Fig. 3

Effect of π-stackings on systemic and cell-to-cell movement, and replication of TVBMV. a Systemic movement of TVBMV-GFP, TVBMV-GFP (ΔY105) and TVBMV-GFP (ΔY147). GFP fluorescence in the systemic leaves of N. benthamiana plants inoculated with the virus was observed under UV light at 7 dpai. Blue and green arrows indicate the infiltrated and systemic leaves, respectively. b Viral accumulation was detected by ELISA in the systemic leaves of N. benthamiana plants inoculated with TVBMV-GFP, one of its 16 mutants derived from aromatic residues (F⁹⁴, W⁹⁷, Y⁹⁸, Y¹⁰⁵, W¹²², W¹³⁶, Y¹⁴⁷, F¹⁶⁰, F¹⁶⁷, Y¹⁷⁴, Y¹⁸⁴, Y¹⁸⁸, Y²⁰², F²⁰⁴, F²⁰⁶ and Y²⁰⁷) that were predicted to form π-stacking, or TVBMV-GFP (ΔF46) at 7 dpai. A sample from a buffer-inoculated plant was used as a negative control (Mock). c Cell-to-cell movement of 16 viral mutants in the infiltrated leaves at 5 dpai. The number of cells showing GFP fluorescence was the means ± SD from 30 infection foci per treatment. d Cell-to-cell movement of TVBMV-GFP, TVBMV-GFP (ΔY105), and TVBMV-GFP (ΔY147). GFP fluorescence was observed under a confocal microscope at 5 dpai. e The replication levels of TVBMV-GFP, viral mutants derived from 16 aromatic residues, and replication-defective mutants TVBMV-GFP (NIbΔGDD) and TVBMV-GFP (CPSTOP). The minus-strand (−)RNA accumulating level in the infiltrated leaf patches was detected by RT-qPCR at 60 hpai. The expression level of NbEF1A was used as an internal control. The values are the means ± SD from three biological replicates per treatment. Treatments marked with the same lowercase letters have no significant statistical difference. The inoculation tests were repeated three times independently. The viral systemic and cell-to-cell movement in the plants inoculated with the other 14 aromatic mutants were the same as those in the plants inoculated with TVBMV-GFP (ΔY105) and TVBMV-GFP (ΔY147) (data are not shown). ‘Δ’ indicates the codon for the residue has been deleted