G-quadruplex in the TMV Genome Regulates Viral Proliferation and Acts as Antiviral Target of Photodynamic Therapy.

Plant viruses seriously disrupt crop growth and development, and classic protein-targeted antiviral drugs could not provide complete protection against them. It is urgent to develop antiviral compounds with novel targets. Photodynamic therapy shows potential in controlling agricultural pests, but nonselective damage from reactive oxygen species (ROS) unexpectedly affects healthy tissues. A G-quadruplex (G4)-forming sequence in the tobacco mosaic virus (TMV) genome was identified to interfere the RNA replication in vitro, and affect the proliferation of TMV in tobacco. N-methyl mesoporphyrin IX stabilizing the G4 structure exhibited inhibition against viral proliferation, which was comparable to the inhibition effect of ribavirin. This indicated that G4 could work as an antiviral target. The large conjugate planes shared by G4 ligands and photosensitizers (PSs) remind us that the PSs could work as antiviral agents by targeting G4 in the genome of TMV. Chlorin e6 (Ce6) was identified to stabilize the G4 structure in the dark and selectively cleave the G4 sequence by producing ROS upon LED-light irradiation, leading to 92.2% inhibition against TMV in vivo, which is higher than that of commercial ningnanmycin. The inhibition of Ce6 was lost against the mutant variants lacking the G4-forming sequence. These findings indicated that the G-quadruplex in the TMV genome worked as an important structural element regulating viral proliferation, and could act as the antiviral target of photodynamic therapy.

An automatic fluorescence phenotyping platform to evaluate dynamic infection process of Tobacco mosaic virus-green fluorescent protein in tobacco leaves.

Tobacco is one of the important economic crops all over the world. Tobacco mosaic virus (TMV) seriously affects the yield and quality of tobacco leaves. The expression of TMV in tobacco leaves can be analyzed by detecting green fluorescence-related traits after inoculation with the infectious clone of TMV-GFP (Tobacco mosaic virus - green fluorescent protein). However, traditional methods for detecting TMV-GFP are time-consuming and laborious, and mostly require a lot of manual procedures. In this study, we develop a low-cost machine-vision-based phenotyping platform for the automatic evaluation of fluorescence-related traits in tobacco leaf based on digital camera and image processing. A dynamic monitoring experiment lasting 7 days was conducted to evaluate the efficiency of this platform using Nicotiana tabacum L. with a total of 14 samples, including the wild-type strain SR1 and 4 mutant lines generated by RNA interference technology. As a result, we found that green fluorescence area and brightness generally showed an increasing trend over time, and the trends were different among these SR1 and 4 mutant lines samples, where the maximum and minimum of green fluorescence area and brightness were mutant-4 and mutant-1 respectively. In conclusion, the platform can full-automatically extract fluorescence-related traits with the advantage of low-cost and high accuracy, which could be used in detecting dynamic changes of TMV-GFP in tobacco leaves.