Award details

Defining and deploying Rpi gene diversity in S. americanum to control late blight in potato

Reference	BB/P019595/1
Principal Investigator / Supervisor	Professor Paul Birch
Co-Investigators / Co-Supervisors
Institution	University of Dundee
Department	School of Life Sciences
Funding type	Research
Value (£)	270,811
Status	Completed
Type	Research Grant
Start date	04/07/2018
End date	29/01/2021
Duration	31 months

Abstract

Wild potato and tomato relatives can show heritable variation in late blight (LB) resistance due to Resistance to P. infestans (Rpi) genes. AIM1 is to clone all the Rpi-amr genes in our Solanum americanum stocks. We developed a new method for fast Rpi gene cloning- sequence capture with long PacBio reads- and used this to clone Rpi-amr3. In unpublished work, we thus cloned at least 3 more. We aim to clone the remaining 3-6 Rpi genes in our S. americanum populations, and provide at least 8 genes that could be deployed in stacks. AIM2 is to test functionality of each Rpi-amr gene in transgenic potato in field environments, and to verify their non-interference in each other's function. AIM3 is to discover P. infestans effectors (AvrAmrs) detected by our cloned Rpi-amr genes. Rpi genes enable plants to sense P. infestans effectors, and upon recognition, activate plant defence. All P. infestans Avr secreted effectors carry an RxLR motif. It is possible to identify all RxLR effectors in a reference genome. Libraries of such effectors have been constructed in DNA vectors that enable them to be transiently expressed in plant cells; co-expression of Rpi gene and recognized effector causes a hypersensitive cell death response; we will screen existing libraries for such Rpi/effector combinations. With the Hein/Birch labs, we will also use sequence capture ("Pathseq") to refine our knowledge of the full RxLR effector repertoire of Pi. New RxLRs revealed by PathSeq will be added to the list of RxLRs tested to discover AvrAmr effectors. In addition, a refined bait library for sequence capture (PathSeq2.0) will be used to screen world-wide Pi diversity. AIM4 of the project is to introduce the Rpi- genes into their orthologous chromosomal positions in potato. These "New Breeding Technologies", taking advantage of CrispR/Cas9 editing methods, may enable less controversial control of LB using genetics.

Summary

Plant disease reduces crop yields, wasting the resources of fertilizer and water applied by farmers, and necessitating regular applications of agrichemicals. These agrichemical applications increase costs and necessitate costly tractor passes that emit CO2. Overall, it is highly desirable to replace chemical control of disease with genetic control. Potato late blight (LB), caused by the fungus-like pathogen Phytophthora infestans (Pi), causes severe losses to potato and tomato production worldwide. Wild relatives of cultivated potato and tomato show heritable variation for LB resistance, and this can often be due to specific Resistance to P. infestans (Rpi) genes. Although some Rpi genes have been used by plant breeders, it is highly desirable for more Rpi genes to be at our disposal. When Rpi genes are deployed by potato breeders, one gene at a time, they are often overcome by new races of LB. However, when such genes are deployed in combination, in "stacks", individual Rpi genes in the stack are in effect "saved" by other genes in the stack, because a new race that cannot overcome all Rpi genes in the stack, cannot overcome any of them. The first aim of this project is to discover all the Rpi genes that can be found in available accessions of the wild potato relative, Solanum americanum, which has several features that render it easy for genetic analysis. We have reported the cloning of one such gene, Rpi-amr3, and in unpublished work we defined at least 3 more. This project aims to clone the remaining 3-6 Rpi genes that we believe to be present in our S. americanum populations, providing a total of at least 8 genes that could be used to protect crops. The second aim of this project is to test function of all these Rpi-amr genes in the field. Each will be deployed in a transgenic (GM) potato, and tested to verify they confer blight resistance not just in the lab, but also in an agricultural environment. Like other resistance genes, Rpi genes work by enabling the plant to (i) sense when the pathogen starts to grow on it, and (ii) activate the plant's powerful defence mechanisms upon recognition. Aim 3 of this project is to discover which pathogen molecules are detected by our 8-10 cloned Rpi-amr genes. Pathogens cause disease in part through suppressing host defences using proteins they deliver into host cells, called effectors. In turn, Rpi genes encode receptors that have evolved to detect one of the many P. infestans effectors. Most recognized effectors from P. infestans carry two amino acid sequence motifs, one (a signal peptide) for export from a P. infestans cell, and an "RxLR" motif that likely plays a role in uptake into cells of infected plants. From genome sequencing, it is possible to identify all the RxLR effectors. Libraries of such effectors have been constructed in DNA vectors that enable them to be transiently expressed in plant cells; a combination of Rpi gene and recognized effector causes cell death. We will search for Rpi-amr/effector gene combinations that trigger cell death. We will also refine our definition of the RxLR repertoire of several P. infestans strains, using a DNA sequence capture method that increases the accuracy with which we can define the full RxLR effector repertoire. We will thus iteratively obtain a progressively better picture of the Pi effector repertoire, and as previously undetected RxLRs are revealed, they will be tested for recognition by our cloned Rpi-amr genes. The 4th aim of the project is to introduce the Rpi- genes into the chromosomal position in potato that corresponds to the potato homolog of each Rpi gene. This use of "New Breeding Technologies" (NBTs), taking advantage of new gene editing methods, will provide a genetic means for controlling LB that may be less controversial.

Impact Summary

Phytophthora infestans causes late blight disease and is the most significant threat to potato production, the world's third largest food crop. Late blight is thus a considerable threat to global food security. Building on established research excellence and jointly developed tools (RenSeq and PathSeq), this proposal will identify and functionally characterise NB-LRR gene resistances from the wild species Solanum americanum (Rpi_amrs). To predict the durability of these Rpi genes, corresponding pathogen avirulence (Avr) genes must be identified. PathSeq is an unpublished tool designed to facilitate the enrichment of pathogen effectors prior to sequencing. A key component of the proposed research is the utilisation of PathSeq to refine the pan-RXLRome of P. infestans effectors and to identify cognate Avr genes for Rpi-amrs. All known avirulence genes belong to this class of effectors, which contain a signal peptide followed by a canonical RXLR motif. A pilot study of PathSeq in six P. infestans isolates including the reference strain T30-4, provided evidence that the currently described RXLRome is under-representative. By using PathSeq in combination with long-read sequencing platforms such as PacBio and further mining existing, non-enriched PacBio sequences from T30-4, 3928A (13_A2), 2010_7822B (6_A1) and 110059 (US-23) and further mining existing, non-enriched PacBio sequences from T30-4, we will refine the RXLR set of P. infestans and develop a new bait library that included all identified RXLRs (PathSeq2.0). This information, alongside the development of PathSeq, will be made available to the research community through open access publications. Similar to the NB-LRR gene re-annotation of the sequenced potato clone DM, which is accessible through a bespoke potato gbrowser hosted at the James Hutton Institute, the refined effector repertoire of P. infestans will be made easily accessible through a customized P. infestans genome browser that will includes a specific, downloadable track for effectors. There will be no identification restrictions for this Internet resource. Based on the refined RXLR analysis, novel core effectors will be synthesised and cloned into a suitable expression system for transient delivery in Solanum species to help elucidating Avr-amrs. We will share this resource with international groups such as Wageningen University (WU) and INRA Avignon who utilize Effectoromics to characterise host and non-host resistances. The pan-RXLRome of P. infestans will be established by analysing the genomes of 24 diverse isolates that represent the most diverse genotypes from America, Europe and Asia. PathSeq reads will be obtained from gDNA and cDNA to identify presence/absence, expression and sequence polymorphisms. The nature of the Avr-amrs and the observed sequence/expression polymorphism for Avr-amrs alongside other, known Avr genes will be shared with the commercial partner, Simplot, and the scientific community to assess the potential durability of Rpi-amrs resistances in different geographic locations. PathSeq will be developed to enable the future use of the technology for rapid and cost-effective analyses of RXLR effectors from diverse P. infestans isolates collected, for example, from the field. As such, PathSeq will complement existing P. infestans genotyping platforms such as currently used SSR markers and will enable a detailed insight into the molecular diversity of infective isolates. This information can be utilised as 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 such changes. In addition, PathSeq could be established, in the future, as a component of precision farming and inform farmers about the necessity to apply chemical controls agents if deployed resistances could be threatened by emerging P. infestans isolates that can evade recognition by Rpi genes.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Crop Science, Microbiology, Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/P021646/1 Defining and deploying Rpi gene diversity in S. americanum to control late blight in potato

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