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

植物病害降低了作物产量，浪费了农民使用的肥料和水资源，并需要定期使用农用化学品。这些农化应用增加了成本，需要昂贵的拖拉机通过排放二氧化碳。由马铃薯晚疫病菌(Phytophthora infestans，PI)引起的马铃薯晚疫病给世界马铃薯和番茄生产造成严重损失。栽培马铃薯和番茄的野生近缘种表现出可遗传的抗性变异，这往往是由于对致病疫霉(P.infestans，RPI)基因的特异性抗性所致。虽然一些RPI基因已经被植物育种者使用，但我们非常希望有更多的RPI基因供我们使用。当马铃薯育种者部署RPI基因时，一次一个基因，它们通常会被新的LB小种所克服。然而，当这些基因组合在一起时，堆栈中的单个RPI基因实际上被堆栈中的其他基因“保存”了，因为一个新的小种不能克服堆栈中的所有RPI基因，不能克服它们中的任何一个。这个项目的第一个目标是发现所有可以在野生马铃薯近亲Solanum americanum的现有材料中找到的RPI基因，它具有几个特征，使得遗传分析变得容易。我们已经报道了一个这样的基因的克隆，RPI-amr3，在未发表的工作中，我们定义了至少3个。该项目旨在克隆剩余的3-6个RPI基因，我们认为这些基因存在于我们的美洲链霉菌种群中，总共提供至少8个可用于保护作物的基因。该项目的第二个目标是在田间测试所有这些RPI-AmR基因的功能。每个RPI基因都将被部署在转基因(GM)马铃薯中，并进行测试，以验证它们不仅在实验室，而且在农业环境中都具有抗疫性。像其他抗性基因一样，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，以及(Ii)在识别后激活植物强大的防御机制。本项目的目标3是发现我们克隆的8-10个RPI-AmR基因能检测到哪些病原体分子。病原体通过向宿主细胞传递被称为效应器的蛋白质来抑制宿主防御，从而导致疾病。反过来，RPI基因编码的受体已经进化成检测许多致病疫霉的效应器之一。大多数已知的致病疫霉的效应器携带两个氨基酸序列基序，一个是从致病疫霉细胞输出的(信号肽)，另一个是“RxLR”基序，可能在被感染植物的细胞吸收中发挥作用。从基因组测序中，有可能识别所有的RxLR效应器。这些效应器的文库已经被构建在DNA载体中，使它们能够在植物细胞中瞬时表达；RPI基因和识别的效应器的组合导致细胞死亡。我们将寻找触发细胞死亡的RPI-AmR/效应器基因组合。我们还将使用一种DNA序列捕获方法来完善我们对几种致病疫霉菌株的RxLR谱系的定义，这种方法可以提高我们定义完整的RxLR效应谱的准确性。因此，我们将迭代地获得PI效应器谱系的逐步更好的图像，并且正如之前未检测到的RxLRs所揭示的那样，它们将被我们克隆的RPI-AmR基因测试识别。该项目的第四个目标是将RPI-基因引入马铃薯中与每个RPI基因的同源物相对应的染色体位置。这种利用新的基因编辑方法的“新育种技术”(NBTS)的使用，将提供一种可能不那么有争议性的控制黑斑病的遗传手段。

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.