Skip to main content
NCBI home page
Frontiers in Microbiology logoLink to Frontiers in Microbiology
. 2018 Oct 4;9:2280. doi: 10.3389/fmicb.2018.02280

Melatonin Treatment Inhibits the Growth of Xanthomonas oryzae pv. oryzae

Xian Chen 1,†, Cheng Sun 2,†, Pedro Laborda 1, Yancun Zhao 1, Ian Palmer 3, Zheng Qing Fu 3, Jingping Qiu 4,*, Fengquan Liu 1,*
PMCID: PMC6180160  PMID: 30337911

Abstract

Xanthomonas oryzae pv. oryzae (Xoo) causes rice bacterial blight (BB), one of the most widespread and destructive diseases in rice-growing regions worldwide. Melatonin enhances pathogen resistance by inducing plant innate immunity, but the direct effect of melatonin on plant pathogenic bacteria is poorly understood. In this study, we investigated the direct effects of melatonin on Xoo. Exogenous melatonin at 200 μg/mL significantly inhibited the proliferation of Xoo and reduced the mRNA expression of five genes involved in cell division. This concentration of melatonin also inhibited the motility and biofilm formation of Xoo. Notably, melatonin was observed to alter the length of Xoo cells. To provide deeper insights into the mechanisms underlying this antibacterial activity, we examined global gene expression changes in Xoo strain PXO99 in response to the application of 200 μg/mL melatonin using RNA sequencing (RNA-Seq). A wide range of differentially expressed genes (DEGs) related to catalytic activity and metal-binding activity were downregulated in Xoo cells in response to the melatonin treatment. In addition, DEGs responsible for carbohydrate and amino acid metabolism were also downregulated. These results suggest that the inhibitory mechanism of melatonin on Xoo proliferation may involve the regulation of cell division in combination with a reduction in the concentration or activity of enzymes involved in metabolism.

Keywords: melatonin, Xanthomonas oryzae pv. oryzae, antibacterial action, growth, transcriptome

Introduction

Melatonin (N-acetyl-5-methoxytryptamine) is a highly evolutionarily conserved molecule that exists in the microbe (Manchester et al., 2015), insect (Vivien-Roels et al., 1984), animal (Menendez-Pelaez and Reiter, 1993), and plant kingdoms (Dubbels et al., 1995). In animals, melatonin was discovered in the bovine pineal gland in 1958 (Lerner et al., 1958). This indoleamine is a well-known animal neurohormone involved in numerous cellular and physiological functions, such as sleep (Garfinkel et al., 1995), circadian rhythms (Jung-Hynes et al., 2010), stem cell differentiation (Radio et al., 2006), and scavenging of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Reiter et al., 2016). In plants, melatonin was simultaneously discovered by two research groups in 1995 (Dubbels et al., 1995; Hattori et al., 1995).

Since then, melatonin has been found in a variety of plant species (Kolar and Machackova, 2005). Plant melatonin is involved in many significant plant processes, including plant growth (Chen et al., 2009; Arnao and Hernández-Ruiz, 2017) and defence against both biotic (Vielma et al., 2014; Shi et al., 2016) and abiotic stresses (Byeon and Back, 2016; Zhang et al., 2017). In microbes, exogenous melatonin acts as a biocide against some fungi and bacteria (Wang et al., 2001; Hu et al., 2017). Melatonin shows antibacterial activity against Gram-positive and Gram-negative bacteria at a low concentration in vitro (Atroshi et al., 1998; Konar et al., 2000; Ozturk et al., 2000; Karakas et al., 2013). In vivo, the exogenous application of melatonin was shown to suppress PstDC3000 propagation in Arabidopsis leaves (Lee et al., 2014). Melatonin may prevent the uptake of free iron by bacteria (Limson et al., 1998; Tekbas et al., 2008), inhibit constitutive bacterial protein secretion (Bubis and Zisapel, 1995), and reduce intracellular substrates that are important for bacterial growth (Tekbas et al., 2008). However, the mechanisms underlying these inhibitory effects of melatonin on bacteria have been little studied.

Bacterial blight (BB) of rice caused by X. oryzae pv. oryzae (Xoo) is one of the most destructive diseases in most rice-growing countries, especially those in Asia (Mansfield et al., 2012). This disease leads to leaf blight during the growing season, hindering photosynthesis and diminishing the production and quality of crops (Mahmood et al., 2006). Despite attempts to control BB by broad-spectrum breeding with high-yield cultivars, this disease remains a major constraint on rice production (Suh et al., 2013). Earlier research demonstrated that N-acetylserotonin methyltransferase (ASMT), the last enzyme involved in the synthesis of melatonin, was induced during Xoo infection (Wei et al., 2016). However, there have been no reports on the relationship between melatonin and Xoo.

In this study, we used Xoo strain PXO99 to determine whether melatonin exhibits antibacterial activity against this pathogen. In addition, the relationships between melatonin and cell division and morphology were investigated. We also used RNA sequencing (RNA-Seq) to explore how melatonin, in its role as an antibiotic, inhibits the growth of PXO99. A genome-wide expression profiling analysis clearly demonstrated that many genes involved in metabolic and transcription processes were downregulated. Our results could help to gain a better understanding of the mechanisms by which melatonin inhibits the proliferation of Gram-negative bacteria.

Materials and Methods

Bacterial Strain and Plants

Xoo strain PX099 was streaked on nutrient agar (NA) medium (beef extract, 3 g/L; yeast extract, 1 g/L; polypeptone, 5 g/L; sucrose, 10 g/L; and agar, 15 g/L) and incubated at 28°C for 2 days. Rice seedlings of the Nipponbare (Oryza sativa spp. Nipponbare) cultivar were germinated and grown in a growth chamber under an alternating 12-h light, 30°C/12-h dark, 28°C cycle with a photon flux density of 200 μmol/m2.s1. Rice leaves were inoculated with Xoo strain PXO99 (race P6) for pathogenicity tests using the leaf clipping method (Kauffman et al., 1973). Tobacco plants (Nicotiana benthamiana, Nb) were grown in a growth chamber under an alternating 12-h light/12-h dark cycle at 25°C with a photon flux density of 120 μmol/m2.s1. Tobacco leaves were inoculated with PXO99 for hypersensitive reaction (HR) assays using the needleless syringe method (Xu et al., 2015). Statistical analyses were performed using SPSS Version 20.0. The variables were analyzed using Student’s t-tests and were tested for significance at the P < 0.05 (∗), P < 0.01 (∗∗), P < 0.001 (∗∗∗), and P < 0.0001 (∗∗∗∗) levels.

Measurement of the Effect of Melatonin on Bacterial Growth

Xoo strain P6 was incubated with shaking in nutrient broth (NB) medium (NA without agar) at 28°C until an OD600 = 1.0 (early logarithmic phase) was reached. The cells were harvested and resuspended in an equal volume of sterilized ddH2O. Next, 0.5 mL of the cell suspension was added to 50 mL of NB liquid medium containing different concentrations of melatonin (0, 200, 400, or 1000 μg/mL). Methanol (MeOH) solvent without melatonin (0 μg/mL) served as a control. All cultures were shaken (200 rpm) at 28°C in the dark, and the OD600 was measured every 3 h until bacterial growth reached the stationary phase. Each experiment was performed three times, with three replicates per experiment.

Transmission Electron Microscope (TEM) Observations

The concentration of fresh bacteria in sterilized ddH2O was adjusted to OD600 = 1.0. Next, 0.5 mL of cell suspension was added into 50-mL fresh NB medium containing different concentrations of melatonin (0, 200, or 400 μg/mL). Methanol (MeOH) solvent without melatonin (0 μg/mL) served as a control. All cultures were grown at 28°C with shaking at 200 rpm for 12 h. Bacterial samples were placed on copper mesh grids with formvar membranes and negatively stained with phosphotungstic acid (2% v/v, pH = 6.7). The samples were then observed using a TEM (Hitachi H-7650) at 80 kV and photographed with a Gatan832 CCD camera (Gatan, Pleasanton, CA, United States).

Determination of Cell Motility and Biofilm Formation

Swimming motility and biofilm formation assays were performed as described previously (Tian et al., 2015). The concentration of fresh bacteria in sterilized ddH2O was adjusted to OD600 = 1.0. Next, a 5-μL aliquot of the bacterial suspension was spotted onto semi-solid NA (0.3% agar) containing different concentrations of melatonin (0, 10, 40, or 250 μg/mL). Methanol (MeOH) solvent without melatonin (0 μg/mL) served as a control. Cell motility was monitored after a 96 h incubation at 28°C in darkness. Each experiment was performed three times, with five replicates per experiment. For the biofilm formation assay, a 30-μL cell suspension was inoculated into 3 mL NB liquid medium containing different concentrations of melatonin (0, 10, 40, or 250 μg/mL). After inoculation, the cultures were incubated at 28°C for 5 days without shaking. After gently removing the cultures, the cells adhered to the culture tubes were stained with two volumes of 10% (w/v) crystal violet solution and incubated at 28°C without shaking for 1 h, followed by gentle washing with sterilized ddH2O three times, and air drying for 1 h. The crystal violet in the stained cells was dissolved using destaining buffer [40% methanol (v/v), 10% glacial acetic acid (v/v), 50% ddH2O (v/v)], and the absorbance at 595 nm (OD595) was measured using a spectrophotometer (Eppendorf, Germany). Each experiment was performed three times, with six replicates each time.

Measurement of Endogenous Melatonin

To determine the melatonin content of Xoo cells, a direct sample extraction method was used. The concentration of fresh bacteria in sterilized ddH2O was adjusted to OD600 = 1.0. Next, 0.5 mL of cell suspension was added to 50 mL of fresh NB medium containing different concentrations of melatonin (0 or 200 μg/mL). Methanol (MeOH) solvent without melatonin (0 μg/mL) served as a control. All cultures were grown at 28°C with shaking at 200 rpm for 24 h. The two cultures were adjusted to the same concentration (OD600 = 1.0) and washed with sterilized ddH2O three times. Next, the cultures were centrifuged and the pellets were suspended in 10 mL of acetonitrile buffer. The bacterial cells were homogenized using a sonicator (Scientz, Ningbo). After centrifugation, the supernatants were subjected to LC-MS as described previously (Huang and Mazza, 2011).

RNA Sequencing and Data Analysis

RNA was extracted from strain PXO99 treated with MeOH (M0) and 200 μg/mL melatonin (M200) and used for RNA sequencing. After a 21 h incubation, bacterial cultures at OD600 = 1.0 (early logarithmic phase) in broth were harvested. Total RNA was extracted from the mock and melatonin-treated samples using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) according to the procedure recommended by the manufacturer. The following steps were then completed by a commercial company (Genepioneer Biotechnologies Corporation, Nanjing, China). Three micrograms of RNA per sample was used for library construction. For direct comparisons, two libraries (M0 and M200) were prepared in the same manner and sequenced on an Illumina HiSeq Xten platform. We selected genes with a log2FC > 2 and p < 0.01 for further analysis. Differentially expressed genes (DEGs) between the melatonin-treated and mock samples were analyzed by Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment.

RNA Extraction and Quantitative Real-Time PCR Analysis

Specific primers for quantitative real-time PCR (qRT-PCR) were designed with Primer 5 (version 5) using the corresponding gene sequences from the NCBI database (Supplementary Table S1). Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) according to the procedure recommended by the manufacturer, treated with DNase I (Takara, Japan) to eliminate genomic DNA, and then converted into cDNA using a PrimeScriptTM RT Reagent Kit (Takara, Japan). Next, qRT-PCR was performed using diluted cDNA and SYBR Green PCR Master Mix (Takara, Japan) on a Quant Studio 6 Real Time PCR system (Thermo Fisher Scientific, United States). The expression data, given as quantification cycle (Cq), were collected and statistically processed using the 2-Δ(ΔCp) method. RecA was used as an internal control, and each experiment was conducted three times with three replicates.

Results and Discussion

Melatonin Inhibits Xoo Growth

Melatonin has been previously observed to play multiple roles in a wide variety of significant processes in plants, animals, and humans (Dollins et al., 1994; Guerrero and Reiter, 2002; Shi et al., 2015; Fu et al., 2017). However, the impact of melatonin on agriculturally relevant bacteria has not been explored. To this end, we assessed the bacterial growth rates of Xoo treated with methanol (mock control) and various concentrations of melatonin (Figure 1A). The OD600 value of PXO99 at 24 h pre-treated with melatonin (200 μg/mL) was approximately 1.0, only half that of the control group (Figure 1B). Thus, 200 μg/mL of melatonin effectively reduced the growth of PXO99. When the concentration was elevated, the bacterial density showed a greater decrease. No PXO99 cells survived 24 h in broth containing 1000 μg/mL melatonin or 50 μg/mL kanamycin. Melatonin represses the growth of human pathogenic bacteria at certain concentrations (Hu et al., 2017), including that of Streptococcus agalactiae at 2 μg/mL (Atroshi et al., 1998) and Saccharomyces cerevisiae at 1000 μg/mL (Konar et al., 2000). Our growth inhibition results showed that melatonin inhibited PXO99 growth in a concentration-dependent manner, and the inhibitory effect may be dose dependent. In plants, we also observed that 200 μg/mL melatonin suppressed the HR induced by PXO99 on tobacco leaves (Supplementary Figure S1). Overexpression of a melatonin-induced gene (OsMAPK12-1) resulted in plants with enhanced disease resistance against PXO99 (Xiao et al., 2017). Melatonin-induced plant resistance is mediated by MAPK signaling (Lee and Back, 2017) Thus, whether the suppression of HR caused by melatonin-induced plant innate immunity or melatonin inhibiting the proliferation of PXO99 caused the observed plant disease resistance still requires further investigation.

FIGURE 1.

FIGURE 1

Open in a new tab

Melatonin inhibits the growth of Xoo. (A) The state of PXO99 under melatonin treatment. (B) Statistical analysis of growth curves of PXO99 treated with different concentrations of melatonin (μg/mL) and cultured for 24 h.

Melatonin Reduces Xoo Swimming Motility but Increases Biofilm Formation

Swimming motility is necessary for biofilm formation and is crucial for bacterial attachment (O’Toole and Kolter, 1998; de Kerchove and Elimelech, 2008). However, the impact of melatonin on bacterial swimming motility has rarely been reported. To study the influence of melatonin on bacterial motility, the swimming motility diameter of Xoo was measured in the presence and absence of melatonin. In initial experiments, we observed that a higher concentration of melatonin disrupted the swimming ability of Xoo. Thus, melatonin was used at no more than 200 μg/mL in subsequent tests. As shown in Figure 2A, the colony diameters in plates with 10 μg/mL melatonin were decreased by more than 30% compared with that observed in the mock control. When the melatonin concentration was increased, the swimming motility diameter was further decreased. The colony diameters in plates with 200 μg/mL melatonin were reduced by more than half compared with that observed in the mock control. Thus, melatonin affected the motility of Xoo in a concentration-dependent manner. These results suggest that the inhibition of bacterial motility by melatonin may occur through increasing cell death, although further investigation of this possibility is necessary.

FIGURE 2.

FIGURE 2

Open in a new tab

Statistical analysis of the swimming motility (A) and biofilm formation of Xoo (B) treated with melatonin. ∗P < 0.05, ∗∗∗P < 0.001.

Biofilm formation plays a crucial role in plant pathogen infections (Parsek and Singh, 2003). Melatonin was reported to inhibit the biofilm formation of Candida parapsilosis and S. aureus ATCC29213 at 2.9 and 340 μg/mL, respectively (Yang et al., 2014; Romic et al., 2016). Biofilm-associated pathogens can form light-colored rings on the wall of a culture tube at the interface between air and broth. To evaluate the effect of melatonin on the attachment of Xoo, the biofilm formation of PXO99 in response to melatonin challenge was analyzed. As shown in Figure 2B, the presence of 10 μg/mL melatonin slightly increased the biofilm formation of PXO99. When the melatonin concentration was increased, the CV absorbance at OD595 showed a greater increase. The observed OD595 value from tubes containing 40 μg/mL melatonin was threefold higher than that of the mock control. However, the opposite effect was observed when melatonin was present at higher concentrations. The OD595 value in the tubes containing melatonin at 200 μg/mL was 40% lower than that of the control. Thus, the effects of melatonin on PXO99 biofilm formation did not resemble the observed effects on swimming motility or growth inhibition. When melatonin was present at a high concentration, no swimming motility, or biofilm formation was observed. Our results suggest that melatonin may induce biofilm formation in Xoo at low concentrations but inhibit its formation at high concentrations. Interestingly, we observed that both the bacterial abundance and lesion length in rice leaves infected with PXO99 treated with melatonin (200 μg/mL) was similar to that of the control group (Supplementary Figure S2). Moreover, the HR in tobacco leaves induced by PXO99 treated with melatonin (200 μg/mL) was similar to that of the control group (Supplementary Figure S3). Thus, the results indicated that melatonin may not affect Xoo pathogenicity.

Xoo Becomes Highly Enriched With Melatonin

Melatonin has been observed to easily pass through cell walls (Tekbas et al., 2008). In this study, we evaluated the content of endogenous melatonin in PXO99 cell treated with melatonin by LC-MS. The endogenous melatonin was 14.43 ng in POX99 cells that were harvested from 30 mL broth cultures after incubating for 24 h. In contrast, 156.13 ng of endogenous melatonin was detected in POX99 cells that were incubated with exogenous melatonin and harvested from 30 mL broth cultures after incubating for 24 h (Figure 3A). The results showed that melatonin can easily pass through the cell wall and become enriched in Xoo cells. The endogenous melatonin detected in the treatment group was approximately 100 times that in the control group (Figure 3B). This disruption in normal endogenous melatonin levels in Xoo may inhibit the proliferation of this bacterium. Because melatonin was detected in PXO99, we can assume that Xoo has the ability to synthesize melatonin and may have a biosynthetic pathway that is similar to that present in plants or animals. However, the function of melatonin in Xoo needs further study.

FIGURE 3.

FIGURE 3

Open in a new tab

Extraction and identification of melatonin in Xoo. (A) Chromatogram corresponding to melatonin. (a) Chromatogram corresponding to melatonin collected from PXO99 cells not treated with melatonin (Sigma). (b) Chromatogram corresponding to melatonin collected from PXO99 cells pre-treated with melatonin (Sigma). (B) Statistical analysis of melatonin (ng/cells) from PXO99 cells. UV response: 280 nm. ∗∗∗P < 0.001.

Melatonin Inhibits Xoo Cell Division

Bacterial cell division occurs by the formation of a Z-ring at the site of division (Lutkenhaus and Addinall, 1997). The dynamics of the Z-ring are regulated by the cell division-related genes ZapE and FtsZ, and the role of ZapA is to recruit ZapB to the inner face of the Z-ring (Galli and Gerdes, 2010; Marteyn et al., 2014). To investigate whether melatonin inhibits bacterial proliferation by disrupting or inhibiting cell division, the mRNA expression of nine cell division-related genes in PXO99 challenged with melatonin (200 g/mL) was analyzed by qRT-PCR. The mRNA expression of many internal genes has been reported to be affected by melatonin treatment (Sheshadri et al., 2018). In preliminary experiments, we tested the stability of two PXO99 internal candidate reference genes and observed that RecA was the more stable of the two in PXO99 cells treated with melatonin. As shown in Figure 4, four cell division-related genes (FetQ, ZapE, FetL, and FetE) were upregulated, and five (ZipA, FetB, ZapA, FetD, and FetZ) were downregulated in Xoo cells treated with melatonin compared to the control cells. Our results indicate that the melatonin treatment resulted in a decrease in Xoo cell division. Because bacterial proliferation depends on the ability of cells to divide (Pardee, 1989), the reduction in cell division could result in an inhibition of Xoo growth.

FIGURE 4.

FIGURE 4

Open in a new tab

qRT-PCR analysis of the mRNA expression of cell-division-related genes in Xoo treated with melatonin. ∗∗∗P < 0.001.

Melatonin Alters Xoo Morphology

A previous study showed that P. infestans cells treated with melatonin exhibited reduced lipid droplet production and inhibited the proliferation of P. infestans (Zhang et al., 2017). In this study, we investigated the effect of melatonin on the cellular morphology of PXO99 by making TEM observations. As shown in Figure 5, both bacterial size and shape were easily distinguished by TEM using a negative staining method. The width and length of individual PXO99 cells ranges from 0.6 to 1.0 μm and from 1.0 to 2.7 μm, respectively, and our observations agreed with these specifications (Figure 5A). By contrast, the width of PXO99 cells treated with melatonin was slightly shorter than in the control, and the length of PXO99 treated with 200 μg/mL of melatonin exhibited a significant reduction (20%) compared to the control (Figure 5B). These data indicate that the reduction in the cell length of PXO99 treated with melatonin may result from the inhibition of Xoo proliferation.

FIGURE 5.

FIGURE 5

Open in a new tab

Observations of Xoo morphology following treatment with melatonin. (A) The morphology of PXO99 cells. (a) The morphology of PXO99 cells not treated with melatonin. (b) The morphology of PXO99 cells treated with melatonin (200 μg/mL). (B) Statistical analysis of the width and length of PXO99 cells. Bar = 0.5 μm.

RNA-Seq Transcriptome Analysis of Melatonin-Treated Xoo

To further investigate the mechanism of the effects of melatonin on Xoo proliferation, total RNA from PXO99 cells that were treated or untreated with melatonin was collected and analyzed by RNA-Seq. An analysis of the gene expression changes obtained from the RNA-Seq assay showed that 138 genes had alterations in mRNA transcript levels in response to a melatonin challenge at 21 h post-treatment (Table 1), corresponding to 2.73% of the Xoo genome. Fourteen genes were upregulated, and 124 genes were downregulated, and these DEGs were characterized both by using the GO database, which provides annotation information regarding cellular components, molecular functions and biological processes, and by using the KEGG database. Of the 14 upregulated genes, four were enriched in flagellar components, four were enriched in transporter activity, and three were involved in metabolic processes Flagella are used for motility in PXO99. Whether the four flagellar genes regulated by melatonin are involved in swimming motility or biofilm formation requires further study. Transporters are well known to play a crucial role in substantial exchanges between cells and the outside environment, and the upregulated genes involved in transporter activity and metabolic processes may help PXO99 survive. Among the observed downregulated genes, a notable overrepresentation of genes associated with membrane and cellular components was observed in the cellular component category (Figure 6A). Moreover, genes encoding catalytic activity-related proteins were overrepresented in the molecular function category (Figure 6B). Consistently, a notable overrepresentation of genes associated with metabolic processes in the biological processes category was observed (Figure 6C). In the metabolic processes, 41 genes were dominant in the biological processes category (Figure 6C). Consistently, 56 and 27 genes involved in catalytic activity and metal-binding activity, respectively, were dominant in the category of molecular functions (Figure 6B). To verify the reliability of the transcriptomes, 18 randomly selected genes were analyzed using qRT-PCR. The results were consistent with the sequencing data (Figure 7). Genes related to oxidative phosphorylation, citrate cycle, protein secretion, and two component systems were downregulated in PXO99 treated with melatonin.

Table 1.

Differentially expressed genes in Xoo treated with melatonin.

No. Gene ID FCa Annotation
Transcription and translation
1 PXO_RS00600 -2.83 Transposase
2 PXO_RS24475 +1.60 Transposase
3 PXO_RS24500 +1.97 Transposase
4 PXO_RS27155 -2.48 Isrso17-ISXo8 transposase protein
5 PXO_RS08305 -1.56 AbrB family transcriptional regulator
6 PXO_RS05275 -2.34 LysR transcriptional regulator
7 PXO_RS17440 -1.84 TetR family transcriptional regulator
8 PXO_RS09765 -2.27 PhoU family transcriptional regulator
9 PXO_RS08695 -1.98 GntR family transcriptional regulator
10 PXO_RS19870 -2.38 PhoB family transcriptional regulator
11 PXO_RS20365 -1.91 AraC family transcriptional regulator
12 PXO_RS13760 -1.88 MarR family transcriptional regulator
13 PXO_RS23615 -3.61 Alkaline phosphatase D
14 PXO_RS23610 -4.78 Alkaline phosphatase
15 PXO_RS18050 -2.20 DNA-binding protein
16 PXO_RS08310 -1.56 DNA-binding protein
17 PXO_RS03000 -1.60 Disulphide-isomerase
18 PXO_RS20265 -2.92 Chlamydia polymorphic membrane family protein
19 PXO_RS07715 -1.80 MFS transporter
Carbon and protein metabolism
20 PXO_RS22365 -2.39 Acetyl-CoA acetyltransferase
21 PXO_RS12655 -2.11 Acetyl-CoA acetyltransferase
22 PXO_RS11725 -2.01 Acetyl-CoA acetyltransferase
23 PXO_RS22370 -2.39 3-Oxoadipate:succinyl-CoA transferase, partial
24 PXO_RS01865 2.03 3-Methylcrotonyl-CoA carboxylase subunit alpha
25 PXO_RS11720 -2.07 3-Hydroxyacyl-CoA dehydrogenase
26 PXO_RS12650 -2.07 3-Hydroxyacyl-CoA dehydrogenase
27 PXO_RS20635 -1.90 2-Methylisocitrate lyase
28 PXO_RS20105 -2.20 Malate dehydrogenase
29 PXO_RS02070 -2.19 NADH-dependent FMN reductase
30 PXO_RS27075 +1.86 Fumarylacetoacetate hydrolase domain protein
31 PXO_RS08315 -2.97 Bifunctional aconitate hydratase 2/2-methylisocitrate dehydratase
32 PXO_RS21360 -2.23 Chorismate mutase
33 PXO_RS20590 -4.82 Glycerophosphodiester phosphodiesterase
34 PXO_RS00340 -2.69 Aldolase
35 PXO_RS00350 -2.69 Aldolase
36 PXO_RS23355 -1.69 Cellulase
37 PXO_RS05615 -3.88 Xylanase
38 PXO_RS01665 -2.32 Xylose isomerase
39 PXO_RS01605 -4.72 Beta-1,4-xylanase
40 PXO_RS19450 +2.54 Glycosidases
41 PXO_RS19890 -2.48 Glycosyl transferase
42 PXO_RS21065 -1.75 Mannose-1-phosphate guanyltransferase
43 PXO_RS23055 +1.61 Fucose permease
44 PXO_RS19900 -2.48 UDP-2,3-diacylglucosamine hydrolase
45 PXO_RS18550 -1.50 Ubiquinol oxidase subunit II
46 PXO_RS22130 -2.61 Lipase
47 PXO_RS15625 -2.94 Peptidase S53
48 PXO_RS06470 -2.30 Peptidase C1
49 MSTRG.1600 +1.94 Pentapeptide repeats family protein
50 PXO_RS16310 -3.64 Oar protein
Signal transduction
51 PXO_RS19895 -2.48 Phosphoesterase
52 PXO_RS05010 -1.93 Phosphoanhydride phosphohydrolase
53 PXO_RS09785 -2.66 Phosphate-binding protein
54 PXO_RS09790 -2.35 Phosphate-binding protein
55 PXO_RS09775 -1.88 Phosphate transporter permease subunit PtsA
56 PXO_RS09780 -1.88 Phosphate ABC transporter permease
57 PXO_RS09770 -2.44 Phosphate ABC transporter ATP-binding protein
58 MSTRG.1547 -2.42 Putative ABC transporter phosphate-binding protein
59 PXO_RS17230 -2.63 Sulfite reductase
60 PXO_RS17465 -1.51 Serine/threonine protein kinase
61 MSTRG.60 -1.83 Serine kinase
62 MSTRG.2902 -1.51 Cytochrome D ubiquinol oxidase subunit II, partial
63 PXO_RS18555 -1.50 Cytochrome bd-type quinol oxidase, subunit 1
Cell structure
64 PXO_RS13030 +1.50 Flagellar biosynthesis
65 PXO_RS12100 +1.68 Flagellar biosynthesis
66 PXO_RS00345 -2.69 Flagellar biosynthesis protein FliP
67 PXO_RS12815 +1.63 Flagellar basal body rod protein FlgB
68 PXO_RS11885 +1.76 Flagellar basal body rod protein FlgB
Pathogenicity
69 PXO_RS19875 -2.08 Two-component system sensor protein
70 PXO_RS08560 -2.18 Type VI secretion protein
71 PXO_RS08540 -2.18 Type VI secretion protein
72 PXO_RS00365 -2.69 Type III secretion system protein
73 PXO_RS23030 -2.97 Type III secretion system effector protein
74 PXO_RS02310 -2.84 Type III secretion system effector protein
75 PXO_RS03830 -2.24 Type III secretion system effector protein
76 PXO_RS00355 -2.69 Type III secretion protein
77 PXO_RS08565 -2.18 Tal3b, TAL effector AvrBs3/PthA family
78 PXO_RS08545 -2.12 Tal3a, TAL effector AvrBs3/PthA family
79 PXO_RS00740 -4.91 Tat pathway signal protein
80 MSTRG.3198 -1.97 TonB-dependent receptor
81 PXO_RS19075 -4.14 TonB-dependent receptor
82 PXO_RS20595 -3.71 TonB-dependent receptor
83 PXO_RS20360 -1.91 TonB-dependent receptor
84 MSTRG.2688 -2.54 TonB-dependent receptor, partial
85 PXO_RS00735 -3.43 TonB-dependent receptor
86 PXO_RS17235 -2.63 TonB-dependent receptor
87 PXO_RS00360 -2.69 Hypersensitivity response secretion protein hrcV
88 PXO_RS00320 -3.97 HrpE
89 PXO_RS00370 -3.09 HPr kinase
90 PXO_RS00330 -2.69 HPr kinase
91 PXO_RS00325 -2.66 HPr kinase
92 PXO_RS00375 -2.63 HPr kinase
93 PXO_RS00315 -3.13 protein HpaB
94 PXO_RS00335 -2.69 Protein HpaA
95 PXO_RS06005 -2.01 Putative sulfotransferase required for AvrXa21 activity ST (raxST)
96 PXO_RS25330 -1.75 Xanthomonadin biosynthesis protein
97 PXO_RS21615 -2.04 Adhesin
98 PXO_RS22720 +1.51 Ankyrin
99 PXO_RS22730 +1.58 Hemolysin D
Stress response
100 PXO_RS05270 -2.49 Protocatechuate degradation protein
101 PXO_RS05265 -2.24 Protocatechuate 3,4-dioxygenase subunit beta
102 PXO_RS22360 -1.98 Protocatechuate 3,4-dioxygenase subunit beta
103 PXO_RS22355 -1.75 Protocatechuate 3,4-dioxygenase subunit alpha
104 PXO_RS13750 -2.23 Multidrug transporter
105 PXO_RS08690 -1.65 Multidrug transporter
106 PXO_RS13755 -1.88 Multidrug RND transporter
107 emrB -1.68 Multidrug resistance protein B
108 PXO_RS27000 -2.18 Multidrug resistance efflux pump
109 PXO_RS09795 -2.78 Porin
Function unknown
110 PXO_RS25045 -3.49 Hypothetical protein
111 PXO_RS24050 -2.67 Hypothetical protein
112 PXO_RS26160 +1.56 Hypothetical protein
113 PXO_RS25955 +1.52 Hypothetical protein
114 PXO_RS00415 -4.29 Hypothetical protein
115 PXO_RS19070 -4.12 Hypothetical protein
116 PXO_RS00605 -3.49 Hypothetical protein
117 PXO_RS00425 -3.28 Hypothetical protein
118 PXO_RS00580 -3.09 Hypothetical protein
119 PXO_RS22960 -3.07 Hypothetical protein
120 PXO_RS20695 -3.03 Hypothetical protein
121 PXO_RS01615 -2.32 Hypothetical protein
122 PXO_RS20585 -2.18 Hypothetical protein
123 PXO_RS00380 -2.10 Hypothetical protein
124 PXO_RS01795 -1.95 Hypothetical protein
125 PXO_RS03845 -1.84 Hypothetical protein
126 PXO_RS09760 -1.73 Hypothetical protein
127 PXO_RS03505 -1.64 Hypothetical protein
128 PXO_RS02075 -1.50 Hypothetical protein
129 PXO_RS22735 +1.62 Hypothetical protein
130 PXO_RS17460 -2.95 Hypothetical protein
131 PXO_RS17455 -2.95 Hypothetical protein
132 PXO_RS06000 -2.85 Hypothetical protein
133 PXO_RS21520 -2.36 Hypothetical protein
134 PXO_RS17445 -2.33 Hypothetical protein
135 PXO_RS06495 -2.32 Hypothetical protein
136 PXO_RS01735 -1.60 Hypothetical protein
137 PXO_RS25040 -2.83 Hypothetical protein
138 PXO_RS25015 -2.62 Hypothetical protein
Open in a new tab

aThe symbol “+” indicates upregulated genes in Xoo cells treated with melatonin, while the symbol “-” indicates downregulated genes in Xoo cells treated with melatonin.

FIGURE 6.

FIGURE 6

Open in a new tab

Classification of differentially expressed genes by gene ontology (GO) enrichment and cellular mapping. (A) Number of differentially expressed genes. (B) Cellular component classifications. (C) Molecular function. (D) Biological process.

FIGURE 7.

FIGURE 7

Open in a new tab

Validation of 18 differentially expressed genes at the mRNA level by qRT-PCR.

Melatonin Regulates Xoo Metabolism

Metabolism is an important characteristic of bacteria, and melatonin was reported to significantly reduce the expression mRNA of genes associated with metabolism in microbes (Zhang et al., 2017). In this study, we observed that genes involved in carbohydrate and amino acid metabolism were enriched (Figure 8). The best carbon and nitrogen sources for Xoo growth are sucrose and glutamate (Singh, 2016). Interestingly, we observed that many genes involved in sucrose and glutamate metabolism were downregulated in PXO99 when challenged with melatonin.

FIGURE 8.

FIGURE 8

Open in a new tab

Classification of differential genes in the metabolism in PXO99 under melatonin treatment.

Bacterial pathogens are known to require iron for replication and infection (Schaible and Kaufmann, 2004; White and Yang, 2009; Skaar, 2010). Xoo requires ferrous sulfate for optimal proliferation and modulates copper redistribution in rice during infection (Yuan et al., 2010). According to the RNA-Seq results, the mRNA expression of the transporter TonB, which is responsible for iron absorption (Yue et al., 2003), was downregulated in PXO99 challenged with melatonin (Table 1). Other DEGs related to metal binding were also downregulated (Figure 6B). The mRNA levels of genes that encode metal-ion binding and cation binding proteins were previously observed to be downregulated in rice leaves treated with melatonin, similar to our results (Liang et al., 2015). The content of endogenous melatonin in melatonin-treated PXO99 was approximately 100 times that of the control group (Figure 3). Interestingly, 18 genes involved in xenobiotic metabolism were downregulated (Figure 8). Melatonin has a strong ability to bind copper and iron(III) (Limson et al., 1998). Thus, we speculate that melatonin can cause a free iron deficiency in bacterial cells and inhibit growth through the metal-binding activity of melatonin or by reducing the concentration and activity of metal-binding enzymes.

Phosphate is most commonly used in energy metabolic processes and serves as a buffering agent in cells (Lardy and Wellman, 1952). In this study, the mRNA expression of the transcription factor PhoU, which function in environmental phosphate (Pi) sensing and transportation (Muda et al., 1992), was reduced in PXO99 cells treated with melatonin (Table 1). In addition, DEGs encoding proteins located on the cell membrane related to phosphate transporter and phosphate binding proteins involved in energy metabolism were both downregulated (Figure 8). In humans, melatonin inhibits cancer cell growth by preventing the cell membrane from assimilating linoleic fatty acid (Blask et al., 1999). The results of this study indicated that the inhibitory mechanism of melatonin on bacterial growth may be related to reducing phosphate levels, although a detailed characterization of these mechanisms will require further investigation.

Conclusion

In this study, we investigated the potential effects of melatonin on X. oryzae pv. oryzae. Our data showed that melatonin can cross the cell wall and become enriched in Xoo cells, inhibiting the cell division and proliferation of this bacterium. Importantly, melatonin altered the cell structure and reduced the motility and attachment ability of Xoo cells. The results of the transcriptome analysis suggest that the inhibitory effects of melatonin on Xoo proliferation may occur through (i) decreasing cell division and (ii) reducing the concentration and activity of enzymes involved in metabolism. This work provides new insights into the inhibitory effect of melatonin on bacterial growth and gene expression.

Author Contributions

FL, ZF, and XC designed the study. XC and CS performed the experiments. XC, CS, and YZ analyzed the data. XC, CS, and PL drafted the manuscript. FL, IP, JQ, and ZF reviewed and edited the manuscript.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The authors thank Professor Jianping CHEN (Ningbo University, China) for his language editing of this manuscript.

Funding. This research was supported by grants from the National Natural Science Foundation of China (31571974), the Special Fund for Agro-scientific Research in the Public Interest (201303015), the Natural Science Foundation of Jiangsu Province of China (BK20170606), and National Key R&D program of China (2017YFD0200900).

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2018.02280/full#supplementary-material

FIGURE S1

The hypersensitive reaction triggered by Xoo on tobacco leaves pretreated with melatonin. The tobacco leaves were inoculated with different concentrations of Xoo (ddH2O, OD600 = 0.2, 0.4, 0.8). (a) Tobacco leaves without the melatonin treatment and (b) tobacco leaves pretreated with 200 μg/mL melatonin for 12 h.

Click here for additional data file. (1.2MB, TIF)
FIGURE S2

Effects of exogenous melatonin on the pathogenicity of PXO99 and the hypersensitive response. (A) Phenotype of rice leaves inoculated with PXO99 treated with melatonin (200 μg/mL). (B) Lesion length on rice leaves inoculated with PXO99 treated with melatonin (200 μg/mL). (C) Bacterial population in rice leaves inoculated with PXO99 treated with melatonin (200 μg/mL). Bar = 2 cm.

Click here for additional data file. (372.7KB, TIF)
FIGURE S3

The hypersensitive reaction of tobacco leaves triggered by Xoo pretreated with melatonin. Xoo cells were pretreated with melatonin (200 μg/mL) for 24 h. (A) The tobacco leaves inoculated with ddH2O, Xoo without melatonin treatment, Xoo pretreated with melatonin (200 μg/mL). (B) The mRNA expression of HrpD6 in response to the melatonin treatment.

Click here for additional data file. (1MB, TIF)
Click here for additional data file. (1MB, doc)

References

  1. Arnao M. B., Hernández-Ruiz J. (2017). Growth activity, rooting capacity, and tropism: three auxinic precepts fulfilled by melatonin. Acta Physiol. Plant 39:127 10.1007/s11738-017-2428-3 [DOI] [Google Scholar]
  2. Atroshi F., Rizzo A., Westermarck T., Ali-vehmas T. (1998). Effects of tamoxifen, melatonin, coenzyme Q10, and L-carnitine supplementation on bacterial growth in the presence of mycotoxins. Pharmacol. Res. 38 289–295. 10.1006/phrs.1998.0363 [DOI] [PubMed] [Google Scholar]
  3. Blask D. E., Sauer L. A., Dauchy R. T., Holowachuk E. W., Ruhoff M. S., Kopff H. S. (1999). Melatonin inhibition of cancer growth in vivo involves suppression of tumor fatty acid metabolism via melatonin receptor-mediated signal transduction events. Cancer Res. 59 4693–4701. [PubMed] [Google Scholar]
  4. Bubis M., Zisapel N. (1995). Facilitation and inhibition of G-protein regulated protein secretion by melatonin. Neurochem. Int. 27 177–183. 10.1016/0197-0186(95)00027-6 [DOI] [PubMed] [Google Scholar]
  5. Byeon Y., Back K. (2016). Low melatonin production by suppression of either serotonin N-acetyltransferase or N-acetylserotonin methyltransferase in rice causes seedling growth retardation with yield penalty, abiotic stress susceptibility, and enhanced coleoptile growth under anoxic conditions. J. Pineal Res. 60 348–359. 10.1111/jpi.12317 [DOI] [PubMed] [Google Scholar]
  6. Chen Q., Qi W. B., Reiter R. J., Wei W., Wang B. M. (2009). Exogenously applied melatonin stimulates root growth and raises endogenous indoleacetic acid in roots of etiolated seedlings of Brassica juncea. J. Plant Physiol. 166 324–328. 10.1016/j.jplph.2008.06.002 [DOI] [PubMed] [Google Scholar]
  7. de Kerchove A. J., Elimelech M. (2008). Bacterial swimming motility enhances cell deposition and surface coverage. Environ. Sci. Technol. 42 4371–4377. 10.1021/es703028u [DOI] [PubMed] [Google Scholar]
  8. Dollins A. B., Zhdanova I. V., Wurtman R. J., Lynch H. J., Deng M. H. (1994). Effect of inducing nocturnal serum melatonin concentrations in daytime on sleep, mood, body temperature, and performance. Proc. Natl. Acad. Sci. U.S.A. 91 1824–1828. 10.1073/pnas.91.5.1824 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dubbels R., Reiter R. J., Klenke E., Goebel A., Schnakenberg E., Ehlers C., et al. (1995). Melatonin in edible plants identified by radioimmunoassay and by high performance liquid chromatography-mass spectrometry. J. Pineal Res. 18 28–31. 10.1111/j.1600-079X.1995.tb00136.x [DOI] [PubMed] [Google Scholar]
  10. Fu J., Wu Y., Miao Y., Xu Y., Zhao E., Wang J., et al. (2017). Improved cold tolerance in elymus nutans by exogenous application of melatonin may involve ABA-dependent and ABA-independent pathways. Sci. Rep. 7:39865. 10.1038/srep39865 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Galli E., Gerdes K. (2010). Spatial resolution of two bacterial cell division proteins: ZapA recruits ZapB to the inner face of the Z-ring. Mol. Microbiol. 76 1514–1526. 10.1111/j.1365-2958.2010.07183.x [DOI] [PubMed] [Google Scholar]
  12. Garfinkel D., Laudon M., Nof D., Zisapel N. (1995). Improvement of sleep quality in elderly people by controlled-release melatonin. Lancet 346 541–544. 10.1016/S0140-6736(95)91382-3 [DOI] [PubMed] [Google Scholar]
  13. Guerrero J. M., Reiter R. J. (2002). Melatonin-immune system relationships. Curr. Top. Med. Chem. 2 167–179. 10.2174/1568026023394335 [DOI] [PubMed] [Google Scholar]
  14. Hattori A., Migitaka H., Iigo M., Itoh M., Yamamoto K., Ohtani-Kaneko R., et al. (1995). Identification of melatonin in plants and its effects on plasma melatonin levels and binding to melatonin receptors in vertebrates. Biochem. Mol. Biol. Int. 35 627–634. [PubMed] [Google Scholar]
  15. Hu W., Deng C., Ma Z., Wang D., Fan C., Li T., et al. (2017). Utilizing melatonin to combat bacterial infections and septic injury. Br. J. Pharmacol. 174 754–768. 10.1111/bph.13751 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Huang X., Mazza G. (2011). Application of LC and LC-MS to the analysis of melatonin and serotonin in edible plants. Crit. Rev. Food Sci. Nutr. 51 269–284. 10.1080/10408398.2010.529193 [DOI] [PubMed] [Google Scholar]
  17. Jung-Hynes B., Reiter R. J., Ahmad N. (2010). Sirtuins, melatonin and circadian rhythms: building a bridge between aging and cancer. J. Pineal Res. 48 9–19. 10.1111/j.1600-079X.2009.00729.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Karakas F., Karakas A., Turker A., Yildirim A. (2013). Antibacterial and antitumor activities of melatonin hormone. Spatula DD 3 33–39. 10.5455/spatula.20130422052142 [DOI] [Google Scholar]
  19. Kauffman H. E., Reddy A. P. K., Hsieh S. P. Y., Merca S. D. (1973). Improved technique for evaluating resistance of rice varieties to Xanthomonas oryzae. Plant Dis. Rep. 57 537–541. [Google Scholar]
  20. Kolar J., Machackova I. (2005). Melatonin in higher plants: occurrence and possible functions. J. Pineal Res. 39 333–341. 10.1111/j.1600-079X.2005.00276.x [DOI] [PubMed] [Google Scholar]
  21. Konar V. V., Yilmaz O., Ozturk A. I., Kirba gbreve S., Arslan M. (2000). Antimicrobial and biological effects of bomphos and phomphos on bacterial and yeast cells. Bioorg. Chem. 28 214–225. 10.1006/bioo.2000.1173 [DOI] [PubMed] [Google Scholar]
  22. Lardy H. A., Wellman H. (1952). Oxidative phosphorylations; role of inorganic phosphate and acceptor systems in control of metabolic rates. J. Biol. Chem. 195 215–224. [PubMed] [Google Scholar]
  23. Lee H. Y., Back K. (2017). Melatonin is required for H2 O2 - and NO-mediated defense signaling through MAPKKK3 and OXI1 in Arabidopsis thaliana. J. Pineal Res. 62:e12379. 10.1111/jpi.12379 [DOI] [PubMed] [Google Scholar]
  24. Lee H. Y., Byeon Y., Back K. (2014). Melatonin as a signal molecule triggering defense responses against pathogen attack in arabidopsis and tobacco. J. Pineal Res. 57 262–268. 10.1111/jpi.12165 [DOI] [PubMed] [Google Scholar]
  25. Lerner A. B., Case J. D., Takahashi Y., Lee T. H., Mori W. (1958). Isolation of melatonin, the pineal gland factor that lightens melanocyteS1. J. Am. Chem. Soc. 80 2587–2587. 10.1021/ja01543a060 [DOI] [Google Scholar]
  26. Liang C., Zheng G., Li W., Wang Y., Hu B., Wang H., et al. (2015). Melatonin delays leaf senescence and enhances salt stress tolerance in rice. J. Pineal Res. 59 91–101. 10.1111/jpi.12243 [DOI] [PubMed] [Google Scholar]
  27. Limson J., Nyokong T., Daya S. (1998). The interaction of melatonin and its precursors with aluminium, cadmium, copper, iron, lead, and zinc: an adsorptive voltammetric study. J. Pineal Res. 24 15–21. 10.1111/j.1600-079X.1998.tb00361.x [DOI] [PubMed] [Google Scholar]
  28. Lutkenhaus J., Addinall S. G. (1997). Bacterial cell division and the Z ring. Annu. Rev. Biochem. 66 93–116. 10.1146/annurev.biochem.66.1.93 [DOI] [PubMed] [Google Scholar]
  29. Mahmood T., Jan A., Kakishima M., Komatsu S. (2006). Proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf blades. Proteomics 6 6053–6065. 10.1002/pmic.200600470 [DOI] [PubMed] [Google Scholar]
  30. Manchester L. C., Coto-Montes A., Boga J. A., Andersen L. P., Zhou Z., Galano A., et al. (2015). Melatonin: an ancient molecule that makes oxygen metabolically tolerable. J. Pineal Res. 59 403–419. 10.1111/jpi.12267 [DOI] [PubMed] [Google Scholar]
  31. Mansfield J., Genin S., Magori S., Citovsky V., Sriariyanum M., Ronald P., et al. (2012). Top 10 plant pathogenic bacteria in molecular plant pathology. Mol. Plant Pathol. 13 614–629. 10.1111/j.1364-3703.2012.00804.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Marteyn B. S., Karimova G., Fenton A. K., Gazi A. D., West N., Touqui L., et al. (2014). ZapE is a novel cell division protein interacting with FtsZ and modulating the Z-ring dynamics. mBio 5:e00022-e14. 10.1128/mBio.00022-14 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Menendez-Pelaez A., Reiter R. J. (1993). Distribution of melatonin in mammalian tissues: the relative importance of nuclear versus cytosolic localization. J. Pineal Res. 15 59–69. 10.1111/j.1600-079X.1993.tb00511.x [DOI] [PubMed] [Google Scholar]
  34. Muda M., Rao N. N., Torriani A. (1992). Role of PhoU in phosphate transport and alkaline phosphatase regulation. J. Bacteriol. 174 8057–8064. 10.1128/jb.174.24.8057-8064.1992 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. O’Toole G. A., Kolter R. (1998). Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 30 295–304. 10.1046/j.1365-2958.1998.01062.x [DOI] [PubMed] [Google Scholar]
  36. Ozturk A. I., Yilmaz O., Kirbag S., Arslan M. (2000). Antimicrobial and biological effects of ipemphos and amphos on bacterial and yeast strains. Cell Biochem. Funct. 18 117–126. [DOI] [PubMed] [Google Scholar]
  37. Pardee A. B. (1989). G1 events and regulation of cell proliferation. Science 246 603–608. 10.1126/science.2683075 [DOI] [PubMed] [Google Scholar]
  38. Parsek M. R., Singh P. K. (2003). Bacterial biofilms: an emerging link to disease pathogenesis. Annu. Rev. Microbiol. 57 677–701. 10.1146/annurev.micro.57.030502.090720 [DOI] [PubMed] [Google Scholar]
  39. Radio N. M., Doctor J. S., Witt-Enderby P. A. (2006). Melatonin enhances alkaline phosphatase activity in differentiating human adult mesenchymal stem cells grown in osteogenic medium via MT2 melatonin receptors and the MEK/ERK (1/2) signaling cascade. J. Pineal Res. 40 332–342. 10.1111/j.1600-079X.2006.00318.x [DOI] [PubMed] [Google Scholar]
  40. Reiter R. J., Mayo J. C., Tan D. X., Sainz R. M., Alatorre-Jimenez M., Qin L. (2016). Melatonin as an antioxidant: under promises but over delivers. J. Pineal Res. 61 253–278. 10.1111/jpi.12360 [DOI] [PubMed] [Google Scholar]
  41. Romic M. D., Klaric M. S., Lovric J., Pepic I., Cetina-Cizmek B., Filipovic-Grcic J., et al. (2016). Melatonin-loaded chitosan/pluronic(R) F127 microspheres as in situ forming hydrogel: an innovative antimicrobial wound dressing. Eur. J. Pharm. Biopharm. 107 67–79. 10.1016/j.ejpb.2016.06.013 [DOI] [PubMed] [Google Scholar]
  42. Schaible U. E., Kaufmann S. H. (2004). Iron and microbial infection. Nat. Rev. Microbiol. 2 946–953. 10.1038/nrmicro1046 [DOI] [PubMed] [Google Scholar]
  43. Sheshadri S. A., Nishanth M. J., Yamine V., Simon B. (2018). Effect of Melatonin on the stability and expression of reference genes in Catharanthus roseus. Sci. Rep. 8 2222. 10.1038/s41598-018-20474-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Shi H., Chen Y., Tan D. X., Reiter R. J., Chan Z., He C. (2015). Melatonin induces nitric oxide and the potential mechanisms relate to innate immunity against bacterial pathogen infection in Arabidopsis. J. Pineal Res. 59 102–108. 10.1111/jpi.12244 [DOI] [PubMed] [Google Scholar]
  45. Shi H., Wei Y., He C. (2016). Melatonin-induced CBF/DREB1s are essential for diurnal change of disease resistance and CCA1 expression in Arabidopsis. Plant Physiol. Biochem. 100 150–155. 10.1016/j.plaphy.2016.01.018 [DOI] [PubMed] [Google Scholar]
  46. Singh B. (2016). Sugar, amino acids and nitrate as nitrogenous source of fertilization influence growth of Xanthomonas oryzae pv. oryzae and Aspergillus species in hypersensitive response development of tobacco crop. Int. J. Plant Reprod. Biol. 8 88–93. 10.14787/ijprb [DOI] [Google Scholar]
  47. Skaar E. P. (2010). The battle for iron between bacterial pathogens and their vertebrate hosts. PLoS Pathog. 6:e1000949. 10.1371/journal.ppat.1000949 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Suh J. P., Jeung J. U., Noh T. H., Cho Y. C., Park S. H., Park H. S., et al. (2013). Development of breeding lines with three pyramided resistance genes that confer broad-spectrum bacterial blight resistance and their molecular analysis in rice. Rice 6:5. 10.1186/1939-8433-6-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tekbas O. F., Ogur R., Korkmaz A., Kilic A., Reiter R. J. (2008). Melatonin as an antibiotic: new insights into the actions of this ubiquitous molecule. J. Pineal Res. 44 222–226. 10.1111/j.1600-079X.2007.00516.x [DOI] [PubMed] [Google Scholar]
  50. Tian Y., Zhao Y., Wu X., Liu F., Hu B., Walcott R. R. (2015). The type VI protein secretion system contributes to biofilm formation and seed-to-seedling transmission of acidovorax citrulli on melon. Mol. Plant Pathol. 16 38–47. 10.1111/mpp.12159 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Vielma J. R., Bonilla E., Chacin-Bonilla L., Mora M., Medina-Leendertz S., Bravo Y. (2014). Effects of melatonin on oxidative stress, and resistance to bacterial, parasitic, and viral infections: a review. Acta Trop. 137 31–38. 10.1016/j.actatropica.2014.04.021 [DOI] [PubMed] [Google Scholar]
  52. Vivien-Roels B., Pevet P., Beck O., Fevre-Montange M. (1984). Identification of melatonin in the compound eyes of an insect, the locust (Locusta migratoria), by radioimmunoassay and gas chromatography-mass spectrometry. Neurosci. Lett. 49 153–157. 10.1016/0304-3940(84)90152-6 [DOI] [PubMed] [Google Scholar]
  53. Wang H. X., Liu F., Ng T. B. (2001). Examination of pineal indoles and 6-methoxy-2-benzoxazolinone for antioxidant and antimicrobial effects. Comp. Biochem. Physiol. C. Toxicol Pharmacol. 130 379–388. 10.1016/S1532-0456(01)00264-2 [DOI] [PubMed] [Google Scholar]
  54. Wei Y., Zeng H., Hu W., Chen L., He C., Shi H. (2016). Comparative transcriptional profiling of melatonin synthesis and catabolic genes indicates the possible role of melatonin in developmental and stress responses in rice. Front. Plant Sci. 7:676. 10.3389/fpls.2016.00676 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. White F. F., Yang B. (2009). Host and pathogen factors controlling the rice-Xanthomonas oryzae interaction. Plant Physiol. 150 1677–1686. 10.1104/pp.109.139360 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Xiao X., Zhijuan T., Xiuqiong L., Yuhui H., Boling L., Wenfang X., et al. (2017). Overexpressing OsMAPK12-1 inhibits plant growth and enhances resistance to bacterial disease in rice. Funct. Plant Biol. 44 694–704. 10.1071/fp16397 [DOI] [PubMed] [Google Scholar]
  57. Xu H., Zhao Y., Qian G., Liu F. (2015). XocR, a LuxR solo required for virulence in Xanthomonas oryzae pv. oryzicola. Front. Cell Infect. Microbiol. 5:37. 10.3389/fcimb.2015.00037 [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Yang H. P., Tsang P. C., Tsang P. W. (2014). Melatonin inhibits biofilm formation in Candida parapsilosis. J. Mycol. Med. 24 360–361. 10.1016/j.mycmed.2014.05.003 [DOI] [PubMed] [Google Scholar]
  59. Yuan M., Chu Z., Li X., Xu C., Wang S. (2010). The bacterial pathogen Xanthomonas oryzae overcomes rice defenses by regulating host copper redistribution. Plant Cell 22 3164–3176. 10.1105/tpc.110.078022 [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yue W. W., Grizot S., Buchanan S. K. (2003). Structural evidence for iron-free citrate and ferric citrate binding to the TonB-dependent outer membrane transporter FecA. J. Mol. Biol. 332 353–368. 10.1016/S0022-2836(03)00855-6 [DOI] [PubMed] [Google Scholar]
  61. Zhang S., Zheng X., Reiter R. J., Feng S., Wang Y., Liu S., et al. (2017). Melatonin attenuates potato late blight by disrupting cell growth, stress tolerance, fungicide susceptibility and homeostasis of gene expression in phytophthora infestans. Front. Plant Sci. 8:1993. 10.3389/fpls.2017.01993 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

FIGURE S1

The hypersensitive reaction triggered by Xoo on tobacco leaves pretreated with melatonin. The tobacco leaves were inoculated with different concentrations of Xoo (ddH2O, OD600 = 0.2, 0.4, 0.8). (a) Tobacco leaves without the melatonin treatment and (b) tobacco leaves pretreated with 200 μg/mL melatonin for 12 h.

Click here for additional data file. (1.2MB, TIF)
FIGURE S2

Effects of exogenous melatonin on the pathogenicity of PXO99 and the hypersensitive response. (A) Phenotype of rice leaves inoculated with PXO99 treated with melatonin (200 μg/mL). (B) Lesion length on rice leaves inoculated with PXO99 treated with melatonin (200 μg/mL). (C) Bacterial population in rice leaves inoculated with PXO99 treated with melatonin (200 μg/mL). Bar = 2 cm.

Click here for additional data file. (372.7KB, TIF)
FIGURE S3

The hypersensitive reaction of tobacco leaves triggered by Xoo pretreated with melatonin. Xoo cells were pretreated with melatonin (200 μg/mL) for 24 h. (A) The tobacco leaves inoculated with ddH2O, Xoo without melatonin treatment, Xoo pretreated with melatonin (200 μg/mL). (B) The mRNA expression of HrpD6 in response to the melatonin treatment.

Click here for additional data file. (1MB, TIF)
Click here for additional data file. (1MB, doc)

Articles from Frontiers in Microbiology are provided here courtesy of Frontiers Media SA

RESOURCES