Sub-cellular localization of suppressor proteins of tomato leaf curl New Delhi virus

Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, is the most important among the 14 species of begomoviruses infecting tomato in Indian subcontinent. Begomovirus is known to  evade RNA silencing of host plants through suppressor proteins. However, in case of ToLCNDV, the suppressor proteins have not been studied well. The objective of the study is to know the sub-cellular localization of three suppressor proteins encoded by AV2, AC2 and AC4 ORFs of ToLCNDV in Nicotiana benthamiana. AV2, AC2 and AC4 ORFs of ToLCNDV were  cloned and sequenced (accession numbers MW423574, MW423576, MW423575, respectively) from a ToLCNDV isolate characterized earlier (accession number MW429271) and GFP tagged constructs were prepared in a plant expressing binary vector pEarleygate103. Bioinformatics analysis using Peptide 2.0 server predicted that all these proteins have more basic amino acid residues then acidic amino acid and AV2 protein has more hydrophobic amino acid residues. ScanProsite server predicted presence of different fuctional motifs in these proteins amongst which presence of kinase motif was observed in all of them. Virus mPLoc server predicted their subcellular localization. The suppressor gene constructs were agroinfiltrated on to leaves of one month old N. benthamiana plants and their subcellular localization has been studied through confocal microscopy. Results have shown that AV2 localizes in the host cell membrane and nucleus, AC2 in the nucleus and AC4 in the host cell membrane. Earlier reports with other begomoviruses also showed similar localization behaviour of these suppressor protein except AV2, where it was shown to be present in cytoplasm. Such localization study will help understand the mechanism of their suppression activity.