A comprehensive phosphoproteomic analysis of infection-related development by the rice blast fungus Magnaporthe oryzae reveals a complex phosphorylation landscape critical for plant infection. The study maps 8,005 phosphosites on 2,062 fungal proteins, highlighting major re-wiring of phosphorylation-based signaling cascades during appressorium development. Comparing phosphosite conservation across 41 fungal species identifies phosphorylation signatures associated with biotrophic and hemibiotrophic fungal infection. Using parallel reaction monitoring (PRM), 32 Pmk1 substrates are identified, with Vts1 requiring Pmk1-dependent phosphorylation for virulence. The study defines the phosphorylation landscape of infection, providing potential therapeutic interventions for plant disease control. Pmk1 is essential for appressorium development and virulence, with its activity regulated by phosphorylation. The phosphorylation landscape reveals key signaling pathways, including those involved in autophagy, lipid metabolism, and melanization, which are crucial for appressorium function and plant infection. The study also identifies conserved phosphosites across fungal species, highlighting the role of Pmk1-related MAPK pathways in fungal pathogenesis. Quantitative phosphoproteomic analysis enables the identification of Pmk1-dependent phosphorylation events, revealing the specific signaling pathways targeted by the MAPK. The study demonstrates the importance of phosphoproteomic changes during infection-related development by a pathogenic fungus, providing insights into the regulatory processes controlled by Pmk1 during infection. The findings underscore the potential of phosphoproteomic datasets as a resource for identifying novel virulence determinants in a wide range of fungal species.A comprehensive phosphoproteomic analysis of infection-related development by the rice blast fungus Magnaporthe oryzae reveals a complex phosphorylation landscape critical for plant infection. The study maps 8,005 phosphosites on 2,062 fungal proteins, highlighting major re-wiring of phosphorylation-based signaling cascades during appressorium development. Comparing phosphosite conservation across 41 fungal species identifies phosphorylation signatures associated with biotrophic and hemibiotrophic fungal infection. Using parallel reaction monitoring (PRM), 32 Pmk1 substrates are identified, with Vts1 requiring Pmk1-dependent phosphorylation for virulence. The study defines the phosphorylation landscape of infection, providing potential therapeutic interventions for plant disease control. Pmk1 is essential for appressorium development and virulence, with its activity regulated by phosphorylation. The phosphorylation landscape reveals key signaling pathways, including those involved in autophagy, lipid metabolism, and melanization, which are crucial for appressorium function and plant infection. The study also identifies conserved phosphosites across fungal species, highlighting the role of Pmk1-related MAPK pathways in fungal pathogenesis. Quantitative phosphoproteomic analysis enables the identification of Pmk1-dependent phosphorylation events, revealing the specific signaling pathways targeted by the MAPK. The study demonstrates the importance of phosphoproteomic changes during infection-related development by a pathogenic fungus, providing insights into the regulatory processes controlled by Pmk1 during infection. The findings underscore the potential of phosphoproteomic datasets as a resource for identifying novel virulence determinants in a wide range of fungal species.