Introduction
============

Melatonin (*N*-acetyl-5-methoxytryptamine) is a highly evolutionarily conserved molecule that exists in the microbe ([@B30]), insect ([@B52]), animal ([@B33]), and plant kingdoms ([@B9]). In animals, melatonin was discovered in the bovine pineal gland in 1958 ([@B25]). This indoleamine is a well-known animal neurohormone involved in numerous cellular and physiological functions, such as sleep ([@B12]), circadian rhythms ([@B17]), stem cell differentiation ([@B39]), and scavenging of reactive oxygen species (ROS) and reactive nitrogen species (RNS) ([@B40]). In plants, melatonin was simultaneously discovered by two research groups in 1995 ([@B9]; [@B14]).

Since then, melatonin has been found in a variety of plant species ([@B20]). Plant melatonin is involved in many significant plant processes, including plant growth ([@B6]; [@B1]) and defence against both biotic ([@B51]; [@B45]) and abiotic stresses ([@B5]; [@B61]). In microbes, exogenous melatonin acts as a biocide against some fungi and bacteria ([@B53]; [@B15]). Melatonin shows antibacterial activity against Gram-positive and Gram-negative bacteria at a low concentration *in vitro* ([@B2]; [@B21]; [@B36]; [@B18]). *In vivo*, the exogenous application of melatonin was shown to suppress *Pst*DC3000 propagation in Arabidopsis leaves ([@B24]). Melatonin may prevent the uptake of free iron by bacteria ([@B27]; [@B49]), inhibit constitutive bacterial protein secretion ([@B4]), and reduce intracellular substrates that are important for bacterial growth ([@B49]). 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 ([@B31]). This disease leads to leaf blight during the growing season, hindering photosynthesis and diminishing the production and quality of crops ([@B29]). Despite attempts to control BB by broad-spectrum breeding with high-yield cultivars, this disease remains a major constraint on rice production ([@B48]). Earlier research demonstrated that *N*-acetylserotonin methyltransferase (ASMT), the last enzyme involved in the synthesis of melatonin, was induced during *Xoo* infection ([@B54]). 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 {#s1}
=====================

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/m^2^.s^1^. Rice leaves were inoculated with *Xoo* strain PXO99 (race P6) for pathogenicity tests using the leaf clipping method ([@B19]). 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/m^2^.s^[1](#fn01){ref-type="fn"}^. Tobacco leaves were inoculated with PXO99 for hypersensitive reaction (HR) assays using the needleless syringe method ([@B57]). 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 OD~600~ = 1.0 (early logarithmic phase) was reached. The cells were harvested and resuspended in an equal volume of sterilized ddH~2~O. 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 OD~600~ 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 ddH~2~O was adjusted to OD~600~ = 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 ([@B50]). The concentration of fresh bacteria in sterilized ddH~2~O was adjusted to OD~600~ = 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 ddH~2~O three times, and air drying for 1 h. The crystal violet in the stained cells was dissolved using destaining buffer \[40% methanol (v/v), 10% glacial acetic acid (v/v), 50% ddH~2~O (v/v)\], and the absorbance at 595 nm (OD~595~) 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 ddH~2~O was adjusted to OD~600~ = 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 (OD~600~ = 1.0) and washed with sterilized ddH~2~O 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 ([@B16]).

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 OD~600~ = 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 log~2~FC \> 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](#TS1){ref-type="supplementary-material"}**). 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 PrimeScript^TM^ 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(ΔC~p~)^ 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 ([@B8]; [@B13]; [@B44]; [@B10]). 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](#F1){ref-type="fig"}**). The OD~600~ 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](#F1){ref-type="fig"}**). 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 ([@B15]), including that of *Streptococcus agalactiae* at 2 μg/mL ([@B2]) and *Saccharomyces cerevisiae* at 1000 μg/mL ([@B21]). 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](#FS1){ref-type="supplementary-material"}**). Overexpression of a melatonin-induced gene (*OsMAPK12-1*) resulted in plants with enhanced disease resistance against PXO99 ([@B56]). Melatonin-induced plant resistance is mediated by MAPK signaling ([@B23]) 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.

![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.](fmicb-09-02280-g001){#F1}

Melatonin Reduces *Xoo* Swimming Motility but Increases Biofilm Formation
-------------------------------------------------------------------------

Swimming motility is necessary for biofilm formation and is crucial for bacterial attachment ([@B35]; [@B7]). 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](#F2){ref-type="fig"}**, 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.

![Statistical analysis of the swimming motility **(A)** and biofilm formation of *Xoo* **(B)** treated with melatonin. ^∗^*P* \< 0.05, ^∗∗∗^*P* \< 0.001.](fmicb-09-02280-g002){#F2}

###### 

Differentially expressed genes in *Xoo* treated with melatonin.

  No.                                 Gene ID       FC^a^   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
                                                            

a

The symbol "+" indicates upregulated genes in

Xoo

cells treated with melatonin, while the symbol "-" indicates downregulated genes in

Xoo

cells treated with melatonin.

Biofilm formation plays a crucial role in plant pathogen infections ([@B38]). Melatonin was reported to inhibit the biofilm formation of *Candida parapsilosis* and *S. aureus* ATCC29213 at 2.9 and 340 μg/mL, respectively ([@B58]; [@B41]). 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](#F2){ref-type="fig"}**, the presence of 10 μg/mL melatonin slightly increased the biofilm formation of PXO99. When the melatonin concentration was increased, the CV absorbance at OD~595~ showed a greater increase. The observed OD~595~ 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 OD~595~ 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](#FS2){ref-type="supplementary-material"}**). 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](#FS3){ref-type="supplementary-material"}**). 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 ([@B49]). 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](#F3){ref-type="fig"}**). 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](#F3){ref-type="fig"}**). 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.

![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.](fmicb-09-02280-g003){#F3}

Melatonin Inhibits *Xoo* Cell Division
--------------------------------------

Bacterial cell division occurs by the formation of a Z-ring at the site of division ([@B28]). 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 ([@B11]; [@B32]). 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 ([@B43]). 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](#F4){ref-type="fig"}**, 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 ([@B37]), the reduction in cell division could result in an inhibition of *Xoo* growth.

![qRT-PCR analysis of the mRNA expression of cell-division-related genes in *Xoo* treated with melatonin. ^∗∗∗^*P* \< 0.001.](fmicb-09-02280-g004){#F4}

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* ([@B61]). In this study, we investigated the effect of melatonin on the cellular morphology of PXO99 by making TEM observations. As shown in **Figure [5](#F5){ref-type="fig"}**, 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](#F5){ref-type="fig"}**). 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](#F5){ref-type="fig"}**). These data indicate that the reduction in the cell length of PXO99 treated with melatonin may result from the inhibition of *Xoo* proliferation.

![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.](fmicb-09-02280-g005){#F5}

RNA-Seq Transcriptome Analysis of Melatonin-Treated *Xoo*
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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](#T1){ref-type="table"}**), 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](#F6){ref-type="fig"}**). Moreover, genes encoding catalytic activity-related proteins were overrepresented in the molecular function category (**Figure [6B](#F6){ref-type="fig"}**). Consistently, a notable overrepresentation of genes associated with metabolic processes in the biological processes category was observed (**Figure [6C](#F6){ref-type="fig"}**). In the metabolic processes, 41 genes were dominant in the biological processes category (**Figure [6C](#F6){ref-type="fig"}**). Consistently, 56 and 27 genes involved in catalytic activity and metal-binding activity, respectively, were dominant in the category of molecular functions (**Figure [6B](#F6){ref-type="fig"}**). 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](#F7){ref-type="fig"}**). Genes related to oxidative phosphorylation, citrate cycle, protein secretion, and two component systems were downregulated in PXO99 treated with melatonin.

![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.](fmicb-09-02280-g006){#F6}

![Validation of 18 differentially expressed genes at the mRNA level by qRT-PCR.](fmicb-09-02280-g007){#F7}

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 ([@B61]). In this study, we observed that genes involved in carbohydrate and amino acid metabolism were enriched (**Figure [8](#F8){ref-type="fig"}**). The best carbon and nitrogen sources for *Xoo* growth are sucrose and glutamate ([@B46]). Interestingly, we observed that many genes involved in sucrose and glutamate metabolism were downregulated in PXO99 when challenged with melatonin.

![Classification of differential genes in the metabolism in PXO99 under melatonin treatment.](fmicb-09-02280-g008){#F8}

Bacterial pathogens are known to require iron for replication and infection ([@B42]; [@B55]; [@B47]). *Xoo* requires ferrous sulfate for optimal proliferation and modulates copper redistribution in rice during infection ([@B59]). According to the RNA-Seq results, the mRNA expression of the transporter TonB, which is responsible for iron absorption ([@B60]), was downregulated in PXO99 challenged with melatonin (**Table [1](#T1){ref-type="table"}**). Other DEGs related to metal binding were also downregulated (**Figure [6B](#F6){ref-type="fig"}**). 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 ([@B26]). The content of endogenous melatonin in melatonin-treated PXO99 was approximately 100 times that of the control group (**Figure [3](#F3){ref-type="fig"}**). Interestingly, 18 genes involved in xenobiotic metabolism were downregulated (**Figure [8](#F8){ref-type="fig"}**). Melatonin has a strong ability to bind copper and iron(III) ([@B27]). 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 ([@B22]). In this study, the mRNA expression of the transcription factor PhoU, which function in environmental phosphate (Pi) sensing and transportation ([@B34]), was reduced in PXO99 cells treated with melatonin (**Table [1](#T1){ref-type="table"}**). 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](#F8){ref-type="fig"}**). In humans, melatonin inhibits cancer cell growth by preventing the cell membrane from assimilating linoleic fatty acid ([@B3]). 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.

**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).

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

<http://www.genome.jp/kegg/>

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>

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The hypersensitive reaction triggered by *Xoo* on tobacco leaves pretreated with melatonin. The tobacco leaves were inoculated with different concentrations of *Xoo* (ddH~2~O, OD~600~ = 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.

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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.

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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 ddH~2~O, *Xoo* without melatonin treatment, *Xoo* pretreated with melatonin (200 μg/mL). **(B)** The mRNA expression of HrpD6 in response to the melatonin treatment.

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[^1]: Edited by: Jack Wong, The Chinese University of Hong Kong, Hong Kong

[^2]: Reviewed by: Maximino Manzanera, Universidad de Granada, Spain; Shahper Nazeer Khan, Aligarh Muslim University, India

[^3]: ^†^These authors have contributed equally to this work
