Fig. 5: The potential binding sites of compound Z9 and N protein significantly affect the replication of RNP and the proliferation of TSWV.

a Analysis of binding sites of compound Z9 and N protein by molecular docking. b Schematic representation of constructs expressing TSWV full-length antigenomic L_(+)opt with optimized RdRp, full-length genomic M_(−)opt with optimized glycoproteins (GP) and the viral movement protein (NSm), full-length antigenomic SR_(+)eGFP^Mut, and the sequence encoding amino acids (R^94A, G^147A, Y^184A, or R^94A&Y^184A) on the N protein gene were mutated to that of alanine. Minus sign (−) and 5′ to 3′ designation indicate the genomic RNA of TSWV. c Constructs (L(_+)opt + SR_(+)eGFP^WT) or its mutant (L(+)opt + SR_(+)eGFP^Mut) with three RNA silencing virus inhibitors VSRs (P19, Hc-Pro, and γb) co-expression in plants together with at least three plants in each experiment, the expression of eGFP protein in leaves was observed by fluorescence microscope on the 5^th day. Bars, 500 μm. d The average fluorescence intensities in infiltrated leaves on the 5^th day) was measured by ImageJ. Error bars indicate ± SD of the mean three independent experiments (n  =  3). Statistical P-values were calculated using the two-tailed independent samples t-tests (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, P > 0.05 not significant). R^94A (P = 0.0064), G^147A (P = 0.3054), Y^184A (P = 0.0001), R^94A&Y^184A (P < 0.0001). e The accumulation level of N protein in the infiltrated leaves was detected by western blot. Staining of RuBisCO with Ponceau S was used as a sample loading control. The experiment was repeated three times with similar results. f Phenotypes of TSWV^WT and TSWV^Mut (TSWV^R94A, TSWV^G147A, TSWV^Y184A, or TSWV^R94A&Y184A) -infected plants on the 21^st day. Constructs (L_(+)opt + SR_(+)eGFP^WT + M_(-)opt) or its mutant (L_(+)opt+ SR_(+)eGFP^Mut + M_(-)opt) with three RNA silencing virus inhibitors VSRs (P19, Hc-Pro, and γb) co-expression in plants together with at least ten plants in each experiment. g The infection rates of TSWV^WT and TSWV^Mut -infected plants on days 14, 17, and 21. Error bars indicate  ±  SD of the mean three independent experiments (n  =  3). h The percentages of TSWV^WT and TSWV^Mut -infected plants with different disease symptom grades. Error bars indicate ± SD of the mean three independent experiments (n  =  3). i The accumulation level of N protein in the systemic leaves was detected by western blot. Staining of RuBisCO with Ponceau S was used as a sample loading control. The experiment was repeated three times with similar results. Source data are provided as a Source Data file.