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.

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.

Screening anti-TMV agents targeting tobacco mosaic virus helicase protein.

Tobacco mosaic virus helicase (TMV-Hel) plays important roles in viral multiplication. TMV-Hel is a potential target of anti-TMV agents. Our previous studies expressed and purified TMV-Hel as target protein for cytosinpeptidemycin. In this study, we preform molecular docking to study the binding sites of commercial antiviral agents with TMV-Hel. Then we verify the interactions between the potential anti-TMV agents and TMV-Hel in vitro using Microscale Thermophoresis experiment and study the inhibiting expression of TMV-Hel with the potential anti-TMV agents in vivo using Western-blot (WB) method. The results showed that ribavirin bound to TMV-Hel with a dissociation constant of 1.55 µM by direct interaction with eight binding sites, which was consistent with the docking studies. Ribavirin inhibited the expression of TMV-Hel in Nicotiana benthamiana. Docking studies combined Microscale Thermophoresis and WB experiment provided a new method to screen anti-TMV agents targeting TMV-Hel.

Discovery of Potent and Novel Quinazolinone Sulfide Inhibitors with Anti-ToCV Activity.

A series of novel quinazolinone sulfide derivatives containing a dithioacetal moiety were designed and synthesized using Tomato chlorosis virus coat protein (ToCVCP) as a potential drug target, and the inhibitory effect of ToCV was systematically evaluated in vitro and in vivo. The experimental results showed that most of the compounds presented a strong affinity. Notably, the binding abilities of compounds D8 and D16 to ToCVCP both reached a micromolar level, which were 0.19 and 0.83 µM, respectively. The relative expression level of ToCVCP gene was detected using real-time quantitative polymerase chain reaction in Nicotiana benthamiana. Compounds D8 and D16 significantly reduced the relative expression level of ToCVCP gene by 93.34 and 83.47%, respectively, which were better than those of conventional antiviral agents. This study lays a good foundation for the structural design and modification of quinazolinone sulfide derivatives as anti-ToCV drugs.

Design, synthesis and anti-TMV activities of novel chromone derivatives containing dithioacetal moiety.

Thirty-five novel chromone derivatives containing dithioacetal moiety were designed, synthesized, and their anti-TMV activities were evaluated through half-leaf method. The results showed compound c23 illustrates highly curative, protective and inactivating activities against TMV at 500 mg/L, with the values of 68.8%, 58.8%, 86.0% respectively, which were superior to that of Ribavirin (42.3%, 49.8%, 68.4%, respectively) and similar to that of Ningnanmycin (59.4%, 52.4%, 88.4%, respectively). The EC50 value of inactivating activities of compound c23 is 9.3 mg/L, which was better than that of Ribavirin (135.2 mg/L), and equivalent to that of Ningnanmycin (8.8 mg/L). Furthermore, compound c23 can destroy the integrity of TMV-CP, resulting in reduced infectivity of TMV. Meanwhile, compound c23 can combine with TMV protein coat and hydrolyze TMV protein coat to impact the process of self-assembling of TMV, with the association constant (Kd) 4.5 mg/L. This finding suggests that chromone derivatives containing dithioacetal moiety can be used as new antiviral agent.

Screening of a potential leafhopper attractants and their applications in tea plantations.

Pheromones can be used as leafhopper attractants. However, commercial pheromone products, such as the Ingle lure, have certain limitations, including poor persistence in the field. In this study, (E)-2-hexenal, (Z)-3-hexen-1-ol, (Z)-3-hexenyl acetate, (E)-ocimene, linalool, and geraniol were selected and behaviorally tested as potential leafhopper attractants. Y-tube olfactometer tests showed that the C2 formulation was more effective than other formulations. In tea field trials, the number of leafhoppers caught by sticky board traps baited with C2 lures was greater than that caught by treatment. The number of leafhoppers attracted by the C2 lures was greater than that attracted by the commercial Ingle lures. Additionally, the total amount of active C2 components on lures was greater than that of the active components on the lure after 14 days. Thus, the results indicated that the C2 formulation may attract leafhoppers and have a greater persistence than other formulations in tea field.

Activation of biochemical factors in CMV-infected tobacco by ningnanmycin.

Cucumber mosaic virus (CMV) is a plant virus with one of the largest host ranges, the widest distribution, and economic importance, and ningnanmycin (NNM) is a commercial antiviral agent. Studies have shown that NNM induces and promotes pathogenesis-related proteins in tobacco mosaic virus-inoculated tobacco. In the present study, the defense enzymes and the biochemical factors of CMV-inoculated tobacco treated with NNM were measured. The biochemical factors of CMV-inoculated tobacco leaves treated with NNM were analyzed. Results showed that the phenylalanine ammonia-lyase, peroxidase, polypheuoloxidase, and superoxide in the CMV-inoculated tobacco leaves treated with NNM were higher than those in non-treated tobacco leaves. Furthermore, NNM activated the oxidation-reduction process, metabolic process, and oxidoreductase activity in the CMV-infected tobacco.

Binding studies between cytosinpeptidemycin and the superfamily 1 helicase protein of tobacco mosaic virus.

Tobacco mosaic virus (TMV) helicases play important roles in viral multiplication and interactions with host organisms. They can also be targeted by antiviral agents. Cytosinpeptidemycin has a good control effect against TMV. However, the mechanism of action is unclear. In this study, we expressed and purified TMV superfamily 1 helicase (TMV-Hel) and analyzed its three-dimensional structure. Furthermore, the binding interactions of TMV-Hel and cytosinpeptidemycin were studied. Microscale thermophoresis and isothermal titration calorimetry experiments showed that cytosinpeptidemycin bound to TMV-Hel with a dissociation constant of 0.24-0.44 µM. Docking studies provided further insights into the interaction of cytosinpeptidemycin with the His375 of TMV-Hel. Mutational and Microscale thermophoresis analyses showed that cytosinpeptidemycin bound to a TMV-Hel mutant (H375A) with a dissociation constant of 14.5 µM. Thus, His375 may be the important binding site for cytosinpeptidemycin. The data are important for designing and synthesizing new effective antiphytoviral agents.