Biology subjects

Crean, E.

Publications and source records attributed to Crean, E..

2 recordsLinked to original sources

bioRxiv2023

Multiplexed effector screening for recognition by endogenous resistance genes using positive defense reporters in wheat protoplasts

O_LIPlant 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. C_LIO_LIHere, 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 fifty Avr candidates. C_LIO_LIWe identified Avr/R-induced defense genes to create 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. C_LIO_LIOur 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. C_LI

plant biology↗

bioRxiv2021

Optimised production of disulfide-bonded fungal effectors in E. coli using CyDisCo and FunCyDisCo co-expression approaches

Effectors are a key part of the arsenal of plant pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognised by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterise effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a co-expression system within the heterologous host E. coli termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalised co-expression approach termed FunCyDisCo (Fungi-CyDisCo) can significantly improve the production yields of numerous disulfide bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallisation of several of these reported fungal effectors. We suggest this approach could be useful when investigating the function and recognition of a broad range of disulfide-bond containing effectors.

biochemistry↗