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

马铃薯晚疫病是马铃薯作物的一种毁灭性疾病，由疫霉（Pi）引起，导致了19世纪40年代爱尔兰马铃薯饥荒。大多数马铃薯品种都易受枯萎病的影响，在英国，用于防治枯萎病的杀菌剂费用约为6000万英镑，在全球范围内约为70亿美元。对枯萎病的遗传抗性将大大减少对农业化学喷雾剂的需求，并节省排放二氧化碳和压实土壤的拖拉机旅行。植物有强大的防御机制，但抵抗的关键是识别。枯萎病是一种快速进化的病原体，有许多不同的种族。与抗生素一样，依赖一种作用模式或一种耐药性来源是有风险的，而且与COVID一样，病原体可以迅速进化以应对耐药性机制。我们已经在一个潜在的新品种（“PiperPlus”）中部署了3个抗Pi （Rpi）基因，但为了预测病原体的进化，也为了加强我们对植物/病原体共同进化和病原体毒力机制的理解，需要更多的Rpi基因。我们的主要目标是了解马铃薯近亲美洲茄（“Sam”）对晚疫病的近乎免疫，美洲茄是广泛分布的英国本土植物黑茄（S. nigrum）的二倍体祖先。我们从世界各地获得了54种不同的菌株，但在田间都具有完全的抗性，尽管有些菌株在促进疾病的实验室条件下表现出易感性，这使我们能够利用遗传学技术克隆出两种抗鼠疫杆菌（Rpi）基因，Rpi-amr1和Rpi-amr3。因为我们已经克隆了被Rpi-amr1和Rpi-amr3识别的Pi分子（Avramr1和Avramr3），我们可以在我们的收集中识别许多额外的“非amr1,3”抗性。我们有大量的Sam基因组序列数据，这对检测和抵抗Pi的遗传变异分析有很大的帮助。这个提议的中心目标是克隆多个额外的抗性，并验证它们对多个派种族的效力。我们有信心在我们的一组材料中至少有两个额外的抗性基因；来自Sam序列SP2275的Rpi-amr5（可能但不一定与SP3370的Rpi-amr12相同），来自SP2300的Rpi-amr13（可能但不一定与SP2298的Rpi-amr15相同）和来自SP1101的Rpi-amr14。鉴定这些新的Rpi基因的遗传作图工作进展良好，将在资助期内完成并发表，并在转基因马铃薯植物中验证功能。由于Sam具有如此强的抗性，它可能有许多不同的识别Pi的方式。我们已经从Pi中确定了另外7个毒力成分，这些毒力成分至少在一个Sam接入中被识别，并且正在确定支撑这些识别能力的Sam基因。其中一个已经被克隆。我们假设这些多重识别能力有助于抵抗。我们将以两种方式对此进行测试(i)我们将测试是否将这些额外的识别能力转移到马铃薯中，单独或组合，可以提高对Pi的抵抗力。（ii）我们将使用最新的“CrispR”技术进行靶向诱变，对加入物SP2271中的三个基因进行突变，并测试识别能力的降低是否会损害抗性并提高易感性。病原体效应物在宿主上生长时已经进化到促进其繁殖成功。效应物通过干扰植物防御反应的一部分机制来提高病原体的毒力。通过识别病原体效应物的宿主目标，我们确定了植物防御机制的关键组成部分。因此，每一个新的抗性基因的分离不仅有助于实现持久的抗病能力，而且还提供了一种途径来识别识别分子，这是一个关键的病原体毒力成分，从而也为它们的植物靶点，使我们能够大大提高我们对植物免疫的理解。

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 susceptibilityPathogen 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.