Zhang, XiaoXiaoNguyen, NealBreen, SusanOutram, MeganDodds, Peter NKobe, BostjanSolomon, PeterWilliams, Simon2021-09-032021-09-031464-6722http://hdl.handle.net/1885/247358Although the lifestyles and infection strategies of plant pathogens are diverse, a prevailing feature is the use of an arsenal of secreted proteins, known as effectors, which 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 characterization of small cysteine-rich (SCR) effectors utilizing 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 crystallization and functional experiments. These results are 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 research was supported by the Australian Research Council (ARC) Discovery Projects DP120100685 and DP120103558. X.Z. was a recipient of an ANZ Trustee’s PhD Scholarship for Medical Research in Queensland. B.K. is a National Health and Medical Research Council (NHMRC) Research Fellow (1003325 and 1110971).application/pdfen-AU© 2016 BSPP AND JOHN WILEY & SONS LTDhttp://creativecommons.org/licenses/by-nc-nd/4.0/effector proteinsplant diseaseprotein biochemistryprotein expressionsmall cysteine-rich proteinsstructural biology201710.1111/mpp.123852020-11-23Creative Commons Attribution Non-Commercial No Derivatives 4.0 International