Designing a synthetic LRR generator for the improvement of plant immunity

由病虫害和植物病原体引起的作物病害对全球粮食安全构成威胁。它们对大量的作物产量损失负有责任；小麦大约22%，大米30%，大豆21%。这对农业生产来说是个问题，到2050年，农业生产必须增长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，疾病就会产生：病原体、易感宿主和有利环境。由于气候变化导致气温上升，枯萎病蔓延到目前温带气候地区的风险正在增加。选择性育种改进了普通作物的基因组，使它们适应人类的食物供应需求。然而，这往往是以致病性抗性的遗传变异为代价的。植物不具有适应性免疫系统，而是依赖于不同的先天免疫机制。这些可分为效应触发免疫（ETI）和模式触发免疫（PTI）。PTI需要跨膜受体介导的对病原体分子（如鞭毛蛋白）的反应。另一方面，ETI提供了对效应分子——在细胞内部署的致病基因产物——的防御。这种免疫模式是由所谓的抗性(R)基因编码的细胞内核苷酸结合的富含亮氨酸的重复受体家族（nlr）介导的。在富含亮氨酸的c末端重复序列（lrr）的帮助下，它们识别病原体的无毒基因（Avr）产物，即效应物，并启动信号级联反应，通常导致超敏反应：局部细胞凋亡，阻止感染在受影响植物中的传播。效应物要么直接与NLR受体相互作用，要么通过辅助因子蛋白作为致病分子和受体之间的介质相互作用。有些可以直接识别一些效应因子，而另一些可以与多个辅助因子相互作用。这突出了NLR受体革命的动态性。植物基因组可以编码数百个这样的基因。问题是，即使这对栽培作物来说是正确的，在病原体继续进化的过程中，在野生环境中发现的同样的选择压力并没有作用于它们的nlr。该项目将专注于使用一个新的定向进化平台来产生新的富含亮氨酸的重复（LRR）蛋白结构域。目的是通过将合成的LRRs引入现有的NLR受体，以扩大和/或提高模式植物拟南芥对病原体的识别特异性，这是一种替代基因堆叠的方法。我们将设计一个合成构造，使合成lrr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变异体进行筛选。然后，通过替换现有的lrr编码序列，将阳性变异引入一组经过充分研究的NLR受体中。我们希望通过基因枪将合成的NLRs转化为拟南芥，并测试其对铜绿假单胞菌等拟南芥常见病原体的抗性。

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.