This study presents a comprehensive phosphoproteomic analysis of infection-related development by the rice blast fungus *Magnaporthe oryzae*, a pathogen that threatens global food security. The research aims to characterize cellular signaling during plant infection to inform future disease control strategies. Key findings include: 1. **Phosphorylation Landscape**: A total of 8,005 phosphosites on 2,062 fungal proteins were identified during infection-related development, revealing significant changes in phosphorylation dynamics, particularly in the early stages of appressorium formation. 2. **Conserved Phosphorylation**: Phosphosite conservation across 41 fungal species was analyzed, revealing phosphorylation signatures associated with biotrophic and hemibiotrophic fungal infections. These signatures highlight conserved signaling pathways and proteins involved in infection structure development and host invasion. 3. **Pmk1 MAPK Substrates**: Parallel reaction monitoring (PRM) was used to identify 32 substrates of the Pmk1 MAPK, a master regulator of infection-related development. This includes transcription factors, kinases, and other regulatory proteins. 4. **Vts1 as a Pmk1 Target**: The study identified Vts1 as a novel Pmk1 target, which is required for rice blast disease. Vts1 is phosphorylated by Pmk1 at specific sites (S175 and S420), and its mutation impairs fungal virulence and appressorium morphogenesis. 5. **Signaling Pathways**: The study mapped differentially phosphorylated residues onto signaling pathways involved in appressorium morphogenesis and plant infection, including the cAMP-dependent protein kinase A pathway, autophagy, and the Vast1 pathway. 6. **Quantitative Phosphoproteomics**: The approach provided detailed insights into the regulatory processes controlled by Pmk1 during infection, highlighting the dynamic nature of phosphorylation in fungal infection. This research underscores the importance of phosphoproteomic analysis in understanding fungal pathogenesis and identifies potential therapeutic targets for controlling plant diseases.This study presents a comprehensive phosphoproteomic analysis of infection-related development by the rice blast fungus *Magnaporthe oryzae*, a pathogen that threatens global food security. The research aims to characterize cellular signaling during plant infection to inform future disease control strategies. Key findings include: 1. **Phosphorylation Landscape**: A total of 8,005 phosphosites on 2,062 fungal proteins were identified during infection-related development, revealing significant changes in phosphorylation dynamics, particularly in the early stages of appressorium formation. 2. **Conserved Phosphorylation**: Phosphosite conservation across 41 fungal species was analyzed, revealing phosphorylation signatures associated with biotrophic and hemibiotrophic fungal infections. These signatures highlight conserved signaling pathways and proteins involved in infection structure development and host invasion. 3. **Pmk1 MAPK Substrates**: Parallel reaction monitoring (PRM) was used to identify 32 substrates of the Pmk1 MAPK, a master regulator of infection-related development. This includes transcription factors, kinases, and other regulatory proteins. 4. **Vts1 as a Pmk1 Target**: The study identified Vts1 as a novel Pmk1 target, which is required for rice blast disease. Vts1 is phosphorylated by Pmk1 at specific sites (S175 and S420), and its mutation impairs fungal virulence and appressorium morphogenesis. 5. **Signaling Pathways**: The study mapped differentially phosphorylated residues onto signaling pathways involved in appressorium morphogenesis and plant infection, including the cAMP-dependent protein kinase A pathway, autophagy, and the Vast1 pathway. 6. **Quantitative Phosphoproteomics**: The approach provided detailed insights into the regulatory processes controlled by Pmk1 during infection, highlighting the dynamic nature of phosphorylation in fungal infection. This research underscores the importance of phosphoproteomic analysis in understanding fungal pathogenesis and identifies potential therapeutic targets for controlling plant diseases.