Optimized Production of Disulfide-Bonded Fungal Effectors in Escherichia coli Using CyDisCo and FunCyDisCo Coexpression Approaches

Daniel S. Yu, Megan A. Outram, Emma Crean, Ashley Smith, Yi Chang Sung, Reynaldi Darma, Xizhe Sun, Lisong Ma, David A. Jones, Peter S. Solomon, Simon J. Williams*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    2 Citations (Scopus)

    Abstract

    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 recognized by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterize 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 coexpression system within the heterologous host Escherichia coli, termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalized coexpression 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 crystallization of several of these reported fungal effectors. We suggest this approach could be broadly useful in the investigation of the function and recognition of a broad range of disulfide bond–containing effectors.

    Original languageEnglish
    Pages (from-to)109-118
    Number of pages10
    JournalMolecular Plant-Microbe Interactions
    Volume35
    Issue number2
    DOIs
    Publication statusPublished - Feb 2022

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