Development of infectious clones of cassava mosaic virus and their validation

Abstract

The study entitled “Development of infectious clones of cassava mosaic virus and their validation” was conducted at the ICAR- Central Tuber Crop Research Institute (ICAR-CTCRI), Sreekariyam, Thiruvananthapuram during 2017- 2018. The major objectives of the study were cloning and characterisation of SLCMV/ICMV infected leaf samples, construction of infectious clones of SLCMV/ICMV and agroinoculation of Nicotiana benthaminana with the partial dimers constructed in order to check the infectiousness of the viral clones. The whole genome amplification of SLCMV/ICMV DNA samples were done and cloned in pUC19 vectors to obtain pSLCMV A7 (2746 bp), pSLCMV B2 (2738 bp) and pICMV A5 (2739 bp) full length clones. The sequence of pSLCMV A7 showed maximum similarity of 99 % with ‘SLCMV-[TVM1]’ sequence in NCBI blast. While the sequence of pSLCMV B2 showed maximum similarity of 99 % with ‘SLCMV-[Ker20]’ sequence in NCBI blast. The sequence of pICMV A5 showed maximum similarity of 95 % with ‘ICMV-[Mah]’ sequence in NCBI blast. In order to develop infectious clones, partial dimers were constructed for SLCMV DNA-A and SLCMV DNA-B and cloned in binary vector pPZP201. These infectious clones were successfully transformed into wild type A. tumefaciens, Ach5 strain by triparental method. Then agroinoculation of N. benthamiana with the constructed partial dimers was found to be successful. After 14 days post inoculation, plants infected with DNA-A + DNA-B partial dimers showed severe symptoms like leaf curling, stunting. The plants infected with partial dimer of DNA-A alone showed mild symptoms like upward leaf curling which confirmed its monopartite lineage. While those plants agroinoculated with partial dimer of DNA-B alone did not show any symptoms. These efficient infectious clones of cassava mosaic virus and their subsequent inoculation technique would provide a major advancement to the resistance development in cassava.