Anti Plant Virus Mod
Anna Pybus
In plants, viral diseases are second only to fungal diseases in terms of occurrence, and cause substantial damage to agricultural crops. The aqueous extracts of shell ginger, Alpinia zerumbet exhibit inhibitory effects against virus infections in belonging to the Solanaceae family. In this study, we isolated an anti-plant-virus molecule from the extracts using a conventional method involving a combination of reversed phase column chromatography, dialysis, and lyophilization. The anti-plant-virus molecule was identified as proanthocyanidin, which mostly consisted of epicatechin and exhibited more than 40 degrees of polymerization.
Recent advances in biotechnology and chemistry of natural products have enabled to make substantial progress in bio-pesticide development. Bio-pesticides have garnered considerable interest in recent years because they are environmentally safe, owing to the fact that they decompose quickly and leave little residue. The production of bio-pesticides is currently growing at a rate of 16% per year, which is approximately three-times higher than that of conventional agrochemicals (5.5% per year, Marrone 2013).
In plants, viral diseases are second only to fungal diseases in terms of occurrence, causing substantial damage to agricultural crops. Globally, plant viruses cause economic losses of approximately $60 billion annually, and the loss of food crops alone account for $20 billion per year (Xie et al. 2009).
It is well known that plant viruses are difficult to control compared to other plant pathogens primarily due to the lack of chemicals that are effective against plant viruses. To limit the damage caused by plant viruses, different management strategies have been applied, including prevention of plant virus spread by limiting the spread of transmitting vectors using insecticides, breeding cultivars that are resistant to virus infection, or simply cutting down infected plants. However, these efforts are still limited, and are not effective enough. Researchers have attempted to identify and develop anti-plant-virus agents, and recently, significant progress has been made in the field of biogenic anti-plant-virus substances (Zhao et al. 2017). Biogenic anti-plant-virus substances include proteins, polysaccharides, and small molecules from plants, microorganisms, algae, and animals. However, the number of anti-plant-virus substances with high efficiency and good economic benefits is still limited (Zhao et al. 2017).
More than 500 compounds have been isolated from 35 Alpinia species, and the major compounds are terpenoids and diarylheptanoids. The crude extracts and identified compounds exhibit a broad spectrum of bioactivities including anti-emetic, anti-ulcer, anti-bacterial, anti-inflammatory, anti-amnesic, and anti-cancer activities (Ma et al. 2017). Shell ginger, A. zerumbet (Pers.) B.L. Burtt & R.M. Smith, a member of the Zingiberaceae family (Teschke and Xuan 2018), grows widely in subtropical and tropical regions in East Asia, including the Okinawa Islands in Japan. A. zerumbet is commonly used in traditional Okinawa cuisine and as a herbal medicine. Bioactive compounds have been isolated and identified from crude A. zerumbet extracts (Tawata et al. 2008). A recent study demonstrated that A. zerumbet substantially increased the lifespan of Caenorhabditis elegans (Upadhyay et al. 2013).
The antiviral assay was performed following a method described in our previous study (Narusaka et al. 2020). To evaluate the inhibitory effect of purified sample on viruses, the sample solution was applied on Nicotiana benthamiana plants (in the third true leaf stage) as foliar sprays only once; three days post treatment, the plants were inoculated with tomato mosaic virus (ToMV). Inoculation of N. benthamiana plants with ToMV was performed as described, with some modifications, including using pTL-derived plasmids (pTLBN.G3), which contain a full-length ToMV cDNA and a gene encoding green fluorescent protein (GFP) (Kubota et al. 2003). In vitro transcription of 2 μg of the template DNA using the AmpliCap-Max T7 High Yield Message Maker Kit (CELLSCRIPT, Madison, WI, USA) was performed at 37 C for 40 min in a 20-μL reaction. Third true leaves of N. benthamiana that were treated with either the sample solution or distilled water (controls) were mechanically inoculated with 20 μL of 40-fold dilution of the transcription mixture. GFP foci were used to detect virus infection, and they were observed under blue light irradiation at 3 days post-inoculation (dpi).
When the aqueous extract of A. zerumbet was fractioned using a reverse-phase chromatography and an antiviral assay was performed (Fig. 1), we found that the antiviral molecule was concentrated in the fraction obtained by elution with 20% acetonitrile. The fraction was then evaporated, dialyzed, and lyophilized. The resultant sample was used as the purified sample. The antiviral activity of the purified sample was confirmed by the inhibition against growth of GFP-tagged ToMV (Fig. 2). The protection value (%) of the purified sample increased as the final concentration of the sample applied to the leaves increased. Thus, we successfully purified an anti-plant-virus molecule from the aqueous extracts of A. zerumbet using a conventional method. To estimate the material balance in each fraction in the purification process, aliquots of each fraction were lyophilized (Table 1). The yield of the final fraction of the water extract was approximately 12% (w/v). This fraction was rich in the active molecule, accounting for ca. 10% (w/v) of the water extract of A. zerumbet.
To the best of our knowledge, the present study is the first to report the identification of an anti-plant-virus molecule, PAC, in A. zerumbet. PACs are synthesized as polymeric end-products of flavan-3-ols (catechin or epicatechin), which are biosynthesized from phenylalanine and malonyl-CoA via the flavonoid pathway. Phenylalanine is derived from the shikimate pathway, and malonyl-CoA is obtained from citrate that is produced by the tricarboxylic acid cycle (Oliveira et al. 2013). However, the polymerization pathway of PACs remains unexplained. We are currently attempting to analyze the genome of A. zerumbet to elucidate biosynthesis pathway of the PAC described in this study. Further studies are necessary to determine the mechanism of anti-plant-virus activity of PAC.
In this study, we identified the anti-plant-virus molecule in A. zerumbet as a PAC by using Nicotiana benthamiana plants and GFP-fused ToMV. The PAC, as the active molecule, was present at high proportions in the 10% (w/v) water extract. In addition, the PAC was categorized as B-type, with more than 40 DP, and almost consisted of epicatechin. Thus, A. zerumbet is a potential bioresource of pesticides against viral diseases in plants.
MU and TH purified the anti-plant-virus molecule from Alpinia zerumbet. YY, YN, and MN conducted the antiviral assays. TH identified the anti-plant-virus molecule and wrote the manuscript. All authors read and approved the final manuscript.
Natural products have long been regarded as a source of inspiration for drug design, providing many unknown chemical scaffolds and pharmacophores (Eschenbrenner-Lux et al., 2014). In addition, natural products readily interact with biological targets, thereby exhibiting specific biological activities (Lowe, 2014; Bauer and Brӧnstrup, 2014). Identifying natural product structure and studying biological activity are of great significance for drug discovery (Chen and Song, 2021; Della-Felice et al., 2022). The discovery of anti-plant virus agents based on natural products is an important research direction in the prevention and control of plant virus diseases and has always attracted much attention (Eckert et al., 2003; Carli et al., 2012; Katayama et al., 2013; Wang et al., 2015). Ningnamycin (Figure 1A), isolated from Strepcomces noursei var xichangensisn for the first time, has broad-spectrum and excellent antiviral activity and is currently the most successful antiviral agent, playing a huge role in the control of plant virus diseases (Han et al., 2014). Ningnanmycin promotes the accumulation of pathogen-related proteins (PRs), a marker of systemic acquired resistance (SAR), by inhibiting the polymerization process of TMV coat protein (TMV-CP) (Han et al., 2014). In addition, ningnamycin can activate redox and metabolic processes in CMV-infected tobacco (Gao et al., 2019).
The EC50 for the curative, protective and inactivating activities of compound 26 (Figure 8) against TMV were 329.5, 269.2 and 48.1 mg/L (Yang Y. et al., 2020). The curative, protective and inactivating activities of compound 27 against TMV were 50.9, 58.9 and 81.8%, respectively. Compound 27 can not only destroy the morphology of TMV particles, but also has a strong binding effect with TMV-CP (Wang et al., 2019). At 500 mg/L, compound 28 exhibited 51.8 and 90.1% of the curative and inactivating activities against TMV, respectively, and it was able to hinder the self-assembly of TMV (Luo et al., 2020). Compound 29 inhibited ToCV infection in the host and decreased the expression level of ToCV-mCP gene (Yang H. et al., 2020). Compound 30 has good curative and protective activities against PVY, CMV and TMV, and can improve the resistance of tobacco to viruses (Chen et al., 2018).
The inactivating, curative, and protective activities of the marine natural product debromohamacanthin A (Figure 10A) against TMV were 53, 51 and 56%, respectively. The inactivating, curative, and protective activities of its derivative 41 against TMV were 60, 59, and 63%, respectively, and the antiviral activity of the compound was improved through structural optimization (Wang T. et al., 2021). In addition, compound 41 can bind to TMV-CP and interfere with the assembly process of TMV-CP and RNA, thus showing antiviral resistance. The inactivating, curative, and protective activities of compound 42 (Figure 10B) against TMV were 51.2, 49.0, and 53.6%, respectively (Wang et al., 2022). The functional groups containing CF2, indole or cyano favored the antiviral activity of 3,3-helix cyclic indole derivatives. At 500 mg/L, the curative, protective, and inactivating activities of compound 43 against TMV were 47, 50, and 51%, respectively (Chen L. et al., 2020). At 500 mg/L, the inactivating, curative, and protective activities of compound 44 were 58, 55.2, and 49.7%, respectively (Chen M. et al., 2016).
aa06259810