Designing a synthetic LRR generator for the improvement of plant immunity

由病虫害和植物病原体引起的农作物病害对全球粮食安全构成威胁。它们对农作物产量的大幅损失负有责任；小麦大约损失22%，水稻大约损失30%，大豆大约损失21%。这对农业生产来说是个问题，到2050年，农业生产必须增加60%-70%才能维持不断增长的人口。当以下三个因素同时存在时，疾病就会发生：病原体、易受感染的宿主和有利的环境。由于气候变化导致气温上升，枯萎病传播到目前更温和的气候地区的风险正在增加。选择性育种提炼了普通作物的基因组，使其适应人类的食物供应需求。然而，这往往是以病原性抗性的遗传变异为代价的。植物不具备适应性免疫系统，而是依赖不同的先天免疫机制。它们可分为效应器触发免疫(ETI)和模式触发免疫(PTI)。PTI需要跨膜受体介导的对鞭毛等病原体分子的反应。另一方面，ETI提供了对效应分子的防御--致病基因产物，尤其是细胞内。这种免疫模式是由由所谓的抗性(R)基因编码的细胞内核苷酸结合的富含亮氨酸的受体重复家族(NLR)介导的。在C端域富含亮氨酸的重复序列(LRR)的帮助下，它们识别病原菌的无毒(AVR)基因产物，称为效应器，并启动一系列信号级联反应，通常导致超敏反应：局部细胞凋亡，以防止感染在受影响植物中的传播。效应器要么直接与NLR受体相互作用，要么通过辅助因子蛋白作为病原分子和受体之间的调节器。一些人直接识别一些效应器，而另一些人则可以与多个辅助因素相互作用。这突显了NLR接受革命的动态性质。植物基因组可以对数百种这样的生物进行编码。问题是，即使这对栽培作物来说可能是真的，当病原体继续进化时，在野外发现的同样的选择压力也不会作用于它们的NLR。该项目将专注于使用一个新的定向进化平台来生成新的富含亮氨酸重复序列(LRR)的蛋白质结构域。目的是通过将合成的LRRs引入到现有的NLR受体中，作为基因堆积的替代方案，拓宽和/或提高模式植物拟南芥的病原体识别特异性。我们将设计一个合成结构，它将使合成LRRS文库的生成成为可能。一旦获得突变体文库，将使用酵母2杂交(Y2H)技术对LRR变体进行筛选，以筛选出一系列常见的效应物。然后，通过替换现有的LRR编码序列，将阳性变体引入一组经过充分研究的NLR受体。我们希望通过基因枪将合成的NLRs转化拟南芥，并测试其对铜绿假单胞菌等拟南芥常见病原菌的抗性。

Crop diseases caused by pests and plant pathogens pose a risk for the global food security. They areresponsible for substantial crop yield losses; roughly 22% in wheat, 30% in rice, and 21% in soybean. Thisis problematic for the agricultural production which must increase by 60-70% by 2050 to sustain the growingpopulations. A disease arises when the following three factors are present simultaneously: a pathogen, asusceptible host, and a favourable environment. Due to climate change caused rise in temperatures, therisk for the spreading of blights into currently more temperate climate zones is increasing. Selectivebreeding has refined the genomes of common crops to tailor them to human food supply demands.However, this often came at the cost of genetic variation in pathogenicity resistance. Plants do not possessan adaptive immune system, but instead rely on different mechanisms of innate immunity. These can beclassified into effector-triggered immunity (ETI) and pattern-triggered immunity (PTI). PTI entails thetransmembrane receptor-mediated responses against pathogen molecules such as flagellin. ETI, on theother hand, provides defences against effector molecules - pathogenic gene products, deployedintracellularly. This mode of immunity is mediated by the intracellular nucleotide-binding leucine-rich repeatfamily of receptors (NLRs) encoded by the so-called resistance (R) genes.With the help of the C-terminal domain leucine-rich repeats (LRRs), they recognise a pathogen's avirulence(Avr) gene products known as effectors and initiate a signalling cascade which usually results in thehypersensitive response: local cell apoptosis which prevents the spread of an infection in the affected plant.An effector either interacts with an NLR receptor directly, or through a cofactor protein which acts as amediator between the pathogenic molecule and the receptor. Some recognise a number of effectorsdirectly, while others can interact with multiple cofactors. This highlights the dynamic nature of NLR receptorevolution. Plants genomes can encode for hundreds of these. The problem is that even if this might be truefor cultivated crops, the same selective pressures found in the wild are not acting upon their NLRs whilethe pathogens continue evolving.The project will focus on using a new directed-evolution platform for generating novel Leucine-rich repeat(LRR) protein domains. The aim is to broaden and/or improve the pathogen recognition specificity of themodel plant organism Arabidopsis thaliana by introducing synthetic LRRs into existing NLR receptors, asan alternative to gene stacking. We will design a synthetic construct which will enable the generation of alibrary of synthetic LRRs. Once the mutant library has been obtained, the LRR variants will be screenedagainst a shortlist of common effectors using the yeast-2-hybrid (Y2H) technique. The positive variants willthen be introduced in a selected set of well-studied NLR receptors by replacing the existing LRR-encodingsequences. We hope to transform Arabidopsis thaliana with the synthetic NLRs via a gene gun and test forresistance against a set of common Arabidopsis pathogens such as Pseudomonas aeruginosa.