Novel sources of disease resistance and effector detection from genetic and genomic analysis of Solanum americanum diversity

Potato late blight, caused by Phytophthora infestans (Pi), is a devastating disease of potato crops and led to the Irish potato famine of the 1840s. Most potato varieties are susceptible to blight, and its control costs ~£60M in fungicide applications in the UK, and ~$7B world-wide.

Genetic resistance to blight would greatly reduce the need for agrichemical sprays and save on tractor journeys that emit CO2 and compact the soil.
Plants have powerful defence mechanisms, but the key to resistance is recognition. Blight is a rapidly evolving pathogen, with many different races. As with antibiotics, reliance on one mode of action or one source of resistance is risky, and as with COVID, pathogens can rapidly evolve to cope with resistance mechanisms. We have deployed a stack of 3 Resistance to Pi (Rpi) genes in a potential new variety ("PiperPlus"), but more Rpi genes are needed in anticipation of pathogen evolution, and also to enhance our understanding of plant/pathogen coevolution, and pathogen virulence mechanisms.

Our primary objective is to understand the near-immunity to late blight of the potato relative Solanum americanum ("Sam"), the diploid ancestor of the widespread UK native plant black nightshade (S. nigrum). We have 54 different accessions from around the world, but all are fully resistant in the field, though some show susceptibility under disease-promoting lab conditions, which enabled us to use genetics to clone two Resistance to P. infestans (Rpi) genes, Rpi-amr1 and Rpi-amr3.
Because we have cloned the Pi molecules (Avramr1 and Avramr3) that are recognized by Rpi-amr1 and Rpi-amr3, we could identify many additional "non-amr1,3" resistances in our collection. We have extensive Sam genome sequence data that greatly helps analysis of genetic variation for detection of and resistance to Pi. A central goal of this proposal is to clone multiple additional resistances and to verify their efficacy against multiple races of Pi. We are confident there at least two additional resistance genes in our set of accessions; Rpi-amr5 from Sam accession SP2275 (perhaps but not necessarily the same as Rpi-amr12 from SP3370), and Rpi-amr13 from SP2300 (perhaps but not necessarily the same as Rpi-amr15 from SP2298) and Rpi-amr14 from SP1101. Genetic mapping to identify these new Rpi genes is well advanced and will be completed and published during the grant period, with function verified in transgenic potato plants
Since Sam is so resistant, it is likely to have many different ways of recognising Pi. We have identified another 7 virulence components from Pi that are recognised in at least one Sam accession, and are well on the way to identifying the Sam gene that underpins each of these recognition capacities. One is already cloned. We hypothesise that these multiple recognition capacities contribute to resistance. We will test this in two ways
(i) we will test if transfer of these additional recognition capacities into potato, alone or in combination, can elevate resistance tp Pi.
(ii) we will use the recent "CrispR" technology for targeted mutagenesis to mutate three of these genes in accession SP2271, and test if reduction in recognition capacity compromises resistance and elevates susceptibility

Pathogen effectors have evolved to promote their reproductive success when growing on their hosts. Effectors contribute to pathogen virulence by interfering with plant mechanisms that are part of the plant defence response. By identifying the host target of a pathogen effector, we identify key components of plant defence mechanisms. Thus, every new resistance gene isolated not only helps enable durable disease resistance, but also provides a route to identifying the recognised molecule that is a key pathogen virulence component, and therefore also their plant targets, enabling us to greatly enhance our understanding of plant immunity.

We aim to understand the near-immunity of Solanum americanum ("Sam") to P infestans (Pi). We will clone at least two "non amr1,3" Rpi genes from our 54 Sam accessions. We have a high-quality reference genome for 3 resistant and 1 susceptible accession, and 10-deep sequence data for all. We clone Rpi genes using sequence capture (RenSeq) to identify expressed cosegregating NLR-encoding genes, transient expression of candidate genes in N. benthamiana (Nb), and verification of function in stable transgenic potato lines.

Aim 1. Clone additional Rpi genes
Aims 1.1 Isolate Rpi-amr5 from SP2275, Rpi-amr12 from SP3370
Rpi-amr5 and Rpi-amr12 map to the Rpi-amr1 haplotype, with ~ 6 expressed cosegregating NLR genes (but mutated Rpi-amr1). We will assess which paralogs confer Pi resistance after transient expression in Nb, and then verify candidates in stable transgenic potato lines.
Aims 1.2, 3, 4, Isolate Rpi-amr13 from SP1101, Rpi-amr14 from SP2300, Rpi-amr15 from SP2298
These Sam accessions carry Rpi-amr1 and Rpi-amr3, and carry distinct non amr1,3 genes. For each, back cross progeny segregate for resistance. We are generating Illumina Renseq data for resistant and susceptible non amr1,3 bulks from SP2300, SP110 and SP2298. We anticipate identifying at least two more Rpi- gene after transient expression in Nb and selecting NLRs to make transgenic potato for Pi resistance assays.

Aim 2. Effectoromics to identify and clone new Sam effector recognition genes with GWAS and genetics.
All Sam lines are completely resistant in the field but show variation in effector responses. From transient expression of 338 effectors in all accessions, 7 Sam loci were revealed; 1 is already cloned, and we aim to clone 5 more in this grant. We will test transgenic potato carrying each recognition for elevated Pi resistance and use CrispR to mutate and test the role of these recognitions in Sam resistance to Pi.