Functional analysis of target of rapamycin in regulating mycelial growth and development in Penicillium italicum

Citrus fruits are highly valued for their nutritional benefits and global economic significance. They are susceptible to many fungal pathogens during postharvest storage. Citrus blue mold, caused by the fungus Penicillium italicum, is a major citrus disease, leading to significant economic losses every year. P. italicum spreads rapidly after infection, and the molecular mechanisms regulating its growth and development are largely unknown. Target of rapamycin (TOR) is a central regulator of cell growth, metabolism, and stress responses in eukaryotes. In this study, we investigated the role of TOR in P. italicum using rapamycin, a specific TOR inhibitor. We found that P. italicum possesses a single TOR gene. Rapamycin treatment significantly inhibited mycelial growth of P. italicum by up to 50%, leading to small, twisted mycelia cells and disordered growth orientation. The distribution of mycelial layers was also affected by rapamycin treatment. In addition, rapamycin strongly suppressed conidia formation, while it did not influence conidial germination. These observations suggest that PiTOR regulates mycelial growth and differentiation, but not conidial germination in P. italicum. Transcriptomic analysis showed that rapamycin treatment led to 322 upregulated genes and 149 downregulated genes. Gene ontology (GO) annotation analysis revealed that many dysregulated genes were involved in membrane integrity, consistent with the observed morphological abnormalities. GO enrichment analysis further showed that downregulated genes were associated with cell wall components, whereas upregulated DEGs were linked to amino acid metabolic and catabolic processes, suggesting a crucial role of PiTOR in regulating cell growth and metabolism. This study enhances our understanding of TOR signaling in phytopathogenic fungi and facilitates the development of potential strategies for controlling citrus blue mold.