Designing a synthetic LRR generator for the improvement of plant immunity

点击翻译按钮获取中文摘要

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.