will benefit those engaged in potato improvement, such as breeders and companies that generate varieties for the UK fresh and processed markets. JHI is ideally placed to exploit this link through its commercial arm, Mylnefield Research Services (MRS), which runs potato breeding programmes for all major national potato producers, enabling ready routes to translate research outcomes to UK industry. In the long term, breeding of other crop species will benefit from generic technologies developed in this proposal.

Our project is unusual in that our industrial partner is a US-based company that has extensive reach into the development of a diverse array of processed potato products. We believe that Simplot has a forward looking strategy for procuring improved raw material for their product range, and are not averse to the use of the latest technologies. We seek the best acceptable route to deploy resistances to important pathogens that will ultimately impact on food security as well as the sustainability of potato production. In the UK at the present time, this equates to conventional potato breeding. However, in the USA other options, including GM, are considered. We believe that the industrial link with Simplot will be very helpful for UK plant breeding and agriculture in the medium to long term. The public and stake holders will benefit from the availability of cultivars that are produced under more environmentally benign farming regimes which will require less pesticide input.

How will they benefit from this research?
In the shorter term potato breeders will have a much improved 'toolbox' for breeding varieties with improved resistances to pests and pathogens that impact on both UK production (e.g. blight, PCN) as well as those affecting countries to which UK, and Scotland, in particular, exports seed (e.g. viruses). JHI is well placed to efficiently exploit this information through its commercial arm, MRS, which operates multiple commercial breeding programmes with industrial partners. The availability of novel resistance genes and markers diagnostic for these genes will allow more rapid development of new resistant varieties. Moreover, varieties developed using these resources will combine resistances to more than one pathogen, something which has been hard to achieve to date.

Stakeholders, including the public and farmers will benefit from improved environmental conditions through reduced use of fungicides, nematicides and insecticides used to control blight, PCN and aphid vectors of viruses. The public will also benefit through availability of fresh produce and process products containing less chemical residues.
 
Description We have three publications one describing the use of large DNA fragments in RenSeq and PacBio long read sequencing, one on further improvements to RenSeq using even longer fragments and PacBio sequencing to even betterdefine NB-LRR gene repertoires.
We have a further publication published benchmarking PacBio and MinION nanopore sequencing technologies, which we believe to be the first publication of a targeted resequencing project on MinIONs.

We have seen that this new RenSeq methodology is now widespread in the agrigenomics community and wider.
Exploitation Route Breeding disease resistant potatoes using GM (which our partner Simplot is doing in the USA) or MAS (in the EU where GM is heavily regulated).
Sectors Agriculture, Food and Drink,Environment

 
Description Our IPA partner Simplot (one of the largest potato breeding companies in the world) is testing new varieties of potatoes based on resistances we have found.
First Year Of Impact 2016
Sector Agriculture, Food and Drink
Impact Types Economic

 
Title targeted capture pipeline 
Description A new pipeline to assemble Resistance Genes in plants. 
Type Of Technology Software 
Year Produced 2016 
Open Source License? Yes  
Impact Improves accuracy of the assembly and makes the process open source. 
URL https://github.com/paajanen/RenSeq
 
Description 1st international RenSeq workshop (Norwich) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Gave a talk about our RenSeq targeted capture protocol, which created interest in the audience to use longer captures to increase the length of the sequence data.
Year(s) Of Engagement Activity 2015
 
Description Illumina users group meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation: "Plant genomics - assembling genomies and understanding haplotypes"
Year(s) Of Engagement Activity 2018
 
Description Public engagement as part of the 1851 Royal Commission even on "Genetic engineering" at the NHM and broadcast on the BBC World Service 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact As part of the reception before the debate on "Genetic Engineering" and broadcast on the BBC, I demonstrated to the public with plant samples how Wheat is a consequence of an historic, and natural, hybridization of three grass species. Furthermore that these events are still occurring and beneficial e.g. Triticale is a hybrid of wheat and rye, and wheat cultivars such as Rialto contain ~250,000,000bp of DNA from rye, which makes them resistant to fungi that otherwise would require extensive use of environmentally damaging pesticides.

With my colleague, Sandra Knapp, we also showed with samples how Solanaceae crop wild relatives can be used to introduce desirable traits controlled by single genes into crops such as potatoes and tomatoes.
Year(s) Of Engagement Activity 2018
URL https://www.bbc.co.uk/mediacentre/proginfo/2018/39/the-weekend-documentary-the-engineers
 
Description TGAC open day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact A poster presentation and lively discussions in the open day of TGAC about Potato research we do. Discussed gene editing, technology, farming policy, with 10 participants, many more glanced through the poster. This increased the visibility and practical impact of our research and affected the opinion on GMO crops.
Year(s) Of Engagement Activity 2015
URL http://us1.campaign-archive1.com/?u=1b391e9b0f556ff8b126a5b6a&id=32850c25d2
 
Description Throwing the kitchen sink at Solanum verrucosum: genome assemblies integrating PacBio, Bionano and other tools 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact I was invited by BioNano Genomics to present on our work comparing different genome assembly methods in plant genomics
Year(s) Of Engagement Activity 2016