The inhibitory effects of metabolites from Bacillus pumilus on potato virus Y and the induction of early response genes in Nicotiana tabacum

The inhibitory effects of metabolites from Bacillus pumilus on potato virus Y and the induction of early response genes in Nicotiana tabacum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Original article The inhibitory effects of metabolites from Bacillus pumilus on potato virus Y and the induction of early response genes in Nicotiana tabacum Shuo Shen, Wei Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-53377/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Aug, 2020 Read the published version in AMB Express → Version 1 posted 5 You are reading this latest preprint version Abstract To develop a new antiviral preparation from a microbial source, the halophilic bacterium Bacillus pumilus E303035 was isolated from a soil sample collected at Qarhan Salt Lake in Qinghai, China. The inhibitory activity of an ethyl acetate extract of its fermentation broth was higher than that of an n-butanol extract. After isolation and purification, 9 compounds were obtained: cyclo(L-Leu-L-Pro) ( 1 ), cyclo(L-Pro-L-Tyr) ( 2 ), Brevianamide F ( 3 ), 2-(3-Indolyl) ethanol ( 4 ), N-[2-(1H-indol-3-yl) ethyl] acetamide ( 5 ), 3, 3-di(1H-indol-3-yl)propane-1,2-diol ( 6 ), Lincomycin B ( 7 ), dibutylphthalate ( 8 ), and p-hydroxyphenethyl alcohol ( 9 ). Compounds 1 , 5 , and 9 showed inhibitory activities against potato virus Y (PVY). Compounds 1 , 4 , and 9 had significant inhibitory activity against genes HC-pro , P3 , and Nib , compound 5 against gene P3 , and compounds 1 and 4 against NIa . Compounds 1 , 4 , 5 , and 9 had significant inhibitory activity against genes VPg and 6K1 . Active compounds 1 , 5 , and 9 had various effects on the expression of viral genes related to pathogenesis. Expression of genes cullin and XTH was up-regulated and CP was down-regulated, compared to the positive control. In conclusion, compounds 1 , 5 , and 9 might be considered as potential antiviral agents for future development. Applied & Industrial Microbiology halophilic bacterium Bacillus pumilus active compounds inhibitory activity Potato Virus Y· genes encoding viral proteins early response genes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The species Potato virus Y (PVY) is the most important viral pathogen affecting potato crops and a serious threat to potato production worldwide (Davie et al. 2017 ; Funke et al. 2017 ). It can infect various crops, including potato, tobacco, tomato, pepper and other crops belonging to the Solanaceae family, and can cause serious damage and significant economic loss (Zhang et al. 2017 ). Currently, few agents are available to effectively control this plant virus, such as ningnanmycin, dufulin, and ribavirin (Xiao et al. 2015 ). The search for new antiviral compounds is critical, by considering new sources and novel antiviral mechanisms. Microbes are a broad and rich resource of natural bioactive products. Bacteria of the genus Bacillus are known to produce a wide range of active substances. They have successfully been used to generate compounds such as amylase, proteases, antibiotics, and surfactants (Caetano et al. 2011 ). The antagonistic effects of Bacillus are due to secondary metabolite production of antibiotics and antimicrobial peptides (Hu et al. 2010 ). In this study, a halophilic Bacillus strain Bacillus pumilus E303035 with antiviral activity against PVY which we have screened in our lab was chosen. 9 compounds were isolated from this strain. The antiviral activities against PVY of fermentation extracts and isolated compounds were evaluated with real-time PCR. In order to illustrate the inhibitory mechanisms of active compounds isolated from strain E303035, the effects were determined on total RNA and on genes encoding the functional proteins of PVY. Materials And Methods Plant and PVY materials Tobacco plants ( Nicotiana tabacum var. Samsun NN) were grown under greenhouse conditions, with 16 h light at 25 ℃ and 8 h darkness at 15 ℃, and PVY was kept at 4 ℃, in the Key Laboratory of Qinghai-Tibet Plateau Biotechnology (KLB), Ministry of Education, Qinghai Province, China. Three or four fully expanded leaves from, approximately, one-month-old plants were used in the experiment. Strains and preparation of fermentation broth Halophilic bacterium Bacillus pumilus Strain E303035 (Genbank accession number: MN238693; GDMCC number: 60077) was isolated from a Qarhan Salt Lake soil sample, and was preserved at 4 ℃ in the KLB. It was cultured at 37 ℃, with a rotation speed of 200 r/min in a shaker cabinet for 7 days, in Erlenmeyer flasks (1 L). Each flask contained 600 mL of ATCC213 medium (10 g MgSO 4 ·7H 2 O, 0.2 g CaCl 2 ·2H 2 O 0.2 g, KCl, 2.5 g peptone, 10 g yeast extract, 30 g NaCl, adding deuterium-depleted water (D.D.W) to 1000 mL, pH 7.2–7.4). Then, the fermentation broth was centrifuged at a rotation speed of 8000 r/min, the supernatant was filtered through a 0.22 µm millipore filter and stored at 4 ℃ for the preparation of crude extracts (Zhang et al. 2017 ). Preparation of crude extracts of fermentation broth of strain E303035 The fermentation broth of strain E303035 was extracted with either ethyl acetate or n-butanol. The two kinds of extract were concentrated in vacuo to yield a residue. The residues were dissolved in distilled water (using DMSO as a vehicle, at a maximum concentration of 0.1%) to prepare concentrations of 1, 5, 10, and 20 mg/ml for antiviral testing against PVY (Zhang et al. 2017 ). Isolation and identification of compounds in strain E303035 1H, 13C, DEPT, 1H-1H-COSY, HSQC, and HMBC NMR spectra were obtained with a JNM-ECA600 spectrometer. FAB-MS spectra were recorded on a JEOL JMS-HX 110 instrument. The chromatographic stationary phases were silica gel (200–300 mesh), Sephadex LH-20 (25–100 µm, Pharmacia) and MCI-gel CHP20P (75–150 mm, Mitsubishi Chemical). Using thin-layer chromatography, compounds were visualized by spraying with 5% H 2 SO 4 , followed by heating (Shen et al. 2013 ). The ethyl acetate extract was dissolved in ethanol and concentrated in vacuo to yield 11 g oily fraction. Afterwards, the fraction was subjected to handera-SI (10 µm), and eluted with n -hexane: isopropanol in the range 80:20 − 50:50, to give Fraction 1 (Fr1) 4.5 g, Fr2 248 mg, Fr3 516 mg, Fr4 597 mg, and Fr5 267 mg. Fr1 was subjected to MCI-gel CHP20P and eluted with 100% MeOH, MeOH:H 2 O (1:2), and 100% acetone to give compound 8 (3900 mg). Fr2 was subjected to Handera C18 (10 µm) and eluted with C 2 H 3 N-H 2 O (1:10–3:10) to give compounds 4 (51 mg) and 9 (10 mg). Fr3 was subjected to Handera C18 (10 µm) and eluted with C 2 H 3 N:H 2 O (1:20–7:20) to give compound 7 (13 mg). Fr4 was subjected to Handera C18 (10 µm) and eluted with C 2 H 3 N:H 2 O (1:20–3:10) to give compounds 1 (86 mg), 5 (78 mg), and 6 (8 mg). Fr5 was subjected to Handera C18 (10 µm) and eluted with C 2 H 3 N:H 2 O (1:20–3:10) to give compounds 2 (25 mg) and 3 (11 mg). Compounds 1–9 were dissolved in distilled water (using DMSO as a vehicle, at a maximum concentration of 0.1%) to prepare the 31, 63, 125, 250, and 500 mg/ml concentrations for antiviral testing against PVY (Zhang et al. 2017 ). PVY inoculation Inoculation with PVY, and with PVY with an equal volume of extract (crude extracts, compounds, or ningnanmycin), was conducted on two top leaves of the tobacco plants, using a conventional friction method with a 10⋅ concentration of inoculums. Phosphate buffer inoculation was used as a control. The two infected leaves were collected from each plant, for RNA extraction, 7 days from when the symptoms appeared (Chen et al. 2017 ). Real-time PCR (qPCR) Analysis The total RNA was extracted using a Total RNA Reagent Kit (Takara) from the leaves of plants that were subjected to PVY infection after treatment with each extract (at concentrations of 1, 5, 10, 20 mg/mL, using DMSO as vehicle at a maximum concentration of 0.1%) or compound (at concentrations of 31, 63, 125, 250, and 500 µg/mL, using DMSO as a vehicle at maximum concentration of 0.1%), in triplicate. Total RNA samples were reverse transcribed using a PrimeScriptTMRT reagent Kit with gDNA Eraser (Takara). Real-time PCR was performed with SYBR Premix Ex Taq Kit (Takara), with β-actin as an internal control. The final volume of the PCR mixture was 20 µL, including 10 µL of the SYBR Green Master mix reagent, 7.8 µL of sterile water, 0.4 µL of DyeII, 1 µL of cDNA, and 0.4 µL (2 mM) of each real-time PCR primer. Primers used were R β-actin (5´-AAGGGATGCGAGGATGGA-3´) and F β-actin (5´-CAAGGAAATCACCGCTTTGG-3´), R PVY (5´-TTCATCTCCATCCATCATAACC-3´) and F PVY (5´-TACAACTTGCATACGACATAGG-3´), R Lhc (5´-TTAAGAGAAGAAGCCGAATGTG-3´)and F Lhc (5´-CCACACTTCAACTTGCTGAG-3´), R PSii (5´- TTTCTCCTCCCTCCCTTTCTCT-3´) and F PSii (5´-TTGCTTGACCGTCGTTGTG-3´), R XTH (5´-GCGAGGATTTGAGGCACAG-3´) and F XTH (5´-GCAACGAGAGGTGGATTAGAGAA-3´), R CPIP3 (5´-GTCAGTACAGCCAGAGCCAGAA-3´) and F CPIP3 (5´-AAGCGACTACCAAAACACACACA-3´), R cullin-1 (5´-GTCGCAGAATGTGGCAAGAA-3´)and F cullin-1 (5´-AGAGAAGAGAGATGTGGTTGGT TTG-3´). The following conditions were used: 95 ℃ for 10 s, 95 ℃ for 50 s, and 35 cycles of both 95 ℃ for 6 s and 62 ℃ for 35 s. Standard curves were constructed using a series of five tenfold dilutions of the cDNA template. The relative expression levels were calculated (Shen et al. 2018 ). PCR Analysis The total RNA was extracted using a Total RNA Reagent Kit (Takara) from the leaves of plants that were subjected to PVY infection after the different treatments of compounds (at the concentrations of 31, 63, 125, 250, and 500 µg/mL, in triplicate. Total RNA samples were reverse transcribed using a PrimeScriptTMRT reagent Kit with gDNA Eraser (Takara). β-actin was used as an internal control. The final volume of the PCR mixture was 20 µL, including 10 µL of 2 × FastTaq PCR Master Mix, 1 µL of cDNA, 8.6 µL of sterile water, and 0.2 µL (2 mM) of each RT-PCR primer. Primers used were R β-actin (5´-AAGGGATGCGAGGATGGA-3´) and F β-actin (5´-CAAGGAAATCACCGCTTTGG-3´), R HC-Pro (5´-ACCAACTCTATAGTGCTTAATGTCAGA-3´)and F HC-Pro (5´-GGAGTTCTAGACTCAATGGTTCAGT-3´), R P1 (5´-TTGTGTAACCTTGGAACGCGC-3´) and F P1 (5´-ATGGCAACTTACATGTCAACGATTC-3´), R P3 (5´-CTGATGCCGCACATTATATTCTTC-3´) and F P3 (5´-GGTATTCCTGGGCATGTCCTG-3´), R NIa (5´-TTGCTCTACAACAACATCATGATCAA-3´) and F NIa (5´-GCCAAATCACTCATGAGAGGTTTAA-3´), R NIb (5´-TTGATGGTGCACTTCATAAGTATCG-3´) and F NIb (5´-GCTAAACATTCTGCGTGGATGTAT-3´), R 6K1 (5´-CTGGTGTTTAACTTCATGATCCATT-3´)and F 6K1 (5´-CGCTCCACACCAGGTGTTAG-3´), R VPg (5´-TTCATGCTCCACTTCCTGTTTTG-3´)and F VPg (5´-GGCAAGAACAAATCCAAAAGAATTC-3´), R CP (5´-CAGTTCTTGACTCCAAGTAGAGTATG-3´)and F CP (5´-GGAAATGACACAATCGATGCAG-3´) (Shen et al. 2018 ). Statistical analysis The EC 50 values were determined from concentration-effect curves by linear regression analysis. Statistical analysis was performed using SPSS 20.0, and data were presented as the arithmetic mean ± standard deviation. Results Antiviral activities of crude extracts against PVY As shown in Table 1 , both ethyl acetate and n-butanol extracts from strain E303035 showed significant antiviral activity against PVY. The inhibitory activity of ethyl acetate extract was higher than that of n-butanol. The inhibition rates of both extracts were concentration-dependent. The ethyl acetate extract possessed its highest inhibitory activity at 10 mg/mL, with an inhibition rate of 99.70%. The n-butanol extract possessed its highest inhibitory activity at 20 mg/mL, with an inhibition rate of 96.43%. The ethyl acetate extract was then isolated to obtain compounds with anti-viral activity against PVY. Table 1 The inhibitory activity of two different extracts from E303035 fermentation against PVY Concentration (mg/mL) Inhibition Rate(%) ethyl acetate n-butanol 1 85.59 ± 1.66 b 41.81 ± 7.89 c 5 94.59 ± 2.03 a 88.89 ± 0.00 b 10 99.70 ± 0.12 a 91.55 ± 10.51 a 20 99.04 ± 0.17 a 96.43 ± 1.86 a Data are expressed as means ± SD from experiments with three replicates. Means with a different superscript letter in the same column are significantly different, by Duncan’s multiple range test (P < 0.05). Isolation of compounds from the halophilic bacterium Bacillus pumilus As shown in Fig. 1 , 9 compounds were isolated from the ethyl acetate extract of strain E303035 and identified as cyclo(L-Leu-L-Pro) ( 1 ) (Yue et al. 2010 ), cyclo(L-Pro-L-Tyr) ( 2 ) (Li et al. 2013 ), Brevianamide F ( 3 ) (Wang et al. 2015 ), 2-(3-Indolyl) ethanol ( 4 ) (Böhlendorf et al. 1996 ), N-[2-(1H-indol-3-yl) ethyl] acetamide ( 5 ) (Anouhe et al. 2015 ), 3, 3-di(1H-indol-3-yl)propane- 1,2-diol ( 6 ) (Zhao et al. 2009 ), Lincomycin B ( 7 ) (Argoudelis et al. 1965 ), dibutylphthalate ( 8 ) (Dai et al. 2006 ), and p-hydroxyphenethyl alcohol ( 9 ) (Lou et al. 2001 ). Cyclo(L-Leu-L-Pro) ( 1 ). Yield: 86 mg; white amorphous powder; molecular formula C 11 H 18 N 2 O 2 . Cyclo(L-Pro-L-Tyr) ( 2 ). Yield: 25 mg; colorless oil; molecular formula C 14 H 16 N 2 O 3 . Brevianamide F ( 3 ). Yield: 11 mg; white powder; molecular formula C 16 H 17 N 3 O 2 . 2-(3-indolyl) ethanol ( 4 ). Yield: 51 mg; white powder; molecular formula C 10 H 11 NO. N-[2-(1H-indol-3-yl) ethyl] acetamide ( 5 ). Yield: 78 mg; yellowish-brown oil; molecular formula C 12 H 13 N 2 O. 3, 3-di(1H-indol-3-yl)propane- 1,2-diol ( 6 ). Yield: 8 mg; white powder; molecular formula C 18 H 18 N 2 O 2 . Lincomycin B ( 7 ). Yield: 13 mg; white powder; molecular formula C 17 H 32 N 2 O 6 S. Dibutylphthalate ( 8 ). Yield: 3900 mg; brownish red oil; molecular formula C 16 H 22 O 4 . P-hydroxyphenethyl alcohol ( 9 ). Yield: 10 mg; colorless needle crystal; molecular formula C 8 H 10 O 2 . The data of 1 H NMR and 13 C NMR of compounds 1 – 9 is in supplementary file. Antiviral activities of compounds isolated from strain E303035 against PVY The antiviral activities, against PVY, of compounds isolated from strain E303035 were determined at 31, 63, 125, 250, and 500 µg/mL. As shown in Table 2 , compounds 1 , 5 , and 9 possessed higher antiviral activity against PVY, with EC 50 values of 210.99, 224.26, and 305.37 µg/mL, respectively. Compound 5 showed significant antiviral activity against PVY at 250 and 500 µg/mL, with inhibition values of 62.40% and 75.99%, respectively. Compound 1 also showed moderate antiviral activity at a concentration of 125 µg/mL, with an inhibition value of 53.87%. Compound 4 showed weak antiviral activity at 250 and 500 µg/mL, with inhibition values of 30.21% and 24.29%, respectively. In summary, we found that compounds 1 , 5 , and 9 had good inhibitory activity against PVY, and compounds 1 , 4 , 5 , and 9 were then used for further research of inhibitory activity against genes encoding functional proteins of PVY. Table 2 The antiviral activity of compounds isolated from strain E303035 against PVY Compound Inhibition rate(%) EC 50 (µg/mL) 500 (µg/mL) 250 (µg/mL) 125 (µg/mL) 63 (µg/mL) 31 (µg/mL) 1 65.48 ± 1.98 b 59.12 ± 2.43 b 53.87 ± 2.47 a 48.24 ± 2.17 a 17.06 ± 1.37 b 210.99 2 -* - - - - - 3 9.68 ± 0.45 d - - - - - 4 30.21 ± 2.29 c 24.29 ± 3.49 d - - - - 5 75.99 ± 0.54 a 62.40 ± 1.28 a 32.34 ± 1.09 c 31.67 ± 2.33 b 31.53 ± 2.15 a 224.26 6 - - - - - - 7 - - - - - - 8 - - - - - - 9 61.75 ± 0.62 b 52.62 ± 3.34 c 45.78 ± 1.15 b 28.51 ± 0.46 c 10.21 ± 2.29 c 305.37 Data are expressed as means ± SD from experiments with three replicates. Means with a different letter in the same column are significantly different, by Duncan’s multiple range test (P < 0.05). * No inhibitory activity. Effects of active compounds on the expression of genes encoding functional proteins The expressions of genes HC-pro , P1 , P3 , NIa , NIb , 6K1 , VPg , and CP under treatment with compounds 1 , 4 , 5 , and 9 at 31, 63, 125, 250, and 500 µg/mL were determined by PCR (Fig. 2 and Fig. 3 ). As shown in Fig. 2 , organizing data by gene, HC-pro and NIb showed no expression under treatment with compounds 1 , 4 , and 9 at all concentrations, and with compound 5 at 125 and 500 µg/mL (Fig. 2 A and Fig. 2 C). Gene P3 showed no expression under treatment with compounds 1 , 4 , 5 , and 9 at all concentrations (Fig. 2 B). Gene NIa showed no expression under treatment with compounds 1 and 4 at all tested concentrations, compound 5 at 125 and 500 µg/mL, and compound 9 at 125, 250, and 500 µg/mL (Fig. 2 D). Organizing data by compound, at all tested concentrations: 1 and 4 significantly inhibited the expressions of genes HC-pro , P3 , NIb , and NIa , compound 5 inhibited gene P3 , and compound 9 inhibited NIb . As shown in Fig. 3 , with data organized by gene, genes VPg and 6K1 showed no expression under treatment with compounds 1 , 4 , 5 , and 9 at all tested concentrations (Fig. 3 A and Fig. 3 C). Gene CP showed no expression under treatment with compound 1 at 63, 125, and 500 µg/mL, compound 4 at 250 and 500 µg/mL, compound 5 at 125 and 500 µg/mL, and compound 9 at 125, 250, and 500 µg/mL (Fig. 3 B). Gene P1 showed no expression under treatment with compound 1 at 63, 125, and 500 µg/mL, compound 4 at 63, 250, and 500 µg/mL, compound 5 at 125, 250, and 500 µg/mL, and compound 9 at 63, 125, 250, and 500 µg/mL (Fig. 3 D). Organizing the data by compound, compounds 1 , 4 , 5 , and 9 significantly inhibited the expressions of genes VPg and 6K1 at all tested concentrations. Effect of active compounds on the expression of early response genes induced by potato virus Y Effects of active compounds on the expression of chlorophyll a-b binding protein ( Lhc ) As shown in Fig. 4 , the expression of gene Lhc under the treatment of compounds 1 , 5 , and 9 at 31, 63, 125, 250, and 500 µg/mL after 2, 4, and 6 days were determined by real-time PCR. For compound 1 , increasing the concentration caused significant up-regulation of the gene Lhc . Over time, the gene was significantly down-regulated at day 4, then up-regulated at day 6, at all concentrations. The expression of gene Lhc was at its lowest at day 4 (Fig. 4 A). For compound 5 , increasing the concentration caused significant down-regulation of the gene Lhc . The gene was significantly up-regulated at day 4, and then down-regulated at day 6, at all concentrations. The expression of gene Lhc was at its highest at day 4 (Fig. 4 B). For compound 9 , increasing the concentration caused significant down-regulation of the gene Lhc . The gene was significantly down-regulated at day 4, and then up-regulated at day 6, at all concentrations. The expression of gene Lhc was at its lowest at day 4 (Fig. 4 C). Effect of active compounds on the expression of photo system II ( PSii ) As shown in Fig. 5 , the expression of gene PSii under treatments with compounds 1 , 5 , and 9 at 31, 63, 125, 250, and 500 µg/mL after 2, 4, and 6 days was determined by real-time PCR. For compounds 1 , 5 , and 9 , increasing the concentration caused significant up-regulation of the gene PSii . The gene was significantly up-regulated at day 4, then down-regulated at day 6, at all concentrations. The expression of gene PSii was at its highest at day 4. Effect of active compounds on the expression of xyloglucan endotransglucosylase hydrolase ( XTH ) As shown in Fig. 6 , the expression of gene XTH under treatments with compounds 1 , 5 , and 9 at 31, 63, 125, 250, and 500 µg/mL after 2, 4, and 6 days was determined by real-time PCR. For compound 1 and 9 , increasing the concentration caused significant down-regulation of the gene XTH . The gene was significantly down-regulated at day 4, and then up-regulated at day 6, at all concentrations. The expression of gene XTH was at its lowest at day 4 (Fig. 6 A, 6 C). For compound 5 , increasing the concentration caused significant up-regulation of the gene XTH . The gene was significantly up-regulated at day 4, and then down-regulated at day 6, at all concentrations. The expression of gene XTH was at its highest at day 4 (Fig. 6 B). Effect of active compounds on the expression of gene CP-interacting protein 3 ( CPIP3 ) As shown in Fig. 7 , the expression of gene CPIP3 under treatment with compounds 1 , 5 , and 9 at 31, 63, 125, 250, and 500 µg/mL after 2, 4, and 6 days was determined by real-time PCR. For compounds 1 and 5 , increasing the concentration caused significant down-regulation of the gene CPIP3 . The gene was significantly up-regulated at day 4, and then down-regulated at day 6, at all concentrations. The expression of gene CPIP3 was at its highest at day 4 (Fig. 7 A, 7 B). For compound 9 , increasing the concentration caused significant up-regulation of the gene CPIP3 . The gene was significantly up-regulated at day 4, and then down-regulated at day 6, at all concentrations. The expression of gene CPIP3 was at its highest at day 4 (Fig. 7 C). Effect of active compounds on the expression of the gene cullin-1 isoform ( cullin-1 ) As shown in Fig. 8 , the expression of gene cullin-1 under treatment with compounds 1 , 5 , and 9 at 31, 63, 125, 250, and 500 µg/mL after 2, 4, and 6 days was determined by real-time PCR. For compound 1 and 5 , increasing the concentration caused significant down-regulation of the gene cullin-1 . The gene was significantly up-regulated at day 4, and then down-regulated at day 6, at all concentrations. The expression of gene cullin-1 was at its highest at day 4 (Fig. 8 A, 8 B). For compound 9 , increasing the concentration caused significant up-regulation of the gene cullin-1 . The gene was significantly up-regulated at day 4, and then down-regulated at day 6, at all concentrations. The expression of gene cullin-1 was the highest at day 4 (Fig. 8 C). Discussion The genome of PVY is composed of a single-stranded RNA, length 9.7 kb, which covalently links with a viral-encoded protein (VPg) at its 5ʹ-end and contains a 3ʹ-polyadenylated tail. The genome of PVY encodes two polyproteins, a larger polyprotein of about 3000 amino acids and a shorter one translated from a 2 + frameshift in the P3 coding region. These polyproteins are cleaved by viral proteases, subsequently generating eleven mature proteins (Kim et al. 2015 ). Compounds 1 and 4 significantly inhibited the expression of gene HC-pro at all concentrations tested. HC-Pro encodes viral helper component-proteinase (HC-Pro), which is a pathogen elicitor. It is also involved in multiple roles in aphid transmission, RNA binding, suppression of gene silencing and protease activity (Chowdhury et al. 2020 ; Kumar et al. 2020 ). Compounds 1 , 4 , 5 and 9 significantly inhibited the expression of gene VPg at all concentrations tested. VPg encodes a viral protein called VPg which sabotages host antiviral RNA silencing to promote viral infection (Cheng et al. 2016). Compounds 1 , 4 , 5 , and 9 significantly inhibited the expression of genes CP and P1 , at some concentrations. CP encodes a viral coat protein and P1 encodes plasma membrane cation-binding protein 1 (PCaP1), which is shown to be important for the intra-cellular movement of two members of the genus Potyvirus , in Arabidopsis (rockcress) and in tobacco plants (Beris et al. 2020 ). Compounds 1 and 4 significantly inhibited the expression of gene NIa at all concentrations tested. NIa encodes the “nuclear inclusion a” (NIa) protease of PVY, which is involved in processing the polyprotein encoded by its positive sense RNA genome. The NIa protein is also responsible for the release of the VPg protein involved in viral RNA replication (Gargouri-Bouzid et al. 2006 ). Compounds 1 , 4 and 9 significantly inhibited the expression of gene NIb at all concentrations tested. NIb is an RNA-dependent RNA polymerase in the PVY genome, which has been reported to confer virulence in infections of pepper plants (Janzac et al. 2010 ). Compounds 1 , 4 and 5 significantly inhibited the expression of gene P3 at all concentrations tested. P3 encodes potyviral membrane protein. Compounds 1 , 4 , 5 , and 9 significantly inhibited the expression of gene 6K1 . The function of 6K1 is currently unknown. In summary, all compounds showed inhibitory activities against various genes encoding functional proteins of PVY. The results provide a starting point for future research into the inhibitory mechanisms and pathways of active compounds against PVY. The Lhc gene encodes chlorophyll a-b binding protein, which takes part in regulating photosynthesis and chlorophyll synthesis. The down-regulation of this gene means a decrease in viral nucleic acid, which is also seen in PVY-resistant plants (Chen et al. 2017 ). Compounds 5 and 9 down-regulated the Lhc gene. There was an inverse relationship between concentration and gene expression. At day 4, the expression of gene Lhc was at its lowest. These results suggest that compounds 5 and 9 elicit their inhibitory activity due to their influence on chlorophyll synthesis and photosynthesis during days 2 − 4. The PSii gene takes part in photosystem II of photosynthesis. A down-regulation of this gene has the same effect as inhibition of Lhc : a decrease in viral nucleic acid. Compounds 1 , 5 , and 9 showed no significant inhibitory effect of this gene, but the expression of PSii gene was at its lowest on day 6. The XTH gene encodes xyloglucan endotransglucosylase hydrolase, which is an essential constituent of the primary cell wall and participates in cell wall elongation and construction (Otulak-Kozieł K et al. 2018a ; Otulak-Kozieł K et al. 2018b ). The cell wall is the first barrier that protects plants against entry of pathogens and other harmful bodies, and offers protection against mechanical stress to plant cells. The expression of cell-wall-related genes, including XTH , has been shown to be regulated by biotic and abiotic stresses (Li et al. 2015). In this study, XTH was up-regulated under treatment with compound 5 as the concentration increased before 4 days. This suggests that compound 5 could increase elongation and construction of plant cell walls at different concentrations, especially at day 4. However, the XTH gene showed down-regulation under treatment with compounds 1 and 9 at day 4 and then up-regulation at day 6. This suggests that both compounds 1 and 9 could in increase elongation and construction of plant cell walls after 4 days, especially at day 6. It affected the plant cell wall later than that of compound 5 . This may indicate that these compounds contribute to induce PVY resistance in Nicotiana. CPIP3 is a stress response gene, encoding CP-interacting protein 3. The interaction between viral coat protein (CP) and host plant CP-interacting protein were shown to be important for the plant defense response, viral propagation, and long distance movement (Li et al. 2005 ; Park et al. 2009 ). A low expression of the CPIP3 gene signifies a low quantity of CP-interacting protein 3 which, to some extent, signifies inhibitory activity against PVY (Li et al. 2005 ). In this study, compounds 1 and 5 significantly down-regulated the gene CPIP3 at 2 days, with their increased concentration. Therefore, compounds 1 and 5 could inhibit the viral multiplication of PVY by inducing the plant defense response in tobacco plants. Although compound 9 was able to up-regulate CPIP3 at days 2 and 4, by day 6 there was significant down-regulation of the gene, at all concentrations tested. The result demonstrated the inhibitory activity of compound 9 against the multiplication of PVY, by inducing the tobacco plant defense response at 6 days. Gene cullin-1 also encodes a stress response protein. The up-regulation of it was reported to be important in the resistance response to pathogen infection and autoimmunity (Cheng et al. 2011 ; Gou et al. 2009 ; Liu et al. 2002 ). In our study, the gene cullin-1 was significantly up-regulated by compounds 1 , 5 , and 9 at days 2 and 4. This meant that all three compounds had significant inhibitory activity against PVY by inducing the resistance response to PVY infection and autoimmunity in tobacco plants at 2–4 days. In detail, compounds 1 and 5 up-regulated cullin-1 at low concentrations of 31, 63, and 125 µg/mL, while compound 9 up-regulated cullin-1 at a high concentration of 500 µg/mL. Taken together, active compounds 1 , 5 , and 9 had significant inhibitory activity against PVY and various effects on the expression of viral genes related to pathogenesis. They might be considered as potential antiviral agents for future development. Declarations Availability of data and materials We admit availability of data and material. Ethical approval and consent to participate This article does not contain any studies with human participants or animals. We declare consent to participate. Competing interests The authors declare that they have no conflict of interest. Funding This research was financed by the Natural Science Fund of Qinghai Province (2019-ZJ-914) and National Natural Science Fund (31860512). Author’s contributions Wei Li and Shuo Shen conceived and designed research. Wei Li and Shuo Shen conducted experiments. Shuo Shen carried out all experiments. Wei Li and Shuo Shen analyzed data. Shuo Shen wrote the manuscript. All authors read and approved the manuscript. Acknowledgements We thank Prof. Bao-An Song and his staff in Guizhou University for technical assistance. References Anouhe JBS, Adima AA, Niamké FB, Stien D, Amian BK, Blandinières PA, Virieux D, Pirat JL, Kati-Coulibaly S, Amusant N (2015) Dicorynamine and harmalan-N-oxide, two new b-carboline alkaloids from Dicorynia guianensis Amsh heartwood. Phytochem Let 12:158–163 Argoudelis AD, Fox JA, Eble TE (1965) A new lincomycin-related antibiotic. Biochem 4:698–703 Beris D, Kotsaridis K, Vakirlis N, Termentzi A, Theologidis I, Moury B, Vassilakos N (2020) The plasma membrane Cation binding protein 1 affects accumulation of Potato virus Y in pepper both at the systemic level and in protoplasts. 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J Agr Food Chem 65(23):4582–4588 Zhao TT, Li XM, Li JM, Li K, Cui CM, Li CS, Wang BG (2009) Chemical constituents of EN-22, an endophytic fungus derived from the marine red alga Polysiphonia urceolata . Mar Sci 33(7):81–86. Chinese Supplementary Files Supplementaryfile.docx Cite Share Download PDF Status: Published Journal Publication published 20 Aug, 2020 Read the published version in AMB Express → Version 1 posted Editorial decision: Accept 10 Aug, 2020 Editor assigned by journal 08 Aug, 2020 Editor invited by journal 07 Aug, 2020 Submission checks completed at journal 04 Aug, 2020 First submitted to journal 03 Aug, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-53377","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Original article","associatedPublications":[],"authors":[{"id":1206126,"identity":"cc4fa83c-1640-43f3-95c8-88c368ab0f5d","order_by":0,"name":"Shuo Shen","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Shuo","middleName":"","lastName":"Shen","suffix":""},{"id":1206127,"identity":"167147c7-78e9-4248-a2dc-f64d2d14aa87","order_by":1,"name":"Wei Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYBACPgYeBmYGBgsGfmbmww+I0sIG0SLBINnOlmZAmhaD8zwKEsRpkcg9+LmwTSJx82EeBgOGGptoIrTkJUvPBGrZdpj3wAOGY2m5DQS1SOcYSPO2SeRuO8yXYMDYcJgoLca/QVo2N/MYSBCrxQxsywZmorXIv0uznnFOon7GYWAgJxDjF36es4dvF5TZGPP3Hz784EONDWEtYMDIBmUkEKUcDP4Qr3QUjIJRMApGIAAAVkU4KOcbHjIAAAAASUVORK5CYII=","orcid":"","institution":"Qinghai University","correspondingAuthor":true,"prefix":"","firstName":"Wei","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2020-08-04 11:03:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-53377/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-53377/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13568-020-01089-1","type":"published","date":"2020-08-20T12:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":1809210,"identity":"ff00bdc2-cc57-46af-a9e6-8d65e6d38981","added_by":"auto","created_at":"2020-08-05 22:34:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":41971,"visible":true,"origin":"","legend":"Structures of compounds 1–9","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-53377/v1/Fig1.png"},{"id":1809211,"identity":"7dc9fa03-0650-4cd7-a208-c5b8c24a21c4","added_by":"auto","created_at":"2020-08-05 22:34:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":163841,"visible":true,"origin":"","legend":"Effect of active compounds on expression of genes encoding PVY functional proteins: HC-pro, P3, NIb and NIa. A: gene HC-Pro; B: gene P3; C: gene NIb; D: gene NIa. M: Marker lane; Series of Lanes 1-5: compound 5; 6-10: compound 1; 11-15: compound 9; 16-20: compound 4. Each series of 5 lanes between markers represents the concentrations used: 500, 250, 125, 63, and 31 μg/mL, from left to right.","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-53377/v1/Fig2.png"},{"id":1809212,"identity":"8ce15b82-5660-4ecc-9f2c-37e910977cd3","added_by":"auto","created_at":"2020-08-05 22:34:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":157273,"visible":true,"origin":"","legend":"Effect of active compounds on expression of genes encoded PVY functional proteins of VPg, CP, 6K1, and P1. A: gene VPg; B: gene CP; C: gene 6K1; D: gene P1. M: Marker lane; Series of Lanes 1-5: compound 5; 6-10: compound 1; 11-15: compound 9; 16-20: compound 4. Each series of 5 lanes between markers represents the concentrations used: 500, 250, 125, 63, and 31 μg/mL, from left to right.\n\n\n","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-53377/v1/Fig3.png"},{"id":1809213,"identity":"b9232a11-03b1-4552-8d94-194c84a89d6d","added_by":"auto","created_at":"2020-08-05 22:34:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":29278,"visible":true,"origin":"","legend":"The effect of active compounds 1, 5, and 9 on the expression of gene Lhc. A: Compound 1; B: Compound 5；C: Compound 9","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-53377/v1/Fig4.png"},{"id":1809214,"identity":"9f79a215-b3bb-4608-a7fa-27a78ec1573d","added_by":"auto","created_at":"2020-08-05 22:34:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29627,"visible":true,"origin":"","legend":"The effect of active compounds 1, 5, and 9 on the expression of gene PSii. A: Compound 1; B: Compound 5；C: Compound 9","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-53377/v1/Fig5.png"},{"id":1809215,"identity":"1e39c1f6-ae42-4941-a9d6-b992189d09cb","added_by":"auto","created_at":"2020-08-05 22:34:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":22184,"visible":true,"origin":"","legend":"The effect of active compounds 1, 5, and 9 on the expression of gene XTH. A: Compound 1; B: Compound 5；C: Compound 9","description":"","filename":"ig6.png","url":"https://assets-eu.researchsquare.com/files/rs-53377/v1/ig6.png"},{"id":1809216,"identity":"c8250299-dab0-48bd-8d50-69533c0843af","added_by":"auto","created_at":"2020-08-05 22:34:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":31328,"visible":true,"origin":"","legend":"The effect of active compounds 1, 5, and 9 on the expression of gene CPIP3. A: Compound 1; B: Compound 5；C: Compound 9","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-53377/v1/Fig7.png"},{"id":1809217,"identity":"699467a7-a166-4562-96b1-25a3fdf640d3","added_by":"auto","created_at":"2020-08-05 22:34:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":30682,"visible":true,"origin":"","legend":"The effect of active compounds 1, 5, and 9 on the expression of gene cullin-1. A: Compound 1; B: Compound 5；C: Compound 9","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-53377/v1/Fig8.png"},{"id":15667999,"identity":"948776ba-09b1-4b6e-8782-0574d743b222","added_by":"auto","created_at":"2021-11-18 13:46:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1062176,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-53377/v1/aeb95ebf-6358-4c59-a338-7263bff3b687.pdf"},{"id":1809219,"identity":"308e2ae8-ad17-4c34-ad79-678600b9b38e","added_by":"auto","created_at":"2020-08-05 22:34:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25316,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-53377/v1/Supplementaryfile.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eThe inhibitory effects of metabolites from \u003cem\u003eBacillus pumilus\u003c/em\u003e on potato virus Y and the induction of early response genes in \u003cem\u003eNicotiana tabacum\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":" \u003cp\u003eThe species \u003cem\u003ePotato virus Y\u003c/em\u003e (PVY) is the most important viral pathogen affecting potato crops and a serious threat to potato production worldwide (Davie et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Funke et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It can infect various crops, including potato, tobacco, tomato, pepper and other crops belonging to the \u003cem\u003eSolanaceae\u003c/em\u003e family, and can cause serious damage and significant economic loss (Zhang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Currently, few agents are available to effectively control this plant virus, such as ningnanmycin, dufulin, and ribavirin (Xiao et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The search for new antiviral compounds is critical, by considering new sources and novel antiviral mechanisms.\u003c/p\u003e \u003cp\u003eMicrobes are a broad and rich resource of natural bioactive products. Bacteria of the genus \u003cem\u003eBacillus\u003c/em\u003e are known to produce a wide range of active substances. They have successfully been used to generate compounds such as amylase, proteases, antibiotics, and surfactants (Caetano et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The antagonistic effects of \u003cem\u003eBacillus\u003c/em\u003e are due to secondary metabolite production of antibiotics and antimicrobial peptides (Hu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, a halophilic \u003cem\u003eBacillus\u003c/em\u003e strain \u003cem\u003eBacillus pumilus\u003c/em\u003e E303035 with antiviral activity against PVY which we have screened in our lab was chosen. 9 compounds were isolated from this strain. The antiviral activities against PVY of fermentation extracts and isolated compounds were evaluated with real-time PCR. In order to illustrate the inhibitory mechanisms of active compounds isolated from strain E303035, the effects were determined on total RNA and on genes encoding the functional proteins of PVY.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant and PVY materials\u003c/h2\u003e \u003cp\u003eTobacco plants (\u003cem\u003eNicotiana tabacum\u003c/em\u003e var. Samsun NN) were grown under greenhouse conditions, with 16\u0026nbsp;h light at 25 ℃ and 8\u0026nbsp;h darkness at 15 ℃, and PVY was kept at 4 ℃, in the Key Laboratory of Qinghai-Tibet Plateau Biotechnology (KLB), Ministry of Education, Qinghai Province, China. Three or four fully expanded leaves from, approximately, one-month-old plants were used in the experiment.\u003c/p\u003e \u003c/div\u003e \n\n\u003ch2\u003eStrains and preparation of fermentation broth \u003c/h2\u003e \u003cp\u003eHalophilic bacterium \u003cem\u003eBacillus pumilus\u003c/em\u003e Strain E303035 (Genbank accession number: MN238693; GDMCC number: 60077) was isolated from a Qarhan Salt Lake soil sample, and was preserved at 4 ℃ in the KLB. It was cultured at 37 ℃, with a rotation speed of 200 r/min in a shaker cabinet for 7 days, in Erlenmeyer flasks (1\u0026nbsp;L). Each flask contained 600\u0026nbsp;mL of ATCC213 medium (10\u0026nbsp;g MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 0.2\u0026nbsp;g CaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO 0.2\u0026nbsp;g, KCl, 2.5\u0026nbsp;g peptone, 10\u0026nbsp;g yeast extract, 30\u0026nbsp;g NaCl, adding deuterium-depleted water (D.D.W) to 1000\u0026nbsp;mL, pH 7.2\u0026ndash;7.4).\u003c/p\u003e \u003cp\u003eThen, the fermentation broth was centrifuged at a rotation speed of 8000 r/min, the supernatant was filtered through a 0.22\u0026nbsp;\u0026micro;m millipore filter and stored at 4 ℃ for the preparation of crude extracts (Zhang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003ch2\u003ePreparation of crude extracts of fermentation broth of strain E303035\u003c/h2\u003e \u003cp\u003eThe fermentation broth of strain E303035 was extracted with either ethyl acetate or n-butanol. The two kinds of extract were concentrated \u003cem\u003ein vacuo\u003c/em\u003e to yield a residue. The residues were dissolved in distilled water (using DMSO as a vehicle, at a maximum concentration of 0.1%) to prepare concentrations of 1, 5, 10, and 20\u0026nbsp;mg/ml for antiviral testing against PVY (Zhang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \n\n\n\u003ch2\u003eIsolation and identification of compounds in strain E303035\u003c/h2\u003e \u003cp\u003e1H, 13C, DEPT, 1H-1H-COSY, HSQC, and HMBC NMR spectra were obtained with a JNM-ECA600 spectrometer. FAB-MS spectra were recorded on a JEOL JMS-HX 110 instrument. The chromatographic stationary phases were silica gel (200\u0026ndash;300 mesh), Sephadex LH-20 (25\u0026ndash;100\u0026nbsp;\u0026micro;m, Pharmacia) and MCI-gel CHP20P (75\u0026ndash;150\u0026nbsp;mm, Mitsubishi Chemical). Using thin-layer chromatography, compounds were visualized by spraying with 5% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, followed by heating (Shen et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe ethyl acetate extract was dissolved in ethanol and concentrated \u003cem\u003ein vacuo\u003c/em\u003e to yield 11\u0026nbsp;g oily fraction. Afterwards, the fraction was subjected to handera-SI (10\u0026nbsp;\u0026micro;m), and eluted with \u003cem\u003en\u003c/em\u003e-hexane: isopropanol in the range 80:20\u0026thinsp;\u0026minus;\u0026thinsp;50:50, to give Fraction 1 (Fr1) 4.5\u0026nbsp;g, Fr2 248\u0026nbsp;mg, Fr3 516\u0026nbsp;mg, Fr4 597\u0026nbsp;mg, and Fr5 267\u0026nbsp;mg.\u003c/p\u003e \u003cp\u003eFr1 was subjected to MCI-gel CHP20P and eluted with 100% MeOH, MeOH:H\u003csub\u003e2\u003c/sub\u003eO (1:2), and 100% acetone to give compound \u003cb\u003e8\u003c/b\u003e (3900\u0026nbsp;mg). Fr2 was subjected to Handera C18 (10\u0026nbsp;\u0026micro;m) and eluted with C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e3\u003c/sub\u003eN-H\u003csub\u003e2\u003c/sub\u003eO (1:10\u0026ndash;3:10) to give compounds \u003cb\u003e4\u003c/b\u003e (51\u0026nbsp;mg) and \u003cb\u003e9\u003c/b\u003e (10\u0026nbsp;mg). Fr3 was subjected to Handera C18 (10\u0026nbsp;\u0026micro;m) and eluted with C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e3\u003c/sub\u003eN:H\u003csub\u003e2\u003c/sub\u003eO (1:20\u0026ndash;7:20) to give compound \u003cb\u003e7\u003c/b\u003e (13\u0026nbsp;mg). Fr4 was subjected to Handera C18 (10\u0026nbsp;\u0026micro;m) and eluted with C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e3\u003c/sub\u003eN:H\u003csub\u003e2\u003c/sub\u003eO (1:20\u0026ndash;3:10) to give compounds \u003cb\u003e1\u003c/b\u003e (86\u0026nbsp;mg), \u003cb\u003e5\u003c/b\u003e (78\u0026nbsp;mg), and \u003cb\u003e6\u003c/b\u003e (8\u0026nbsp;mg). Fr5 was subjected to Handera C18 (10\u0026nbsp;\u0026micro;m) and eluted with C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e3\u003c/sub\u003eN:H\u003csub\u003e2\u003c/sub\u003eO (1:20\u0026ndash;3:10) to give compounds \u003cb\u003e2\u003c/b\u003e (25\u0026nbsp;mg) and \u003cb\u003e3\u003c/b\u003e (11\u0026nbsp;mg).\u003c/p\u003e \u003cp\u003eCompounds \u003cb\u003e1\u0026ndash;9\u003c/b\u003e were dissolved in distilled water (using DMSO as a vehicle, at a maximum concentration of 0.1%) to prepare the 31, 63, 125, 250, and 500\u0026nbsp;mg/ml concentrations for antiviral testing against PVY (Zhang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\n\n\u003ch2\u003ePVY inoculation\u003c/h2\u003e \u003cp\u003eInoculation with PVY, and with PVY with an equal volume of extract (crude extracts, compounds, or ningnanmycin), was conducted on two top leaves of the tobacco plants, using a conventional friction method with a 10\u0026sdot; concentration of inoculums. Phosphate buffer inoculation was used as a control. The two infected leaves were collected from each plant, for RNA extraction, 7 days from when the symptoms appeared (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \n\n\u003ch2\u003eReal-time PCR (qPCR) Analysis \u003c/h2\u003e \u003cp\u003eThe total RNA was extracted using a Total RNA Reagent Kit (Takara) from the leaves of plants that were subjected to PVY infection after treatment with each extract (at concentrations of 1, 5, 10, 20\u0026nbsp;mg/mL, using DMSO as vehicle at a maximum concentration of 0.1%) or compound (at concentrations of 31, 63, 125, 250, and 500\u0026nbsp;\u0026micro;g/mL, using DMSO as a vehicle at maximum concentration of 0.1%), in triplicate. Total RNA samples were reverse transcribed using a PrimeScriptTMRT reagent Kit with gDNA Eraser (Takara). Real-time PCR was performed with SYBR Premix Ex Taq Kit (Takara), with \u003cem\u003eβ-actin\u003c/em\u003e as an internal control. The final volume of the PCR mixture was 20\u0026nbsp;\u0026micro;L, including 10\u0026nbsp;\u0026micro;L of the SYBR Green Master mix reagent, 7.8\u0026nbsp;\u0026micro;L of sterile water, 0.4\u0026nbsp;\u0026micro;L of DyeII, 1\u0026nbsp;\u0026micro;L of cDNA, and 0.4\u0026nbsp;\u0026micro;L (2\u0026nbsp;mM) of each real-time PCR primer. Primers used were R\u003cem\u003eβ-actin\u003c/em\u003e (5\u0026acute;-AAGGGATGCGAGGATGGA-3\u0026acute;) and F\u003cem\u003eβ-actin\u003c/em\u003e (5\u0026acute;-CAAGGAAATCACCGCTTTGG-3\u0026acute;), R\u003cem\u003ePVY\u003c/em\u003e (5\u0026acute;-TTCATCTCCATCCATCATAACC-3\u0026acute;) and F\u003cem\u003ePVY\u003c/em\u003e (5\u0026acute;-TACAACTTGCATACGACATAGG-3\u0026acute;), R\u003cem\u003eLhc\u003c/em\u003e (5\u0026acute;-TTAAGAGAAGAAGCCGAATGTG-3\u0026acute;)and F\u003cem\u003eLhc\u003c/em\u003e (5\u0026acute;-CCACACTTCAACTTGCTGAG-3\u0026acute;), R\u003cem\u003ePSii\u003c/em\u003e (5\u0026acute;- TTTCTCCTCCCTCCCTTTCTCT-3\u0026acute;) and F\u003cem\u003ePSii\u003c/em\u003e (5\u0026acute;-TTGCTTGACCGTCGTTGTG-3\u0026acute;), R\u003cem\u003eXTH\u003c/em\u003e (5\u0026acute;-GCGAGGATTTGAGGCACAG-3\u0026acute;) and F\u003cem\u003eXTH\u003c/em\u003e (5\u0026acute;-GCAACGAGAGGTGGATTAGAGAA-3\u0026acute;), R\u003cem\u003eCPIP3\u003c/em\u003e (5\u0026acute;-GTCAGTACAGCCAGAGCCAGAA-3\u0026acute;) and F\u003cem\u003eCPIP3\u003c/em\u003e (5\u0026acute;-AAGCGACTACCAAAACACACACA-3\u0026acute;), R\u003cem\u003ecullin-1\u003c/em\u003e (5\u0026acute;-GTCGCAGAATGTGGCAAGAA-3\u0026acute;)and F\u003cem\u003ecullin-1\u003c/em\u003e (5\u0026acute;-AGAGAAGAGAGATGTGGTTGGT TTG-3\u0026acute;). The following conditions were used: 95 ℃ for 10\u0026nbsp;s, 95 ℃ for 50\u0026nbsp;s, and 35 cycles of both 95 ℃ for 6\u0026nbsp;s and 62 ℃ for 35\u0026nbsp;s. Standard curves were constructed using a series of five tenfold dilutions of the cDNA template. The relative expression levels were calculated (Shen et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \n\n\u003ch2\u003ePCR Analysis\u003c/h2\u003e \u003cp\u003eThe total RNA was extracted using a Total RNA Reagent Kit (Takara) from the leaves of plants that were subjected to PVY infection after the different treatments of compounds (at the concentrations of 31, 63, 125, 250, and 500\u0026nbsp;\u0026micro;g/mL, in triplicate. Total RNA samples were reverse transcribed using a PrimeScriptTMRT reagent Kit with gDNA Eraser (Takara). β-actin was used as an internal control. The final volume of the PCR mixture was 20\u0026nbsp;\u0026micro;L, including 10\u0026nbsp;\u0026micro;L of 2\u0026thinsp;\u0026times;\u0026thinsp;FastTaq PCR Master Mix, 1\u0026nbsp;\u0026micro;L of cDNA, 8.6\u0026nbsp;\u0026micro;L of sterile water, and 0.2\u0026nbsp;\u0026micro;L (2\u0026nbsp;mM) of each RT-PCR primer. Primers used were R\u003cem\u003eβ-actin\u003c/em\u003e (5\u0026acute;-AAGGGATGCGAGGATGGA-3\u0026acute;) and F\u003cem\u003eβ-actin\u003c/em\u003e (5\u0026acute;-CAAGGAAATCACCGCTTTGG-3\u0026acute;), R\u003cem\u003eHC-Pro\u003c/em\u003e (5\u0026acute;-ACCAACTCTATAGTGCTTAATGTCAGA-3\u0026acute;)and F\u003cem\u003eHC-Pro\u003c/em\u003e (5\u0026acute;-GGAGTTCTAGACTCAATGGTTCAGT-3\u0026acute;), R\u003cem\u003eP1\u003c/em\u003e (5\u0026acute;-TTGTGTAACCTTGGAACGCGC-3\u0026acute;) and F \u003cem\u003eP1\u003c/em\u003e (5\u0026acute;-ATGGCAACTTACATGTCAACGATTC-3\u0026acute;), R\u003cem\u003eP3\u003c/em\u003e (5\u0026acute;-CTGATGCCGCACATTATATTCTTC-3\u0026acute;) and F\u003cem\u003eP3\u003c/em\u003e (5\u0026acute;-GGTATTCCTGGGCATGTCCTG-3\u0026acute;), R\u003cem\u003eNIa\u003c/em\u003e (5\u0026acute;-TTGCTCTACAACAACATCATGATCAA-3\u0026acute;) and F\u003cem\u003eNIa\u003c/em\u003e (5\u0026acute;-GCCAAATCACTCATGAGAGGTTTAA-3\u0026acute;), R\u003cem\u003eNIb\u003c/em\u003e (5\u0026acute;-TTGATGGTGCACTTCATAAGTATCG-3\u0026acute;) and F\u003cem\u003eNIb\u003c/em\u003e (5\u0026acute;-GCTAAACATTCTGCGTGGATGTAT-3\u0026acute;), R\u003cem\u003e6K1\u003c/em\u003e (5\u0026acute;-CTGGTGTTTAACTTCATGATCCATT-3\u0026acute;)and F\u003cem\u003e6K1\u003c/em\u003e (5\u0026acute;-CGCTCCACACCAGGTGTTAG-3\u0026acute;), R\u003cem\u003eVPg\u003c/em\u003e (5\u0026acute;-TTCATGCTCCACTTCCTGTTTTG-3\u0026acute;)and F\u003cem\u003eVPg\u003c/em\u003e (5\u0026acute;-GGCAAGAACAAATCCAAAAGAATTC-3\u0026acute;), R\u003cem\u003eCP\u003c/em\u003e (5\u0026acute;-CAGTTCTTGACTCCAAGTAGAGTATG-3\u0026acute;)and F\u003cem\u003eCP\u003c/em\u003e (5\u0026acute;-GGAAATGACACAATCGATGCAG-3\u0026acute;) (Shen et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe EC\u003csub\u003e50\u003c/sub\u003e values were determined from concentration-effect curves by linear regression analysis. Statistical analysis was performed using SPSS 20.0, and data were presented as the arithmetic mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAntiviral activities of crude extracts against PVY\u003c/h2\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, both ethyl acetate and n-butanol extracts from strain E303035 showed significant antiviral activity against PVY. The inhibitory activity of ethyl acetate extract was higher than that of n-butanol. The inhibition rates of both extracts were concentration-dependent. The ethyl acetate extract possessed its highest inhibitory activity at 10\u0026nbsp;mg/mL, with an inhibition rate of 99.70%. The n-butanol extract possessed its highest inhibitory activity at 20\u0026nbsp;mg/mL, with an inhibition rate of 96.43%. The ethyl acetate extract was then isolated to obtain compounds with anti-viral activity against PVY.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe inhibitory activity of two different extracts from E303035 fermentation against PVY\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eConcentration (mg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eInhibition Rate(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eethyl acetate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003en-butanol\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e85.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.81\u0026thinsp;\u0026plusmn;\u0026thinsp;7.89\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e94.59\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e88.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e91.55\u0026thinsp;\u0026plusmn;\u0026thinsp;10.51\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eData are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from experiments with three replicates. Means with a different superscript letter in the same column are significantly different, by Duncan\u0026rsquo;s multiple range test (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \n\n\n\u003ch2\u003eIsolation of compounds from the halophilic bacterium Bacillus pumilus\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, 9 compounds were isolated from the ethyl acetate extract of strain E303035 and identified as cyclo(L-Leu-L-Pro) (\u003cb\u003e1\u003c/b\u003e) (Yue et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), cyclo(L-Pro-L-Tyr) (\u003cb\u003e2\u003c/b\u003e) (Li et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), Brevianamide F (\u003cb\u003e3\u003c/b\u003e) (Wang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), 2-(3-Indolyl) ethanol (\u003cb\u003e4\u003c/b\u003e) (B\u0026ouml;hlendorf et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), N-[2-(1H-indol-3-yl) ethyl] acetamide (\u003cb\u003e5\u003c/b\u003e) (Anouhe et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), 3, 3-di(1H-indol-3-yl)propane- 1,2-diol (\u003cb\u003e6\u003c/b\u003e) (Zhao et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), Lincomycin B (\u003cb\u003e7\u003c/b\u003e) (Argoudelis et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1965\u003c/span\u003e), dibutylphthalate (\u003cb\u003e8\u003c/b\u003e) (Dai et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and p-hydroxyphenethyl alcohol (\u003cb\u003e9\u003c/b\u003e) (Lou et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eCyclo(L-Leu-L-Pro)\u003c/em\u003e (\u003cb\u003e1\u003c/b\u003e). Yield: 86\u0026nbsp;mg; white amorphous powder; molecular formula C\u003csub\u003e11\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCyclo(L-Pro-L-Tyr)\u003c/em\u003e (\u003cb\u003e2\u003c/b\u003e). Yield: 25\u0026nbsp;mg; colorless oil; molecular formula C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBrevianamide F\u003c/em\u003e (\u003cb\u003e3\u003c/b\u003e). Yield: 11\u0026nbsp;mg; white powder; molecular formula C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003e2-(3-indolyl) ethanol\u003c/em\u003e (\u003cb\u003e4\u003c/b\u003e). Yield: 51\u0026nbsp;mg; white powder; molecular formula C\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e11\u003c/sub\u003eNO.\u003c/p\u003e \u003cp\u003e \u003cem\u003eN-[2-(1H-indol-3-yl) ethyl] acetamide\u003c/em\u003e (\u003cb\u003e5\u003c/b\u003e). Yield: 78\u0026nbsp;mg; yellowish-brown oil; molecular formula C\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e \u003cp\u003e \u003cem\u003e3, 3-di(1H-indol-3-yl)propane- 1,2-diol\u003c/em\u003e (\u003cb\u003e6\u003c/b\u003e). Yield: 8\u0026nbsp;mg; white powder; molecular formula C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLincomycin B\u003c/em\u003e (\u003cb\u003e7\u003c/b\u003e). Yield: 13\u0026nbsp;mg; white powder; molecular formula C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003eS.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDibutylphthalate\u003c/em\u003e (\u003cb\u003e8\u003c/b\u003e). Yield: 3900\u0026nbsp;mg; brownish red oil; molecular formula C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eP-hydroxyphenethyl alcohol\u003c/em\u003e (\u003cb\u003e9\u003c/b\u003e). Yield: 10\u0026nbsp;mg; colorless needle crystal; molecular formula C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe data of \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR of compounds \u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e9\u003c/b\u003e is in supplementary file.\u003c/p\u003e \u003ch2\u003eAntiviral activities of compounds isolated from strain E303035 against PVY\u003c/h2\u003e \u003c/p\u003e \u003cp\u003eThe antiviral activities, against PVY, of compounds isolated from strain E303035 were determined at 31, 63, 125, 250, and 500\u0026nbsp;\u0026micro;g/mL. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, compounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e possessed higher antiviral activity against PVY, with EC\u003csub\u003e50\u003c/sub\u003e values of 210.99, 224.26, and 305.37\u0026nbsp;\u0026micro;g/mL, respectively. Compound \u003cb\u003e5\u003c/b\u003e showed significant antiviral activity against PVY at 250 and 500\u0026nbsp;\u0026micro;g/mL, with inhibition values of 62.40% and 75.99%, respectively. Compound \u003cb\u003e1\u003c/b\u003e also showed moderate antiviral activity at a concentration of 125\u0026nbsp;\u0026micro;g/mL, with an inhibition value of 53.87%. Compound \u003cb\u003e4\u003c/b\u003e showed weak antiviral activity at 250 and 500\u0026nbsp;\u0026micro;g/mL, with inhibition values of 30.21% and 24.29%, respectively. In summary, we found that compounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e had good inhibitory activity against PVY, and compounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e4\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e were then used for further research of inhibitory activity against genes encoding functional proteins of PVY.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe antiviral activity of compounds isolated from strain E303035 against PVY\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eInhibition rate(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003cp\u003e(\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e250\u003c/p\u003e \u003cp\u003e(\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e125\u003c/p\u003e \u003cp\u003e(\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e63\u003c/p\u003e \u003cp\u003e(\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e31\u003c/p\u003e \u003cp\u003e(\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e65.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e48.24\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e210.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.21\u0026thinsp;\u0026plusmn;\u0026thinsp;2.29\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.29\u0026thinsp;\u0026plusmn;\u0026thinsp;3.49\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e31.53\u0026thinsp;\u0026plusmn;\u0026thinsp;2.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e224.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52.62\u0026thinsp;\u0026plusmn;\u0026thinsp;3.34\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.21\u0026thinsp;\u0026plusmn;\u0026thinsp;2.29\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e305.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eData are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from experiments with three replicates. Means with a different letter in the same column are significantly different, by Duncan\u0026rsquo;s multiple range test (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). * No inhibitory activity.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003ch2\u003eEffects of active compounds on the expression of genes encoding functional proteins\u003c/h2\u003e \u003c/p\u003e \u003cp\u003eThe expressions of genes \u003cem\u003eHC-pro\u003c/em\u003e, \u003cem\u003eP1\u003c/em\u003e, \u003cem\u003eP3\u003c/em\u003e, \u003cem\u003eNIa\u003c/em\u003e, \u003cem\u003eNIb\u003c/em\u003e, \u003cem\u003e6K1\u003c/em\u003e, \u003cem\u003eVPg\u003c/em\u003e, and \u003cem\u003eCP\u003c/em\u003e under treatment with compounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e4\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e\u0026nbsp;at 31, 63, 125, 250, and 500\u0026nbsp;\u0026micro;g/mL were determined by PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, organizing data by gene, \u003cem\u003eHC-pro\u003c/em\u003e and \u003cem\u003eNIb\u003c/em\u003e showed no expression under treatment with compounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e4\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e\u0026nbsp;at all concentrations, and with compound \u003cb\u003e5\u003c/b\u003e\u0026nbsp;at 125 and 500\u0026nbsp;\u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Gene \u003cem\u003eP3\u003c/em\u003e showed no expression under treatment with compounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e4\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e\u0026nbsp;at all concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Gene \u003cem\u003eNIa\u003c/em\u003e showed no expression under treatment with compounds \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e4\u003c/b\u003e\u0026nbsp;at all tested concentrations, compound \u003cb\u003e5\u003c/b\u003e\u0026nbsp;at 125 and 500\u0026nbsp;\u0026micro;g/mL, and compound \u003cb\u003e9\u003c/b\u003e\u0026nbsp;at 125, 250, and 500\u0026nbsp;\u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Organizing data by compound, at all tested concentrations: \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e4\u003c/b\u003e significantly inhibited the expressions of genes \u003cem\u003eHC-pro\u003c/em\u003e, \u003cem\u003eP3\u003c/em\u003e, \u003cem\u003eNIb\u003c/em\u003e, and \u003cem\u003eNIa\u003c/em\u003e, compound \u003cb\u003e5\u003c/b\u003e inhibited gene \u003cem\u003eP3\u003c/em\u003e, and compound \u003cb\u003e9\u003c/b\u003e inhibited \u003cem\u003eNIb\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, with data organized by gene, genes \u003cem\u003eVPg\u003c/em\u003e and \u003cem\u003e6K1\u003c/em\u003e showed no expression under treatment with compounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e4\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e\u0026nbsp;at all tested concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Gene \u003cem\u003eCP\u003c/em\u003e showed no expression under treatment with compound \u003cb\u003e1\u003c/b\u003e\u0026nbsp;at 63, 125, and 500\u0026nbsp;\u0026micro;g/mL, compound \u003cb\u003e4\u003c/b\u003e\u0026nbsp;at 250 and 500\u0026nbsp;\u0026micro;g/mL, compound \u003cb\u003e5\u003c/b\u003e\u0026nbsp;at 125 and 500\u0026nbsp;\u0026micro;g/mL, and compound \u003cb\u003e9\u003c/b\u003e\u0026nbsp;at 125, 250, and 500\u0026nbsp;\u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Gene \u003cem\u003eP1\u003c/em\u003e showed no expression under treatment with compound \u003cb\u003e1\u003c/b\u003e\u0026nbsp;at 63, 125, and 500\u0026nbsp;\u0026micro;g/mL, compound \u003cb\u003e4\u003c/b\u003e\u0026nbsp;at 63, 250, and 500\u0026nbsp;\u0026micro;g/mL, compound \u003cb\u003e5\u003c/b\u003e\u0026nbsp;at 125, 250, and 500\u0026nbsp;\u0026micro;g/mL, and compound \u003cb\u003e9\u003c/b\u003e\u0026nbsp;at 63, 125, 250, and 500\u0026nbsp;\u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Organizing the data by compound, compounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e4\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e significantly inhibited the expressions of genes \u003cem\u003eVPg\u003c/em\u003e and \u003cem\u003e6K1\u003c/em\u003e at all tested concentrations.\u003c/p\u003e \u003ch2\u003eEffect of active compounds on the expression of early response genes induced by potato virus Y\u003c/h2\u003e \u003ch2\u003eEffects of active compounds on the expression of chlorophyll a-b binding protein (\u003cem\u003eLhc\u003c/em\u003e)\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the expression of gene \u003cem\u003eLhc\u003c/em\u003e under the treatment of compounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e\u0026nbsp;at 31, 63, 125, 250, and 500\u0026nbsp;\u0026micro;g/mL after 2, 4, and 6 days were determined by real-time PCR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor compound \u003cb\u003e1\u003c/b\u003e, increasing the concentration caused significant up-regulation of the gene \u003cem\u003eLhc\u003c/em\u003e. Over time, the gene was significantly down-regulated at day 4, then up-regulated at day 6, at all concentrations. The expression of gene \u003cem\u003eLhc\u003c/em\u003e was at its lowest at day 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eFor compound \u003cb\u003e5\u003c/b\u003e, increasing the concentration caused significant down-regulation of the gene \u003cem\u003eLhc\u003c/em\u003e. The gene was significantly up-regulated at day 4, and then down-regulated at day 6, at all concentrations. The expression of gene \u003cem\u003eLhc\u003c/em\u003e was at its highest at day 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFor compound \u003cb\u003e9\u003c/b\u003e, increasing the concentration caused significant down-regulation of the gene \u003cem\u003eLhc\u003c/em\u003e. The gene was significantly down-regulated at day 4, and then up-regulated at day 6, at all concentrations. The expression of gene \u003cem\u003eLhc\u003c/em\u003e was at its lowest at day 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003ch2\u003eEffect of active compounds on the expression of photo system II (\u003cem\u003ePSii\u003c/em\u003e)\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the expression of gene \u003cem\u003ePSii\u003c/em\u003e under treatments with compounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e\u0026nbsp;at 31, 63, 125, 250, and 500\u0026nbsp;\u0026micro;g/mL after 2, 4, and 6 days was determined by real-time PCR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor compounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e, increasing the concentration caused significant up-regulation of the gene \u003cem\u003ePSii\u003c/em\u003e. The gene was significantly up-regulated at day 4, then down-regulated at day 6, at all concentrations. The expression of gene \u003cem\u003ePSii\u003c/em\u003e was at its highest at day 4.\u003c/p\u003e \u003ch2\u003eEffect of active compounds on the expression of xyloglucan endotransglucosylase hydrolase (\u003cem\u003eXTH\u003c/em\u003e)\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the expression of gene \u003cem\u003eXTH\u003c/em\u003e under treatments with compounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e\u0026nbsp;at 31, 63, 125, 250, and 500\u0026nbsp;\u0026micro;g/mL after 2, 4, and 6 days was determined by real-time PCR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor compound \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e9\u003c/b\u003e, increasing the concentration caused significant down-regulation of the gene \u003cem\u003eXTH\u003c/em\u003e. The gene was significantly down-regulated at day 4, and then up-regulated at day 6, at all concentrations. The expression of gene \u003cem\u003eXTH\u003c/em\u003e was at its lowest at day 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eFor compound \u003cb\u003e5\u003c/b\u003e, increasing the concentration caused significant up-regulation of the gene \u003cem\u003eXTH\u003c/em\u003e. The gene was significantly up-regulated at day 4, and then down-regulated at day 6, at all concentrations. The expression of gene \u003cem\u003eXTH\u003c/em\u003e was at its highest at day 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003ch2\u003eEffect of active compounds on the expression of gene CP-interacting protein 3 (\u003cem\u003eCPIP3\u003c/em\u003e)\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the expression of gene \u003cem\u003eCPIP3\u003c/em\u003e under treatment with compounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e\u0026nbsp;at 31, 63, 125, 250, and 500\u0026nbsp;\u0026micro;g/mL after 2, 4, and 6 days was determined by real-time PCR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor compounds \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e, increasing the concentration caused significant down-regulation of the gene \u003cem\u003eCPIP3\u003c/em\u003e. The gene was significantly up-regulated at day 4, and then down-regulated at day 6, at all concentrations. The expression of gene \u003cem\u003eCPIP3\u003c/em\u003e was at its highest at day 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFor compound \u003cb\u003e9\u003c/b\u003e, increasing the concentration caused significant up-regulation of the gene \u003cem\u003eCPIP3\u003c/em\u003e. The gene was significantly up-regulated at day 4, and then down-regulated at day 6, at all concentrations. The expression of gene \u003cem\u003eCPIP3\u003c/em\u003e was at its highest at day 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003ch2\u003eEffect of active compounds on the expression of the gene cullin-1 isoform (\u003cem\u003ecullin-1\u003c/em\u003e)\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, the expression of gene \u003cem\u003ecullin-1\u003c/em\u003e under treatment with compounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e\u0026nbsp;at 31, 63, 125, 250, and 500\u0026nbsp;\u0026micro;g/mL after 2, 4, and 6 days was determined by real-time PCR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor compound \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e, increasing the concentration caused significant down-regulation of the gene \u003cem\u003ecullin-1\u003c/em\u003e. The gene was significantly up-regulated at day 4, and then down-regulated at day 6, at all concentrations. The expression of gene \u003cem\u003ecullin-1\u003c/em\u003e was at its highest at day 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFor compound \u003cb\u003e9\u003c/b\u003e, increasing the concentration caused significant up-regulation of the gene \u003cem\u003ecullin-1\u003c/em\u003e. The gene was significantly up-regulated at day 4, and then down-regulated at day 6, at all concentrations. The expression of gene \u003cem\u003ecullin-1\u003c/em\u003e was the highest at day 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e "},{"header":"Discussion","content":" \u003cp\u003eThe genome of PVY is composed of a single-stranded RNA, length 9.7\u0026nbsp;kb, which covalently links with a viral-encoded protein (VPg) at its 5ʹ-end and contains a 3ʹ-polyadenylated tail. The genome of PVY encodes two polyproteins, a larger polyprotein of about 3000 amino acids and a shorter one translated from a 2\u003csup\u003e+\u003c/sup\u003e frameshift in the P3 coding region. These polyproteins are cleaved by viral proteases, subsequently generating eleven mature proteins (Kim et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCompounds \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e4\u003c/b\u003e significantly inhibited the expression of gene \u003cem\u003eHC-pro\u003c/em\u003e at all concentrations tested. \u003cem\u003eHC-Pro\u003c/em\u003e encodes viral helper component-proteinase (HC-Pro), which is a pathogen elicitor. It is also involved in multiple roles in aphid transmission, RNA binding, suppression of gene silencing and protease activity (Chowdhury et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCompounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e4\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e and \u003cb\u003e9\u003c/b\u003e significantly inhibited the expression of gene \u003cem\u003eVPg\u003c/em\u003e at all concentrations tested. \u003cem\u003eVPg\u003c/em\u003e encodes a viral protein called VPg which sabotages host antiviral RNA silencing to promote viral infection (Cheng et al. 2016).\u003c/p\u003e \u003cp\u003eCompounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e4\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e significantly inhibited the expression of genes \u003cem\u003eCP\u003c/em\u003e and \u003cem\u003eP1\u003c/em\u003e, at some concentrations. \u003cem\u003eCP\u003c/em\u003e encodes a viral coat protein and \u003cem\u003eP1\u003c/em\u003e encodes plasma membrane cation-binding protein 1 (PCaP1), which is shown to be important for the intra-cellular movement of two members of the genus \u003cem\u003ePotyvirus\u003c/em\u003e, in \u003cem\u003eArabidopsis\u003c/em\u003e (rockcress) and in tobacco plants (Beris et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCompounds \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e4\u003c/b\u003e significantly inhibited the expression of gene \u003cem\u003eNIa\u003c/em\u003e at all concentrations tested. \u003cem\u003eNIa\u003c/em\u003e encodes the \u0026ldquo;nuclear inclusion a\u0026rdquo; (NIa) protease of PVY, which is involved in processing the polyprotein encoded by its positive sense RNA genome. The NIa protein is also responsible for the release of the VPg protein involved in viral RNA replication (Gargouri-Bouzid et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCompounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e4\u003c/b\u003e and \u003cb\u003e9\u003c/b\u003e significantly inhibited the expression of gene \u003cem\u003eNIb\u003c/em\u003e at all concentrations tested. \u003cem\u003eNIb\u003c/em\u003e is an RNA-dependent RNA polymerase in the PVY genome, which has been reported to confer virulence in infections of pepper plants (Janzac et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCompounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e4\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e significantly inhibited the expression of gene \u003cem\u003eP3\u003c/em\u003e at all concentrations tested. \u003cem\u003eP3\u003c/em\u003e encodes potyviral membrane protein.\u003c/p\u003e \u003cp\u003eCompounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e4\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e significantly inhibited the expression of gene \u003cem\u003e6K1\u003c/em\u003e. The function of \u003cem\u003e6K1\u003c/em\u003e is currently unknown. In summary, all compounds showed inhibitory activities against various genes encoding functional proteins of PVY. The results provide a starting point for future research into the inhibitory mechanisms and pathways of active compounds against PVY.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eLhc\u003c/em\u003e gene encodes chlorophyll a-b binding protein, which takes part in regulating photosynthesis and chlorophyll synthesis. The down-regulation of this gene means a decrease in viral nucleic acid, which is also seen in PVY-resistant plants (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Compounds \u003cb\u003e5\u003c/b\u003e and \u003cb\u003e9\u003c/b\u003e down-regulated the \u003cem\u003eLhc\u003c/em\u003e gene. There was an inverse relationship between concentration and gene expression. At day 4, the expression of gene \u003cem\u003eLhc\u003c/em\u003e was at its lowest. These results suggest that compounds \u003cb\u003e5\u003c/b\u003e and \u003cb\u003e9\u003c/b\u003e elicit their inhibitory activity due to their influence on chlorophyll synthesis and photosynthesis during days 2\u0026thinsp;\u0026minus;\u0026thinsp;4.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ePSii\u003c/em\u003e gene takes part in photosystem II of photosynthesis. A down-regulation of this gene has the same effect as inhibition of \u003cem\u003eLhc\u003c/em\u003e: a decrease in viral nucleic acid. Compounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e showed no significant inhibitory effect of this gene, but the expression of \u003cem\u003ePSii\u003c/em\u003e gene was at its lowest on day 6.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eXTH\u003c/em\u003e gene encodes xyloglucan endotransglucosylase hydrolase, which is an essential constituent of the primary cell wall and participates in cell wall elongation and construction (Otulak-Kozieł K et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e; Otulak-Kozieł K et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e). The cell wall is the first barrier that protects plants against entry of pathogens and other harmful bodies, and offers protection against mechanical stress to plant cells. The expression of cell-wall-related genes, including \u003cem\u003eXTH\u003c/em\u003e, has been shown to be regulated by biotic and abiotic stresses (Li et al. 2015). In this study, \u003cem\u003eXTH\u003c/em\u003e was up-regulated under treatment with compound \u003cb\u003e5\u003c/b\u003e as the concentration increased before 4 days. This suggests that compound \u003cb\u003e5\u003c/b\u003e could increase elongation and construction of plant cell walls at different concentrations, especially at day 4. However, the \u003cem\u003eXTH\u003c/em\u003e gene showed down-regulation under treatment with compounds \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e9\u003c/b\u003e\u0026nbsp;at day 4 and then up-regulation at day 6. This suggests that both compounds \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e9\u003c/b\u003e could in increase elongation and construction of plant cell walls after 4 days, especially at day 6. It affected the plant cell wall later than that of compound \u003cb\u003e5\u003c/b\u003e. This may indicate that these compounds contribute to induce PVY resistance in \u003cem\u003eNicotiana.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eCPIP3\u003c/em\u003e is a stress response gene, encoding CP-interacting protein 3. The interaction between viral coat protein (CP) and host plant CP-interacting protein were shown to be important for the plant defense response, viral propagation, and long distance movement (Li et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Park et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). A low expression of the \u003cem\u003eCPIP3\u003c/em\u003e gene signifies a low quantity of CP-interacting protein 3 which, to some extent, signifies inhibitory activity against PVY (Li et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In this study, compounds \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e significantly down-regulated the gene \u003cem\u003eCPIP3\u003c/em\u003e at 2 days, with their increased concentration. Therefore, compounds \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e could inhibit the viral multiplication of PVY by inducing the plant defense response in tobacco plants. Although compound \u003cb\u003e9\u003c/b\u003e was able to up-regulate \u003cem\u003eCPIP3\u003c/em\u003e at days 2 and 4, by day 6 there was significant down-regulation of the gene, at all concentrations tested. The result demonstrated the inhibitory activity of compound \u003cb\u003e9\u003c/b\u003e against the multiplication of PVY, by inducing the tobacco plant defense response at 6 days.\u003c/p\u003e \u003cp\u003eGene \u003cem\u003ecullin-1\u003c/em\u003e also encodes a stress response protein. The up-regulation of it was reported to be important in the resistance response to pathogen infection and autoimmunity (Cheng et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Gou et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In our study, the gene \u003cem\u003ecullin-1\u003c/em\u003e was significantly up-regulated by compounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e\u0026nbsp;at days 2 and 4. This meant that all three compounds had significant inhibitory activity against PVY by inducing the resistance response to PVY infection and autoimmunity in tobacco plants at 2\u0026ndash;4 days. In detail, compounds \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e up-regulated \u003cem\u003ecullin-1\u003c/em\u003e\u0026nbsp;at low concentrations of 31, 63, and 125\u0026nbsp;\u0026micro;g/mL, while compound \u003cb\u003e9\u003c/b\u003e up-regulated \u003cem\u003ecullin-1\u003c/em\u003e\u0026nbsp;at a high concentration of 500\u0026nbsp;\u0026micro;g/mL.\u003c/p\u003e \u003cp\u003eTaken together, active compounds \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e had significant inhibitory activity against PVY and various effects on the expression of viral genes related to pathogenesis. They might be considered as potential antiviral agents for future development.\u003c/p\u003e "},{"header":"Declarations","content":" \u003cp\u003e \u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eWe admit availability of data and material.\u003c/p\u003e \u003ch2\u003eEthical approval and consent to participate\u003c/h2\u003e \u003cp\u003eThis article does not contain any studies with human participants or animals. We declare consent to participate.\u003c/p\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was financed by the Natural Science Fund of Qinghai Province (2019-ZJ-914) and National Natural Science Fund (31860512).\u003c/p\u003e \u003ch2\u003eAuthor\u0026rsquo;s contributions\u003c/h2\u003e \u003cp\u003eWei Li and Shuo Shen conceived and designed research. Wei Li and Shuo Shen conducted experiments. Shuo Shen carried out all experiments. Wei Li and Shuo Shen analyzed data. Shuo Shen wrote the manuscript. All authors read and approved the manuscript.\u003c/p\u003e \u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe thank Prof. Bao-An Song and his staff in Guizhou University for technical assistance.\u003c/p\u003e "},{"header":"References","content":"\u003col\u003e\u003cli\u003e \u003cspan\u003eAnouhe JBS, Adima AA, Niamk\u0026eacute; FB, Stien D, Amian BK, Blandini\u0026egrave;res PA, Virieux D, Pirat JL, Kati-Coulibaly S, Amusant N (2015) Dicorynamine and harmalan-N-oxide, two new b-carboline alkaloids from Dicorynia guianensis Amsh heartwood. Phytochem Let 12:158\u0026ndash;163\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eArgoudelis AD, Fox JA, Eble TE (1965) A new lincomycin-related antibiotic. 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Chinese\u003c/span\u003e \u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"amb-express","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ambe","sideBox":"Learn more about [AMB Express](http://amb-express.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/AMBE/default.aspx","title":"AMB Express","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"halophilic bacterium Bacillus pumilus, active compounds, inhibitory activity, Potato Virus Y· genes, encoding viral proteins, early response genes","lastPublishedDoi":"10.21203/rs.3.rs-53377/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-53377/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo develop a new antiviral preparation from a microbial source, the halophilic bacterium \u003cem\u003eBacillus pumilus\u003c/em\u003e E303035 was isolated from a soil sample collected at Qarhan Salt Lake in Qinghai, China. The inhibitory activity of an ethyl acetate extract of its fermentation broth was higher than that of an n-butanol extract. After isolation and purification, 9 compounds were obtained: cyclo(L-Leu-L-Pro) (\u003cstrong\u003e1\u003c/strong\u003e), cyclo(L-Pro-L-Tyr) (\u003cstrong\u003e2\u003c/strong\u003e), Brevianamide F (\u003cstrong\u003e3\u003c/strong\u003e), 2-(3-Indolyl) ethanol (\u003cstrong\u003e4\u003c/strong\u003e), N-[2-(1H-indol-3-yl) ethyl] acetamide (\u003cstrong\u003e5\u003c/strong\u003e), 3, 3-di(1H-indol-3-yl)propane-1,2-diol (\u003cstrong\u003e6\u003c/strong\u003e), Lincomycin B (\u003cstrong\u003e7\u003c/strong\u003e), dibutylphthalate (\u003cstrong\u003e8\u003c/strong\u003e), and p-hydroxyphenethyl alcohol (\u003cstrong\u003e9\u003c/strong\u003e). Compounds \u003cstrong\u003e1\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e, and \u003cstrong\u003e9\u003c/strong\u003e showed inhibitory activities against potato virus Y (PVY). Compounds \u003cstrong\u003e1\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, and \u003cstrong\u003e9\u003c/strong\u003e had significant inhibitory activity against genes \u003cem\u003eHC-pro\u003c/em\u003e, \u003cem\u003eP3\u003c/em\u003e, and \u003cem\u003eNib\u003c/em\u003e, compound \u003cstrong\u003e5 \u003c/strong\u003eagainst gene \u003cem\u003eP3\u003c/em\u003e, and compounds \u003cstrong\u003e1\u003c/strong\u003e and \u003cstrong\u003e4\u003c/strong\u003e against \u003cem\u003eNIa\u003c/em\u003e. Compounds \u003cstrong\u003e1\u003c/strong\u003e,\u003cstrong\u003e 4\u003c/strong\u003e,\u003cstrong\u003e 5\u003c/strong\u003e, and\u003cstrong\u003e 9\u003c/strong\u003e had significant inhibitory activity against genes \u003cem\u003eVPg\u003c/em\u003e and \u003cem\u003e6K1\u003c/em\u003e. Active compounds \u003cstrong\u003e1\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eand \u003cstrong\u003e9 \u003c/strong\u003ehad various effects on the expression of viral genes related to pathogenesis. Expression of genes \u003cem\u003ecullin\u003c/em\u003e and \u003cem\u003eXTH \u003c/em\u003ewas up-regulated and \u003cem\u003eCP\u003c/em\u003e was down-regulated, compared to the positive control. In conclusion, compounds \u003cstrong\u003e1\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e, and \u003cstrong\u003e9\u003c/strong\u003e might be considered as potential antiviral agents for future development.\u003c/p\u003e","manuscriptTitle":"The inhibitory effects of metabolites from Bacillus pumilus on potato virus Y and the induction of early response genes in Nicotiana tabacum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-08-05 22:34:56","doi":"10.21203/rs.3.rs-53377/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2020-08-10T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-08-08T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-08-07T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-08-04T12:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-08-03T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"amb-express","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ambe","sideBox":"Learn more about [AMB Express](http://amb-express.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/AMBE/default.aspx","title":"AMB Express","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"24178fcf-9089-4ff0-b8c4-5cec41643e30","owner":[],"postedDate":"August 5th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":248416,"name":"Applied \u0026 Industrial Microbiology"}],"tags":[],"updatedAt":"2020-08-25T17:11:37+00:00","versionOfRecord":{"articleIdentity":"rs-53377","link":"https://doi.org/10.1186/s13568-020-01089-1","journal":{"identity":"amb-express","isVorOnly":false,"title":"AMB Express"},"publishedOn":"2020-08-20 12:00:00","publishedOnDateReadable":"August 20th, 2020"},"versionCreatedAt":"2020-08-05 22:34:56","video":"","vorDoi":"10.1186/s13568-020-01089-1","vorDoiUrl":"https://doi.org/10.1186/s13568-020-01089-1","workflowStages":[]},"version":"v1","identity":"rs-53377","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-53377","identity":"rs-53377","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}