The elucidation of the methylation status of ToCSV and the identification of microRNAs in susceptible and resistant tomato lines

Abstract: Tomato curly stunt virus (ToCSV) is one of the most important plant viruses affecting tomato production in South Africa and tomato-growing fields affected by ToCSV can have as high as 95% crop losses. The molecular interactions between a plant host and viral pathogen are complex and successful infection often depends on the intricate interplay between the plant genotype, the virus strain, the insect vector and environmental conditions. The underlying mechanisms and dynamics underlying ToCSV infection in tomato plants has not been fully elucidated. The main aim of this study was to investigate the underlying mechanisms that may be contributing to the complexities of the ToCSV-tomato pathosystem. In particular, the objectives of this study were to (1) investigate the methylation status of CpGs on the ToCSV genome post infection in a susceptible (NIL395), versus a resistant line (NIL396) of tomato and (2); to investigate the role of conversed miRNAs and their target genes in ToCSV-infected susceptible and resistant leaf tissue. The first objective involved an infectivity study monitoring ToCSV replication and progression over a 35-day period. In brief, NIL395 (S-plants) and NIL396 (R-plants) seedlings were infected with infectious clones of ToCSV via the Agrobacterium-based inoculation method. ToCSV infection and symptom development was monitored at 8 dpi (early infection), 15dpi (onset of symptoms) and 35 dpi (fully symptomatic) using conventional PCR and the Disease Severity Index (DSI), respectively. Additionally, ToCSV titer was measured relative to the expression of the 18S housekeeping genes using quantitative (qPCR) and the relative delta Ct method. ToCSV infection was positively confirmed at all three time points in both S-plants and R-plants leaf tissue where a 10.94-fold increase in viral load observed between 8 and 15 dpi, and a 1.89-fold change observed between 15 and 35 dpi in the R-plants. In S-plants, infection was confirmed at all three time points with a 264.79-fold change between 8 and 15 dpi and a 11.69-fold change between 15 and 35 dpi with a statistical significance at p < 0.05. Positively infected leaf tissue for R-plants and S-plants was then used for further downstream methylation and miRNA investigation. For the methylation study, CpG island prediction using the full genome of ToCSV was conducted using MethPrimer, MethPrimer 2.0, Emboss and Sequence manipulation suite software tool...M.Sc. (Biochemistry