Multiplexed effector screening for recognition by endogenous resistance genes using positive defense reporters in wheat protoplasts.
Salome WilsonBayantes DagvadorjRita TamLydia MurphySven Schulz-KroenertNigel HengEmma E CreanJulian R GreenwoodJohn P RathjenBenjamin SchwessingerPublished in: The New phytologist (2024)
Plant resistance (R) and pathogen avirulence (Avr) gene interactions play a vital role in pathogen resistance. Efficient molecular screening tools for crops lack far behind their model organism counterparts, yet they are essential to rapidly identify agriculturally important molecular interactions that trigger host resistance. Here, we have developed a novel wheat protoplast assay that enables efficient screening of Avr/R interactions at scale. Our assay allows access to the extensive gene pool of phenotypically described R genes because it does not require the overexpression of cloned R genes. It is suitable for multiplexed Avr screening, with interactions tested in pools of up to 50 Avr candidates. We identified Avr/R-induced defense genes to create a promoter-luciferase reporter. Then, we combined this with a dual-color ratiometric reporter system that normalizes read-outs accounting for experimental variability and Avr/R-induced cell death. Moreover, we introduced a self-replicative plasmid reducing the amount of plasmid used in the assay. Our assay increases the throughput of Avr candidate screening, accelerating the study of cellular defense signaling and resistance gene identification in wheat. We anticipate that our assay will significantly accelerate Avr identification for many wheat pathogens, leading to improved genome-guided pathogen surveillance and breeding of disease-resistant crops.
Keyphrases
- genome wide
- genome wide identification
- bioinformatics analysis
- high throughput
- cell death
- dna methylation
- crispr cas
- transcription factor
- genome wide analysis
- copy number
- escherichia coli
- public health
- high glucose
- diabetic rats
- oxidative stress
- single cell
- cell proliferation
- gene expression
- single molecule
- innate immune
- regulatory t cells
- living cells
- fluorescent probe
- cell cycle arrest