Expression and purification of Austropuccinia psidii effector proteins in Escherichia coli

The plant disease myrtle rust is caused by the fungus Austropuccinia psidii. It has led to functional myrtaceous species extinctions in Australia and is a significant threat to other species globally. During infection, A. psidii secretes effector proteins that manipulate the host plant's defences. Numerous putative effectors are encoded in this pathogen's genome, some being expressed early during urediniospore germination and initial invasion of plant tissues. Four putative effector proteins (AP1260, AP5292, AP10948, and AP143) were found to be differentially expressed in the first 24-48 h of infection, suggesting that they play important roles in the infection process. As in other rust fungi, these effector proteins are small and cysteine-rich, often forming disulfide bonds, and their isolation for biophysical characterisation can be challenging. AlphaFold3 models predict that AP1260, AP5292, AP10948, and AP143 form disulfide bonds, while disorder analysis indicates the presence of intrinsically disordered regions. The four putative A. psidii effector proteins were recombinantly produced using SHuffle Escherichia coli cells with an adapted co-expression vector, 'ApFunCyDisCo'. Three of the effectors were successfully produced, but were insoluble. The fourth effector, AP1260, was successfully produced in the soluble fraction and purified using a four-step process: immobilised metal affinity chromatography, desalting, anion exchange chromatography, and size exclusion chromatography. Circular dichroism spectroscopy revealed that AP1260 has a mainly random coil character, but also has both β-strand and α-helical content. This first successful production and isolation of an A. psidii protein provides a foundation for future investigation of the molecular mechanisms of A. psidii pathogenicity.