Matrine-Type Alkaloids with Anti-Tomato Spotted Wilt Virus Activity from the Root of Sophora tonkinensis Gagnep.

As a continuation of our research on the development of pesticide active quinolizidine alkaloids (QAs) from the family Fabaceae, the chemical constituents of the root of Sophora tonkinensis Gagnep. were systematically investigated. Seventeen new matrine-type alkaloids (1-17), including one new naturally occurring compound (17), along with 20 known ones were isolated from the EtOH extract of S. tonkinensis. Notably, compound 5 possessed an unprecedented 6/6/5/4/6/6 hexacyclic system. Their structures were confirmed via comprehensive spectroscopic data analysis (IR, UV, NMR, HRESIMS), ECD calculation, and X-ray crystallography. Biological tests indicated that compounds 1, 4, 10, 12, 13, and 30 displayed significant anti-tomato spotted wilt virus (TSWV) activities compared with the positive control ningnanmycin. Moreover, compound 12 strongly inhibited the expression of the TSWV N, NSs, and NSm genes and TSWV NSs protein in plant host. Furthermore, compounds 4, 10, 12, 20, and 22 exhibited moderate insecticidal activities against TSWV thrip vector (Frankliniella occidentalis).

Discovery of novel chromone derivatives containing a sulfonamide moiety as potential anti-TSWV agents.

A number of chromone derivatives containing sulfonamide structure were designed and synthesized. Firstly, the target compounds were evaluated for anti-TSWV activities in vivo by the half-leaf method. We found that most of the compounds had good anti-TSWV activities. Among them, compound 12B had excellent anti-TSWV inactivating activity with an EC50 of 80.5 µg/mL, which was significantly better than xiangcaoliusuobingmi (765.7 µg/mL). Secondly, TSWV nucleocapsid protein (N) was expressed and purified, and the affinity between the compounds and TSWV N was tested by microscale thermophoresis (MST). Compound 12B had a good affinity for TSWV N with a Kd value of 5.02 µM, which was superior to xiangcaoliusuobingmi (29.83 µM). Finally, in order to study the mode of interaction between the compound 12B and TSWV N, we carried out molecular docking. The results indicated that compound 12B might inactivate the virus by destroying the TSWV N oligomer structure. These results lay a solid foundation for the further discovery of chromone derivatives containing sulfonamide structure with high anti-TSWV activities.

Induction of Plant Resistance in Tobacco (Nicotiana tabacum) against Tomato Spotted Wilt Orthotospovirus through Foliar Application of dsRNA.

Thrips-transmitted tomato spotted wilt orthotospovirus (TSWV) continues to be a constraint to peanut, pepper, tobacco, and tomato production in Georgia and elsewhere. TSWV is being managed by an integrated disease management strategy that includes a combination of cultural practices, vector management, and growing virus-resistant varieties where available. We used a non-transgenic strategy to induce RNA interference (RNAi)-mediated resistance in tobacco (Nicotiana tabacum) plants against TSWV. Double-stranded RNA (dsRNA) molecules for the NSs (silencing suppressor) and N (nucleoprotein) genes were produced by a two-step PCR approach followed by in vitro transcription. When topically applied to tobacco leaves, both molecules elicited a resistance response. Host response to the treatments was measured by determining the time to symptom expression, and the level of resistance by absolute quantification of the virus. We also show the systemic movement of dsRNA_N from the inoculated leaves to younger, non-inoculated leaves. Post-application, viral siRNAs were detected for up to nine days in inoculated leaves and up to six days in non-inoculated leaves. The topical application of dsRNAs to induce RNAi represents an environmentally safe and efficient way to manage TSWV in tobacco crops and could be applicable to other TSWV-susceptible crops.

Diterpenoid compounds from Wedelia trilobata induce resistance to Tomato spotted wilt virus via the JA signal pathway in tobacco plants.

Tomato spotted wilt virus (TSWV) causes major losses of many crops worldwide. Several strategies have been attempted to control disease caused by TSWV. However, many challenges for the effective control of this disease remain. A promising approach is the use of abiotic or biotic inducers to enhance plant resistance to pathogens. We screened a diterpenoid compound from Wedelia trilobata, 3α-Angeloyloxy-9ß-hydroxy-ent-kaur-16-en-19-oic acid (AHK), which had higher curative and protective effects against TSWV than the ningnanmycin control. The rapid initiation of the expression of all the TSWV genes was delayed by more than 1d in the curative assay, and the expression of the NSs, NSm and RdRp genes was inhibited. In addition, the replication of all TSWV genes in systemic leaves was inhibited in the protective assay, with an inhibition rate of more than 90%. The concentrations of jasmonic acid (JA) and jasmonic acid isoleucine (JA-ILE) in the AHK-treated and systemic leaves of the treated plants were significantly higher than those observed in the control. The results suggested that AHK can induce systemic resistance in treated plants. The transcription of the NtCOI1 gene, a key gene in the JA pathway, was significantly higher in both the inoculated and systemic leaves of the AHK-treated plants compared to the control. The AHK-induced resistance to TSWV in Nicotiana benthamiana could be eliminated by VIGS-mediated silencing of the NtCOI1 gene. These results indicated that AHK can activate the JA pathway and induce systemic resistance to TSWV infection.

Trichoderma L-Lysine-α-Oxidase Producer Strain Culture Fluid Inhibits Impatiens Necrotic Spot Virus.

A method for PCR diagnosis of impatiens necrotic spot virus is developed. Concentrated culture fluid with active L-lysine-α-oxidase (0.54 U/ml) from Trichoderma harzianum Rifai fungus producer strain F-180 inhibits vitally hazardous impatiens necrotic spot phytovirus.

Modified expression of alternative oxidase in transgenic tomato and petunia affects the level of tomato spotted wilt virus resistance.

BACKGROUND: Tomato spotted wilt virus (TSWV) has a very wide host range, and is transmitted in a persistent manner by several species of thrips. These characteristics make this virus difficult to control. We show here that the over-expression of the mitochondrial alternative oxidase (AOX) in tomato and petunia is related to TSWV resistance. RESULTS: The open reading frame and full-length sequence of the tomato AOX gene LeAox1au were cloned and introduced into tomato 'Healani' and petunia 'Sheer Madness' using Agrobacterium-mediated transformation. Highly expressed AOX transgenic tomato and petunia plants were selfed and transgenic R1 seedlings from 10 tomato lines and 12 petunia lines were used for bioassay. For each assayed line, 22 to 32 tomato R1 progeny in three replications and 39 to 128 petunia progeny in 13 replications were challenged with TSWV. Enzyme-Linked Immunosorbent Assays showed that the TSWV levels in transgenic tomato line FKT4-1 was significantly lower than that of wild-type controls after challenge with TSWV. In addition, transgenic petunia line FKP10 showed significantly less lesion number and smaller lesion size than non-transgenic controls after inoculation by TSWV. CONCLUSION: In all assayed transgenic tomato lines, a higher percentage of transgenic progeny had lower TSWV levels than non-transgenic plants after challenge with TSWV, and the significantly increased resistant levels of tomato and petunia lines identified in this study indicate that altered expression levels of AOX in tomato and petunia can affect the levels of TSWV resistance.

Biological and molecular analyses of the acibenzolar-S-methyl-induced systemic acquired resistance in flue-cured tobacco against Tomato spotted wilt virus.

Tomato spotted wilt virus (TSWV) is an economically important virus of flue-cured tobacco. Activation of systemic acquired resistance (SAR) by acibenzolar-S-methyl (ASM) in flue-cured tobacco was studied under greenhouse conditions by challenge inoculation with a severe isolate of TSWV. ASM restricted virus replication and movement, and as a result reduced systemic infection. Activation of resistance was observed within 2 days after treatment with ASM and a high level of resistance was observed at 5 days onward. Expression of the pathogenesis-related (PR) protein gene, PR-3, and different classes of PR proteins such as PR-1, PR-3, and PR-5 were detected at 2 days post-ASM treatment which inversely correlated with the reduction in the number of local lesions caused by TSWV. Tobacco plants treated with increased quantities of ASM (0.25, 0.5, 1.0, 2.0, and 4.0 g a.i./7,000 plants) showed increased levels of SAR as indicated by the reduction of both local and systemic infections by TSWV. The highest level of resistance was at 4 g a.i., but this rate of ASM also caused phytotoxicity resulting in temporary foliar spotting and stunting of plants. An inverse correlation between the TSWV reduction and phytotoxicity was observed with the increase of ASM concentration. ASM at the rate of 1 to 2 g a.i./7,000 plants activated a high level of resistance and minimized the phytotoxicity. Use of gibberellic acid in combination with ASM reduced the stunting caused by ASM. Present findings together with previous field experiments demonstrate that ASM is a potential option for management of TSWV in flue-cured tobacco.

Peptide-mediated broad-spectrum plant resistance to tospoviruses.

Plant viruses have a significant impact on agronomic losses worldwide. A new strategy for engineering virus-resistant plants by transgenic expression of a dominant interfering peptide is presented here. This peptide of 29 aa strongly interacts with the nucleocapsid proteins (N) of different tospoviruses. Transgenic Nicotiana benthamiana lines expressing the peptide fused to a carrier protein were challenged with five different tospoviruses that have a nucleocapsid protein interacting with the peptide. In the transgenic plants, strong resistance to tomato spotted wilt virus, tomato chlorotic spot virus, groundnut ring spot virus, and chrysanthemum stem necrosis virus was observed. This therefore demonstrates the feasibility of using peptide "aptamers" as an in vivo tool to control viral infection in higher plants.

Immunoprecipitation of a 50-kDa protein: a candidate receptor component for tomato spotted wilt tospovirus (Bunyaviridae) in its main vector, Frankliniella occidentalis.

A 50-kDa protein that binds to viral particles in solid-phase assays and that is recognized by anti-idiotypic antibodies made against anti-viral glycoproteins G1/G2 (anti-Ids) has been proposed as a receptor candidate for tomato spotted wilt tospovirus (TSWV) in its main thrips vector, Frankliniella occidentalis Pergande (Bandla et al., 1998. Phytopathology 88, 98-104). Here we show the immunoprecipitation of the 50-kDa protein by anti-Ids and by an anti-G1/G2-TSWV conjugate - a new immunoprecipitation method. In addition, we show that anti-Ids made against anti-G1 (anti-IdG1) block virus replication in an insect tissue replication assay. The results indicate that (a) the TSWV-50-kDa protein interaction occurs in solution, as it must do in vivo; (b) G1 is a viral attachment protein; and (c) the 50-kDa protein is a candidate host factor essential for TSWV entry. These results provide additional support for the role of the 50-kDa thrips protein as a viral receptor. Additionally these experiments provide the basis for testing saturable binding and represent an important step toward the first cloning and identification of a cellular receptor for a plant virus.