Cucumber mosaic virus (CMV) and Alternaria spp. are among important and devastating plant pathogens with a wide host range. However, mixed infection of CMV and Alternaria species, is more common in greenhouses and fields. 2b is an important CMV gene that plays a critical role in CMV-host interactions and bZIP60 is among the plant genes that affect by biotic stresses especially some plant viruses. In this research, the effect of silencing of 2b on bZIP60 expression and resistance to A. atra was evaluated. The results data showed induction of bZIP60 in CMV infected plants by 18 folds while six-fold downregulation by A. atra compared to the healthy plants and also it showed a reduction in bZIP60 expression to 1/2 fold in mixed infection plants in comparison with healthy plants. But transiently expression of 2b silencing gene construct in CMV infected plants reduced Alternaria necrosis leaf spot area and reduced the expression of bZIP60 to 40 fold in mixed infected plants. However, the results of this research suggested the application of CMV-2b silencing construct for induction of resistance to both pathogens.