Development of Infectious Clones of Tomato Leaf Curl Karnataka Virus (ToLCKV) and Study of Pathogenicity on Tomato (Solanum Lycopersicum L.) through Agroinoculation
R. K. Likhith
Department of Plant Biotechnology, University of Agricultural Sciences, GKVK, Bangalore, 560065, India.
Anitha Peter *
Department of Plant Biotechnology, University of Agricultural Sciences, GKVK, Bangalore, 560065, India.
*Author to whom correspondence should be addressed.
Abstract
Tomato leaf curl disease (ToLCD) caused by Tomato leaf curl Karnataka virus (ToLCKV) is a serious threat to tomato production. It is a Begomovirus (Geminiviridae) with monopartite single stranded DNA and transmitted by whitefly. In this study, we have amplified the whole genome of ToLCKV by rolling circle amplification (RCA) using the bacteriophage Phi 29 DNA polymerase. The infectious clones (ICs) were generated by monomerising the RCA products by partial digestion using a single restriction enzyme followed by cloning into a single digested binary vector. The resultant recombinant vector was sequenced, which revealed the genome to be 2663 bp in length, with the consensus nonanucleotide found in all geminiviruses. The sequence information was used for recombination analysis using Recombination Detection Program (RDP). The recombination analysis using RDP method and gene conversion method showed the virus to be recombinant. The genome was moved to Agrobacterium for agroinoculation of tomato plants. Susceptible variety and a resistant hybrid of tomato were agroinoculated to study the pathogenicity of the ICs. The susceptible variety showed leaf curling and yellowing symptoms 15 days post inoculation (dpi), with resistant hybrid not showing much symptoms except on the lower leaves, confirming that monopartite component is sufficient for successful infection.
Graphical Abstract
Keywords: Rolling circle amplification (RCA), infectious clones (ICs), Tomato leaf curl Karnataka virus (ToLCKV), Agroinoculation, Recombination detection program (RDP), Solanum lycopersicum