Molecular Cloning and Characterization of a Serotonin N-Acetyltransferase Gene, xoSNAT3, from Xanthomonas oryzae pv. oryzae

Rice bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the top ten bacterial plant diseases worldwide. Serotonin N-acetyltransferase (SNAT) is one of the key rate-limiting enzymes in melatonin (MT) biosynthesis. However, its function in pathogenic bacteria remains unclear. In this study, a Xoo SNAT protein (xoSNAT3) that showed 27.39% homology with sheep SNAT was identified from a collection of 24 members of GCN5-related N-acetyltransferase (GNAT) superfamily in Xoo. This xoSNAT3 could be induced by MT. In tobacco-based transient expression system, xoSNAT3 was found localized on mitochondria. In vitro studies indicated that xoSNAT3 showed the optima enzymatic activity at 50 °C. The recombinant enzyme showed Km and Vmax values of 709.98 μM and 2.21 nmol/min/mg protein, respectively. Mutant △xoSNAT3 showed greater impaired MT biosynthesis than the wild-type strain. Additionally, △xoSNAT3 showed 14.06% less virulence and 26.07% less biofilm formation. Collectively, our results indicated that xoSNAT3 services as a SNAT involved in MT biosynthesis and pathogenicity in Xoo.


Introduction
Melatonin (N-acetyl-5-methoxytryptamine, MT) is an evolutionary ancient and ubiquitous molecule present in animals, plants and microorganisms [1]. MT was first identified in animals in 1958 [2]. Thirty-six years later, MT was also identified in both plant and bacteria kingdoms [3,4]. As a well-known hormone, MT has been thoroughly studied in humans [5]. MT is synthesized and secreted by pineal gland in vertebrates, and its changes are related to the circadian rhythm, aging and immunity [6][7][8][9]. MT is a super-radical scavenger, not only because of the its structural characteristics, but also because MT has the ability to regulate the activity of antioxidant enzymes [10], and its antioxidant effects have been reported to be a beneficial treatment for COVID-19 patients [11]. MT also plays a key role as a master regulator in plant growth and stress responses [7]. It has been speculated that MT may act as a hormone in plants, and the first MT receptor has been recently identified in Arabidopsis [8]. Despite mentioned advances in animals and plants, the number of studies regarding the biological roles of MT in bacteria are limited and, thus, its function in bacteria is still poorly understood.
Systematic analysis of MT metabolic network may help to achieve a better understanding of its biological function. In vertebrates, MT is synthesized from tryptophan following four consecutive enzymatic steps [9]. Firstly, tryptophan is converted to 5-hydroxy tryptophan by the tryptophan hydroxylase (TPH). Then, this intermediate is converted to serotonin via an aromatic amino acid decarboxylase (AADC). Next, serotonin is further converted to N-acetylserotonin by the serotonin N-acetyltransferase (SNAT). Finally, N-acetylserotonin is converted to MT by the caffeic acid O-methyltransferase (COMT), also known as N-acetylserotonin O-methyltransferase (ASMT). Previous studies have reported that SNAT plays a critical role in the regulation of MT's biosynthesis [10,11]. In many vertebrates, SNAT is localized in mitochondria and is believed to be the key rate-limiting enzyme in MT's biosynthesis [12].
In plants, the biosynthesis of MT is quite different from that in vertebrates. Firstly, tryptophan is converted to tryptamine by the tryptophan decarboxylase (TDC) [13]. In the second step, this intermediate is converted to serotonin by the tryptophan 5-hydroxylase (T5H). Next, serotonin is O-methylated to 5-methoxytryptamine (5-MT) by ASMT/COMT. Finally, 5-MT is N-acetylated to MT by SNAT [14]. SNAT was reported to be localized in the chloroplasts, while T5H was located in the endoplasmic reticulum (ER), and TPH, ASMT/COMT and AADC/TDC were distributed in the cytoplasm [14,15]. TDC is also one of the key rate-limiting enzyme in plant MT's biosynthesis [16]. Overexpression of OsTDC significantly enhances the content of MT in rice plants [17]. Although the biosynthetic steps of MT's biosynthesis in vertebrates and plants have been thoroughly studied, research on the bacterial MT biosynthetic pathway are very limited. The changes in MT's concentration have been correlated to the bacterial circadian rhythm [18]. The synthetic intermediates of serotonin, 5-hydroxytryptophan and N-acetylserotonin were identified in Pseudomonas fluorescens RG11, an endophytic bacterium isolated from grapevine roots [19]. In the cyanobacterium Synechocystis sp. PCC 6803, SNAT was reported to be a thermo-tolerant enzyme with 56% homology compared to rice OsSNAT [20].
In previous studies, our group reported that exogenous MT had strong antibacterial activity against Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight (BB) [21]. Xoo is considered one of the top ten most dangerous bacterial pathogen in plant pathology [22]. In this study, endogenous MT was detected in Xoo, and the predicted xoSNAT3 was proved to be involved in MT biosynthesis. Additionally, the role of xoSNAT3 in the Xoo pathogenicity was characterized.

General Information and Bacterial Strain
All the chemicals and reagents were used as received from commercial suppliers without further purification. Xoo strain PXO99 (P6) was grown in nutrient broth (NB) medium (5 g peptone, 1 g yeast extract, 3 g beef extract paste and 10 g sucrose, pH 7.0-7.2, in 1 L of distilled water) or NA agar medium (NB broth with 17 g agar) [23]. E. coli DH5α and BL21 were grown in lysogeny broth (LB) medium (5 g of yeast extract, 10 g of triptone and 10 g of sodium chloride, pH 7.0-7.2, in 1 L of distilled water) or LB solid medium (LB liquid medium with 20 g agar).

Identification of MT in Xoo
To detect MT in Xoo, the bacterial metabolites were extracted using the water-ethyl acetate method. Firstly, bacterial cells in NB medium were harvested and washed with ddH 2 O, and the cells was adjusted to OD 600 = 0.8~1.0. Then, 2 mL of standard bacterial was added to fresh 200 mL NB broth medium and all cultures were incubated at 28 • C and 200 rpm. The cells were harvested after 24 h and washed with ddH 2 O twice. Then, the cells were re-suspended in 5 mL ddH 2 O. Next, suspension cells were mixed with an equal volume of ethyl acetate, and mixed vigorously on a vortex for 2 min. The mixture was centrifuged at 10,000 rpm for 3 min, and the supernatant (organic phase) was transfer to a new 15 mL tube and dried with a nitrogen evaporator with nitrogen flow. The dried samples were reconstituted in 200 µL methanol, and shaken in a vortex at 2000 rpm for 1 min. After centrifuging at 10,000 rpm for 3 min, the mixture was filtered through a 0.1 µm membrane and analyzed using a liquid chromatography instrument with a Time of Flight Mass Spectrometer (TOF, Applied Biosystems Sciex, triple TOF 5600). The metabolites were identified using a Liquid Chromatograph Mass Spectrometer (LC-MS/MS, Agilent Poroshell 120 EC-C18, 2.7 µm, 3.0 mm × 100 mm). Pure ddH 2 O containing 0.1% trifluoracetic acid and methanol were used as the A and B mobile phases, respectively. The gradient program was performed at a flow rate of 0.3 mL/min. All samples were performed in triplicate, and the results were presented as the mean ± standard deviation.

Phylogenetic Analysis of xoSNAT3 from Xoo Strain PXO99
In order to predict a Xoo gene encoding a serotonin N-acetyltransferase enzyme, the nine full-length proteins belonging to the GNAT family (Table 1), which were downloaded from the NCBI website (https://www.ncbi.nlm.nih.gov/ (accessed on 8 October 2022)), were performed to find SNAT homologs in Xoo strain PXO99A protein database (https: //www.ncbi.nlm.nih.gov (accessed on 8 October 2022)). Amino acid sequences of xoSNAT1 (WP_027703221.1), xoSNAT2 (WP_011258206.1) and xoSNAT3 (WP_027703680.1) were download from NCBI. Multiple amino acid sequences alignments were performed using the MEGA 6 software (Version 6.0), based on the cloning of SNATs from rice plant, sheep and other species. The phylogenetic analysis was performed with MEGA 6 by using the neighbor joining method with 1000 bootstrap replicates.

Cloning of xoSNAT3 from Xoo Strain PXO99
To obtain the genomic DNA from Xoo, 0.5 mL of bacterial solution (OD 600 = 1.0) was added to fresh 50 mL NB broth medium, and the culture was shaken at 28 • C and 200 rpm for 24 h. Then, 6 mL of Xoo was centrifuged at 10,000 rpm for 3 min, and the genomic DNA was isolated by using the TIANamp Bacteria DNA Kit (Cat. NO. DP302-02, Beijing, China). To amplify the xoSNAT3 from PXO99, polymerase chain reaction (PCR) primers were designed based on the annotated sequence information of putative N-acyl-transferase (GenBank accession no. PXO_RS04110) using the forward primer 5 -ATGTCCACCACAGCCCT-3 , and reverse primer 5 -CAAAGGAGCCGCGCCGGCA-3 . The resulting PCR product was purified using the DNA Clean-Up kit (Cat. NO. CW2301M, CWBIO, Taizhou, China), and cloned into the PJET1.2 vector (Cat. NO. K1231, Thermo Fisher, Waltham, MA, USA). The map of PJET1.2 vector was available in addgene (https: //www.addgene.org/124439/ (accessed on 8 October 2022)). The insert fragment was verified via sequencing analysis by Tsingke Biotechnology Co., Ltd. (Nanjing, China).

Subcellular Localization of xoSNAT3 in Tobacco Leaves
The full-length coding regions of xoSNAT3 in PJET1.2 vector were amplified using restriction enzymes for the respective forward (BamHI) 5 -CGGGATCCATGTCCACCACAGCCCT-3 , and reverse primers (KpnI) 5 -GGGGGTACCAGGAGCCGCGCCGGCA-3 . After digestion, the PCR product with restriction enzyme site were fused in frame with GFP in the PCV-eGFP-N 1 vector [24]. Then, the sequenced plasmid was introduced into Agrobacterium tumefaciens EHA105, and then transferred to 3-weeks-old Nicotiana benthamiana leaves, using the transient expression method. Briefly, EHA105 cells with PCV-eGFP-xoSNAT3 were harvested at 16 h post-inoculation. The harvest cells were then re-suspended in soaking solution [containing 10 mM MgCl 2 , 10 mM MES (pH 5.6), 200 µM acetylsyringone (As)], and the bacterial mixture was adjusted to OD 600 = 0.8~1.0. EHA105 with PCV-eGFP-N 1 vector was used as mock control. Finally, the mixtures were slowly penetrated into the back of N. benthamiana leaves. These tobacco plants were culture for 2 days at 25 • C. The GFP signal was excited at 395 nm and observed at 450-490 nm with LSM 710 (ZEISS, Jena, Germany) for confocal imaging.

Measurement of xoSNAT3 Enzymatic Activity In Vitro
The full-length coding regions of xoSNAT3 in PJET1.2 was amplified using restriction enzymes with the respective the forward (BamHI) 5 -CGGGATCCTCCACCACAGCCCT-3 , and reverse primers (NotI) 5 -GCGGCCGCAGGAGCCGCGCCGGCA-3 . After digestion, the PCR product with restriction enzyme site was fused in frame with GST in pGEX-6p-1 vector. Then, the sequenced plasmid was introduced into E. coli BL21. Then, the expression of GST-xoSNAT3 fusion protein was induced by 0.4 mM IPTG and examined by western blot. Next, GST-xoSNAT3 fusion protein was purified by GSTsep glutathione agarose resin, according to the manufacturer's instructions. Then, the concentration of purified protein was measured using the Bradford method.
For the enzymatic activity assay, GST-xoSNAT3 and GST were added to 100 µL reaction buffer (0.5 mM serotonin, 0.5 mM acetyl-CoA and 100 mM potassium phosphate, pH = 8.8), respectively. The mixture was incubated at 30 • C for 30 min, and the reaction was stopped by adding 25 µL methanol. Next, a 20 µL aliquot of the reaction samples was analyzed by HPLC to determine the substrates. To investigate the substrate affinity (K m ) and V max values of xoSNAT3, different concentrations of xoSNAT3 were added to the above-described buffer, serotonin and N-acetylserotonin were both determined by LC-MS/MS. The mixture were filtered through a 0.1 µm membrane and analyzed by liquid chromatography in tandem with a Time of Flight Mass Spectrometer (TOF, Applied Biosystems Sciex, triple TOF 5600). The metabolites were identified using a Liquid Chromatograph Mass Spectrometer (LC-MS/MS, Agilent Poroshell 120 EC-C18, 2.7 µm, 3.0 mm × 100 mm). Pure ddH 2 O containing 0.1% trifluoracetic acid and methanol were used as the A and B mobile phases, respectively. The compounds were eluted at a flow rate of 0.3 mL/min. All samples were performed in triplicate, and the results were presented as the mean ± standard deviation.

Generation of xoSNAT3 Deletion Mutant
In Xoo, xoSNAT3 (nucleotides 901096-901596) is located 251 bp downstream of PXO_ RS04100 and 6 bp upstream of PXO_RS04115. To generate a nonpolar mutation in xoSNAT, fragments located 509 bp upstream and 510 bp downstream of xoSNAT were amplified from Xoo genomic DNA using the primers snat up-F (BamHI): 5 -CGGGATCCTCACGCACG ACGACGTGCG-3 , snat up-R: 5 -CATGCGAACTCCAAAGGAGGGTGGACATCACCG CATGA-3 , snat D-F: 5 -T CATGCGGTGATGTCCACCCTCCTTTGGAGTTCGCATG-3 , and snat D-R (XbaI): 5 -GCTCTAGACACCTGCGTACGGGTACGC-3 , respectively. Then, the upstream and downstream PCR productions were combined together using the PCR fusion method. Briefly, upstream (1 µL) and downstream (1 µL), 2× PCR Mix (10 µL, AS102-01, TRAN) and ddH 2 O (7.6 µL) were mixed together in a 200 µL PCR tube. The PCR mixture were performed on a S1000 PCR system (Bio-rad, Hercules, CA, USA), and PCR conditions were as follows: 98 • C for 3 min, then 10 cycles of 98 • C for 5 s, 60 • C for 10 s, 68 • C for 20 s, with a final 68 • C for 2 min. The resulting PCR product was cloned into vector pMDT18-T, and verified by DNA sequencing. The construct was digested with BamHI and XbaI to release the cloned fragment, and then ligated into the vector pK18mobsacB. The recombinant plasmid was introduced into Xoo by electroporation. Briefly, 10 µL recombinant plasmid mixed with 100 µL Xoo cells, and placed on ice for 10 min. Then, this mixture was transfer into electric shock cup, and electric shock by electric shock apparatus (Bio-rad, MicroPulser, Hercules, CA, USA). A mutant of lacking xoSNAT3 was initially obtained in NAN medium, and then on the NA medium following the procedure. Firstly, the transconjugants were selected on NA plates with kanamycin (Km, 50 µg/mL) in the absent of sucrose. Then, positive colonies were selected on NA plates with 10% (w/v) sucrose to generate the in-frame deletion mutant via allelic homologous recombination. The resulting mutant, containing the xoSNAT in-fame deletion, was further confirmed by PCR and quantitative RT-PCR. Three of the confirmed mutants, named xoSNAT3, were selected for further study.

Complementation of the xoSNAT3 Mutant
For the complementation of xoSNAT3, a 489 bp DNA of the entire coding region of snat was amplified from the Xoo strain PXO99 genomic DNA using the forward (EcoRI): 5 -CGGAATTCATGTCCACCACAGCCCTCCCT-3 and reverse primers (BamHI): 5 -CGGGAT CCCAAAGGAGCCGCGCCGGCAGG-3 . The PCR product was cloned into the vector pMDT18-T and verified by sequencing. The construct was digested with EcoRI and BamHI to release the PCR-fragment, and then ligated into the vector pUFR034. The complemented plasmid pUFR-sant was transformed into the competent cell of xoSNAT by electroporation. Finally, one representative complemented strain, named xoSNAT, was selected on NA plates with Km (50 µg/mL), verified by PCR and used in the subsequent studies.

RNA Extraction and Quantitative RT-PCR Analysis
Special primers for qRT-PCR of xoSNAT were designed using PRIMER 5 (v. 5) software: forward primer 5 -GTCCACCACAGCCCTCCCT-3, and reverse primer 5 -GTAGCTTTGCCGTCCAGTTCC-3. The housekeeping gene 16S rRNA (forward primer: 5 -CAAGGCGCTGCTGATGGTCG-3; reverse primer: 5 -CGTCGCAAGATCGCGTTGACC-3 ), and recA (forward primer: 5 -AATGCCTTGAAGTTCTACGCC-3, reverse primer: 5 -TTCGGTCACGACCTGCTTG-3 ) were used as internal controls. To obtain the RNA from Xoo, xoSNAT and its complementation, 0.5 mL of bacterial solutions (OD 600 = 1.0) were added to fresh 50 mL NB and the culture was shaken at 28 • C and 200 rpm for 24 h. Then, 3-5 mL of bacterial suspension was centrifuged at 10,000 rpm for 3 min, and the total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was treated with DNase I (Takara) to eliminate genomic DNA and the first strand cDNA was synthesized by using the cDNA Synthesis kit (Takara, Bio, Nojihigashi, Kusatsu, Japan) following the manufacturer's instructions. The qRT-PCR was performed on a QuantStudio 6 Real-Time PCR system (Applied Biosystems, Waltham, MA, USA), using diluted cDNA and the SYBR Green PCR Master Mix (Takara, Nojihigashi, Kusatsu, Japan). The expression data, in terms of quantification cycles threshold (Ct), were collected and statistically processed using the 2 − Ct method. Each experiment was conducted three times with three replicates. The variables were analyzed via Student's t test and tested for significance at p < 0.05, p < 0.01, p < 0.001 and p < 0.0001 levels.

Analysis of Pathogenicity and Biofilm Formation
Pathogenicity assays were performed in a glasshouse. Xoo, xoSNAT and its complementation were cultivated in NB medium at 28 • C and 200 rpm for 24 h. The cells were collected and resuspended in sterilized ddH 2 O to OD 600 = 1.0. For pathogenicity assay, the strains were inoculated into the leaves of 4-to 5-week-old rice plants (variety Nipponbare, which is susceptible to BB), using the leaf clipping method [25]. The lesion length was measured at 7 days post inoculation. Thirty leaves were treated with each strain. The experiments were conducted three times. The variables were analyzed via Student's t test and tested for significance at p < 0.05, p < 0.01, p < 0.001 and p < 0.0001 levels.
Biofilm formation was measured as previously described [21,26]. Briefly, Xoo, xoSNAT and its complementation were cultivated in NB medium with shaking at 28 • C and 200 rpm for 24 h. The cells were collected and resuspended in sterilized ddH 2 O to OD 600 = 1.0. Next, 30 µL cell suspension was inoculated into 3 mL NB liquid broth medium and placed in darkness without shaking for 5 days. After gently removing the cultures, the cells adhered to the tubes were stained with crystal violet method and the absorbance of OD 595 was measured using a spectrophotometer (Eppendorf Biophotometer Plus, Hamburg, Germany). Each experiment was performed three times, with seven replicates each time. The variables were analyzed via Student's t test and tested for significance at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 levels.

Phylogenetic Analysis of xoSNAT3
It is well known that finding synthetic genes is the most difficult and crucial step in synthetic biology. The most common and effective way to identify proteins in different species is by homologous comparison. In order to identify the SNAT-ecnoding genes from Xoo, seven full-length proteins belonging to GNAT superfamily (pfam00583) ( Table 1), were submitted to BLAST search in Xoo protein database (https://www.ncbi.nlm.nih.gov/ (accessed on 8 October 2022)). Fortunately, three potential xoSNATs proteins were found in Xoo (Figure 3A,B). Phylogenetic analysis showed that Sheep SNAT and OsSNATs are divided into two different subfamilies ( Figure 3B). The protein sequences of the above three Xoo SNATs and SNATs protein from other species were presented in Figure 3A. Protein annotation showed that xoSNAT1 (WP_027703221.1), xoSNAT2 (WP_011258206.1), xoSNAT3 (WP_027703680.1) and Sheep SNAT (gi|11387097) belonged to the GNAT superfamily. Compared with xoSNAT1 and xoSNAT2, xoSNAT3 showed a closer evolutionary relationship compared with theSNATs from animal and plant kingdoms ( Figure 3B). Sheep SNAT showed 32.36% (xoSNAT1), 25.15% (xoSNAT2) and 22.96% (xoSNAT3) homology, respectively. Homology analysis revealed that xoSNAT3 shared high identity with SaSNAT1 (48.56%), while the homology was only 31.09% and 27.37% when comparing SaSNAT1 with xoSNAT1 and xoSNAT2, respectively. This suggested that xoSNAT3 may encode a SNAT protein.

Enzymatic Activity Analysis of xoSNAT3
The full length of xoSNAT3 contained 501 bp, thus encoding a protein with 166 amino acids. Protein molecular weight of xoSNAT3 and GST is 17.95 and 26 KDa, respectively. It was predicted that xoSNAT3 contained an acetyltransferase (GNAT) domain by Pfam and Uniprot database scanning. To confirm xoSNAT3 activity, its frame was fused into pGEX-6P-1 vector with N-terminal glutathione-S-transferase-tagging. After 12-16 h post IPTG induction, the purified SNAT3-GST fusion proteins were analyzed by SDS-PAGE and western blot, and its enzymatic activity and kinetic parameters were analyzed in vitro. As shown in Figure 4, GST-xoSNAT3 (43.95 KDa) and GST (26 KDa) proteins were successfully induced by IPTG, and further confirmed by western blotting (Figure 4A-C). The optima enzymatic activity of GST-xoSNAT3 was detected at 50 • C ( Figure 4D). The values for K m and V max using serotonin as the subtrate were 709.98 µM and 2.21 nmol/min/mg protein, respectively ( Figure 4E).

Enzymatic Activity Analysis of xoSNAT3
The full length of xoSNAT3 contained 501 bp, thus encoding a protein with 166 amino acids. Protein molecular weight of xoSNAT3 and GST is 17.95 and 26 KDa, respectively. It was predicted that xoSNAT3 contained an acetyltransferase (GNAT) domain by Pfam and Uniprot database scanning. To confirm xoSNAT3 activity, its frame was fused into pGEX-6P-1 vector with N-terminal glutathione-S-transferase-tagging. After 12-16 h post IPTG induction, the purified SNAT3-GST fusion proteins were analyzed by SDS-PAGE and western blot, and its enzymatic activity and kinetic parameters were analyzed in vitro. As shown in Figure 4, GST-xoSNAT3 (43.95 KDa) and GST (26 KDa) proteins were successfully induced by IPTG, and further confirmed by western blotting ( Figure  4A-C). The optima enzymatic activity of GST-xoSNAT3 was detected at 50 °C ( Figure 4D). The values for Km and Vmax using serotonin as the subtrate were 709.98 μM and 2.21 nmol/min/mg protein, respectively ( Figure 4E).

Location of xoSNAT3 in Tobacco Cells
Protein functions are related to its subcellular localization [27]. It was predicted that xoSNAT3 had 60.9% likely to be located in mitochondria by PSORT II database scanning. To further investigate the subcellular localization of xoSNAT3, the recombinant 35: GFP plasmid with full length ORF (Opening Reading Frame) of xoSNAT3 was transferred into tobacco leaves, and the GFP fluorescent signals were detected by confocal microscope. As shown in Figure 5, the GFP signal in the absence of xoSNAT3 was detected both in the plasma membrane and in the cell nucleus of tobacco tissue. The GFP fluorescent signals of xoSNAT3-GFP was observed only in mitochondria, similar with PSORTII result.

Role of xoSNAT3 in MT Biosynthesis in Xoo
To investigate whether xoSNAT3 is induced in response to MT treatment, qRT-PCR was performed to analyze its transcription. The qRT-PCR analysis revealed that the mRNA level of xoSNAT3 was higher after treatment with 1000 ng/mL MT, while its expression was down-regulated when treating with 100 ng/mL MT ( Figure 6A). The results suggested that xoSNAT3 can be induced when applying high MT concentrations.
To further examine the role of xoSNAT3 in MT biosynthesis, the knock-out strain xoSNAT3 was constructed by a two-step homologous recombination approach. The mRNA of xoSNAT3 was only detected in the wild-type strain PXO99 and in the complemented xoSNAT3(xoSNAT3) strain, but not in xoSNAT3 ( Figure 6B). To further investigate the relationship between MT biosynthesis and xoSNAT3 gene expression, the concentration of MT was measured by LC-MS/MS in all strains. MT's concentrations in the wild-type strain PXO99 and complemented strain xoSNAT3(xoSNAT3) were 68.75 ng/50 mL and 99.25 ng/50 mL, respectively, whereas MT production level in xoSNAT3 mutant strain was only 3.75 ng/50 mL, which is consistent with the qRT-PCR analysis ( Figure 6C). These results suggested that xoSNAT3 is critical for MT biosynthesis in Xoo. xoSNAT3 had 60.9% likely to be located in mitochondria by PSORT II database scanning. To further investigate the subcellular localization of xoSNAT3, the recombinant 35: GFP plasmid with full length ORF (Opening Reading Frame) of xoSNAT3 was transferred into tobacco leaves, and the GFP fluorescent signals were detected by confocal microscope. As shown in Figure 5, the GFP signal in the absence of xoSNAT3 was detected both in the plasma membrane and in the cell nucleus of tobacco tissue. The GFP fluorescent signals of xoSNAT3-GFP was observed only in mitochondria, similar with PSORTII result.

Role of xoSNAT3 in MT Biosynthesis in Xoo
To investigate whether xoSNAT3 is induced in response to MT treatment, qRT-PCR was performed to analyze its transcription. The qRT-PCR analysis revealed that the mRNA level of xoSNAT3 was higher after treatment with 1000 ng/mL MT, while its expression was down-regulated when treating with 100 ng/mL MT ( Figure 6A). The results suggested that xoSNAT3 can be induced when applying high MT concentrations.
To further examine the role of xoSNAT3 in MT biosynthesis, the knock-out strain △xoSNAT3 was constructed by a two-step homologous recombination approach. The mRNA of xoSNAT3 was only detected in the wild-type strain PXO99 and in the complemented △xoSNAT3(xoSNAT3) strain, but not in △xoSNAT3 ( Figure 6B). To further investigate the relationship between MT biosynthesis and xoSNAT3 gene expression, the concentration of MT was measured by LC-MS/MS in all strains. MT's concentrations in the wild-type strain PXO99 and complemented strain △xoSNAT3(xoSNAT3) were 68.75 ng/50 mL and 99.25 ng/50 mL, respectively, whereas MT production level in △xoSNAT3 mutant strain was only 3.75 ng/50 mL, which is consistent with the qRT-PCR analysis ( Figure 6C). These results suggested that xoSNAT3 is critical for MT biosynthesis in Xoo. The control experiment was carried out using Xoo strain PXO99 without MT treatment. PXO99 refers to the wild-type strain. pFUR034 refers to Xoo strain carrying mentioned blank plasmid as a mock control. Asterisks indicate statistically significant differences determined using Student's t-test (* p < 0.05; *** p < 0.001; **** p < 0.0001).

Role of xoSNAT3 in Xoo Pathogenicity
To examine the role of xoSNAT3 in Xoo virulence, wild-type, △xoSNAT3 mutant and complemented strain △xoSNAT3(xoSNAT3) were inoculated in rice Nipponbare leaves. As shown in Figure 7A, the lesion length produced by the wild-type and the complemented strain △xoSNAT3(xoSNAT3) were 7.97 and 7.48 cm, respectively. In contrast, the lesion length when using △xoSNAT3 mutant was only 6.69 cm. Thus, xoSNAT3 seems to be involved in the pathogenicity of Xoo.
Biofilm formation is crucial for bacterial colonization and virulence [26]. Biofilm-associated pathogens can form light-colored rings on the wall of a culture tube at the interface between air and broth. To further evaluate the effect of xoSNAT3 on Xoo virulence, the biofilm formation of wild-type strain, △xoSNAT3 mutant strain and complemented strain △xoSNAT3(xoSNAT3) were analyzed. As shown in Figure 7B, the crystal violet (CV) observation at OD595 of wild-type strain and complemented strain △xoSNAT3(xoSNAT3) were 0.45 and 0.47, respectively, while the CV value of △xoSNAT3 mutant strain was only 0.33. This indicated that xoSNAT3 is involved in biofilm formation in Xoo. PXO99 refers to the wild-type strain. pFUR034 refers to Xoo strain carrying mentioned blank plasmid as a mock control. Asterisks indicate statistically significant differences determined using Student's t-test (* p < 0.05; *** p < 0.001; **** p < 0.0001).

Role of xoSNAT3 in Xoo Pathogenicity
To examine the role of xoSNAT3 in Xoo virulence, wild-type, xoSNAT3 mutant and complemented strain xoSNAT3(xoSNAT3) were inoculated in rice Nipponbare leaves. As shown in Figure 7A, the lesion length produced by the wild-type and the complemented strain xoSNAT3(xoSNAT3) were 7.97 and 7.48 cm, respectively. In contrast, the lesion length when using xoSNAT3 mutant was only 6.69 cm. Thus, xoSNAT3 seems to be involved in the pathogenicity of Xoo.

Discussion
Previous reports have indicated that MT provides a main m/z peak in positive mode at 233 [28][29][30], which is in agreement with the mass spectra obtained in this study. This confirms for the first time that MT is produced by Xoo, and probably by other Xanthomonas strains. The presence of MT in Xoo raises numerous questions regarding the potential role of this molecule in plant-Xanthomonas interactions. It is possible that Xoo-secreted MT is able to modify plant metabolism and may be related to Xoo-infection process. The role of MT in Xoo quorum sensing is also an important area that must be addressed in future studies.
MT synthetic routes in animals and plants have been well studied, while its synthetic pathway in microorganisms is still not well understood [9]. Tryptophan is the unique precursor of MT. Thus, the identification of intermediates may help to explore the bacterial MT biosynthetic pathway. In animals, the first step of MT synthesis pathway consists of the conversion of tryptophan into 5-hydroxytryptamine [9,31]. However, the first step of MT biosynthesis in the plant kingdom is the conversion of tryptophan into trypamine [14]. No study regarding the MT biosynthetic pathway in bacteria was reported until date. It has been reported to investigate MT intermediates by the N15 labelled tryptophan mediated isotopic tracer Method [24]. Interestingly, 5-hydroxytryptamine, instead of tryptamine, was detected in Pseudomonas fluorescens RG11, an endophytic bacterium isolated from grapevine roots. The 5-hydroxytryptamine is only consist in animal MT synthesis pathway. Therefore, the author speculates that the synthetic pathway of MT in RG11 may be similar to that reported in animals. Interestingly, tryptamine, 5-hydroxytryptamine, Nacetylserotonin and MT were also identified in Xoo by LC-MS/MS. Moreover, the highest content of tryptamine was 639 ng in 50 mL NA at 24 h post inoculation, while the lowest content of serotonin was 63 ng in 50 mL. It is well known that tryptamine is an intermediate of plant MT biosynthesis, whereas 5-hydroxytryptophan is involved in animal MT synthesis [14,18]. Surprisingly, tryptamine and 5-hydroxytryptophan were both detected Biofilm formation is crucial for bacterial colonization and virulence [26]. Biofilmassociated pathogens can form light-colored rings on the wall of a culture tube at the interface between air and broth. To further evaluate the effect of xoSNAT3 on Xoo virulence, the biofilm formation of wild-type strain, xoSNAT3 mutant strain and complemented strain xoSNAT3(xoSNAT3) were analyzed. As shown in Figure 7B, the crystal violet (CV) observation at OD 595 of wild-type strain and complemented strain xoSNAT3(xoSNAT3) were 0.45 and 0.47, respectively, while the CV value of xoSNAT3 mutant strain was only 0.33. This indicated that xoSNAT3 is involved in biofilm formation in Xoo.

Discussion
Previous reports have indicated that MT provides a main m/z peak in positive mode at 233 [28][29][30], which is in agreement with the mass spectra obtained in this study. This confirms for the first time that MT is produced by Xoo, and probably by other Xanthomonas strains. The presence of MT in Xoo raises numerous questions regarding the potential role of this molecule in plant-Xanthomonas interactions. It is possible that Xoo-secreted MT is able to modify plant metabolism and may be related to Xoo-infection process. The role of MT in Xoo quorum sensing is also an important area that must be addressed in future studies.
MT synthetic routes in animals and plants have been well studied, while its synthetic pathway in microorganisms is still not well understood [9]. Tryptophan is the unique precursor of MT. Thus, the identification of intermediates may help to explore the bacterial MT biosynthetic pathway. In animals, the first step of MT synthesis pathway consists of the conversion of tryptophan into 5-hydroxytryptamine [9,31]. However, the first step of MT biosynthesis in the plant kingdom is the conversion of tryptophan into trypamine [14]. No study regarding the MT biosynthetic pathway in bacteria was reported until date. It has been reported to investigate MT intermediates by the N15 labelled tryptophan mediated isotopic tracer Method [24]. Interestingly, 5-hydroxytryptamine, instead of tryptamine, was detected in Pseudomonas fluorescens RG11, an endophytic bacterium isolated from grapevine roots. The 5-hydroxytryptamine is only consist in animal MT synthesis pathway. Therefore, the author speculates that the synthetic pathway of MT in RG11 may be similar to that reported in animals. Interestingly, tryptamine, 5-hydroxytryptamine, N-acetylserotonin and MT were also identified in Xoo by LC-MS/MS. Moreover, the highest content of tryptamine was 639 ng in 50 mL NA at 24 h post inoculation, while the lowest content of serotonin was 63 ng in 50 mL. It is well known that tryptamine is an intermediate of plant MT biosynthesis, whereas 5-hydroxytryptophan is involved in animal MT synthesis [14,18]. Surprisingly, tryptamine and 5-hydroxytryptophan were both detected in Xoo cells. However, no protein with high homology compared to reported TDCs was found in Xoo genome [18]. Further research is necessary in order to confirm if both pathways are present in Xoo.
Mature proteins must be correctly located in specific subcellular structures to perform their corresponding biological functions. Subcellular localization of rice OsSNAT indicated that this protein was located in chloroplast, while oocytes SNATs were localized in the mitochondria [15,32]. Our result suggested xoSNAT3 was located in mitochondria in tobacco leaves cells, which is a similar to that observed in animal cells. An important limitation in this study is that Xoo is a bacterium and, for this reason, does not contain mitochondria. Unfortunately, methods for confirming the location of proteins in Xanthomonas are lacking. The developed method using tobacco cells can be used in further experiments to examine co-localization of xoSNAT3 with bio-markers of plasma membrane, mitochondria and chloroplast, respectively. Despite the obtained results, further research is necessary to confirm the location of xoSNAT3 in bacterial cells.
The enzymatic activity of proteins is closely related to its biological function [33]. SNAT is the key rate-limiting enzyme that catalyzes the penultimate step in MT biosynthesis [34]. Sheep SNAT was identified in 1998 [35], while rice SNAT was cloned and characterized in 2013 [36]. OsSNAT showed K m and V max values of 270 µgM and 3.3 nmol/min/mg protein, respectively; while the values of K m and V max of the cSNAT from Synechocystis sp. PCC 6803 were 823 µgM and 1.6 nmol/min/mg protein, respectively [15,20]. In this study, enzymatic activity assay indicated that GST and xoSNAT2 have no SNAT activity, while xoSNAT1 showed only low activity ( Figure S2). The optimum temperatures of xoSNAT3 were similar to those observed when studying OsSNATs [15].
It is possible that Xoo has evolved to adapt to the same environmental conditions as rice during the epidemic season. Surprisingly, the V max of OsSNAT was 1.49 times higher than that of xoSNAT3. The obtained results revealed that xoSNAT3 is involved in Xoo pathogenicity and biofilm formation. However, further research is necessary to understand how MT is involved in these factors.

Conclusions
In this study, MT was firstly identified in Xoo by mass spectrometry. Our data showed that xoSNAT3 had SNAT activity in vitro. Subcellular localization using tobacco cells showed that oxSNAT3 was located in the mitochondria. Knocking out of xoSNAT3 in Xoo strain PXO99 showed impaired MT production and reduced pathogenicity and biofilm formation than the wild-type strain. This study reveals for the first time the ability of Xanthomonas to synthesize MT, and provides new insights on the biological roles of MT in pathogenic bacteria.