Functional Characterization of Deubiquitinase UBP Family and Proteomic Analysis of Aaubp14-Mediated Pathogenicity Mechanism in Alternaria alternata

The <i>Alternaria alternata</i> tangerine pathotype causes Alternaria brown spot, a devastating disease of susceptible tangerine varieties and their hybrids. <i>Alternaria citri</i> toxin (ACT) is the primary virulence factor, but the regulatory mechanisms governing ACT synthesis remain unclear. Deubiquitinating enzymes maintain ubiquitination homeostasis and regulate fungal pathogenicity, yet their role in <i>A. alternata</i> remains unexplored. We characterized 13 ubiquitin-specific protease (UBP) family members in <i>A. alternata</i> tangerine pathotype. Six UBP genes (<i>Aaubp2</i>, <i>Aaubp3</i>, <i>Aaubp4</i>, <i>Aaubp6</i>, <i>Aaubp14</i>, and <i>Aaubp15</i>) regulated mycelial growth. <i>Aaubp14</i> deletion abolished sporulation, while mutations of <i>Aaubp3</i>, <i>Aaubp4</i>, <i>Aaubp6</i>, <i>Aaubp8</i>, and <i>Aaubp15</i> altered conidial morphology. qRT-PCR demonstrated distinct host-induced expression patterns among <i>Aaubp</i> genes. Pathogenicity tests showed that Δ<i>Aaubp6</i>, Δ<i>Aaubp14</i>, and Δ<i>Aaubp15</i> mutants failed to produce lesions on <i>Citrus reticulata</i> cv. Hongjv leaves. Moreover, <i>Aaubp14</i> deletion significantly suppressed ACT biosynthesis gene expression and blocked ACT production. Comparative proteomics showed Aaubp14 regulates ACT biosynthesis by modulating protein ubiquitination in metabolic pathways and controls pathogenicity via a complex network. Our findings elucidate <i>Aaubp</i> gene function in development and pathogenicity, particularly the Aaubp14-mediated regulation mechanism, providing insights into ubiquitination-mediated pathogenicity in phytopathogenic fungi.