Infectivity and Mutational Analysis of Two Tomato Infecting Geminiviruses
The present work is aimed toward the study of infectivity and mutational analysis of two geminviruses that infect tomatoes, Pepper golden mosaic virus (PePGMV) and Tomato mottle virus (ToMoV) along with its potential to be used as delivery vector. Infectious viral clones of PepGMV and ToMoV were developed and used to infect Nicotiana benthamiana and tomato plants via agroinoculation. Plants inoculated with the DNA-A and DNA-B components of PepGMV developed systemic infection with characteristic yellow mosaic patterns on leaves in an average of 15 days. Southern blot hybridization of DNA extracts from symptomatic plants revealed different viral DNA forms suggesting active replication of the virus. The replication kinetics of ToMoV cloned into different vectors was assessed by agroinfusion of N. benthamiana or tomato plants with 1.5 copies of a tandemly repeating DNA-A component of ToMoV. Viral DNA was detectable at 3 days post infusion (dpi) in N. benthamiana using all three vectors. However, viral DNA was detectable at 3 days post infusion (dpi) in tomato using the pLSU vectors only. Another aim of the study was to develop a vector to assess the potential use of the virus to deliver a cargo. A PepGMV clone containing GFP instead of CP was able to express GFP in plants but was unable to start an infection even in the presence of DNA-B. The viral DNAs couldn't be detected in DNA extracts obtained from inoculated plants. Lastly, a method was developed to study the mutation in GFP gene delivered by the PepGMV virus. A high rate of mutation was observed based on the positive samples we had, although the number of sequenced clones needs to be increased. Together the experiments in this study have determined that both PePGMV and ToMoV can potentially be used to further develop a viral delivery system to introduce beneficial traits into plants.