Resveratrol inhibits the growth of Xanthomonas oryzae pv. oryzae and the metabolomic analysis

Background Xanthomonas oryzae pv. oryzae ( Xoo ) can cause destructive bacterial leaf blight in rice. As an antibacterial, resveratrol may inhibit Xoo growth. This study focused on the potential structural-activity relationship of resveratrol and its derivatives against Xoo growth, and 1 H-NMR-based metabolomic analysis was applied to investigate the global metabolite changes in Xoo after resveratrol treatment. Results Resveratrol showed the strongest inhibitory effects on Xoo growth compared with its derivatives, which lacked double bonds (compounds 4 – 6 ) or hydroxyls were substituted with methoxyls (compounds 7 – 9 ). The IC 50 of resveratrol against Xoo growth was 11.67 ± 0.58 μg/mL. Results indicated that the double bond of resveratrol contributed to its inhibitory effects on Xoo growth, and hydroxyls were vital for this inhibition. Interestingly, resveratrol also signicantly inhibited Xoo agellum growth. Based on 1 H-NMR global metabolic analysis, a total of 30 Xoo metabolites were identied, the changes in the metabolic prole indicated that resveratrol could cause oxidative stress as well as disturb energy, purine, amino acid, and NAD + metabolism in Xoo , resulting in the observed inhibitory effects on growth. Conclusions This study showed that the double bond of resveratrol contributed to its inhibitory effects on Xoo growth, and hydroxyls were also the important active groups. Resveratrol could cause oxidative stress of Xoo cells, and disturb the metabolism of energy, purine, amino acid and NAD +, thus inhibit Xoo growth.

Resveratrol, as a "model stilbene" and an important phytoalexin, is isolated from grapes, berries, peanuts, pines, and Polygonum cuspidatum Sieb [8]. Additionally, resveratrol can be synthesized easily with a high yield, which make it possible to obtain large scale in low cost [9]. Resveratrol exhibits antioxidant, antiviral, anti-in ammatory, anti-fungal, and anticancer bioactivities and is a known quorum sensing inhibitor (QSI) that can inhibit Pseudomonas aeruginosa virulence [10]. Additionally, resveratrol can be used as a potent antibacterial to inhibit the growth of Xanthomonas [11] and was rescreened in the current study.
To the best of our knowledge, the main active groups of resveratrol, as well as its effects on molecular metabolic pro les and the potential inhibition mechanism against Xanthomonas, remain unknown. With the development of omics technologies, metabolomic has been applied to study metabolic pro les and molecular mechanisms. In this research, we investigated the potential structure-activity relationship of resveratrol and its derivatives and their inhibitory effects on Xoo growth. The metabolic changes in Xoo and underlying inhibition mechanism were also evaluated after treatment with resveratrol using 1 H-NMRbased metabolomics [10]. Results indicated that the double bond of resveratrol contributed to its inhibition of Xoo growth, with hydroxyls found to be the vital active group. Furthermore, our results suggested that resveratrol could disturb energy, purine, amino acid, and NAD + metabolism in Xoo cells, resulting in the observed inhibitory effects on growth.

Metabolite Identi cation and Multivariate Statistical Analyses
Typical 500 MHz CPMG 1 H-NMR spectra for the resveratrol-treated (T-group) and control groups (C-group) are shown in Fig. 4, with a total of 30 metabolites assigned. Detailed information on the 30 metabolites is shown in Table 3. The STOCSY technique, which computes correlation among the intensities of all peaks in a matrix, was used for the assignment of metabolites, such as glutamate, succinate, tyrosine, and histidine ( Fig. 5).
Principal component analysis (PCA) was rst used to obtain an overview of variation between the Cgroup and T-group. Quite signi cant overlap was observed between the PCA score plot of two groups in Fig. 6. OSC-PLS-DA, a supervised pattern recognition technique, was used to identify the metabolic differences between the two groups. In the OSC-PLS-DA score plot (Fig. 7A), the T-group was signi cantly separated from the C-group. The corresponding S-plot (Fig. 7D) and the color-coded loading plots (Fig.  7B,C) were used to identify the contributions of variables between the groups. In addition, changes in metabolites were directly visualized as fold-changes in these plots, and were color-coded according to the differences of p-values between the groups (Fig. 8). Compared with the C-group, cholate, succinate, taurine, NAD + , NADP + , IMP, AMP, and formate signi cantly increased, whereas valine, suberate, lysine, glutamate, 2-aminoadipate, glutamine, glutathione, choline, glycine, uridine, and fumarate markedly decreased in the T-group. The assigned metabolites, their fold change values, and p-values are shown in Table 3.

Discussion
Resveratrol, known as a natural phytoalexin, is found in some medicinal and edible plants, and shows considerable antibacterial, antioxidant, anti-in ammatory, and anticancer activities [10]. Wang reported that resveratrol can inhibit the growth of Xanthomonas oryzae [11]. However, the potential structureactivity relationships among resveratrol derivatives against Xoo growth and the in uence of resveratrol on the metabolic system of Xoo are not known. In this study, the structure-activity relationship and the in uence of resveratrol on the metabolic system of Xoo were investigated by 1 H-NMR-based metabolomics. We found that: (1) The double bond between two benzene rings in resveratrol was essential to inhibit the growth of Xoo; (2) The hydroxyls in these benzene rings might be the main activity group involved in the inhibition of Xoo growth; and (3) resveratrol might cause oxidative stress, as well as disturb energy, nucleic acid, amino acid, and NAD + metabolism, in Xoo cells.
Inhibitory effects on growth of Xoo and the potential structure-activity relationship Compounds (1-3) showed strong inhibition activity on the growth of Xoo, with resveratrol demonstrating the strongest activity among the tested compounds. Even though resveratrol exhibits antibacterial activity and can inhibit the growth of Xanthomonas [11,12], the metabolomic mechanisms of the inhibition are still unknown. It was reported that the mechanisms were complex and different for different inhibitors. Di cidin and bacilysin could in uence the protein and cell wall synthesis and cell division to inhibit the growth of Xanthomonas [14]. Phenazine-1-carboxylic acid in uenced the energy metabolism and disturbed the redox balance in Xoo [15,16]. So as a famous antioxidant, resveratrol maybe had effects on the energy metabolism and redox balance of Xoo. Subsequently, we investigated the structure-activity relationships among resveratrol and its derivatives. Compared with compounds (1-3), compounds (4-6), which lacked the double bond, showed weaker inhibitory effects on the growth of Xoo. Thus, the double bond in the skeleton of compounds (1-3) likely played an important role in the inhibition of Xoo growth. Compared with compounds (1-3), all hydroxyls were substituted by methoxyls on the benzene rings in compounds (7)(8)(9). Interestingly, compounds (7)(8)(9) had no inhibitory effects on Xoo growth, indicating that the hydroxyls were the vital active group for the inhibitory effects on Xoo growth observed for compounds (1)(2)(3). For many inhibitors, the double bond and the hydroxyls played important roles in the anti-Xoo activity, such as sphaeropsidin A, the main phytotoxin produced by Diplodia cupressi [17], and it was reported that stilbenoids with at least one free hydroxyl group as a common structural feature suggested an association with the antimicrobial activity, such as against Bacillus subtilis and Pseudomonas syringae [18]. In addition, the literature also showed that the hydroxyls and double bond of stilbenoids were the vital active group for quorum sensing inhibiting activity [8].

Effects of resveratrol on the agella of Xoo
Flagella are essential for the virulence of organisms such as Pseudomonas and Xanthomonas, and play an important role in the pathogenesis of other mucosal infections as they are directly related to chemotaxis and motility [19]. Comparison of the control and resveratrol-treated groups ( Fig. 3) indicated that resveratrol signi cantly inhibited agellum growth of Xoo. This lack of agella could caused e ciency of chemotaxis and motility, resulting in a decrease in virulence and pathogenicity [19,20].

Oxidative stress
Oxidative stress re ects an imbalance between the generation of reactive oxygen species (ROS) and antioxidant defense, with antioxidative enzymes playing a central role in determining individual risk of developing oxidative stress. The glutathione (GSH) redox system is an important antioxidant defense mechanism, in which glutathione peroxidase (GPx) and glutathione reductase (GR) catalyze the interconversion between reduced glutathione (GSH) and glutathione disul de (GSSG) [10]. The level of GSH decrease indicated that it was excessive consumption to counteract oxidative stress because of its natural antioxidant in the cells. Additionally, GSH can clear electrophilic exogenous substances and in uence detoxi cation under catalyzing by glutathione S-transferase (GST), which is an adaptive mechanism to reduce toxic effects. After Xoo was treated by resveratrol, the detoxi cation mechanism may have exacerbated the depletion of GSH, resulting in accelerated synthesis and signi cantly decreased levels of precursors such as glutamate and glutamine [21].
Cell membranes are susceptible to oxidative damage due to unsaturated fatty acids [22]. Choline and phosphocholine are crucial for the structural integrity of cell membranes. In the present study, choline was remarkably decreased in the resveratrol-treated group, indicating that choline was excessively consumed to repair damaged membranes caused by ROS. Because of the strong antioxidant activity and protective effects, taurine showed protective effects against oxidative stress [23]. The increase in taurine indicated its requirement was improving to protect the cell from oxidative stress after treatment with resveratrol [24,25]. Thus, the decrease in choline and increase in taurine indicated that Xoo underwent severe oxidative damage after treatment with resveratrol.

Energy metabolism
Compared with the control group, the level of succinate was signi cantly increased, whereas the level of fumarate was remarkably decreased after Xoo was treated with resveratrol. Both succinate and fumarate are intermediates of the tricarboxylic acid (TCA) cycle, and succinate can be converted to fumarate with succinate dehydrogenase [10]. In normal conditions, most energy is produced through the TCA cycle under aerobic respiration [24]. The increase in succinate and decrease in fumarate indicated that the TCA cycle was disrupted and energy metabolism was disturbed, which, in turn, inhibited growth of Xoo.
In addition, adenosine monophosphate (AMP) showed a remarkable increase in the resveratrol-treated group. AMP-activated protein kinase (AMPK) can regulate multiple biological processes regarding cell growth, especially cellular energy homeostasis [26]. Therefore, the change in AMP level might disturb the AMPK pathway and in uence the energy metabolism balance.

Purine metabolism
Inosine, used for nucleoside synthesis with inosine monophosphate (IMP), adenosine monophosphate (AMP), and guanosine monophosphate (GMP), provides substrates for the enzymatic biosynthesis of DNA and RNA [21]. The inosine level did not signi cantly increase, whereas the levels of IMP and AMP did show remarkable increase. ATP hydrolyzes into ADP or AMP to produce energy for the cell, and IMP can convert into AMP by adenylosuccinate lyase [27]. Interestingly, the ATP level did not signi cantly increase, so the AMP/ATP ratio increased after treatment with resveratrol, indicating that the balance between the production and consumption of ATP was disturbed [28].

Amino acids metabolism
Valine showed a remarkable decrease in the resveratrol-treated group. Valine is a branched-chain amino acid (BCAA, including leucine, isoleucine, and valine) [29]. BCAAs are essential amino acids in vivo and act as vital substrates to regulate protein synthesis [10]. The decrease in valine observed in the present study suggests that the normal protein synthesis of Xoo broke-down due to resveratrol treatment. As proteins are vital substrates for organisms, the growth of Xoo was inhibited after exposure to resveratrol.
In addition, some evidence indicates that BCAAs are also nutrient signals that regulate many cellular functions, including cell growth, protein transcription, autophagy, and proliferation [29,30,31].
Compared with the control group, the level of lysine was signi cantly decreased in the resveratrol-treated group. Lysine is an essential amino acid and cannot be synthesized with in the cell. Lysine plays a crucial role in the production of carnitine, which can facilitate the oxidization of fatty acids into acetyl CoA, which then enters the TCA cycle. The decrease in lysine in the resveratrol-treated group suggests a facilitated conversion to carnitine, which led to the decrease of growing in extreme condition [24].

NAD + metabolism
The levels of NAD + and NADP + were signi cantly increased in the resveratrol-treated group, indicating that the metabolism of NAD + was disturbed. NAD + was either de novo synthesized from tryptophan or through the niacin salvage pathway. For the niacin pathway, nicotinamide (NAM) was converted to nicotinamide ribotide (NMN) by nicotinamide phosphoribosyl transferase (Nampt), then NMN was converted to NAD + by the catalysis of nicotinamide mononucleotide adenylyl transferase (Nmnat) [32].
The signi cant increase in NAD + observed in the current study indicated that resveratrol enhanced the activity of Nmnat. Through increasing levels of NAD + , Nampt-mediated NAD + biosynthesis could in uence metabolic responses, stress resistance, and cellular differentiation in different cell types [33], and thereby regulate the activity of NAD + -consuming enzymes to improve cellular resistance to damage and stress, and enhance the ability of cells to survive stressful conditions [34]. In conclusion, the increase in NAD + re ects a self-repair mechanism of cells to counteract resveratrol-induced damage.

Conclusion
Resveratrol showed the strongest inhibition on the growth of Xoo among the nine resveratrol derivatives examined. The potential structure-activity relationship indicated that the double bond of resveratrol contributed to its inhibitory effects on Xoo growth, and the hydroxyls were the vital active group for the inhibitory effects. In addition, resveratrol signi cantly inhibited Xoo agella, which might cause chemotaxis and motility de ciency, and thus a decrease in virulence and pathogenicity. The 1 H NMRbased metabolomics approach was applied to study the mechanism of resveratrol against Xoo growth. A total of 30 metabolites were identi ed and assigned. Multivariate statistical analysis highlighted the altered metabolites and indicated that resveratrol could cause oxidative stress and disturb energy, purine, amino acid, and NAD + metabolism in Xoo cells, thus inhibiting Xoo growth. The 1 H-NMR-based metabolomics approach is a rapid and convenient tool for investigating the mechanism of resveratrol against Xoo growth.

Bacterial Strains and Culture Conditions
The Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99 A was kindly provided by Prof. G. L. Qian (Nanjing Agricultural University) [35]. Xoo was cultivated at 28℃ on nutrient broth agar (NA) medium in plates or in nutrient broth (NB) medium in asks. NA medium consists of 10 g of sucrose, 5 g of peptone, 3 g of beef extract, 1 g of yeast powder, and 15 g of agar powder per liter of distilled water. NB medium contained the same components but lacked agar powder.

Antibacterial investigation of compounds (1-9) against Xoo
The antibacterial activity of compounds (1-9) against Xoo was investigated as per previous research, with some modi cations [37]. Brie y, overnight cultures of Xoo (1%, v/v) were resuspended in the fresh NB medium supplemented with samples at concentration gradients in test tubes, then incubated at 28 °C and 180 rpm for 18 h. The concentrations of compounds (1-6) were 0, 2.5, 5, 10, 25, 50, and 100 μg/mL, and compounds (7-9) were 0, 50, 100, 200, 500, and 1000 μg/mL. The same amount of DMSO was used as the control. Then cultures were analyzed for antibacterial activity at 600 nm by a spectrophotometer (BioTek, Vermont, USA). Percentage inhibition was calculated as follows: where A i is the OD 600 of the cultures with compounds (1-9) and A 0 is the OD 600 of the control culture.

Bacterial growth measurement
The effects of resveratrol on the Xoo growth were measured by following the previous methods, with some modi cations [38]. Brie y, overnight culture of Xoo (1%, v/v) were resuspended in the fresh NB medium supplemented with resveratrol at different concentrations (0, 2.5, 5, 10, 25, 50, and 100 μg/mL), then incubated at 28 °C and 180 rpm. The same amount of DMSO was used as the control. The OD 600 values of the culture were measured every 2 h for up to 24 h by a microplate reader (BioTek, Vermont, USA). The Xoo growth was evaluated by plotting the values of OD 600 against time. The results were shown in Fig. S1.
Transmission electron microscope of the Xoo agella Xoo agella were detected by transmission electron microscopy (TEM) following the methods, with some modi cations [20]. Each Xoo overnight culture was diluted into fresh NB medium at 1% (v/v), then incubated at 28 °C and 180 rpm for 18 h. Resveratrol was added at 11.67 μg/mL to ensure 50% effect on growth. The suspension was deposited onto grids, then stained with 2% uranyl acetate for 30 s and dried for 10 min at room temperature, with the agella then observed by TEM (Tecnai 12, Philips, Holland).

Extraction of Xoo metabolite
Metabolites of Xoo were extracted according to our previous study, with some modi cations [10]. Each Xoo overnight culture was diluted into 30 mL of fresh NB medium at 1% (v/v) in Erlenmeyer asks, then incubated at 28 °C and 180 rpm for 18 h (OD 600 ≈ 0.82). Resveratrol was added at 11.67 μg/mL to ensure 50% effect on growth (OD 600 ≈ 0.41). To ensure bacterial equality, two Erlenmeyer asks of culture were combined to obtain one treated group sample. The same amount of DMSO was used in the control group. Fourteen biological replicates were used for the treatment and control groups, respectively. After incubation, the cell culture was chilled by brief incubation on ice. The cell pellet was obtained by centrifugation at 12,000 rpm for 15 min at 4 °C. Subsequently, the cell pellet was washed three times with phosphate-buffered saline (PBS), and then transferred to a 10-mL microtube equipped with 3.8 mL of precooled methanol/water (1/0.9, v/v), and stored at 4 °C until use. Mixtures were then extracted with a homogenizer for 5 min on ice, with 2 mL of chloroform added. After vortexing, place the mixtures on ice for 10 min, then centrifuged at 12,000 rpm for 15 min at 4 °C. Subsequently, the supernatants were transferred to new centrifugal tubes and treated under vacuum with a Speed-Vac Concentrator (Thermo SAVANT, SC110A-230) to completely remove methanol. The supernatants were stored at -80 °C overnight, and then lyophilized in a freeze drier. All samples were stored at -80 °C for further analysis.
The 1 H-NMR spectra of samples were recorded on a Bruker AVANCE III 500 MHz NMR spectrometer at 298 K. D 2 O was used for eld frequency locking, TSP was used as the chemical shift reference ( 1 H, 0.00 ppm). A transverse relaxation-edited Carr-Purcell-Meiboom-Gill (CPMG) sequence [90(τ-180-τ) nacquisition] with a total spin-echo delay (2 nτ) of 40 ms was used to suppress the signals of proteins.
The spectra were Fourier transformed after multiplying the free induction decays (FIDs) by an exponential weighting function corresponding to a line-broadening of 0.5 Hz.

Data Preprocessing and Peak Assignments
Before analysis, the 1 H-NMR spectrum was manually phased and baseline corrected using Bruker Topspin 3.0 software (Bruker GmbH, Karlsruhe, Germany) and referenced to TSP at 0.0 ppm. Subsequently, the ASCII format les were obtained by the convert of MestReNova (Version 8.0.1, Mestrelab Research SL). And then these les were read into R software (http://cran.rproject.org) for multivariate analysis. The spectra between 0.2 and 10 ppm were segmented with an average binning of 0.005 ppm. The regions in uenced by the residual resonance of water was cut off between 4.4 and 5.175 ppm. Then, all spectra were conducted probabilistic quotient normalization and mean-centered before multivariate statistical analysis.

Multivariate Data Analysis
Multivariate statistical analysis was applied to the NMR data, and included principal component analysis (PCA) and supervised orthogonal signal correction partial least-squares discriminant analysis (OSC-PLS-DA). Unsupervised PCA was rst used to reduce the dimensionality of the imported NMR data, and some new latent variables, principal components, were obtained, with such components smaller than variables before transformation. And then lter out irrelevant effects and maximize the discrimination of inter group differences by supervised OSC-PLS-DA. The OSC was applied prior to PLS-DA to lter out unrelated variables not concerning class discrimination to minimize the in uence of unrelated signals.
The quality of the OSC-PLS-DA model was evaluated by repeated two-fold cross-validation. The R 2 and Q 2 parameters re ected the prediction ability and the goodness-of-t of the constructed models. In order to the further validate the supervised model, a permutation test (2000 times) was performed [39]. And color-coded loading plots were constructed to reveal variables that contributed to group separation. The fold-change values of metabolites and their associated p-values corrected by the Benjamini and Hochberg-adjusted method were calculated and visualized in colored tables [40]. In addition, receiver operating characteristic (ROC) curves were used to verify the classi er performance of the established OSC-PLS-DA models after 200 times repeated two-fold cross-validation [10].

Univariate Statistical Analysis
Univariate analyses, including nonparametric Mann-Whitney tests and the parametric Student's t-tests [10], which were used to detect difference in crucial metabolites between groups. The fold-change values of the identi ed metabolites as well as p-values between groups were calculated. Then false discovery rate was controlled by adjusting p-values according to the Benjamini-Hochberg method when proceeding with multiple comparisons.

Statistical Analysis
All experiments were run in triplicate, and experimental results were expressed as means ± standard deviation or averages. Data were analyzed by one-way analysis of variance (ANOVA) and Duncan's multiple range test were performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA) statistical software with a signi cant P value of p < 0.05.

Availability Of Data And Materials
All data generated or analysed of this study are described in this paper.   8  nd  9 nd nd, not detected.

Abbreviations
IC 50 values were obtained by interpolation from linear regression analysis. Values are presented as mean ± SD (n = 3), and means in the same column with different lower case letters (a, b, c) are significantly different (p < 0.05).      Typical 500 MHz CPMG 1H-NMR spectra of Xoo from resveratrol-treated (red line) and control groups (black   PCA score plot of 1H-NMR data for Xoo. Two PCs explained 48.63 and 12.19% of total variances in Xoo. Ellipses represent 95% con dence interval for each group.