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

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 We have reported the cloning of one such gene, Rpi-amr3, and in unpublished work we defined at least 3 more.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