A recombination-based platform for generating LRR domain libraries to find new plant pathogen effector binders
File(s)
Author(s)
Katalinic, Jan
Type
Thesis
Abstract
Plant pathogens pose a growing risk to food security by causing significant yield losses in staple food crops around the globe, expected to become exacerbated by climate change. Current methods for managing plant diseases include chemical control, which provides temporary protection and is associated with multiple negative impacts on the environment, and resistance breeding which is a slow and often inefficient process.
Recent efforts have shifted towards engineering plant immune receptors – particularly the intracellular nucleotide-binding leucine-rich repeat receptors (NLRs), to enable specific recognition of pathogenic virulence factors, called effectors, and downstream activation of an immune response. Current approaches to NLR engineering mostly rely on previous knowledge of receptor structures and effector binding mechanisms thus limiting efforts to the transfer of interactions from one receptor to another.
Our approach simulates the recombination-based evolution of the leucine-rich repeat (LRR) domain of NLRs which is commonly involved in the recognition of invading effectors. Here, we designed, built and tested the LRR shufflon, a DNA construct which shuffles LRRs in vivo and produces vast DNA libraries of variants via Rci recombinase-induced inversions. By incorporating intronic cis-regulatory elements in the shufflon, we enabled alternative splicing of our DNA libraries in the human cell which expands the sequence diversity further and produces functional LRR domain variants (vLRRs). These yielded promising protein folding predictions and are ready for screening against effector targets. We also discovered and tested Rci recombinase homologs and designed a reporter shufflon for comparing shuffling activites of these unusual site-specific recombinases...
Recent efforts have shifted towards engineering plant immune receptors – particularly the intracellular nucleotide-binding leucine-rich repeat receptors (NLRs), to enable specific recognition of pathogenic virulence factors, called effectors, and downstream activation of an immune response. Current approaches to NLR engineering mostly rely on previous knowledge of receptor structures and effector binding mechanisms thus limiting efforts to the transfer of interactions from one receptor to another.
Our approach simulates the recombination-based evolution of the leucine-rich repeat (LRR) domain of NLRs which is commonly involved in the recognition of invading effectors. Here, we designed, built and tested the LRR shufflon, a DNA construct which shuffles LRRs in vivo and produces vast DNA libraries of variants via Rci recombinase-induced inversions. By incorporating intronic cis-regulatory elements in the shufflon, we enabled alternative splicing of our DNA libraries in the human cell which expands the sequence diversity further and produces functional LRR domain variants (vLRRs). These yielded promising protein folding predictions and are ready for screening against effector targets. We also discovered and tested Rci recombinase homologs and designed a reporter shufflon for comparing shuffling activites of these unusual site-specific recombinases...
Version
Open Access
Date Issued
2023-12-20
Date Awarded
2024-03-01
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Windbichler, Nikolai
Southall, Tony
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)