Nicotiana benthamiana plants transformed with the plum pox virus helicase gene are resistant to virus infection

Nicotiana benthamiana Domin. plants were transformed with the cytoplasmic inclusion protein (CI) gene of plum pox potyvirus (PPV) to investigate, whether this non-structural protein would be able to confer resistance. The CI protein is an RNA helicase, which contains a conserved nucleotide binding motif (NTBM) and plays an important role in viral replication. Two gene constructions were developed for plant transformation. The first contains the original coding sequence of the CI gene under the control of 35S-promoter and nos terminator signal, the second is mutated in the NTBM region. Several transgenic plant lines were obtained following Agrobacterium tumefaciens-mediated transformation. The integration of the viral genes into the plant genome was confirmed using the polymerase chain reaction and the transgene derived mRNAs were detected by Northern blot hybridization. The CI protein in the transgenic plants could not be detected by Western blot analyses. One transgenic line containing the mutated CI gene remained completely symptomless after PPV infection, indicating that the putative defective helicase gene was capable of eliciting virus resistance.