Production of small cysteine‐rich effector proteins in Escherichia coli for structural and functional studies

Xiaoxiao Zhang, Neal Nguyen, Susan Breen, Megan A. Outram, Peter N. Dodds, Bostjan Kobe, Peter S. Solomon, Simon J. Williams

While the life-styles and infection strategies of plant pathogens are diverse, a prevailing feature is the use of an arsenal of secreted proteins, known as effectors that aid in microbial infection. In the case of eukaryotic filamentous pathogens such as fungi and oomycetes, effector proteins are typically dissimilar, at the protein sequence level, to known protein families and functional domains. Consequently, we currently have a limited understanding of how fungal and oomycete effectors promote disease. Protein biochemistry and structural biology are two methods that can contribute greatly to the understanding of protein function. Both techniques are dependent on obtaining proteins that are pure and functional, and generally require the use of heterologous recombinant protein expression systems. Here we present a general scheme and methodology for the production and characterisation of small cysteine-rich (SCR) effectors utilising Escherichia coli expression systems. Using this approach, we successfully produced cysteine-rich effectors derived from the biotrophic fungal pathogen Melampsora lini and the necrotrophic fungal pathogen Parastagonospora nodorum. Access to functional recombinant proteins facilitated crystallisation and functional experiments. These results will be discussed in the context of a general workflow that may serve as a template for others interested in understanding the function of SCR effector(s) from their plant pathogen(s) of interest. This article is protected by copyright. All rights reserved.