Rewinding the tape: historical contingency and functional constraints have shaped the evolution of APikL virulence effectors in the blast fungus

Protein evolution is influenced by historical contingencies and functional constraints, but their combined impact on rapidly diversifying pathogen virulence effectors remains poorly understood. Here, we combined ancestral state reconstructions and functional assays to recapitulate the evolution of the MAX-fold effector protein APikL2 of the plant pathogenic blast fungus Magnaporthe (syn. Pyricularia) oryzae, focusing on the ancestral and functionally critical amino acid residue D66 (Asp, codon: GAT). 'Rewinding the tape' experiments based on ancestral sequence resurrection revealed that, out of the seven potential amino acid substitutions derived from single nucleotide polymorphisms, only the naturally occurring D66N (Asp to Asn, GAT to AAT) expanded the binding spectrum to host plant proteins of the heavy metal associated (HMA) family. In contrast, three of the non-synonymous substitutions were deleterious resulting in loss of binding to HMA proteins. Additionally, we identified three cases of homoplasy in the APikL effector family, involving HMA-binding interfaces, indicating recurrent convergent evolution. Our findings suggest an experimental framework for predicting evolutionary outcomes of pathogen effector-host target interactions with implications for plant disease resistance breeding.