Comparative ubiquitinome analysis reveals the roles of protein ubiquitination in the heat stress response from <i>Metarhizium robertsii</i>

Entomopathogenic fungi such as <i>Metarhizium robertsii</i> are widely deployed as environmentally friendly biocontrol agents, yet their field performance is often limited by exposure to fluctuating and elevated temperatures. Although ubiquitination, a reversible post-translational modification that regulates protein stability, localization, and activity, is well known to orchestrate eukaryotic stress responses, its function in fungal heat adaptation has not been explored. To address this gap, we generated the proteome-wide ubiquitinome atlas of <i>M. robertsii</i> under thermal stress, cataloging modified sites across diverse metabolic and signaling pathways. Building on this global dataset, we demonstrate that ubiquitination of a key protein (phosphoenolpyruvate carboxykinase) involved in pyruvate homeostasis is essential for conidial thermotolerance in <i>M. robertsii</i>, thereby contributing to our understanding of the mechanism of heat stress adaptation in fungi. These findings provide a rich dataset that will inform future functional studies and guide the rational engineering or selection of more robust fungal strains for sustainable pest management.