Myricetin Derivative LP11 Targets Cucumber Mosaic Virus 2b Protein to Achieve In Vivo Antiviral Activity in Plants.

Cucumber mosaic virus (CMV) 2b protein plays a key role in the process of CMV infecting plants and symptom formation and is a potential molecular target for the control of this important plant virus. The exploitation of antiviral compounds is one of the strategies with the highest input: output ratio in plant protection. In this study, the CMV 2b recombinant protein was cloned, purified, and identified as the target protein by mass spectrometry. Subsequently, we carried out preliminary functional screening of the LP series of myricetin derivatives designed and synthesized in our laboratory and commercial antiviral compounds by microscale thermophoresis (MST), which showed that LP compounds LP4, LP11, LP13, and LP20 interacted well with CMV 2b, with dissociation constant (Kd) values of 1.39, 0.88, 1.52, and 1.77 µM, respectively. Among the commercially available antiviral compounds, ningnanmycin (NNM) was the most active, with a Kd value of 4.09 µM. Then, the strongest binding force to CMV 2b was identified to be from LP11 by isothermal titration calorimetry (ITC) experiments, with a Kd of 1.19 µM. Among the commercial compounds, NNM had the strongest binding force with CMV 2b, with a Kd of 4.62 µM. Through the screening of commercial compounds and LP series compounds by MST and ITC, LP11, NNM (positive control), LP16 (negative control), and the blank control group were selected to test the in vivo impact of LP11 on CMV. Specifically, the screened compounds were sprayed onto CMV-inoculated Nicotiana benthamiana plants to determine their impact on the regulation of CMV pathogenic gene expression, symptoms, and virus titer. The results showed that LP11 had a strong ability to inhibit CMV infection of tobacco at the transcriptional and translational levels. By mutating the CMV 2b protein, the 15th amino acid leucine and the 18th amino acid methionine at the N-terminal region were shown to be potential sites for binding to compound LP11. This finding provided a theoretical basis for screening and developing anti-CMV agents.

Methyl Eugenol Binds Recombinant Gamma-Aminobutyric Acid Receptor-Associated Protein from the Western Flower Thrips Frankliniella occidentalis.

The western flower thrips (Frankliniella occidentalis) is a major pest insect in agriculture. However, few insecticides are effective for their control. The recombinant gamma-aminobutyric acid receptor-associated protein (rGABARAP) was examined as a potential target of the monoterpenoids responsible for their insecticidal activities. The insecticidal activity of anethole, linalool, and methyl eugenol (ME) was evaluated in the laboratory. The half-maximum lethal concentration (LC50) of ME against second-instar nymphs of F. occidentalis was 5.5 mg/L using membrane and leaf immersion methods, while that of spinosyn A was 1.0 mg/L. The dissociation constants of ME binding to rGABARAP were 1.30 and 4.22 µmol/L, respectively, according to microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) measurements. A molecular docking study showed interactions between ME and Tyr174 via π-π stacking. The MST and ITC experiments showed loss of specific binding between ME and the rGABARAPY174A mutant. Therefore, Tyr174 is a key amino acid residue of rGABARAP involving ME binding. The results revealed GABARAP as a potential target for the development of monoterpenoid insecticides.

Interactions between stipuol enantiomers and human serum albumin.

Natural polyacetylenes occur in food and herbal plants, have a wide range of bioactivities, and are recognized as important nutraceuticals. Stipuol is a natural polyacetylene present in the edible plant Panax notoginseng. The present study was aimed to study interactions of rac-stipuol and its enantiomers with human serum albumin (HSA) using multi-spectroscopic, molecular modeling and microscale thermophoresis. Steady-state and time-resolved fluorescence spectra manifest that the fluorescence quenching mechanism is mainly static in type. The bindings of (S)-stipuol, (R)-stipuol, rac-stipuol lead to some microenvironmental and slight conformational changes of HSA. Competitive ligand displacement experiments and molecular modeling studies revealed that stipuol enantiomers bind to HSA at subdomain III (site IIA). The calculated values of Ka and Kd showed that (R)-stipuol had a stronger binding affinity than (S)-stipuol. The results are informative for use of stipuol as a nutraceutical to improve human health.

Insights into a rapid screening method for anti-cucumber mosaic virus compounds.

Cucumber mosaic virus (CMV) is a detrimental plant virus in agricultural production. Traditionally, the half-leaf method using Nicotiana glutinosa has been used for screening agrochemicals to control CMV. However, this forms a time-consuming experimental bottleneck. In this study, we constructed a rapid screening model for anti-CMV compounds using CMV. In short, purified CMV particles were labeled through amine reactions and then subjected to binding studies with commercial compounds. The relative gene expression levels were then confirmed. Additionally, the rapid screening model results were verified using synthesized compounds. The commercial compounds ningnanmycin, ribavirin, and moroxydine hydrochloride bound to CMV with dissociation constants of 0.012, 2.870, and 0.069 µM, respectively, and they significantly inhibited expression of the gene for the CMV coat protein in CMV-infected tobacco leaves. This rapid screening model was assessed using our synthetic compounds N12, N16, and N18 through binding, which were shown to have dissociation constants 0.008, 0.025, and 70.800 µM, respectively, as well as via gene expression studies. Thus, a rapid method for screening anti-CMV commercial compounds and our synthetic compounds was constructed and confirmed.

Research on the Interaction Mechanism Between α Mino-Phosphonate Derivative Q-R and Harpin-Binding Protein 1 in Tobacco (Nicotiana tabacum) Plants.

Amino-phosphonate derivative R-diphenyl-1-(4-methylbenzothiazole-2-amino)-1-(thiphene-2-yl)-methylphosphonate (Q-R) has a high protective anti-tobacco mosaic virus (TMV) activity. However, the mechanism responsible for Q-R's effect on TMV infection is largely unknown. Here, we studied the expression levels of harpin-binding protein 1 (HrBP1) and pathogenesis-related protein-1a (PR-1a) in TMV-infected tobacco plants by using reverse transcription quantitative real-time PCR. Then, we verified the interactions between Q-R and the HrBP1 protein from Escherichia coli using isothermal titration calorimetry and studied the Q-R-associated assembly of HrBP1 using size-exclusion chromatography. The results showed that the expression levels of HrBP1 and PR-1a genes were significantly increased by Q-R at the transcriptional level in TMV-infected tobacco plants, and the E. coli-expressed HrBP1 protein was assembled into oligomers by Q-R via binding to HrBP1 with a dissociation constant of 1.19 µM. We, therefore, concluded that Q-R activated the HrBP1 and PR-1a genes and enhanced the ability of HrBP1 to assemble in tobacco plants.

Data-independent acquisition proteomic analysis of biochemical factors in rice seedlings following treatment with chitosan oligosaccharides.

Chitosan oligosaccharides (COS) can elicit plant immunity and defence responses in rice plants, but exactly how this promotes plant growth remains largely unknown. Herein, we explored the effects of 0.5 mg/L COS on plant growth promotion in rice seedlings by measuring root and stem length, investigating biochemical factors in whole plants via proteomic analysis, and confirming upregulated and downregulated genes by real-time quantitative PCR. Pathway enrichment results showed that COS promoted root and stem growth, and stimulated metabolic (biosynthetic and catabolic processes) and photosynthesis in rice plants during the seedling stage. Expression levels of genes related to chlorophyll a-b binding, RNA binding, catabolic processes and calcium ion binding were upregulated following COS treatment. Furthermore, comparative analysis indicated that numerous proteins involved in the biosynthesis, metabolic (catabolic) processes and photosynthesis pathways were upregulated. The findings indicate that COS may upregulate calcium ion binding, photosynthesis, RNA binding, and catabolism proteins associated with plant growth during the rice seedling stage.