A pipeline of resistance genes to Phytophthora infestans from wild Solanum species and their accelerated isolation using Illumina sequencing methods

Late blight of potato caused by Phytophthora infestans (Pi) is one of the world's most important crop diseases. UK farmers spray 10-15 times per year for blight control, and in developing countries, late blight also dramatically reduces potato yields. In the wet summer of 2007, blight pressure in Europe was so severe that demand for blight control chemicals exceeded supply. Potato breeding is extremely slow and inefficient- the most popular UK variety (Maris Piper) is over 30 years old- so the best way to control potato blight is to isolate multiple Resistance to P. infestans (Rpi) genes from wild potato relatives and introduce these Rpi genes into popular varieties. It is not clear if the most durable strategy will involve stacking multiple Rpi genes in the same variety, or planting mixtures of the same variety with different Rpi genes, or using different Rpi genes each year. Either strategy requires the isolation of multiple Rpi genes. Isolation of Rpi genes involves making crosses between blight resistant and blight sensitive plants, and scoring the segregation of resistance and sensitivity in the next generation. Using new sequencing methods, we will be able to identify candidate resistance genes that are always associated with resistance by their sequence similarity to known resistance genes. These candidate genes will be transformed (using Agrobacterium) into blight sensitive potato variety Desiree, and transformed plants tested for blight resistance. We have isolated two Rpi genes, Rpi-mcq1 and Rpi-oka1.1 in previous work funded by EU and by BBSRC. To fully explore this approach, we will isolate multiple additional Rpi genes. We are mapping more Rpi genes from additional Solanum species, creating a pipeline for future Rpi gene isolation and deployment, with the goal of providing durable blight resistance to the potato crop. Our work will shed light on how Rpi genes evolve, and also enable us to identify the pathogen molecules that these Rpi genes recognize and confer responsiveness to. We plan to carry out small scale field trials of GM blight resistant potatoes during the course of this project.

We previously identified what we believed to be three segregating Resistance genes to P. infestans (Rpi-oka1, Rpi-oka2 and Rpi-neo1=Rpi-nrs1) in wild Solanum species, as well as Rpi-mcq1 and Rpi-bst1 (now isolated under EU funding). Rpi-oka1.1 was isolated under BBSRC grant BB/C007522/1; however, there is clearly at least one additional linked Rpi gene (Rpi-oka1.2) at the locus, which we have recombined away from Rpi-oka1.1 and crossed to a susceptible line. We will isolate Rpi-oka1.2 in this project. We will also isolate Rpi-ber1 and Rpi-ber2 from Solanum berthaultii, and a 1:1 segregating Rpi gene from variety Sarpo Mira which carries elevated blight resistance compared to most varieties. In collaboration with a Polish group, we will also investigate and seek to isolate Rpi-ngr genes from S. nigrum. We will use the Illumina GA2 next-generation sequencing (NGS) instrument, and Velvet software, to sequence and assemble cDNA sequences from disease-resistant and -sensitive bulked RNA preps. By analysis of assembled cDNA sequences, we will identify alleles of NB-LRR-encoding Rpi gene candidates that cosegregate with a genetically targeted locus, and after high-resolution mapping, will introduce cDNAs for these candidate genes into a binary vector to make transgenic potato lines and test them for blight resistance. Both Sophien Kamoun at the SL, and Paul Birch in SCRI, are developing libraries of P. infestans effectors in plant viral expression vectors, and we plan to use transient co-delivery of Rpi and effector candidate gene in Nicotiana benthamiana to identify the effectors recognized by the Rpi genes. The ultimate aim of the project is to deploy these multiple Rpi genes in the field and to investigate the best strategy for conferring durable resistance against this very destructive, highly variable and rapidly evolving pathogen.