Neofusicoccum parvum can cause twig blight of the walnut (Juglans spp.), resulting in great economic losses and ecological damage. We performed proteomic tandem mass tags (TMT) quantification of two Neofusicoccum parvum strains with different substrates, BH01 in walnut substrate (SW) and sterile water (SK), and BH03 in walnut substrate (WW) and sterile water (WK), in order to identify differentially expressed proteins. We identified 998, 95, and 489 differentially expressed proteins (DEPs) between the SK vs. WK, SW vs. SK, and WW vs. WK comparison groups, respectively. A phylogenetic analysis was performed to classify the ABC transporter proteins annotated in the TMT protein quantification into eight groups. Physicochemical and structural analyses of the 24 ATP-binding cassette (ABC) transporter proteins revealed that 14 of them had transmembrane structures. To elucidate the functions of these transmembrane proteins, we determined the relative expression levels of ABC transporter genes in strains cultured in sodium chloride, hydrogen peroxide, copper sulfate, and carbendazim mediums, in comparison with pure medium; analysis revealed differential upregulation. To verify the expression results, we knocked out the NpABC2 gene and compared the wild-type and knockout mutant strains. The knockout mutant strains exhibited a higher sensitivity to antifungal drugs. Furthermore, the virulence of the knockout mutant strains was significantly lower than the wild-type strains, thus implying that NpABC2 plays a role in the drug resistance of N. parvum and affects its virulence
