eIF4E family is well known for recessive resistance gene of potyvirus in many crops. And Turnip mosaic virus (TuMV) is one of the major viruses in Brassicaceae crops which belong to the genus Potyvirus. To elucidate the key amino acids in the interaction between TuMV VPg and Brassica eIF(iso)4E, amino acids of eIF(iso)4E were mutated. Seven amino acids in cap binding pocket were chose for the candidate amino acid that may play a role in the interaction of TuMV VPg. We demonstrated that a single amino acid mutation in cap binding pocket of Brassica eIF(iso)4E can abolish the interaction with TuMV VPg. eIF(iso)4E which has a mutation at each W49, W95 and K150 positions impaired in its interaction with VPg prominently according to the yeast two hybrid analysis. Complementation of an eIF4E knockout yeast strain by mutated eIF(iso)4E proteins showed that all eIF(iso)4E mutants were able to complement eIF4E of yeast. To find out if these mutations affect the susceptibility of Chinese cabbage, transformant analysis was performed. eIF(iso)4E W95L, W95L/K150E and susceptible wild type were over-expressed in susceptible Chinese cabbage. According to the TuMV screening result of T1 and T2 transformants, over-expression of the eIF(iso)4E mutants showed resistance to four TuMV strains (CHN2, 3, 4 and 5). Our results support that the mutations in eIF(iso)4E may control the broad spectrum TuMV resistance.