, an ortholog of small ubiquitin-like modifier, is essential for growth, organelle function, virulence, and antiviral defense in .

Introduction: SUMOylation is an important post-translational modification that regulates the expression, localization, and activity of substrate proteins, thereby participating in various important cellular processes such as the cell cycle, cell metabolism, gene transcription, and antiviral activity. However, the function of SUMOylation in phytopathogenic fungi has not yet been adequately explored.

Methods: A comprehensive analysis composed of proteomics, affinity pull-down, molecular and cellular approaches was performed to explore the roles of SUMOylation in , the fungal pathogen responsible for chestnut blight.

Results And Discussion: , the gene encoding the SUMO protein CpSmt3 in was identified and characterized. Deletion of the gene resulted in defects in mycelial growth and hyphal morphology, suppression of sporulation, attenuation of virulence, weakening of stress tolerance, and elevated accumulation of hypovirus dsRNA. The Δ deletion mutant exhibited an increase in mitochondrial ROS, swollen mitochondria, excess autophagy, and thickened cell walls. About 500 putative SUMO substrate proteins were identified by affinity pull-down, among which many were implicated in the cell cycle, ribosome, translation, and virulence. Proteomics and SUMO substrate analyses further revealed that deletion of reduced the accumulation of CpRho1, an important protein that is involved in TOR signal transduction. Silencing of resulted in a phenotype similar to that of Δ, while overexpression of CpRho1 could partly rescue some of the prominent defects in Δ. Together, these findings demonstrate that SUMOylation by CpSmt3 is vitally important and provide new insights into the SUMOylation-related regulatory mechanisms in .