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

植物病害降低作物产量，浪费农民施用的肥料和水资源，并且需要定期施用农用化学品。这些农用化学品的应用增加了成本，并且需要排放二氧化碳的昂贵的拖拉机通行证。马铃薯晚疫病（Potato Late Blight，LB）是由马铃薯晚疫病菌（Phytophthora infestans，Pi）引起的一种病害，严重影响马铃薯和番茄的生产。栽培马铃薯和番茄的野生亲缘植物显示出LB抗性的可遗传变异，这通常是由于对致病疫霉（P. infestans）（Rpi）基因的特异性抗性。尽管一些Rpi基因已被植物育种者使用，但我们非常希望有更多的Rpi基因可供我们使用。当马铃薯育种者一次一个基因地部署Rpi基因时，它们通常会被LB的新种族所克服。然而，当这样的基因以组合的方式部署在“堆叠”中时，堆叠中的单个Rpi基因实际上被堆叠中的其他基因“保存”，因为不能克服堆叠中的所有Rpi基因的新种族不能克服它们中的任何一个。其具有使其易于遗传分析的几个特征。我们已经报道了一个这样的基因Rpi-amr 3的克隆，在未发表的工作中，我们定义了至少3个。这个项目的目的是克隆剩下的3-6个Rpi基因，我们相信这些基因存在于我们的S。本项目的第二个目的是在田间测试所有这些Rpi-amr基因的功能。每一个都将被应用到转基因马铃薯中，并进行测试，以验证它们不仅在实验室中，而且在农业环境中赋予枯萎病抗性。与其他抗性基因一样，Rpi基因的工作原理是使植物能够（i）感知病原体何时开始在其上生长，（ii）在识别后激活植物强大的防御机制。本项目的目的3是发现哪些病原体分子被我们克隆的8-10个Rpi-amr基因检测到。病原体通过向宿主细胞输送蛋白质（称为效应子）来抑制宿主防御，从而部分导致疾病。反过来，Rpi基因编码的受体已经进化到检测许多致病疫霉效应子之一。大多数公认的来自致病疫霉的效应子携带两个氨基酸序列基序，一个（信号肽）用于从致病疫霉细胞输出，以及“RxLR”基序，其可能在被感染植物的细胞摄取中起作用。通过基因组测序，可以鉴定所有的RxLR效应子。已经在DNA载体中构建了这些效应子的文库，使它们能够在植物细胞中瞬时表达; Rpi基因和识别的效应子的组合导致细胞死亡。我们将寻找触发细胞死亡的Rpi-amr/效应基因组合。我们还将使用DNA序列捕获方法来完善我们对几种致病疫霉菌株的RxLR库的定义，该方法可以提高我们定义完整RxLR效应库的准确性。因此，我们将迭代地获得Pi效应子库的逐渐更好的图片，并且由于先前未检测到的RxLR被揭示，它们将被我们克隆的Rpi-amr基因识别。这种利用新基因编辑方法的“新育种技术”（NBT）的使用将提供一种控制LB的遗传手段，这种遗传手段可能不那么有争议。

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.