A single amino acid polymorphism in a conserved effector of the multihost blast fungus pathogen expands host-target binding spectrum

Accelerated gene evolution is a hallmark of pathogen adaptation and specialization following host-jumps. However, the molecular processes associated with adaptive evolution between host-specific lineages of a multihost plant pathogen remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), host specialization on different grass hosts is generally associated with dynamic patterns of gain and loss of virulence effector genes that tend to define the distinct genetic lineages of this pathogen. Here, we unravelled the biochemical and structural basis of adaptive evolution of APikL2, an exceptionally conserved paralog of the well-studied rice-lineage specific effector AVR-Pik. Whereas AVR-Pik and other members of the six-gene AVR-Pik family show specific patterns of presence/absence polymorphisms between grass-specific lineages of M. oryzae, APikL2 stands out by being ubiquitously present in all blast fungus lineages from 13 different host species. Using biochemical, biophysical and structural biology methods, we show that a single aspartate to asparagine polymorphism expands the binding spectrum of APikL2 to host proteins of the heavy-metal associated (HMA) domain family. This mutation maps to one of the APikL2-HMA binding interfaces and contributes to an altered hydrogen-bonding network. By combining phylogenetic ancestral reconstruction with an analysis of the structural consequences of allelic diversification, we revealed a common mechanism of effector specialization in the AVR-Pik/APikL2 family that involves two major HMA-binding interfaces. Together, our findings provide a detailed molecular evolution and structural biology framework for diversification and adaptation of a fungal pathogen effector family following host-jumps. Author summary Plant pathogens secrete effector proteins inside the host where they interact with host target proteins to alter cellular processes and enable infection and disease progression. These effector proteins evolve rapidly to adapt to changing host targets and to avoid recognition by the plant immune system, especially in multihost pathogens that frequently undergo host jumps or host-range expansions. The blast fungus Magnaporthe oryzae is one of the most destructive disease of cereals. Host specialization of M. oryzae lineages is generally associated with gains and losses of effector genes and selection of adaptive mutations. However, the molecular mechanisms of how effector proteins adapt to host targets is poorly understood. Here, we unravelled the biochemical and structural basis underlying adaptive evolution of the effector APikL2. APikL2 belongs to a six gene family of sequence related effectors including the well-studied effector AVR-Pik. APikL2 is exceptionally conserved across host-specific lineages but displays host-specific polymorphisms. We combine biochemical, structural and evolutionary biology methods to provide a molecular evolutionary framework how this effector family adapts to changing host targets. In APikL2, a single amino acid change expands binding to a host-target of the heavy-metal associated domain containing protein family. By reconstructing the evolutionary history of the effector family, we show that this amino acid polymorphism is a derived trait. We combine the knowledge gained from our evolutionary and structural biology analyses to unravel a common mechanism of adaptation in the APikL effector family that involves diversification of two major target-binding interfaces. Together, our findings expand our knowledge about how pathogen effectors adapt to changing host targets following host jumps.

Automated summary

Accelerated gene evolution is a hallmark of pathogen adaptation following host-jumps. In this study, the researchers unraveled the basis of adaptive evolution of APikL2, an effector gene in the blast fungus Magnaporthe oryzae. They found that a single amino acid change in APikL2 expanded its binding spectrum to host proteins, shedding light on a common mechanism of effector specialization in the AVR-Pik/APikL2 family. This research provides a detailed molecular evolution and structural biology framework for understanding how fungal pathogens adapt and diversify following host jumps.