To explore the active metabolites from Aspergillus, we have discovered five undescribed tricyclic or tetracyclic indole alkaloids aspergillines K-O (1-5) and four known analogues (6-9) from the strain of Nicotiana tabacum-derived Aspergillus versicolor YNCA0363. The alpha-amino group derived from tryptophan in isolates 1-6 was changed to an oxygen atom, which is uncommon in the reported analogues. To the best of our knowledge, 4 and 5 represent the first examples of indole alkaloids possessing an isochromene moiety. Compounds 1 and 3 showed obvious anti-tobacco mosaic virus (TMV) activities with inhibition rates of 43.6% +/- 2.2 and 58.5% +/- 2.8 at the concentration of 20 mu M, respectively, which were significantly higher than that of the positive control. Compound 3 also showed excellent potential of inducing systemic acquired resistance. Furthermore, compound 3 can increase the activities of two reactive oxygen species-scavenging enzymes, peroxidase and superoxide dismutase, and enhance tobacco resistance to TMV. Molecular docking results showed that compound 3 exhibits a stronger interaction with TMV-helicase, and showed higher antiviral activities than others. This work revealed that the above indole alkaloids, especially 3, could induce resistance in tobacco plants against the viral pathogen TMV.