Fig 1.
Distribution and pattern conservation of G-PQSs in the genome of TMV.
The length of the dash arrows represents the relative pattern conservation of the PQSs in the positive strand.
Fig 2.
PQS5 exhibits typical G4 characteristics in the solution.
(A) CD spectra of PQSs (15 μM) in the genome of TMV. (B) Fluorescence response of NMM to different PQSs. The concentration of NMM and RNA were 5 μM. (C) Fluorescence response of NMM (1.5 μM) to PQS5 with increasing concentrations (0–15 μM), λex = 400 nm.
Fig 3.
G4 Formation by PQS5 interferes with RNA synthesis in vitro.
(A) Workflow of RNA stop assay. (B) Denaturing PAGE of the extended RNAs. Lane 1, FAM labeled RNA markers (S2 Table); lane 2 and lane 7, negative control without RdRP 3Dpol; lane 3 to 6, RNA synthesis with template TMV PQS5 in absence or presence of potassium at 1 mM, 10 mM, 100 mM; lane 8 to 11, RNA synthesis with template TMV PQS5 mutant in absence or presence of potassium at 1 mM, 10 mM, 100 mM.
Fig 4.
TMV PQS5 folding inhibits GFP expression from TMV genome.
(A) Mutant base sites of TMV PQS5 in different expression vectors. (B) Diagram of inoculation of TMV–GFP and tmv–gfp-mut. (C-E) Fluorescence signals of different expression vectors [(C) TMV–GFP and tmv–gfp-m1, (D) TMV–GFP and tmv–gfp-m2, and (E) TMV–GFP and tmv-gfp-m3] in leaves at 67, 79, and 91 hours post-inoculation (hpi) and corresponding plots of the fluorescence area changes with the prolong infiltration time. The ordinate values were derived from the ratio of the actual fluorescence area at different hours post inoculation to the maximum fluorescence area of the corresponding mutant strain.
Fig 5.
NMM exhibits strong inhibitory effects against TMV.
(A) Green fluorescence of TMV-GFP in N. benthamiana leaves treated by NMM at 5 μM, 10μM. (B) Western blot analysis for GFP expression in the N. benthamiana leaves treated by NMM at 5 μM, 10 μM. (C) TMV genomic RNA level in the N. benthamiana leaves treated by 50 μΜ NMM. Relative mRNA level of TMV cp gene was detected by qRT-PCR with the cDNA that was reverse transcript from the genomic RNA with TMV specific primer.
Fig 6.
Photosensitive Ce6 and Ce6TME can bind with TMV PQS5.
Chemical structures of Ce6 (A) and Ce6TME (B). Fluorescence responses of Ce6 (C) and Ce6TME (D) (1.5 μM) to increasing amounts of TMV PQS5 from 0 to 8 μM at 25°C. The spectra of the mixture with 8 μM TMV PQS5 and 1.5 μM Ce6 were highlighted in red, λex = 405 nm.
Fig 7.
ROS generated from the solution with Ce6 upon photoirradiation.
(A) Fluorescence changes in solution of 10 μM DCFH-DA in the presence of 10 μM Ce6 or Ce6TME with or without light irradiation at 25°C. Fluorescence signals of 10 μM DCFH-DA works as an ROS indicator. (B) Fluorescence intensity changes of 10 μM Ce6 in the absence or presence of 10 mM NAC upon photoirradiation at 25°C. λex = 488 nm, λem = 523 nm.
Fig 8.
Ce6 decreases the amount of TMV PQS5 upon photo-irradiation.
Denaturing polyacrylamide gel electrophoresis (20%) of 0.3 μM TMV PQS5 or the ssRNA control in the presence of 100 μM Ce6, Ce6TME after photo-irradiation for the indicated periods (0, 15, 30, 60, 90, 120 min) at 25°C (A), and in the presence of Ce6, Ce6TME with different concentrations (0, 2, 10, 25, 50, and 100 μM) after photo-irradiation for 120 min at 25°C (B). The lane of “D120” means that the samples were treated under dark for 120 min. Change of residual intact RNA of TMV PQS5 or Control ssRNA with the increasing irradiation time (C), and change of residual intact RNA of TMV PQS5 or Control ssRNA with compounds at different concentrations (0–100 μM) (D). Error bars represent mean ± SD; n = 3. (E) 20% denaturing polyacrylamide gel electrophoresis of 0.3 μM TMV PQS5 mixing with 100 μM Ce6 in the absence or presence of 10 mM NAC, followed by photo-irradiation for 120 min at 25°C. (F) Residual intact TMV PQS5 RNA from denaturing polyacrylamide gel after photo-irradiation. Error bars represent mean ± SD; n = 3.
Fig 9.
QRT-PCR assay of relative expression of the TMV cp gene in BY-2 cells.
BY-2 cells were pre-infected with TMV virions for 3 h, and then treated with Ce6, followed by being irradiated with white light or in the dark for 0.5 h. In the treatment of “10 + NAC”, BY-2 cells infected with TMV virions were treated with 10 μM Ce6 and 10 mM NAC.
Table 1.
Anti-TMV activity of Ce6 under normal-light and LED-light irradiation in vivo.
Fig 10.
Ce6 inhibits TMV proliferation via binding to RNA G4 in viral genome.
(A) Fluorescence signals of different expression vectors in leaves treated with 10 μM Ce6 in left-half and 1% DMSO in right-half at 60, 75, 90, and 105 hpi. (B) Plots of the fluorescence area changes of different expression vectors with/without Ce6 at different hpi.