Transcriptome analysis of Xanthomonas oryzae pv. oryzicola exposed to H2O2 reveals horizontal gene transfer contributes to its oxidative stress response.

Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of bacterial leaf streak, is one of the most severe seed-borne bacterial diseases of rice. However, the molecular mechanisms underlying Xoc in response to oxidative stress are still unknown. In this study, we performed a time-course RNA-seq analysis on the Xoc in response to H2O2, aiming to reveal its oxidative response network. Overall, our RNA sequence analysis of Xoc revealed a significant global gene expression profile when it was exposed to H2O2. There were 7, 177, and 246 genes that were differentially regulated at the early, middle, and late stages after exposure, respectively. Three genes (xoc_1643, xoc_1946, xoc_3249) showing significantly different expression levels had proven relationships with oxidative stress response and pathogenesis. Moreover, a hypothetical protein (XOC_2868) showed significantly differential expression, and the xoc_2868 mutants clearly displayed a greater H2O2 sensitivity and decreased pathogenicity than those of the wild-type. Gene localization and phylogeny analysis strongly suggests that this gene may have been horizontally transferred from a Burkholderiaceae ancestor. Our study not only provides a first glance of Xoc's global response against oxidative stress, but also reveals the impact of horizontal gene transfer in the evolutionary history of Xoc.

Introduction

Xanthomonas consists of a large number of phytopathogenic bacteria that infect more than 400 host species, including a wide variety of economically important plants, such as rice, citrus, banana, cabbage, and bean [1, 2]. One of them, Xoc, is the causal agent of bacterial leaf streak (BLS) of rice (Oryza sativa), and is considered one of the most destructive seed-borne bacterial diseases of rice in many Asian countries and parts of Africa [3]. The interaction complexity between Xoc and rice results from the outcome of a longstanding and ongoing evolutionary battle, in which the bacteria attempts to invade and multiply, while the rice plant’s cells attempt to recognize and defend against this invasion. Of these plant responses, oxidative burst is one of the initial response against pathogens [4].

Oxidative burst is a process in which high concentrations of reactive oxygen species (ROS) are produced at the plasma membrane in the vicinity of a pathogen [4, 5]. This activity in plants may directly kill the pathogen, slows its growth by producing toxins, or even act as a signaling cascade leading to various defenses, including the hypersensitive response (HR) and cell wall modifications [6]. Since ROS activity is a common feature of plant defense systems and the mechanism involved in pathogen cell death, any pathogen that is able to resist this effect is likely privileged. These mechanisms require complex and well-orchestrated reactions involving both radical scavenging and enzymatic repair activities [7]. The cellular response to oxidative stress in the model organism, Escherichia coli, has been largely elucidated. When E. coli cells are treated with a low dose of H2O2, growth arrest occurs immediately and the expression of approximately 30 genes is maximally induced within 10–30 minutes of treatment [8]. Among them, the oxidized form of the transcriptional regulator, OxyR, induces many genes such as katG (encoding catalase G), ahpCF (encoding alkyl hydroperoxide reductase), and trxC (encoding reduced thioredoxin) to remove intracellular H2O2, maintain redox homeostasis and ultimately enable cells to resume growth [9, 10]. These studies demonstate that the genes involved in oxidative-stress response are vital for bacteria survival. However, there is limite knowledge on oxidative response network of phytobacteria using time-course system analysis although it is important to elucidatethe molecualr mechanisms that plant pathogens use to resist oxidative stress.

RNA-seq is widely used to investigate differential gene expression in many bacteria [8]. Most of the oxidative stress research using this technology were carried out on human pathogens and environmental bacteria [11, 12]. The application of RNA-seq to Xoc on oxidative stress may provide new insights into molecular mechanisms on oxidative response network.

BLS256, the representative genome of Xoc, has been whole-genome sequenced [13]. Interestingly, more than 30% of the coding genes in this genome are hypothetical genes.

In this study, we identified differentially expressed genes in the Xoc strain BLS256 in response to a time-course H2O2 treatment (early, middle, and late) using the Illumina RNA-Seq platform. Furthermore, we discovered one hypothetical protein, in addition to the genes previously confirmed to be triggered by oxidative stress, which showed high-fold differential expression compared with the control. This finding strongly suggests that this gene could have been horizontally transferred from other microbe organism.

Materials and methods

Strains, plasmids and culture conditions

The bacterial strains and plasmids used in this study are listed in S1 Table. Escherichia coli strains were cultivated at 37 °C in Luria-Bertani (LB) medium or on LB agar plates. Unless otherwise specified, Xoc strains were grown at 28 °C in nutrient broth (NB) (0.1% yeast extract, 0.3% beef extract, 0.5% polypeptone, and 1% sucrose), nutrient agar (NA) (NB with 15 g L-1 agar), NA without sucrose (NAN), NA with 10% sucrose (NAS), and NB without beef extract and sucrose (NY). In some experiments, strains were grown in MMX minimal medium [0.5% glucose, 0.2% (NH4)2SO4, 0.1% trisodium, citrate dihydrate, 0.4% K2HPO4, 0.6% KH2PO4, 0.02% MgSO4·7H2O]. Kanamycin (Km) and Ampicillin (Amp) were added in concentration of 50μg mL-1 and 100μg mL-1, respectively, as required.

Oxidative stress treatments and total RNA preparation

H2O2 resistance assays were performed as described previously described [14] with some modifications. Xoc strains were cultured to the mid-log phase (OD 600 = 1.0 ~ 1.2) in NB medium. Cultures were treated with 0.1mM H2O2 (Fluka) and constantly stirred at 330 rpm in a 28 °C shaking incubator. At time of 0, 7, 15, and 45 min, aliquots were withdrawn and pelleted by centrifugation at 4 °C. Cell The cell pellets was were washed two times with cold PBS and the total RNA immediately extracted using fthe RNeasy Protect Bacteria Mini Kit (QIAGEN) protocal. Two biological replicates were performed in this experiment.

mRNA purification and cDNA synthesis

Ten micrograms from each total RNA sample were treated with the MICROBExpress Bacterial mRNA Enrichment kit and RiboMinus™ Transcriptome Isolation Kit (Bacteria) (Invitrogen). Bacterial mRNAs were chemically fragmented to the size range of 200–250 bp using 1 X fragmentation solution for 2.5 min at 94°C. cDNA was generated according to instructions given in the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen). Each mRNA sample was mixed with 100 pmol of random hexamers, incubated at 65 °C for 5 min, and chilled on ice for 2 min. 4 μL of First-Strand Reaction Buffer (Invitrogen), 2 μL of 0.1 M DTT, 1 μL of 10 mM RNase-free dNTPmix, 1 μL of SuperScript III reverse transcriptase (Invitrogen) were then added to each sample and incubated at 50 °C for 1 h. To generate the second strand, the following Invitrogen reagents were then added: 51.5 μL of RNase-free water, 20 μL of second-strand reaction buffer, 2.5 μL of 10 mM RNase-free dNTP mix, 50 U E. coli DNA Polymerase, and 5 U E. coli RNase H, and incubated at 16 °C for 2.5 h.

Library construction and Illumina sequencing

The Illumina Paired End Sample Prep kit was used for RNA-Seq library creation according to the manufacturer’s instructions. Fragmented cDNA was end-repaired, ligated to Illumina adaptors, and amplified by 18 cycles of PCR. Paired-end 100-bp reads were generated by high-throughput sequencing with the Illumina Hiseq2000 Genome Analyzer instrument.

RNA-Seq data analysis

After removing the low quality reads and adaptors, pair-end reads were mapped to the reference genome BLS256 using Bowtie1.1.1 with default parameters [15]. If reads mapped to more than one location, then only the reading havingthe highest alignment score was kept. Reads mapping to rRNA and tRNA regions were removed from further analysis. Four time-points samples were prepared in this study: 0min, 7min, 15min and 45min, defined as WT, early, middle and late stage, respectively. All samples were compared with the 0 min samples to detect the differential expressed genes (DEGs). After obtaining the number of reads for every sample, the edgeR with TMM normalization method [16] was used to determine the DEGs [17]. FDR value < 0.05 was selected as the cutoff for further analysis. Cluster 3.0 and TreeView were used to represent the cluster of DEGs over time-series samples [18].

Quantitative real-time PCR

Bacterial total RNA was extracted following RNeasy Protect Bacteria Mini Kit (QIAGEN) instructions and used in generating the first strand complementary DNA (cDNA) per Takara PrimeScript RT reagent Kit with gDNA Eraser’s (Takara) protocol. After detecting RNA quantity and quality by Nanodrop ND1000 spectrophotometer V 3.5.2 (NanoDrop Technologies, Wilmington, DE, USA), 1 μg of the total RNA was incubated at 42 °C for 20 mins to eliminate the gDNA by gDNA Eraser before obtaining cDNA. The reverse transcription reaction was accomplished by incubated at 37 °C for 15 min and then 85 °C for 5 s in the presence of random RT primers. The cDNA was then used directly as the template for qRT-PCR using a SYBR Green master mix (Applied Biosystems) on an ABI Prism 7000 sequence detection system (Applied Biosystems). Normalized expression levels of the target gene transcripts were calculated relative to the rRNA using the ΔΔCT method, where CT is the threshold cycle [19]. Three biological replicates were carried out in this experiment.

Construction of defective deletion mutant and complementation

To investigate the role of interested genes in Xoc, In-frame deletion mutations were constructed in BLS256 using homologous recombination. Briefly, two fragments flanking the left and right of corresponding genes were amplified from the wide-type genomic DNA with primer pairs listed in S2 Table. The amplified fragments were cloned into pMD18-T vectors (TaKaRa), confirmed by sequence analysis, and then digested and subcloned into vector pKMS1 [20] at BamHI and PstI (or SalI) sites. The resultant recombinant plasmids were introduced into BLS256 by electroporation, and transformants were plated on NAN plates supplemented with kanamycin. Colonies resulting from a single homologous crossover (integration of deletion construct at either the left or right border of target gene) were then transferred to NBN broth, grown for 12 h at 28°C, and then plated on NAS plates for sucrose-positive deletion mutant selection. Sucrose resistant colonies were visible within 3 to 4 days and then transferred to NA plates and NA plus kanamycin plates. Since kanamycin-sensitive colonies could be mutants containing a second homologous crossover, these were further examined by PCR amplification with the primer pairs.

In order to complement the deletion mutants, the full-length of corresponding genes including promoter regions were amplified using primer pairs listed in S2 Table. The amplified DNA fragments were cloned into pUFR034 vectors [20] at the BamHI and PstI (or SalI) sites to create the recombinant plasmids. The recombinant plasmids were transferred into corresponding mutants by electroporation, and transformants were screened on NA plates with kanamycin.

H2O2 resistance assay

The assay plates were prepared by adding H2O2 to the sterilized NB medium in concentrations of 0, 0.1 and 0.25 mM, respectively. Strains were cultured to the mid-log phase (OD 600 = 1.0) in NB medium, and three-fold and nine-fold dilutions were made. 5-μL aliquots of the initial culture and diluted cultures for each strain were spotted onto NB agar plates (in triplicate) and cultured for 36 h at 28 °C [21].

Pathogenicity assays

Bacteria were prepared based on previous reported method [21]. Briefly, Xanthomonas cells were grown in NB broth at 28°C and 200 rpm for 16 h, when cells approached the exponential phase of growth. Bacterial cells were then harvested by centrifugation, washed twice, and resuspended in sterile water to an optical density at 600 nm (OD600) of 0.3 (approximately 1×108 CFU/mL). Bacteria were inoculated into leaves of adult rice plants (Oryza sativa cv. IR24, susceptible to Xoc, 2 months old) using leaf piercing for lesion length measurement for evaluating water-soaked symptoms. All plants were maintained in a greenhouse as described previously [13]. Five leaves were inoculated for each independent experiment, and each treatment was repeated at least three times.

Phylogenetic analysis

Interested gene was first compared against non-redundant database for homologs searching with E<0.0001 as cutoff. Next, 40% alignment similarity and 80% coverage parameter was used to define the real homologue genes. MAFFT was used to generate the multiple sequences alignment [22]. Maximum Likelihood phylogeny was finally evaluated and built by PhyML [23] using a JTT model and a gamma distribution with eight rate categories. We performed 1,000 bootstraps to gain branch support values.

Results

Global overview of the Xoc transcriptome in response to H2O2

In this study, 100-bp paired-end deep sequencing was performed on all the tested samples. In general, more than 40M reads were generated from each single sample. After adaptor removal, quality control, and removal of reads, mapped to ribosomal RNA, 7M to 10M confident reads remained. The sequencing depth in this experiment was more than 150 X, which is sufficient for further statistical analysis. In general, 7, 177, and 246 genes were differentially regulated in the early, middle, and late stages, respectively (Fig 1, S3 Table). The overall number of differentially expressed genes (DEGs) was similar to the number found in recent research related to human pathogens and environmental bacteria [24, 25], with the exception of DEGs in the early state (7 min), which were almost 10 times smaller than in our study. This number, indicates the oxidative response difference that may occur between plant pathogen versus other bacteria.

Fig 1

Time-course transcriptome study of Xoc (Xanthomonas oryzae pv. oryzicola) resistance to an oxidative stress response.

Hundreds of genes are differentially expressed at different time points (early, middle, and late) during the H2O2 treatment when compared with the 0-min sample. These differentially expressed genes can be classified into different clusters based on their expression patterns. The line from -8.0 to 8.0 represents the Log2 fold change value.

All of the RNA-seq data has been deposited in BioProject database (https://www.ncbi.nlm.nih.gov/bioproject) and the accession number is PRJNA350867.

General gene expression kinetics among the time-course samples

Previous research indicates that bacteria require dynamic regulatory networks at different time-points when they are exposed to environmental stress [24, 26]. To capture this variable activity, gene expression among the time-course samples was generated and results are shown in Fig 1. In general, clusters 1 and 2 had a decreased induction whereas clusters 4 and 5 had an increased induction through time (Fig 1). By contrast, the clusters 3 and 6 consisted of those genes whose expression peaked at the middle time stage.

Gene Ontolog (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were carried out based on the STRING V10 database [27], with a p < 0.01 after a Bonferroni correction [28] set as the cutoff. The GO enrichment analysis revealed the categories of transport (GO:0006810), cell outer membrane (GO:0009279), and acyl-CoA dehydrogenase activity (GO:0003995) as significant regulation based on this cutoff for clusters 1 and 2 (Table 1). The regulation of cellular process (GO:0050794), chemotaxis (GO:0006935), single organism signaling (GO:0044700), response to external stimulus (GO:0009605), molecular transducer activity (GO:0060089), and signal transducer activity (GO:0004871) were significant for clusters 4 and 5 (Table 1). Membrane protein (GO:0016020), outer membrane protein (GO:0019867), and cellular component (GO:0005575) were significant for clusters 3 and 6 (Table 1). The KEGG enrichment results suggested that only valine, leucine, and isoleucine degradation was significant for clusters 1 and 2 (Table 1). Interestingly, we found many TonB-dependent receptors (TBDRs) that were differentially expressed in all the three time stages, indicating their importance in oxidative stress (S3 Table).

Table 1

The functional groups and their significance in different gene clusters.

Functional group	Clusters			p value&
	1 and 2	4 and 5	3 and 6
GO category
Transport (GO:0006810)	+			6.50E-02
Cell outer membrane (GO:0009279)	+			3.13E-02
Acyl-CoA dehydrogenase activity (GO:0003995)	+			1.61E-03
Regulation of cellular process (GO:0050794)		+		1.97E-03
Chemotaxis (GO:0006935)		+		1.60E-02
Single organism signaling (GO:0044700)		+		5.32E-02
Response to external stimulus (GO:0009605)		+		9.49E-02
Molecular transducer activity (GO:0060089)		+		5.94E-03
Signal transducer activity (GO:0004871)		+		7.08E-02
Membrane (GO:0016020)			+	1.09E-03
Outer membrane (GO:0019867)			+	5.20E-02
Cellular component (GO:0005575)			+	7.46E-02
KEGG category
Valine, leucine and isoleucine degradation	+			4.06E-02

&: Bonferroni test

Gene clusters involved in the DEGs

Since those genes forming a bacterial gene cluster are always involved in a common pathway or function [29], the DEGs located in the gene clusters we identified may play a fundamental role in oxidative stress. Based on our criterion—that at least three tandem genes must be differentially expressed to constitute a gene cluster—we identified all the possible clusters and listed them in S4 Table. Not surprisingly, many reported oxidative stress-associated gene clusters were found involved, such as those for the alkyl hydroperoxide reductase, suf operon and pst operon [30, 31]. Furthermore, we also found a large gene cluster encoding ribosomal proteins that were up-regulated in our study (S3 Table). In addition, we found an F1F0 ATPase complex cluster that was up-regulated under oxidative stress.

Quantitative real time PCR experiments confirms the gene expression profiles

The RNA-seq results were validated with a qRT-PCR analysis of four selected genes (xoc_1643, xoc_1946, xoc_2868, and xoc_3249) that showed different levels of expression at 7, 15, and 45 minutes (S3 Table). The mRNA abundance of these transcripts at these three time-points after the H2O2 treatment followed a profile similar to that of the microarray dataset (S3 Table), thus validating the quality of our assay. The Pearson correlation test of the microarray against the qRT-PCR measurements yielded a correlation coefficient of R2 = 0.81 (n = 4), suggesting that RNA-seq dataset correlated positively and tightly with the qRT-PCR quantification (Fig 2).

Fig 2

Real-time quantitative PCR analysis.

Transcript levels of the four candidates under oxidative stress at 7, 15, and 45 min after H2O2 treatment. Values given are the means of five replicate measurements from a representative experiment. The experiment was repeated five times, and similar results were obtained. Columns with the same letters are not significantly different from each other by t-test (i.e., P ≥ 0.05). Error bars represent the standard deviations.

A hypothetical gene plays some role in the oxidative stress response in Xoc

Sensing, detoxification, and adaptation to oxidative stress play a critical role during successful pathogen infection and pathogenesis by Xanthomonas [30]. The XOC_1643 (outer membrane channel protein), XOC_3249 (membrane protein YnfA) and XOC_2868 (hypothetical protein) and mutants clearly displayed a greater sensitivity to H2O2 than did the BLS256 wild-type and complemented strains (Fig 3). Not surprisingly, these three mutants showed decreased pathogenicity when compared with the wild-type (Fig 4). However, the XOC_1946 (TonB-dependent receptor) mutants showed a greater resistance to H2O2 but a decreased pathogenicity when compared with the wild-type (Fig 4). TonB-dependent receptor was proven to be involved in the transport of plant-derived molecules such as sucrose and maltodextrins in previous studies and yet delayed the disease symptom development in plants to some extent [31, 32]. Our studies show similar results with earlier researches, suggests that XOC_1946 is involved in the transport of H2O2. As genes xoc_1643, xoc_1946 and xoc_3249 have proven relationships with an oxidative stress response, this result confirmed the accuracy of our RNA-seq result in this study. Interestingly, XOC_2868, a hypothetical protein, was also involved in the stress response. Sequence analysis revealed that this gene has a MntR domain, thus indicating its potential role in superoxide resistance regulation [33].

Fig 3

Gene mutations changed the resistance to H2O2 in Xanthomonas oryzae pv. oryzicola (Xoc).

Xoc strains, including the wild-type strain BLS256, the gene deletion mutants Δ1643, Δ1946, Δ2868, and Δ3249, and their complemented strains Δ1643 (1643), Δ1946 (1946), Δ2868 (2868), and Δ3249 (3249), were grown on nutrient broth agar (NA) plates with 0 mM H2O2, 0.1 mM H2O2, or 0.25 mM H2O2. Three replicates for each treatment were used, and the experiment was repeated three times. “1x”: original cultures; “3x”: three-fold dilutions of cultures; “9x”: nine-fold dilutions of cultures.

Fig 4

Gene mutations changed the virulence and growth of X. oryzae pv. oryzae in planta.

(A) Symptoms induced by different X. oryzae pv. oryzae strains inoculated to leaves of 2-month-old rice plants (IR24, a susceptible cultivar). Photographs were taken at 14 dpi. Three replicates for each treatment were used, and the experiment was repeated three times. (B) Lesion lengths on the leaves of adult susceptible rice (cv. IR24, 2months old) inoculated with the Xoc strains. Different letters above the data bars indicate a significant difference between the wild-type strain and deletion mutants or complemented strains (P < 0.05; t-test).

xoc_2868 is a horizontally transferred gene

A comprehensive BLAST sequence analysis revealed that this MntR-like gene exists widely in Xoc but not in other Xanthomonas species (Fig 5). Interestingly, homologs of this gene were found to present in many Burkholderiaceae family bacteria, such as Burkholderia, Ralstonia, and Cupriavidus (Fig 5). The phylogenetic analysis further suggested that this gene in Xoc might have originated from a transfer from a Burkholderiaceae ancestor over the course of evolutionary history, an inference with high bootstrapping support (Fig 5). Notably, many genes located adjacent to Xoc_2868 are transposase (i.e., from Xoc_2859 to Xoc_2865). Ochman et al. suggested that HGT is mediated by bacteriophage integrases or mobile element transposases, while Keeling et al. suggested that a phylogenetic tree is the gold standard by which to identify HGT [34, 35]. The results presented here strongly suggest that xoc_2868 is a horizontally transferred gene.

Fig 5

A phylogeny of the Xoc_2868, a MntR-like gene.

The phylogenetic tree shown was calculated using the maximum likelihood (ML) program in PhyML [23]. Only the ML values ≥ 50% were shown. Bar = 0.1 substitution per site.

Discussion

In this study, we demonstrated, for the first time, that TonB-dependent receptors (TBDRs) is involved in resistance to H2O2 and virulence in Xoc. Indeed, the trigger of these TBDRs is reportedly involved in many different kinds of stress responses such as oxidative stress, iron stress and zinc stress [36]. Researchers also found that TBDRs are required for full virulence of Xanthomonas campestris pv. campestris to Arabidopsis [30]. We may infer that TBDRs may play some role in the oxidative response in Xoc. Hence, the function of one TBDRs gene Xoc_1946 was confirmed by H2O2 resistance assays and pathogenicity assays. Our results show that Xoc_1946 did play a role in oxidative response and pathogenicity. However, the role of the TBDRs in these bacterial species remains to be investigated.

There are many oxidative-associated gene clusters were found involved in DEGs on our study. Alkyl hydroperoxide reductase have been reported responsible for alky1 peroxide metabolism in Xanthomonas. It is the best characterized microbial enzyme involved in organis peroxide metabolism[30]. suf operon, which consists of a list of cysteine desulfurase encoding genes, is involved in the assembly of [Fe–S] clusters under oxidative stress, it is also known as being necessary for virulence of the plant pathogen Erwinia chrysanthemi[31]. Another operon pst, which has been found differentially expressed in this study, was also been considered to improve heat, oxygen and starvation stress resisitance in Lactococcus lactis[37]. Our results indicate that the role of these clusters in bacterial resistance to oxidative-stress might not be diverse in different bacterial species.

Prior research has demonstrated that H2O2 causes a slower rate of ribosomal run-off, while the expression of several ribosomal proteins associated with the translation of the stress response-associated genes was increased [38]. Our findings show that there is a gene cluster encoding ribosomal proteins were up-regulated, this result indicate that this gene cluster contributes to the translation of oxidative stress associated genes in our Xoc strain.

It is well-known that the oxidative stress generated in the plant response to pathogens will decrease the intracellular pH [38]. Although direct evidence is lacking for Xanthomonas species, the mutants of these genes from several other bacteria showed clear growth defects under low pH, thus indicating the F1F0 ATPase complex cluster which was found up-regulated in our study is important for maintain the ΔpH [39].

It is now widely appreciated that a time-course transcriptome analysis can help us to better understand how organisms react to stress conditions over time [40]. Here, we set three time points corresponding to the early, middle, and late response stages. Very few genes were significantly differentially expressed in the early stage (S3 Table). Interestingly, however, we did find that one gene encoding SoxR, a redox-sensitive transcriptional activator, was the highest up-regulated gene in this time stage. In E. coli, soxR and soxS were shown to control the superoxide response regulon of E. coli [41]. Since Xoc lacks the homolog of soxS, and soxR is the only transcriptional regulator, we may infer that this gene triggers the Xoc oxidative stress response.

Conclusions

In this research, gene expressions of Xoc strain BLS256 in response to a time-series H2O2 treatment have been presented by RNA-Seq analysis. In general, 7, 177, and 246 genes were differentially regulated in the early, middle, and late stages, respectively. The soxR gene was highly up-regulated in the early stage, indicating that this gene triggers the Xoc oxidative stress response. In addition, the sensitivity to H2O2 and pathogenicity of four DEGs’ mutants were investigated, and the results prove strong relationships between these DEGs and oxidative stress response. Interestingly, the results about a hypothetical protein XOC_2868 presented here strongly suggest that it is encoded by a horizontally transferred gene.

Strains and plasmids used in this study.

(DOCX)

Click here for additional data file.

Primers used in this study.

(DOCX)

Click here for additional data file.

Differential expressed genes in the early, middle and late stage.

(XLSX)

Click here for additional data file.

Gene clusters that were differential expressed in the early (red color), middle (yellow color) and late (blue color) stages.

(XLSX)

Click here for additional data file.

24 Jul 2019

PONE-D-19-16123

Transcriptome analysis of Xanthomonas oryzae pv. oryzicola exposed to H2O2 reveals horizontal gene transfer contributes to its oxidative stress response

PLOS ONE

Dear Mr. Rao,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Sep 07 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Kandasamy Ulaganathan

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1) Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2) We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

3) Please amend the manuscript submission data (via Edit Submission) to include author Xin Dedong.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript described a time-resolved RNA-seq result that reveals Xoc transcriptome change related to oxidative stress response during exposed to hydrogen peroxide. Following the differentially expressed gene analysis, further investigations were carried out to determine 4 DEGs’ role in terms of antioxidation and virulence. This study provided useful information to related professional researcher. However, according to this manuscript, it is regretful to learn that part of result could not sufficient support the conclusion made by the authors.

Major issues

1. The virulence assay result could not support the conclusion made by the authors.

According to Fig 4, I assumed the authors using leaf piercing method to inoculate Xoc on rice leaf. But the authors did not clearly describe this in manuscript, the inoculate process was missing. By using leaf piercing method, the virulence should be evaluated by either calculating the means of lesion length (n >= 10), or establishing bacterial growth curves, or both. But in Fig 4, authors only showed a typical lesion caused by leaf piercing inoculation for each Xoc strain, which is not enough to represent their virulence.

2. To demonstrate whether a gene is required for full virulence of Xoc to rice, the virulence mutant should be assayed along with its complementation strain, which have not been showed in this manuscript.

3. The GEO data is not accessible. The authors declared all raw RNA-seq data is available on GEO at line 315-318. But the given accession numbers are invalid.

Minor issues

1. The organization of Introduction part is poor. And I suggest authors describe some known oxidative stress response process in microbe as part of background.

2. Some bioinformatic methods mentioned in Materials and Methods need proper citation. e.g. in line 135, the TMM method, and in line 155, the 2(-ΔΔC(T)) method, both need a proper citation.

3. At line 101, author mentioned “Cultures were treated with 0.1mM H2O2 in a 28 ℃ shaking incubator.” without describing how to conduct H2O2 treatment. I assumed the authors were closely following the method in Upadhya at.el work. I recommended the authors to provide more details in this part to make it more readable.

4. At line 146, the total RNA extraction process is missing.

5. At line 182, more details for how to interduce H2O2 into NB agar plates is required.

6. At line 190 to 193. author mentioned “Plant phenotypes were scored 24 h post-inoculation for the HR in tobacco, 3 days post-inoculation (dpi) for water-soaked symptoms, and 14 dpi for lesion length.” But there is not any hypersensitive reaction or water-soaked symptom result in this manuscript. Please make sure only the involved methods were included in this manuscript.

7. At line 203 to 206, the result indicated there were about 75% clear reads mapped on ribosomal RNA. Is this a typical rRNA/mRNA ratio for RiboMinus treated sample?

8. At line 235, authors should provide the full form of GO and KEGG.

9. At line 266, what’s the mean of “encompassed a range of expression levels at 7, 15, and 45 minutes”?

10. At line 267-272, author mentioned a microarray dataset but never described it. I assume author intend to refer the RNA-seq data in this study.

11. In Fig 3, the authors did not describe the meaning of captions “1x”, “3x”, and “9x”.

12. At line 315-318, the authors should not put the accession numbers in the main part of manuscript. The authors should list all accession numbers instead of an accession number range.

13. At line 347-358, the phylogenetic analysis result should be moved to the Result section.

14. The order of supporting information label incorrect.

15. The S1 Table, titled as “Differential expressed genes in the early, middle and late stage.”, missing heading at column one.

16. The S4 Table missing its heading.

Overall, the writing of this manuscript is very poor. The authors should proof the writing of the manuscript seriously, or consider hiring a professional copyeditor to handle it.

Reviewer #2: In the manuscript ‘Transcriptome analysis of Xanthomonas oryzae pv. oryzicola exposed to H2O2 reveals horizontal gene transfer contributes to its oxidative stress response’ the authors study the gene expressions of XoC strain BLS256 in response to time series H2O2 treatment, following RNA-Seq analysis. They claim in the Abstract, that ‘soxR triggers and regulates XoC oxidative stress response in early infection, gene expression kinetics among the time-series samples namely for TonB-dependent receptors and suf and pst operons. However, there is no clear data in the Results to corroborate these claims.

The finding that a MntR-like hypothetical protein is highly upregulated at a later time-point and that it is likely to be a horizontally transferred gene is interesting.

However, the English and the overall presentation of the manuscript needs to be improved considerably.

The descriptions of the Supporting data do not match the actual Supporting Tables. Hence,

In lines 249, 261 and 340: S3 Table needs to be replaced by S1 Table.

The authors have validated the role of 4 selected genes for their role in H2O2 sensitivity and pathogenicity by mutant generation and their complementation. It might be better to perform the experiments with at least two-independently generated mutants for each gene.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

6 Sep 2019

When submitting your revision, we need you to address these additional requirements.

1) Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: we have checked our manuscript carefully, it meets PLOS ONE's style requirements.

2) We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Response: Thank you for your suggestion, the phrase “data not shown” has been removed in revised manusscript.

3) Please amend the manuscript submission data (via Edit Submission) to include author Xin Dedong.

Response: Sorry for the mistake, the information of author Xin Dedong will be added when resubmitting.

Reviewer #1:

Major issues

1. The virulence assay result could not support the conclusion made by the authors.

According to Fig 4, I assumed the authors using leaf piercing method to inoculate Xoc on rice leaf. But the authors did not clearly describe this in manuscript, the inoculate process was missing. By using leaf piercing method, the virulence should be evaluated by either calculating the means of lesion length (n >= 10), or establishing bacterial growth curves, or both. But in Fig 4, authors only showed a typical lesion caused by leaf piercing inoculation for each Xoc strain, which is not enough to represent their virulence.

Response: We thank the reviewer for the suggestions. We did inoculate plants by leaf piercing. Sorry for the negligence. In the revised manuscript, The inoculation process was presented. Which is “Briefly, Xanthomonas cells were grown in NB broth at 28°C and 200 rpm for 16 h, when cells approached the exponential phase of growth. Bacterial cells were then harvested by centrifugation, washed twice, and resuspended in sterile water to an optical density at 600 nm (OD600) of 0.3 (approximately 1�108 CFU/mL). Bacteria were inoculated into leaves of adult rice plants (Oryza sativa cv. IR24, susceptible to Xoc, 2 months old) using leaf piercing for lesion length measurement for evaluating water-soaked symptoms.”

We realized there was not enough to represent the virulence only by a typical lesion in Fig 4, so we add the results of the lesion length to evaluate the virulence together with the symptoms of leaf lesion (which is shown in Fig 4).

2. To demonstrate whether a gene is required for full virulence of Xoc to rice, the virulence mutant should be assayed along with its complementation strain, which have not been showed in this manuscript.

Response: Thank you for the excellent question. Actually we have tested the virulence of these complementation strains. We have put the results in our revised manuscript and Fig 4.

3. The GEO data is not accessible. The authors declared all raw RNA-seq data is available on GEO at line 315-318. But the given accession numbers are invalid.

Response: Sorry for the mistake. We have corrected this in the new version. “All the RNA-seq data has been deposited in BioProject database (https://www.ncbi.nlm.nih.gov/bioproject) and the accession number is PRJNA350867.”

Minor issues

1. The organization of Introduction part is poor. And I suggest authors describe some known oxidative stress response process in microbe as part of background.

Response: Great suggestion. As suggested, we reorganized the order of Introduction part. Xanthomonas was introduced at first, then we descibe the oxidative stress response of microorganisms. Subsequently, RNA-seq and BLS256 were briefly introduced. At last, the purpose of our study was clearly presented.

Also, we add a part of description on oxidative stress response of microorganisms. “Oxidative burst is a process in which high concentrations of reactive oxygen species (ROS) are produced at the plasma membrane in the vicinity of a pathogen. This activity in plants may directly kill the pathogen, slows its growth by producing toxins, or even act as a signaling cascade leading to various defenses, including the hypersensitive response (HR) and cell wall modifications. Since ROS activity is a common feature of plant defense systems and the mechanism involved in pathogen cell death, any pathogen that is able to resist this effect is likely privileged. These mechanisms require complex and well-orchestrated reactions involving both radical scavenging and enzymatic repair activities. The cellular response to oxidative stress in the model organism, Escherichia coli, has been largely elucidated. When E. coli cells are treated with a low dose of H2O2, growth arrest occurs immediately and the expression of approximately 30 genes is maximally induced within 10–30 minutes of treatment. Among them, the oxidized form of the transcriptional regulator, OxyR, induces many genes such as katG (encoding catalase G), ahpCF (encoding alkyl hydroperoxide reductase), and trxC (encoding reduced thioredoxin) to remove intracellular H2O2, maintain redox homeostasis and ultimately enable cells to resume growth. These studies demonstate that the genes involved in oxidative-stress response are vital for bacteria survival.”

2. Some bioinformatic methods mentioned in Materials and Methods need proper citation. e.g. in line 135, the TMM method, and in line 155, the 2(-ΔΔC(T)) method, both need a proper citation.

Response: Thank you for your suggestion. The proper citation have been added in revised manuscript.

3. At line 101, author mentioned “Cultures were treated with 0.1mM H2O2 in a 28 ℃ shaking incubator.” without describing how to conduct H2O2 treatment. I assumed the authors were closely following the method in Upadhya at.el work. I recommended the authors to provide more details in this part to make it more readable.

Response: Thank you for your suggestion. I have made some modifications in revised manuscript. “Cultures were treated with 0.1mM H2O2 (Fluka) and constantly stirred at 330 rpm in a 28 ℃ shaking incubator.”

4. At line 146, the total RNA extraction process is missing.

Response: Sorry for the mistake. “Total RNA immediately extracted using fthe RNeasy Protect Bacteria Mini Kit (QIAGEN) protocal.”

1. At line 182, more details for how to interduce H2O2 into NB agar plates is equired.

Response: Thank you for your suggestion. The assay plates were prepared by adding H2O2 to the sterilized NB medium in concentrations of 0, 0.1 and 0.25 mM, respectively.

6. At line 190 to 193. author mentioned “Plant phenotypes were scored 24 h post-inoculation for the HR in tobacco, 3 days post-inoculation (dpi) for water-soaked symptoms, and 14 dpi for lesion length.” But there is not any hypersensitive reaction or water-soaked symptom result in this manuscript. Please make sure only the involved methods were included in this manuscript.

Response: Sorry for the mistake. There is no hypersensitive reaction in our study, we have correted this sentences.

7. At line 203 to 206, the result indicated there were about 75% clear reads mapped on ribosomal RNA. Is this a typical rRNA/mRNA ratio for RiboMinus treated sample?

Response: Great Question. It’s not a typical rRNA/mRNA ratio for RiboMinus treated sample. Xanthomonas oryzae pv. oryzicola is not a regular bacteria that in the recommended list of the RiboMinus kit. So the effect of removing is not so good.

8. At line 235, authors should provide the full form of GO and KEGG.

Response: Sorry for the typo. The full form of GO and KEGG have been added in revised manuscript. There are “Gene Ontolog (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)”.

9. At line 266, what’s the mean of “encompassed a range of expression levels at 7, 15, and 45 minutes”?

Response: Thank you for your question. It is really an ambiguous writing. We have correct this sentence by “showed different levels of expression at 7, 15, and 45 minutes”.

10. At line 267-272, author mentioned a microarray dataset but never described it. I assume author intend to refer the RNA-seq data in this study.

Response: That’s right. It refered to the S3 Table here.

11. In Fig 3, the authors did not describe the meaning of captions “1x”, “3x”, and “9x”.

Response: Sorry for the negligence. I have added the explanations at the end of the Fig 3 legends. “1x”: original cultures; “3x”: three-fold dilutions of cultures; “9x”: nine-fold dilutions of cultures.

12. At line 315-318, the authors should not put the accession numbers in the main part of manuscript. The authors should list all accession numbers instead of an accession number range.

Response: Thank you for your suggestion. I have correct the accession number and put this sentences in the chapter of Resluts.

13. At line 347-358, the phylogenetic analysis result should be moved to the Result section.

Response: Thank you for your suggestion. The phylogenetic analysis has been moved to the Results section.

14. The order of supporting information label incorrect.

Response: Sorry for the mistake. We have placed them in correct order in the revised manuscript.

15. The S1 Table, titled as “Differential expressed genes in the early, middle and late stage.” missing heading at column one.

Response: Sorry for the mistake. I have added the heading in column one.

16. The S4 Table missing its heading.

Response: Sorry for the mistake. I have added the heading in column one.

Overall, the writing of this manuscript is very poor. The authors should proof the writing of the manuscript seriously, or consider hiring a professional copyeditor to handle it.

Response: We have checked the grammar of the manuscript carefully and the English in the revised manuscript has been edited and improved by a native English speaker. We hope it will reach the standards of the journal for publication.

Reviewer #2:

In the manuscript ‘Transcriptome analysis of Xanthomonas oryzae pv. oryzicola exposed to H2O2 reveals horizontal gene transfer contributes to its oxidative stress response’, the authors study the gene expressions of Xoc strain BLS256 in response to time series H2O2 treatment, following RNA-Seq analysis. They claim in the Abstract, that ‘soxR triggers and regulates Xoc oxidative stress response in early infection, gene expression kinetics among the time-series samples namely for TonB-dependent receptors and suf and pst operons’. However, there is no clear data in the Results to corroborate these claims.

Response: Thank you for your good suggestion. It is a hypothesis that ‘soxR triggers and regulates Xoc oxidative stress response in early infection, gene expression kinetics among the time-series samples namely for TonB-dependent receptors and suf and pst operons’. It will be confirmed in subsequent research. So I have rewrited the Abstract in revised manuscript, this sentence has been deleted.

The finding that a MntR-like hypothetical protein is highly upregulated at a later time-point and that it is likely to be a horizontally transferred gene is interesting.

However, the English and the overall presentation of the manuscript needs to be improved considerably.

Response: We have checked the grammar of the manuscript carefully and the English in the revised manuscript has been edited and improved by a native English speaker. We hope it will reach the standards of the journal for publication.

The descriptions of the Supporting data do not match the actual Supporting Tables. Hence, In lines 249, 261 and 340: S3 Table needs to be replaced by S1 Table.

Response: Sorry for the mistake. I have corrected it in revised manuscript.

The authors have validated the role of 4 selected genes for their role in H2O2 sensitivity and pathogenicity by mutant generation and their complementation. It might be better to perform the experiments with at least two-independently generated mutants for each gene.

Response: Thank you for your good suggestion. Actually, we got at least five mutants for each gene, the results they presented were consistent.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

23 Sep 2019

Transcriptome analysis of Xanthomonas oryzae pv. oryzicola exposed to H2O2 reveals horizontal gene transfer contributes to its oxidative stress response

PONE-D-19-16123R1

Dear Dr. Rao,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Kandasamy Ulaganathan

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Please carry out the minor corrections pointed by the Reviewer

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The manuscript looks considerably better and improved.

Following are some minor corrections:

Line 70: limited instead of limite

line 71: space between elucidate and the

line 105: delete Cell

line 106: delete was

line 133: space between having and the

line 276: remove additional space between different and levels

line 362: organic instead of organis

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

24 Sep 2019

PONE-D-19-16123R1

Transcriptome analysis of Xanthomonas oryzae pv.  oryzicola  exposed to H2O2 reveals horizontal gene transfer contributes to its oxidative stress response

Dear Dr. Rao:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Kandasamy Ulaganathan

Academic Editor

PLOS ONE