Designing a synthetic LRR generator for the improvement of plant immunity

Crop diseases caused by pests and plant pathogens pose a risk for the global food security. They are
responsible for substantial crop yield losses; roughly 22% in wheat, 30% in rice, and 21% in soybean. This
is problematic for the agricultural production which must increase by 60-70% by 2050 to sustain the growing
populations. A disease arises when the following three factors are present simultaneously: a pathogen, a
susceptible host, and a favourable environment. Due to climate change caused rise in temperatures, the
risk for the spreading of blights into currently more temperate climate zones is increasing. Selective
breeding 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 possess
an adaptive immune system, but instead rely on different mechanisms of innate immunity. These can be
classified into effector-triggered immunity (ETI) and pattern-triggered immunity (PTI). PTI entails the
transmembrane receptor-mediated responses against pathogen molecules such as flagellin. ETI, on the
other hand, provides defences against effector molecules - pathogenic gene products, deployed
intracellularly. This mode of immunity is mediated by the intracellular nucleotide-binding leucine-rich repeat
family 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 the
hypersensitive 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 a
mediator between the pathogenic molecule and the receptor. Some recognise a number of effectors
directly, while others can interact with multiple cofactors. This highlights the dynamic nature of NLR receptor
evolution. Plants genomes can encode for hundreds of these. The problem is that even if this might be true
for cultivated crops, the same selective pressures found in the wild are not acting upon their NLRs while
the 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 the
model plant organism Arabidopsis thaliana by introducing synthetic LRRs into existing NLR receptors, as
an alternative to gene stacking. We will design a synthetic construct which will enable the generation of a
library of synthetic LRRs. Once the mutant library has been obtained, the LRR variants will be screened
against a shortlist of common effectors using the yeast-2-hybrid (Y2H) technique. The positive variants will
then be introduced in a selected set of well-studied NLR receptors by replacing the existing LRR-encoding
sequences. We hope to transform Arabidopsis thaliana with the synthetic NLRs via a gene gun and test for
resistance against a set of common Arabidopsis pathogens such as Pseudomonas aeruginosa.