An enduring pipeline to identify and utilize durable late blight disease resistance in potato

Lead Research Organisation: University of East Anglia
Department Name: Sainsbury Laboratory

Abstract

Phytophthora infestans causes late blight, the most devastating disease of potato which is the world's third most important food crop. Consequently, late blight is a threat to global food security. Recently, we have witnessed a dramatic shift in the European P. infestans population, which is now dominated by an aggressive strain that overcomes many resistances, including that deployed in the number one organic potato, Lady Balfour. Moreover, many pesticides that farmers rely on to prevent late blight may soon be banned in the EU. Agriculture and science must respond to rapid pathogen population changes to secure food supply with minimal impact on natural resources. This project aims to provide a powerful and enduring pipeline, driven by understanding pathogen population dynamics, to exploit naturally existing biodiversity in wild Solanum species, and rapidly identify and deploy durable late blight resistance (Rpi) genes. The genome sequence of P. infestans is very large, 74% of which is non-coding DNA repeats. It has revealed more than 500 candidate virulence genes encoding effector proteins with an amino acid motif (RXLR) required for their movement into plant cells. All P. infestans effectors recognised by potato Rpi proteins found so far contain this delivery motif. RXLR genes are located in regions of repetitive DNA, which may be subject to higher rates of evolution, perhaps explaining why this pathogen has so readily overcome deployed resistances. Our first key objective aims to identify, using microarray gene expression profiling, a 'core set' of RXLRs that are actively used for infection by a diverse worldwide collection of P. infestans strains, including those currently threatening European crops. Such universally-expressed RXLRs provide 'targets' for resistances that are more likely to be durable. By sequencing these core RXLRs from the diverse strains, we will reveal variant forms which may have evolved to evade resistance. The core set of RXLRs, and variant forms of their sequences, will be used to screen for potentially durable late blight resistance. A large collection of wild potato relatives is maintained at SCRI, providing a living library of genetic variation to seek durable Rpi genes from the >180 candidates in the potato genome. This collection and previously identified resistant species will be screened with diverse contemporary P. infestans isolates. Genetic crosses involving this resistant germplasm will be screened for responses to core RXLRs, seeking correspondence in segregation between resistance and effector recognition. Durability of resistance will be assessed by screening with core RXLR allelic variants. Next-generation sequencing and DNA capture array technologies will be used to rapidly identify candidate Rpi genes specifically from resistant offspring in genetic crosses. Rpi candidates will be expressed in susceptible plants, and tested for their ability to convey resistance to P. infestans. Durable Rpi genes will provide markers to more rapidly breed them into modern potato cultivars within the SCRI mutlitrait breeding programme. Moreover, cloned Rpi genes can be introduced into commercially important potato and tomato cultivars using transgenic technology. We will seek to actively engage with the public to discuss perceptions of GMOs. This project provides a collaborative platform and strategy to yield novel, durable Rpi genes as markers for accelerated breeding, and as genes that could be deployed transgenically in finished cultivars. To defeat the high evolutionary potential of P. infestans, many Rpi genes may be needed and these must be rapidly and appropriately deployed as we detect changes in the P. infestans population. We thus envisage an Rpi discovery pipeline that will continue to exploit the potato biodiversity at our disposal beyond the completion of this project.

Technical Summary

An early objective will draw on an existing pilot study, using a proprietary array containing all candidate RXLR genes from P. infestans, to identify a 'core' effector set that is universally-expressed across 20 diverse isolates that currently threaten world crops. Such 'indispensable' RXLRs must be important for pathogenicity, providing good targets for durable late blight resistance (Rpi) genes. Assessment of sequence diversity across the core RXLRs will provide alleles to test for their ability to evade candidate resistances. We have identified wild Solanum accessions with resistance to diverse isolates, including the prevalent Genotype 13_A2. From these we will identify potentially durable resistances showing correspondence between resistance and recognition of 'core' P. infestans RXLRs. Crucially, we will extend this to genetic crosses, seeking co-segregation between resistance and RXLR recognition. Bulks of resistant (BR) and susceptible (BS) plants from segregating crosses will be analysed in two ways. Firstly, sequencing normalised BR and BS cDNA will use the Illumina NGS platform to rapidly identify NB-LRR genes as genetic markers and candidate Rpi genes. Secondly, proven DNA sequence capture technology will enrich candidate Rpi genes from genomic DNA of BR and BS bulks. The potato genome assembly and cloned potato R genes will be mined to provide a bespoke potato 'resistance gene assembly' to generate a tiled NB-LRR 'capture array'. Captured Rpi candidates will be sequenced using Illumina from BR and BS bulks to identify candidate Rpi alleles. A pilot study will compare the two methods. The most reproducible will be utilised for this project. Rpi candidates will be tested for cosegregation with resistance. They will provide markers to breed identified resistances into modern cultivars. Rpi candidates absolutely linked to resistance will be functionally tested by transient expression in N. benthamiana and stable potato transformation.

Planned Impact

Pathogen attack of crop plants is a key challenge to agricultural sustainability in terms of yield loss due to disease and environmental impact due to fungicide application. Phytophthora infestans is the most significant pathogen of potato, the world's third largest food crop, responsible for large yield losses through late blight disease. Costs associated with chemical control exceed £3.5 billion globally per year. Late blight is thus a considerable threat to global food security. Genetic resistance via resistance to P. infestans genes, and control chemicals, have been deployed with limited success, as both have been readily overcome by variation in pathogen populations. This proposal aims to exploit the P. infestans genome to seek universally-expressed, essential and conserved components of its pathogenicity arsenal that can be targeted by Rpi genes for durable, sustainable potato protection. Specifically, we will identify sources of durable disease resistance in wild Solanum genotypes, from which Rpi genes will be rapidly isolated using state-of-the-art next-generation sequencing and DNA capture technologies that exploit the availability of the potato genome sequence. Rpi genes will provide markers for marker-assisted selection (MAS) to rapidly introgress these durable resistances into new potato cultivars. Such markers will be utilized immediately in the SCRI multitrait (MT) breeding programme, which combines resistances to late blight, potato cyst nematodes and viruses, with other commercially desirable traits. This programme is supported throughout by industrial sponsorship and has delivered finished cultivars for various potato markets, including Lady Balfour, the number one organic potato. However, initially durable late blight resistances in the MT programme have recently succumbed to new virulent strains of P. infestans, prompting the further surveys of the Commonwealth Potato Collection, and the strategy to search for more durable Rpi genes, that are the subject of this proposal. All novel sources of resistance, and the Rpi gene sequences as MAS markers, will thus be offered to the SCRI MT breeding programme. In addition, durably-resistant germplasm and markers will be offered to commercial potato breeders with whom the partners in this grant collaborate. This project will also provide information on a 'core set' of P. infestans RXLR effectors that will facilitate other researchers, particularly our collaborators in Wageningen University (WU), who utilize similar screens for durable resistance on complementary collections of wild germplasm. Researchers in WU will closely collaborate to exploit the findings and methods developed in this proposal and also have commercially-funded potato breeding programmes to develop potato cultivars with durable late blight resistance. In addition to breeding, potentially durable Rpi genes can be judiciously combined using transgenic approaches to more rapidly deploy resistances within commercially important potato and tomato cultivars. Whilst this is not currently legislated for within the EU, it nevertheless remains a most likely route to sustaining resistance in the face of the dramatic, recently witnessed P. infestans population changes. Some Ag-Biotech companies in Europe are considering such approaches and these will be engaged during this project to assess the utility of resistances we discover, which may be deployed in countries outside the EU if GMO legislation remains unlikely to change within it. Critically, we will engage the public, through various means elaborated in the impact plan, to educate about the benefits and potential disadvantages of GMO resistance solutions. Finally, sequence analyses of RXLR effectors in diverse P. infestans isolates will provide a diagnostic to chart future changes in P. infestans populations, allowing growers to deploy appropriate resistances to sustain durable disease resistance in the face of changes in pathogen populations.

Publications

10 25 50
 
Description We made excellent progress on this grant. We have generated multiple populations from crosses between Solanum americanum accessions segregating for late blight resistance. We identified a BAC clone carrying a cosegregating marker for one of them (Rpi-amr1). The clone carries 9 NB-LRR gene homologs, 5 of which are potentially functional. This work has led (partially with subsequent funding) to the cloning of another gene Rpi-amr3, and more recently to finally defining Rpi-amr1e. Two additional blight resistance genes are well advanced.

In addition, we have established and published a broadly useful new method for accelerating resistance gene cloning called Renseq. This uses 120mer oligonucleotides directed against the NB-LRR gene complement to specifically direct Illumina sequencing capacity to the R gene repertoire. We have now established this method with the Miseq and also PacBio sequencing platforms. This method is being widely adopted in TSL for cloning R genes from other plants, including leguminosae and triticaceae. Overall, then, progress is very satisfactory.

Jupe F, Witek K, Verweij W, Etherington G, Pritchard L, MacLean D, Sliwka J, Milne L, Cock P, Bryan G, Hein I, JDG Jones (2013) RenSeq: sequencing DNA enriched for plant resistance genes offers re-annotation of sequenced genomes and rapid mapping of resistance loci in segregating populations Plant Journal 76: 530-544

Andolfo, G., Jupe F, Witek K, Etherington G, Ercolano M, JDG Jones (2014) Defining the full tomato NB-LRR resistance gene repertoire using genomic and cDNA RenSeq Accepted to BMC Plant Biology April 2014

see also Witek et al https://www.ncbi.nlm.nih.gov/pubmed/27111721
Exploitation Route We have licensed Rpi-vnt1 for commercialization in the US in transgenic potato. We are attempting to deploy a triple stack of Rpi-vnt1, Rpi-amr3 and Rpi-amr1e in the UK and have conducted field trials for the last 2 years. We recently applied for followon funding in partnership with UK start up company Biopotatoes to commercialize the traits
Sectors Agriculture, Food and Drink

URL http://www.ncbi.nlm.nih.gov/pubmed/23937694
 
Description we have cloned the Rpi-vnt1 gene that is being licensed to a commercial partner, Simplot, to protect potatoes from blight in the US- these potatoes are now grown commercially. We also obtained funding for a joint project funded in the HAPI program with that partner; this has resulted in Maris Piper potatoes that carry Rpi-vnt1, Rpi-amr1 and Rpi-amr3 and are completely resistant to current blight strains in the UK
First Year Of Impact 2018
Sector Agriculture, Food and Drink
Impact Types Cultural,Economic

 
Description Controlling important diseases in potato by cloning functional NB-LRR-type resistance genes
Amount £366,264 (GBP)
Funding ID BB/L009293/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2014 
End 08/2017
 
Description Defining and deploying Rpi gene diversity in S. americanum to control late blight in potato
Amount £777,912 (GBP)
Funding ID BB/P021646/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description New UK potato varieties with late blight and potato cyst nematode resistance, reduced bruising and improved processing quality
Amount £474,002 (GBP)
Funding ID BB/M017834/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2015 
End 09/2020
 
Title The RenSeq method 
Description Sequence capture of R genes (RenSeq) is being broadly applied across multiple plant species to expand knowledge of plant immune repertoires. In updated methodology, we combined RenSeq with PacBio sequencing to achieve even better definition of angiosperm immune receptor repertoires 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact Many genes that confer stem rust resistance in wheat have been cloned using this method. There was also a recent submission to Bioarxiv detailing the pan NLRome of Arabidopsis thalian- the corresponding paper has now been submitted to Cell - see https://www.biorxiv.org/content/10.1101/537001v1 
 
Description TSL/Simplot/James Hutton/U Dundee partnership for understanding and deploying late blight resistance genes 
Organisation James Hutton Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution we collaborated with Ingo Hein to develop and deploy Renseq for resistance gene analysis
Collaborator Contribution we collaborated with Ingo Hein to develop and deploy Renseq for resistance gene analysis
Impact Simplot is commercializing a resistance gene we cloned, Rpi-vnt1, for blight control in the US It has now received FDA, USDA and EPA approval
Start Year 2012
 
Title LATE BLIGHT RESISTANCE GENE FROM SOLANUM AMERICANUM AND Methods of Use 
Description We map-based cloned the Rpi-amr3 gene from Solanum americanum that confers late blight resistance in potato 
IP Reference WO2016182881 
Protection Patent application published
Year Protection Granted
Licensed Yes
Impact None yet, beyond licensing to Simplot We are using Rpi-amr3 in our HAPI grant
 
Title LATE BLIGHT RESISTANCE GENES AND METHODS OF USE 
Description Compositions and methods and for enhancing the resistance of plants to a plant disease caused by a Phytophthora species are provided. The compositions comprise nucleic acid molecules encoding resistance (R) gene products and variants thereof and plants, seeds, and plant cells comprising such nucleic acid molecules. The methods for enhancing the resistance of a plant to a plant disease caused by a Phytophthora species comprise introducing a nucleic acid molecule encoding an R gene product into a plant cell. Additionally provided are methods for using the plants in agriculture to limit plant disease. 
IP Reference WO2018112356 
Protection Patent application published
Year Protection Granted 2018
Licensed Yes
Impact we have carried out field trials with Rpi-amr1e
 
Description 'How plants can be tweaked to 'naturally' fight disease', Genetic Literacy Project, January 2017 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact 'How plants can be tweaked to 'naturally' fight disease', Genetic Literacy Project, January 2017
Year(s) Of Engagement Activity 2016
URL https://geneticliteracyproject.org/2017/01/16/talking-biotech-plants-can-tweaked-naturally-fight-dis...
 
Description Contribution to a Wellcome Trust project to generate teaching materials for A Level students 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Contribution to a Wellcome Trust project to generate teaching materials for A Level students https://thecrunch.wellcome.ac.uk/schools-and-colleges/feeding-a-growing-world
Year(s) Of Engagement Activity 2016
URL https://thecrunch.wellcome.ac.uk/schools-and-colleges/feeding-a-growing-world
 
Description Food Thinkers Series: 'Dysfunctional regulation of GM crops; scope for improvement post-Brexit?', Presentation for Food Research Collaboration, Centre for Food Policy (CFP) City University London, February 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Food Thinkers Series: 'Dysfunctional regulation of GM crops; scope for improvement post-Brexit?', Presentation for Food Research Collaboration, Centre for Food Policy (CFP) City University London, February 2017
Year(s) Of Engagement Activity 2016
URL http://foodresearch.org.uk/food-thinkers-dysfunctional-regulation-of-gm-crops-scope-for-improvement-...
 
Description Interview on Radio 4 Today program re GM crop regulation post Brexit 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Interview on Today program re GM crop regulation post Brexit
Year(s) Of Engagement Activity 2019
 
Description Interview on Talking Biotech podcast with Paul Vincelli 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact http://www.talkingbiotechpodcast.com/065-plant-r-genes-and-their-applications/
Year(s) Of Engagement Activity 2017
URL http://www.talkingbiotechpodcast.com/065-plant-r-genes-and-their-applications/
 
Description Keynote speaker at ISMPMI meeting Glasgow 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I was plenary speaker at ISMPMI meeting Glasgow 2019
Year(s) Of Engagement Activity 2019
 
Description News about potato GM field trial Summer 2017 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact update on results of field trial
Year(s) Of Engagement Activity 2017
URL https://storify.com/SainsburyLab/early-success-for-late-blight-resistance-trial-in-
 
Description Royal Society pairing with Norman Lamb 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Dr. Marina Pais participated in the Royal Society Pairing Scheme 2017 and was paired with Norman Lamb, MP for North Norfolk and chair of the House of Commons' Science and Technology committee:
Year(s) Of Engagement Activity 2018
URL http://www.tsl.ac.uk/news/lab-bench-back-bench-tsl-scientist-visits-seat-power/