Comparative ubiquitinome analysis reveals the roles of protein ubiquitination in the heat stress response from Metarhizium robertsii.

Protein ubiquitination plays a vital role in the stress response of diverse filamentous fungi. However, few reports are available on fungal insect pathogens, including Metarhizium. Here, we report a comparative ubiquitylome analysis of Metarhizium robertsii exposed to heat stress. The growth of M. robertsii was suppressed, and protein ubiquitination levels were markedly promoted during heat stress. Compared to the control treatment, there were 4,674 sites with differential ubiquitination, of which 3,419 lysine ubiquitination sites across 1,344 proteins were significantly upregulated, and 1,255 sites on 750 proteins were downregulated under heat stress. Further analysis showed that these proteins with upregulated modified sites were preferentially enriched in the phenylalanine, tyrosine, and tryptophan biosynthesis, pantothenate and CoA biosynthesis, and O-glycan biosynthesis pathways. Proteins with downregulated modified sites were significantly enriched in different pathways, including alanine, aspartate, and glutamate metabolism, pyruvate metabolism, and fatty acid biosynthesis. In particular, a key protein (phosphoenolpyruvate carboxykinase, MrPCK1, a central enzyme in gluconeogenesis and pyruvate metabolism) with five ubiquitination sites was identified, and functional analysis further revealed its regulatory role in heat stress tolerance of M. robertsii. Taken together, our findings suggest that M. robertsii may respond to heat stress not only through the canonical pathway of the proteasome but also by modulating specific metabolic pathways, including pyruvate metabolism (notably via MrPCK1) and potentially fatty acid biosynthesis. The results provide insights into the molecular mechanisms by which ubiquitination regulates the heat stress response in M. robertsii and contribute to our understanding of thermotolerance in filamentous fungi. IMPORTANCE: Entomopathogenic fungi such as Metarhizium robertsii 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 M. robertsii 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 M. robertsii, 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.