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

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 We have reported the cloning of one such gene, Rpi-amr3, and in unpublished work we defined at least 3 more.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.

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.

Immediate beneficiaries of this research will be other academic researchers, as described in detail above.

Our industrial partner, Simplot, is a US-based company that has extensive reach into the development of diverse processed potato products. Simplot is very forward-looking and is an early adopter of new technologies; it has already brought "Innate" non-bruising and low acrylamide potatoes to market in the US. We believe that the industrial link with Simplot will be very helpful for UK plant breeding and agriculture in the medium to long term.

One of the main outputs of this project will be an expanded repertoire of resistance genes from S. americanum available for deployment in potato by Simplot. Simplot has a world-wide exclusive license to Rpi-amr genes, except in the UK, where its license is non-exclusive. Simplot is already taking to market in the US a blight-resistant potato carrying Rpi-vnt1 that we cloned previously on BBSRC funding. We have also field tested Rpi-vnt1 in the UK, where it functions well in the field. Their potato variety carrying our gene has received USDA and FDA approval, but still awaits a verdict from the EPA; commercialization is expected in 2017. We have an additional collaboration with Simplot and UK-based BioPotatoes, under the HAPI program, in which we bring a stack of three Rpi genes (Rpi-vnt1, Rpi-amr3 and Rpi-amr1e) to field trials in UK variety Maris Piper; this project is still in its first year. Since pathogens can evolve to overcome resistances (though this process is expected to be much slower on multigene stacks), it is highly desirable in the longer term to have additional sources of resistance that can deployed in the same market-favored variety. If this proposal is funded, together with work expected to be completed on current funding, I expect we will deliver at least 6 and possibly as many as 8 Rpi-amr genes in addition to Rpi-amr3 and Rpi-amr1E.

Despite the broad uncertainty and potentially damaging consequences of Brexit for UK science, the UK might be able to establish a better mechanism for regulating GM crops than the EU, and thus bring useful products such as GM blight-resistant potato to market more quickly. Thus an important impact will be as an example of what the technology can do if regulation is rationalized.

As an additional method to accelerate deployment, we will establish and refine methods for "Knock-In Exchange" of resistance-conferring alleles of Rpi genes into the orthologous position in potato. Repeated deployment of these methods should result in lines identical to Maris Piper but carrying different repertoires of resistance-conferring Rpi gene alleles. It is hoped that this method will render the resulting lines more acceptable to regulatory authorities and the public. Demonstration of the efficacy of these methods will have broad public impact.

Stakeholders, including the public and farmers, will benefit from reduced use of fungicides to control blight. The public will also benefit through availability of fresh produce and processed products containing less chemical residues.

JJ has actively engaged with the public regarding GM crops and their potential benefits, and will continue to do so.