Identifying a possible compensatory mutation in an engineered cucumber mosaic virus isolate

Cucumber mosaic virus has probably one of the largest host ranges of any known plant virus, affecting many agriculturally important crops. It can be transmitted by more than 50 species of aphid. In order to examine the molecular requisites for virus transmission, an amino acid change at the quasi-threefold axes of the virus coat protein had been engineered to alanine to produce the mutant K101A. In my project, this mutant was demonstrated to be defective for infection in tobacco (Nicotiana tabacum), but through successive inoculations onto N.tabacum, the infectivity of K101A increased, thus suggesting a compensatory amino acid change had occurred allowing for the change in phenotype. Plasmids containing the original viral sequence were extracted, linearized, transcribed, and then inoculated onto healthy plants. RNA was extracted from the symptomatically infected plants, amplified using RT-PCR and then sequenced. The sequences were then compared with the original engineered mutant. The results of these experiments will help us to gain a better understanding of the role of the virus coat protein in viral transmission and infection.