Transcriptome analysis reveals defense-related genes and pathways against Xanthomonas campestris pv. vesicatoria in pepper (Capsicum annuum L.)

Bacterial spot (BS), incited by Xanthomonas campestris pv. vesicatoria (Xcv), is one of the most serious diseases of pepper. For a comparative analysis of defense responses to Xcv infection, we performed a transcriptomic analysis of a susceptible cultivar, ECW, and a resistant cultivar, VI037601, using the HiSeqTM 2500 sequencing platform. Approximately 120.23 G clean bases were generated from 18 libraries. From the libraries generated, a total of 38,269 expressed genes containing 11,714 novel genes and 11,232 differentially expressed genes (DEGs) were identified. Functional enrichment analysis revealed that the most noticeable pathways were plant-pathogen interaction, MAPK signaling pathway—plant, plant hormone signal transduction and secondary metabolisms. 1,599 potentially defense-related genes linked to pattern recognition receptors (PRRs), mitogen-activated protein kinase (MAPK), calcium signaling, and transcription factors may regulate pepper resistance to Xcv. Moreover, after Xcv inoculation, 364 DEGs differentially expressed only in VI037601 and 852 genes in both ECW and VI037601. Many of those genes were classified as NBS-LRR genes, oxidoreductase gene, WRKY and NAC transcription factors, and they were mainly involved in metabolic process, response to stimulus and biological regulation pathways. Quantitative RT-PCR of sixteen selected DEGs further validated the RNA-seq differential gene expression analysis. Our results will provide a valuable resource for understanding the molecular mechanisms of pepper resistance to Xcv infection and improving pepper resistance cultivars against Xcv.


Introduction
Bell pepper (Capsicum annuum L.), an important member of the Solanaceae family, is one of the most important vegetable crops in China and many other countries [1]. It is rich in

Plant materials and pathogen inoculation
Two bell pepper genotypes, VI037601 and Early Calwonder (ECW), were used for transcriptomic analysis provided by World Vegetable Center, Thailand (https://avrdc.org/). Plants were grown under standard glasshouse conditions for 16 h lighting at 25˚C/ 8 h darkness at 20˚C in a relative humidity of approximately 60%. Xcv strain 23-1 was grown at 28˚C on nutrient agar medium for two days, and then scraped into sterile water to make a suspension with a concentration of 2×10 8 cfu/ml. The suspension was inoculated using a syringe on the abaxial leaf surface near the midrib of the third to fifth pepper leaves to form a 1.5-2cm diameter watersoaked area, when plants were at the five leaves stage. The leaf fragments within 2 cm of the Xcv infection site, were collected for RNA isolation at 0h, 6h and 24h post-inoculation (hpi), respectively. Pepper leaves of respective varieties at 0 hpi were used as control. Samples were collected from ECW and VI037601 leaves 0-2 cm away from the inoculation point at three different inoculation time points (0, 6, and 24 hours), which were named as ECW_0H, ECW_6H, ECW_24H, VI037601_0H, VI037601_6H, and VI037601_24H, respectively. The samples were immediately placed in liquid nitrogen and stored at -80˚C for RNA extraction and further analysis. Fifteen leaves randomly selected from five different plants were polled as a biological replicate. Three independent biological replicates were prepared for each treatment.

RNA extraction, library construction and transcriptome sequencing
Total RNA was extracted from 18 leaf tissue samples, including 3 replicates of each treatment condition (3 time points × 2 genotypes), using the Trizol Reagent (Life Technologies, California, USA) according to the manufacturer's instructions, and then treated with TURBO DNase I (Promega, Beijing, China) to remove genomic DNA contamination. The integrity and concentration of all RNA samples were examined by the 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) and 1.2% agarose gel electrophoresis. The prepared total RNA samples were sent to Frasergen Bioinformatics Co., Ltd (Wuhan, China) where the cDNA library was constructed using NEBNext1 Ultra™ RNA Library Prep Kit for Illumina1 (NEB, E7530) according to the manufacturer's instructions. In brief, the first-strand and the secondstrand cDNA were synthesized using approximately 250~300 bp RNA inserts, which were fragmented by the enriched mRNA. After end-repair/dA-tail and adaptor ligation, the suitable fragments of double-strand cDNA were isolated by Agencourt AMPure XP beads (Beckman Coulter, Inc.), and then enriched by PCR amplification. Finally, the purity and quality of the libraries were measured by Agilent 2100 Bioanalyzer and Qubit 2.0. The eighteen cDNA libraries prepared were sequenced by Biomarker Technologies (Wuhan, China) using the Illumina HiSeq 2500 platform with pair-end 150 nt. RNA-seq was performed as previously described [23]. The transcriptome sequencing data from this study were available from the NCBI SRA database under BioProject accession number PRJNA693027.

Transcriptome analysis using reference genome-based reads mapping
The quality check was performed to eliminate low quality reads with the only adaptor, unknown nucleotides> 5%, or Q20< 20% using SOAPnuke-2.1.0 [28]. The high-quality clean reads that were filtered from the raw reads were mapped to the reference genome of cultivated pepper Zunla-1 (C. annuum L.) (https://www.pnas.org/content/111/14/5135) with TopHat 2.0 software [29][30][31]. Potential duplicate molecules were removed by examining aligned records from the aligners in BAM/SAM format [32]. Fragments per kilobase of transcript per million fragments mapped read (FPKM) values were used to calculate the gene expression levels based on Cufflinks software [33].

Identification and functional analysis of DEGs
DEGs were identified using DEGseq2 in the four comparisons of ECW_6H-vs-ECW_0H, ECW_24H-vs-ECW_0H, VI037601_6H-vs-VI037601_0H and VI037601_24H-vs-VI037601_0H [34]. The fold change of genes was calculated based on the ratio of the FPKM values. The genes with an absolute value of |log2(fold change)|�1 and the false discovery rate (FDR) values <0.05 were accepted to represent significant DEGs, which were used for further analysis.

Identification of transcription factors (TFs)
Transcription factors were identified using PlantTFDB (http://planttfdb.gao-lab.org/), which included the sequences of 58 plant transcription factor families from 165 plant species [37]. The unigene sequence was compared with the transcription factor database by BLASTx alignment, and the gene with the best E-value less than 10 −5 was selected as the annotation information of the unigenes.

Quantitative RT-PCR (qRT-PCR) analysis
To validate the RNA-Seq data, the relative expression levels of randomly selected DEGs were examined by quantitative Real-time PCR (qRT-PCR). The corresponding mRNA sequences of the selected genes were searched from the Sol Genomics Network (SGN) (https://www.solgenomics.net/). All primers for qRT-PCR were designed according to the transcript sequences using Primer Premier 5.0 (http://www.premierbiosoft.com/). The primers used in this experiment are listed in S11 Table. Approximately 2 μg of total RNA was isolated from infected leaves of ECW and VI037601 by TRIzol reagent, which was used to synthesize the cDNA through the cDNA synthesis kit (TransGen, Beijing, China) according to the manufacturer's instructions. Quantitative RT-PCR (qRT-PCR) was performed in 96-well plates on Thermo Fisher Scientific Biosystems QuantStudio 5 Real-Time PCR system (Applied Biosystem, MA, USA) using SYBR Premix Ex Taq™ Kit (Takara, Dalian, China). The protocols of qRT-PCRs were used as follows: 95˚C for 5 min, followed by 40 cycles of 95˚C for 10 s, 58˚C for 20 s, and 72˚C for 15 s, plus melting curves to verify PCR products. Ubiquitin-conjugating protein CaUbi3 (Accession Number: AY486137.1) was used as an internal reference [38]. Samples were collected as previous in this study, and three independent biological replicates were analyzed. The relative expression level of the selected genes was calculated with the 2 -ΔΔCT method [39].

RNA sequencing of pepper leaves after Xcv infection and assembly of transcriptome
First, to confirm the resistance of ECW and VI037601 to Xcv, an injection inoculation was carried out. The results showed that the symptoms were similar between resistant and susceptible plants by 24 hours post inoculation (hpi) (Fig 1). However, a hypersensitive response (HR) symptom was observed in VI037601 containing the R gene Bs1 at 24 hpi, whereas cultivar ECW presented no HR symptom at either timepoints, indicating that VI037601 and ECW may have different responses to Xcv infection at the transcriptome level (Fig 1).
To accurately evaluate the comparative expression of genes in ECW and VI037601, eighteen cDNA libraries were used for RNA sequencing. Approximately 120.23 G clean bases were generated using an Illumina HiSeq 2500 sequencing platform. After quality control, each library contained between 22,079,427 to 27,158,399 clean read pairs. GC contents were ranged from

Expression analysis and identification of differentially expressed genes
To investigate the expression patterns of genes in pepper leaves during the different stages after Xcv infection, a total of 11,232 DEGs were identified in ECW and VI037601 at 6 hpi and 24 hpi, including 3,361 novel differentially expressed genes (S4 Table). Among them, 1,306 (1,186 up regulated and 120 down regulated) and 8,006 (4,328 up regulated and 3,678 down regulated) DEGs were found at 6 hpi and 24 hpi in ECW, respectively. 3,229 (2,713 up regulated and 516 down regulated) and 7,562 (3,984 up regulated and 3,578 down regulated) DEGs were identified at 6 hpi and 24 hpi in VI037601, respectively (Fig 2A and 2B and S4 Table). However, 999 (920 commonly up regulated and 73 commonly down regulated) and 2,274 (1,953 commonly up regulated and 290 commonly down regulated) DEGs overlapped at 6 hpi and 24 hpi in ECW and VI037601, respectively (Fig 2A and S4 Table). Interestingly, 852 overlapping DEGs were found in ECW and VI037601 post Xcv inoculation (Fig 2A and S5 Table), and 364 DEGs were specific differentially expressed in VI037601 post Xcv inoculation at different time points (Fig 2A and S5 Table). Overall, the resistant genotype, VI037601, had greater number of DEGs at 6 hpi, especially up regulated DEGs whereas the susceptible genotype ECW had greater number of DEGs at 24 hpi (Fig  2A and 2B) suggesting that the host plant response to Xcv infection is different between the two genotypes. Moreover, these DEGs might contain the disease resistance gene(s), such as Bs1, which conferred resistance to Xcv in VI037601.

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Transcriptome analysis of pepper in response to Xanthomonas campestris pv. vesicatoria

Functional enrichment analysis of DEGs
In total, 6,334 of 11,232 DEGs in the four comparisons (ECW_6H-vs-ECW_0H, ECW_24Hvs-ECW_0H, VI037601_6H-vs-VI037601_0H and VI037601_24H-vs-VI037601_0H) were annotated with GO terms and assigned to three categorie (Fig 3 and S6 Table). The DEGs in VI037601_6H-vs-VI037601_0H were most enriched in defense response (GO:0006952), protein phosphorylation (GO:0006468), protein modification process (GO:0036211) in BP categories, integral component of membrane (GO:0016021) and intrinsic component of membrane (GO:0031224) in CC categories, protein kinase activity (GO:0004672), protein serine/threonine kinase activity (GO:0004674), and transcription factor activity, sequence-specific DNA binding (GO:0003700) in MF categories ( Fig 3C and S6 Table). GO functional enrichment analysis of the ECW_6H-vs-ECW_0H group revealed a similar classification as the VI037601_6H-vs-VI037601_0H group (Fig 3A and S6 Table). The DEGs in VI037601_24Hvs-VI037601_0H were most enriched in single-organism process (GO:0044699), single-organism metabolic process (GO:0044710), carbohydrate metabolic process (GO:0005975), secondary metabolic process (GO:0019748) in BP categories ( Fig 3B). Catalytic activity (GO:0003824) and oxidoreductase activity (GO:0016491) dominated MF categories (Fig 3D and S6 Table). The similar GO enrichment classification to VI037601_24H-vs-VI037601_0H groups were also found in ECW_24H-vs-ECW_0H (Fig 3B and S6 Table). These processes associated with disease resistance were enriched, indicating that the corresponding genes of these significant terms might play important roles in resistance to Xcv inoculation.

Transcriptional changes in response to Xcv infection
Many genes play a critical role in recognizing pathogen-associated molecular patterns (PAMPs) and subsequently activating plant defense mechanisms in response to pathogen attacks, such as kinases, pathogenesis-related (PR) protein, oxidoreductase and E3 ubiquitinprotein ligase [23,40]. In this study, 541 pattern recognition receptors (PRRs), 30 MAPK, 246 resistance proteins (R Proteins), and 83 calcium signaling genes were identified by searching

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Transcriptome analysis of pepper in response to Xanthomonas campestris pv. vesicatoria the keywords in the gene annotation (S8 Table). Among these DEGs, 24 protein kinases, 13 disease resistance proteins and 4 receptor-like proteins were specific differentially expressed in VI037601 post Xcv inoculation (S9 Table). We also found that Capana00g000272 (calcineurin B-like protein), Capana04g001405 (carboxylesterase), Capana09g000319 (aldehyde dehydrogenase), and Capana09g000326 (glycosyltransferase) were significantly differentially expressed in VI037601 post Xcv inoculation, but almost not expressed in ECW (S5 Table). Moreover, 83 overlapping differentially expressed kinase response genes, including 13 LRR receptor-like ser/ thr protein kinase, were identified and up regulated in ECW and VI037601 at 6 hpi and 24 hpi (Fig 5A and S9 Table). Besides that, 30 common DEGs encoding other disease response

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proteins were also identified in ECW and VI037601 at 6 hpi and 24 hpi, such as disease resistance proteins, pathogenesis-related proteins and receptor-like proteins (Fig 5A and S9 Table). Interestingly, all of these DEGs were up-regulated at different time points after Xcv inoculation in ECW and VI037601 (Fig 5A and S9 Table). Other overlapping BS disease response genes, including 21 DEGs encoding cytochrome P450, 9 DEGs encoding E3 ubiquitin-protein ligase, 3 DEGs encoding oxidoreductase and 11 DEGs encoding chitinase were also differentially expressed and their expression levels increased after Xcv infection in ECW and VI037601 ( Fig  4B and S9 Table). However, the expression analysis of Bs resistance genes showed that bs2 (Capana09g000438) and bs3 (Capana02g001306) were not/hardly expressed in pepper leaves before and after Xcv inoculation. Furthermore, the expression of their homologs did not change significantly (S3 Table).

The response of differentially expressed transcription factors to Xcv infection
In plants, transcription factors (TFs) play important roles in the regulation of different physiological and biochemical programs in response to plant-pathogen interaction [23]. In our study, 551 DEGs involving 53 TF families were identified, which included 69 zinc finger proteins (ZFPs), 65 ethylene-responsive transcription factor (ERFs), 64 MYB, 32 NAC, and 46 WRKY TFs (S10 and S12 Tables). Among them, most DEGs were up regulated in the four groups (S10 Table). Moreover, 21 TFs, including ZFPs, MYB, WRKY and ERFs were specific differentially expressed in VI037601 post Xcv inoculation (S9 Table). These identified TFs might be likely to perform an important role in pepper -Xcv interaction. Besides that, 63 putative overlapping TFs were differentially expressed in ECW and VI037601 after Xcv infection (Fig 6 and S9 Table). Interestingly, among these differentially expressed TFs, all DEGs encoding MYB, WRKY, ethylene responsive factor (ERFs), HSF, MYB, and bHLH TFs were up regulated after Xcv infection in ECW and VI037601, except for Capana06g001119 (ERF), Capana09g000142 (ERF), and Capana02g003201(ZFPs) (Fig 6 and S9 Table). Thus, enhanced activity of these up-regulated TFs suggests that they may be regulated in multiple ways by cis-acting sequences in response to Xcv infection. Nevertheless, different down regulated DEGs might also play an important role by negatively regulating the pepper immunity upon Xcv infection in ECW and VI037601.

Validation of RNA-seq data by qRT-PCR
To confirm the accuracy of RNA-seq data, transcriptional levels of 16 randomly selected DEGs representing a wide range of expression levels and patterns were detected in ECW and VI037601 post Xcv inoculation by qRT-PCR analysis (Fig 7). Among these 16 selected genes, majority of these DEGs were associated with massive defense response processes including receptor kinase (Capana01g001931 and Capana09g001638), protein kinase (Capana00g002502 and Capana03g000831), pathogenesis-related genes (Capana03g004445 and Capa-na04g001453), ERF (Capana01g000661), MYB TF (Capana05g002225), NAC TF (Capa-na07g001015), WRKY TF (Capana09g001251 and Capana00g000056), zinc finger protein transcription factor (Capana12g000773), disease resistance protein (Capana12g002356), and secondary metabolite biosynthesis (Capana01g001748, Capana04g000463 and Capa-na10g002483) (Fig 7). The fold changes varied in RNA-Seq and qPCR analyses. However, the expression data provided by qRT-PCR were following the profiles detected by RNA-seq at all time points in ECW and VI037601. These results suggested the reliability of RNA-seq to analyze the transcriptome of resistant and susceptible plants after Xcv infection.

Discussion
Plants are exposed to a myriad of pathogenic microorganisms during their lifespan, including bacteria, fungi, viruses and nematodes, all of which try to acquire nutrients from the host plant for their advantage [41]. BS caused by Xcv is a very serious global disease, which has caused enormous yield and economic losses in pepper production, especially in regions with a warm and humid climate. In response to bacterial attack, plants deployed various defense responses, which were mainly activated by two branches of their immune system. One was the transmembrane pattern recognition receptors (PRRs) that initiated immune responses upon recognition of extracellular pathogen-associated molecular patterns (PAMPs). It was common to many kinds of microbes. The other was the products of resistance (R) genes that specifically recognized corresponding pathogen effectors, which were regarded as avirulence (Avr) factors [42]. Previous study revealed that cultivar carried Bs1 was considered as a resistant material to BS

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Transcriptome analysis of pepper in response to Xanthomonas campestris pv. vesicatoria due to hypersensitive response reaction to Xcv containing avrBs1, while non-hypersensitive reaction in response to infiltration of the bacterial suspension into leaf tissues appeared in ECW [43], which was consistent with the results in this study (Fig 1). However, the molecular mechanism of VI037601 and ECW in response to Xcv infection was unclear. In this study,

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RNA-seq technique was used to identify the DEGs associated with disease response during Xcv infection in the leaves of ECW and VI037601. The Q30 of clean reads in 18 samples were at least 89.3% and the mapping ratio of samples to the reference genome was above 91% (S1 Table), indicating that the quality of sequencing data was reliable. Here, the average products of each sample was 6.9 Gb exceeding the sample with 4.9 Gb successfully used to gain insight into CMV infection genes [23], indicating that the sequencing depth was sufficient for the transcriptome coverage. Although the fold change of the gene in qRT-PCR analysis was inconsistent with that in RNA-Seq. The trend of change was consistent in qRT-PCR and RNA-Seq, which indicated that the RNA-seq data was reliable for analyzing the transcriptome of resistant and susceptible plants after Xcv inoculation (Fig 7). Therefore, our transcriptome data is competent to analyze the defense-related genes and pathways against to Xcv in pepper.
Plants have an innate immunity system to defend themselves against pathogens by a number of mechanisms, such as hypersensitive response (HR), induction of genes encoding PR and/or induced biosynthesis of secondary metabolites [44,45]. The functional analysis of DEGs in ECW and VI037601 post Xcv inoculation demonstrated that many biological processes were influenced by pathogen infection (Fig 4 and S7 Table). Many disease resistance pathways were most enriched at 6 hpi in both ECW and VI037601, such as defense response (GO:0006952), protein phosphorylation (GO:0006468), protein modification process (GO:0036211), protein kinase activity (GO:0004672), protein serine/threonine kinase activity (GO:0004674), and transcription factor activity, sequence-specific DNA binding (GO:0003700). Moreover, many secondary metabolic pathways such as oxidoreductase activity (GO:0016684), acting on peroxide as acceptor (GO:0016684), peroxidase activity (GO:0004601), chitinase activity (GO:0004568), chitin binding (GO:0008061), chitin catabolic process (GO:0006032), and chitin metabolic process (GO:0006030) were enriched at 24 hpi both in ECW and VI037601 (Fig 3 and S6 Table). Similar results were obtained in the previous study of tomato in response to infection by Xanthomonas perforans Race T3 [27]. The expression of these defense response genes induced the synthesis of secondary metabolites, which could inhibit the spread of Xcv in peppers. However, the number of enriched DEGs in VI037601 were more than twice the number of enriched DEGs in ECW in many significantly enriched GO terms, such as defense response, protein phosphorylation, protein modification process, protein serine/threonine kinase activity and transcription factor activity, sequence-specific DNA binding (Fig 3 and S6 Table). These DEGs might conferred the resistance of VI037601 to Xcv.
In plants, HR is a form of programmed cell death (PCD) at the site of pathogen infection, which is closely related to active resistance [46]. Previous studies showed that Bs2 and Bs3 were only expressed in BS resistant pepper post Xcv inoculation, which could trigger HR [16,17]. Here, transcriptome profiling analysis results showed that bs2 (Capana09g000438) and bs3 (Capana02g001306) were not/hardly expressed in pepper leaves before and after Xcv inoculation (S3 Table). Moreover, the expression of their homologs also did not change significantly (S3 Table). The results indicated that Bs1 gene that conferred resistance to Xcv in VI037601 may not be a homolog of Bs2 and Bs3. However, many proteins kinases/enzymes encoded by DEGs were involved in defense-related gene induction and innate immunity, such as those that activate genes coding for the receptor-like kinases (RLKs), NAC TFs, WRKY TFs, pathogenesis-related protein and chitinase, as reported previously [23,[47][48][49]. In this study, 1,599 potentially defense-related genes linked to pattern recognition receptors (PRRs), mitogen-activated protein kinase (MAPK), calcium signaling, and transcription factors may regulate pepper resistance to Xcv. Moreover, 364 DEGs including protein kinase, oxidordeuctase, TFs and uncharacterized proteins were specific differentially expressed in VI037601 post Xcv inoculation, such as Capana02g003523 (receptor-like protein kinase), Capana02g000918 (WRKY), Capa-na12g000410 (peroxidase) and Capana01g000533 (uncharacterized protein) (S5 and S9 Tables).
Interestingly, Capana00g000272 (calcineurin B-like protein), Capana04g001405 (carboxylesterase), Capana09g000319 (aldehyde dehydrogenase) and Capana09g000326 (glycosyltransferase) were also specifically expressed in VI037601, and the expression of which was significantly upregulated after Xcv inoculation (S5 Table), indicating that they might play an important role in response to Xcv infection in VI037601. Receptor-like kinases are key pattern recognition receptors in response to pathogens [50]. Our findings also showed that many receptor-like kinases were significantly differentially expressed in ECW and VI037601, such as G-type lectin S-receptor-like serine/threonine-protein kinase (Capana07g002260) and LRR receptor-like serine/threonine-protein kinase (Capana03g000831), which were up regulated in ECW and VI037601 post Xcv inoculation (Fig 4 and S5 and S8 Tables). RLKs were important signaling components that played key roles in adapting to numerous biotic and abiotic stresses as well as in regulating plant growth and development [42,51]. Generally, the mitogen-activated protein kinase (MAPK) cascades were initiated by the stimulated receptors. After a series of cascades reactions, activated MAPKs phosphorylated their substrates, most of which were enzymes and transcription factors, thereby triggering downstream responses [52].
WRKY TFs as the substrates of MAPKs can be regulated by MAPKs at transcriptional and/ or post-translational levels [53][54][55]. For instance, OsWRKY53 was activated by OsMPK3 and OsMPK6 through transcriptional induction and phosphorylation in the process of pathogen infection, thereby enhancing rice resistance to pathogens [52,56]. Here, 17 WRKY TFs were also up-regulated in ECW and VI037601 post Xcv inoculation (Fig 5 and S5, S9 and S10 Tables), which might be induced by MAPKs. Up-regulated expression of these WRKY TFs could activate downstream disease response genes or hormones pathway-related genes to protect against pathogen infection [40]. Besides, many studies showed that TFs that contain the NAC domain played pivotal roles in the regulation of the transcriptional reprogramming associated with plant stress responses, such as abiotic stress response and pathogen defense [57]. These NAC proteins might positively regulate plant defense responses by activating PR genes. One such example is ATAF1, which positively regulated penetration resistance to biotrophic fungus Blumeria graminis f.sp. hordei (Bfh) [58]. OsNAC6, ONAC066, ONAC122, ONAC131, and OsNAC4 have been validated to be involved in defense responses against pathogen attack [59][60][61][62]. Here, the homologs of these NAC TFs were upregulated post Xcv inoculation in ECW and VI037601, such as Capana06g001739, Capana05g000569 and Capana11g001813 (S9 and S10 Tables). MYB TFs also played important roles in response to pathogen infection. Overexpression of SmMYB44 in eggplant increased the resistance to bacterial wilt [63]. Previous study showed that TFs from the stress-related families ERFs, bZip, MYB and WRKY closely associated with the non-host response to Xanthomonas campestris pv. vesicatoria in Citrus sinensi [48]. Here, two MYB TFs were differentially expressed in VI037601, while three MYB TFs were up-regulated in both VI037601 and ECW post Xcv inoculation (Fig 6 and S9 and S10 Tables), which might play pivotal roles in non-host response to Xcv.
Moreover, phytohormones serve as key factors in plant responsiveness to stresses, and hormone-responsive genes are often used to qualitatively and quantitatively evaluate disease resistance responses during pathogen infection [64][65][66][67][68]. Plant resistance to biotrophic pathogens is positively regulated by ethylene and is negatively regulated by the auxin signal transduction pathway [64,65]. Ethylene-responsive transcription factors mediated disease resistance was demonstrated in Arabidopsis and tomato against Botrytis cinerea and Ralstonia solanacearum, respectively [66][67][68]. Phytohormones were also involved in the non-host response of Citrus sinensis to Xanthomonas campestris pv. vesicatoria [69]. Here, 112 DEGs involved in plant hormone signal transduction were identified (S8 Table), and the results showed that almost DEGs involved in ABA, ETH, GA, and SA signal transduction were up regulated, while most DEGs participated to IAA and CTK signal transduction were down regulated after Xcv inoculation (S8 Table). Thus, these DEGs might implicate their roles in the regulation of transcriptional reprogramming associated with the response to Xcv in pepper.

Conclusions
In this study, we performed a transcriptome analysis to reveal the defense related genes and pathways of resistant and susceptible pepper varieties after Xcv inoculation. A total of 120.23 Gb clean bases were generated and 11,232 DEGs were identified in 18 libraries. DEGs involved in PRRs, MAPK signaling, calcium signaling, phytohormone signaling pathways, TFs pathways and secondary metabolism, which were reported previously as relevant to defense response, were explored. To our best knowledge, this is the first study that examined global transcriptional changes in pepper plants infected with Xcv, which provides new knowledge and ideas for improving of peppers to avoid BS.
Supporting information S1