Anthracnose of poplar caused by is a leaf disease that seriously affects poplar growth. The pathogen invades the host in the form of adherent cells, which generate turgor pressure through the metabolism of intracellular substances prior to penetrating the epidermis of poplar leaves. In this study, the expansion-related pressure of the mature appressorium of the wild-type was approximately 13.02 ± 1.54 MPa at 12 h, whereas it was 7.34 ± 1.23 MPa and 9.34 ± 2.22 MPa in the melanin synthesis-related gene knockout mutants Δ and Δ , respectively. The and genes were highly expressed at 12 h in the wild-type control, implying that the DHN melanin biosynthesis pathway may play an important role in the mature appressorium stage. The transcriptome sequencing analysis indicated that the upregulated melanin biosynthesis genes in , such as , , , , and , are involved in specific KEGG pathways (i.e., fatty acid biosynthesis, fatty acid metabolism, and biotin metabolism). Therefore, we speculate that the melanin synthesis-related genes and fatty acid metabolism pathway genes contribute to the regulation of the turgor pressure in the mature appressorium, ultimately leading to the formation of infection pegs that enter plant tissues. These observations may reflect the co-evolution of and its host.