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

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.


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 Open Access *Correspondence: lwbabylw@163.com 3 Key Laboratory of Potato Breeding of Qinghai Province, Xining 810016, Qinghai, China Full list of author information is available at the end of the article 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.

Plant and PVY materials
Tobacco plants (Nicotiana tabacum var. Samsun NN) were grown under greenhouse conditions, with 16 h light at 25 °C and 8 h darkness at 15 °C, and PVY was kept at 4 °C, 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.
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 °C 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).
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 Rβ-actin 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 PrimeScriptT-MRT 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× Fast-Taq PCR Master Mix, 1µL of cDNA, 8.6 µL of sterile water, and 0.2µL (2 mM

Statistical analysis
The EC 50 values were determined from concentrationeffect curves by linear regression analysis. Statistical analysis was performed using SPSS 20.0, and data were presented as the arithmetic mean ± standard deviation.

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 concentrationdependent. 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.
The data of 1 H NMR and 13 C NMR of compounds 1-9 is in Additional file 1.

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.
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. 2a, c). Gene P3 showed no expression under treatment with compounds 1, 4, 5, and 9 at all concentrations (Fig. 2b). 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. 2d). 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.

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 upregulated at day 6, at all concentrations. The expression of gene Lhc was at its lowest at day 4 (Fig. 4a).
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 downregulated at day 6, at all concentrations. The expression of gene Lhc was at its highest at day 4 (Fig. 4b).
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. 4c).

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 downregulated 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. 6a,  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. 6b).

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. 7a, 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 downregulated at day 6, at all concentrations. The expression of gene CPIP3 was at its highest at day 4 (Fig. 7c).
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. 8a, 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 downregulated at day 6, at all concentrations. The expression of gene cullin-1 was the highest at day 4 (Fig. 8c).

Discussion
The genome of PVY is composed of a single-stranded RNA, length 9.7 kb, which covalently links with a viralencoded 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.   Fig. 6 The effect of active compounds 1, 5, and 9 on the expression of gene XTH. a Compound 1; b Compound 5; c Compound 9 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. 2005). 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   Fig. 8 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