The study of effector proteins is essential for understanding the molecular mechanisms that enable plant pathogens to infect host species. Austropuccinia psidii, the causal agent of myrtle rust, utilises a repertoire of effector proteins to manipulate host plant cellular processes. Given that A. psidii has been observed to infect over 500 species within the Myrtaceae family, understanding its infection mechanism is critical for developing effective treatments.
Characterising effector proteins often requires a multidisciplinary approach that combines cell biology, structural biology, and protein biochemistry techniques. These techniques depend on obtaining pure protein in milligram quantities, typically achieved through recombinant expression in Escherichia coli. However, as is the case with A. psidii, obtaining large quantities of proteins with disulfide bonds has proven challenging, with previous efforts often resulting in poor yield and solubility. The development of SHuffle T7 competent E. coli cells was a significant step toward optimizing disulfide bond formation without the need for a periplasmic tag. The coexpression vector CyDisCo (cytoplasmic disulfide bond formation in E. coli), along with its naturalised version FunCyDisCo (Fungal CyDisCo), has further improved yields of disulfide-bonded effector proteins in E. coli.
In this study, we aim to enhance the naturalisation of this system by integrating A. psidii Protein Disulfide Isomerase and Sulfhydryl Oxidase into the coexpression system to improve the folding, solubility, and yield of A. psidii effector proteins. A PaRTI FunCyDisCo (Austropuccinia psidii Recombinantly Integrated FunCyDisCo) system demonstrates the versatility of the FunCyDisCo platform for studying effector proteins from rust fungi plant pathogens.