The polypeptide extract of the dry mycelium of Penicillium chrysogenum (PDMP) can protect tobacco plants from tobacco mosaic virus (TMV), although the potential mechanism underlying PDMP-mediated TMV resistance remains unknown. In our study, we analyzed this potential mechanism via RNA sequencing (RNA-seq) and found that the ABA biosynthetic pathway and β-1,3-glucanase, a callose-degrading enzyme, might play an important role in the PDMP-induced priming of resistance to TMV. To test our hypothesis, we successfully generated a Nicotiana benthamiana ABA biosynthesis mutant and evaluated the role of the ABA pathway in PDMP-induced callose deposition during plasmodesmata resistance to TMV infection via aniline blue staining, quantitative real-time polymerase chain reaction, western blotting, and immunoelectron microscopy. Our results suggested that PDMP can induce callose priming to defend against TMV movement. PDMP inhibited TMV movement by increasing callose deposition around plasmodesmata, but this phenomenon did not occur in the ABA biosynthesis mutant; moreover, these effects of PDMP on callose deposition could be rescued by treatment with exogenous ABA. Our results suggested that callose deposition around plasmodesmata in wild-type plants is mainly responsible for the restriction of TMV movement during the PDMP-induced defensive response to TMV infection and that ABA biosynthesis apparently plays a crucial role in PDMP-induced callose priming for enhancing defense against TMV.