Megan.outram@csiro.au

Jana.Sperschneider@csiro.au

Rust fungi cause severe diseases in plants and are a major threat to wheat production and global food security. Central to the success of rust infection is the secretion of effector proteins, which function to manipulate the host plant. Due to evolutionary pressures, pathogen effector proteins are often sequence diverse, but some are known to adopt conserved structural folds. To investigate conserved structural features of rust effectors that could be targeted for improved disease resistance we used AlphaFold2 to predict the structures of all secreted proteins from five rust species: wheat stem rust (Puccinia graminis f. sp. tritici (Pgt), wheat leaf rust (P. triticina), wheat tripe rust (P. striiformis), oat crown rust (P. coronata f. sp. avenae) secretomes, and flax rust (Melampsora lini).

As observed in flax rust, all six known Pgt avirulence effector proteins belong to different structural classes. This suggests that rusts employ a more structurally diverse set of effectors than oomycetes and powdery mildew pathogens, whose effectors predominantly belong to a single class (WY and RALPH, respectively). Recently, we experimentally determined the structure of the avirulence effector AvrSr27 from Pgt, which has a novel zinc-bound, duplicated domain structure.

We compared the AlphaFold2 predicted structure against our experimentally determined structure of AvrSr27 and demonstrated the prediction accuracy of AlphaFold2 for detecting zinc-binding sites. To determine if zinc binding is prevalent in rust secretomes we employed an established in silico screen, which revealed several other putative metal ion binding effector families, including those containing the recently cloned Pgt avirulence effectors, AvrSr13 and AvrSr22.

To validate our findings, we developed a workflow for the biochemical characterisation of metal-binding in rust effectors and showed that both AvrSr13 and AvrSr22 preferentially bind zinc ions. According to our predictions, zinc binding proteins account for about 13% of the secreted proteins of Pgt, and ~5-10% for the other studied rust species, suggesting that this may be a common feature in rust effectors.