Analysis of the Defence Responses to Tomato Spotted Wilt Orthotospovirus (TSWV) and Hippeastrum Chlorotic Ringspot Orthotospovirus (HCRV) Infection in Capsicum Annuum Based on Transcriptome

Background: Orthotospovirus has a wide range of hosts, widely distributed in southwestern China and have serious harm to crop production. Tomato spotted wilt orthotospovirus (TSWV) and Hippeastrum chlorotic ringspot orthotospovirus (HCRV) are seriously harms the quality of pepper (Capsicum annuum L.). However, the detailed molecular mechanism of pepper disease caused by TSWV and HCRV remains obscure. Method: Transcriptome analysis (RNA-seq) based on high-throughput sequencing, with TSVV, HCRV and mock-inoculated plants as controls, to investigate and compare the gene expression changes in pepper leaves, and analyze the plants invaded by orthotospoviruses defensive response. Results: The RNA-Seq results of pepper 1 day after inoculation (1 dpi) showed that the number of differentially expressed genes(DEG) after HCRV infection was greater than that after TSWV infection. Gene Ontology enrichment and KEGG pathway analyses were performed on the two viruses, and the KEGG results showed that DEG were mainly enriched in plant-pathogen interactions, plant hormone signal transduction, and defence processes. Conclusion: The conclusion of this study is that after orthotospovirus infects pepper, the DEG changes in the processes of plant-pathogen interaction, plant hormone and phenylpropanoid synthesis. In addition, transcription factors such as WRKY and MYB changed signi�cantly. These factors comprehensively affect the plant's defence response and ultimately enhance the plant's resistance to viruses. Findings of present study will signi�cantly help enhance our understanding of the complicated defense process of plant responses to orthotospoviruses infection.

High-throughput technology has been widely used in the study of orthotospoviruses and plant interactions.Microarray and high-throughput sequencing technologies expressing sequence tags were used to study the molecular responses of INSV and TSWV host plants.The results showed that INSVinfected tobacco (Nicotiana benthamiana) involved plant defence after INSV infection compared with the control, with upregulation of genes involved in metabolic processes, such as signal transduction and transcription, and downregulation of genes involved in histone expression [11].TSWV infection also resulted in the upregulation of ethylene-mediated signal transduction pathways and chitin response genes in Dendranthema morifolium (Ramat.)Tzvelev and was involved in the downregulation of genes in DNA metabolism, such as DNA replication, histone modi cation and cytokinesis [12].After TZSV infects pepper (Capsicum annum L., 'Zunla-1') and N. benthamiana, the accumulation of jasmonic acid (JA) increases, and salicylic acid (SA) decreases.It was also proven that JA and SA inhibited the tness of western ower thrips (Frankliniella occidentalis), and 2102 differentially expressed genes (DEG) were identi ed after tobacco infection.These DEG are mainly related to biological functions such as metabolic processes, redox processes, and protein kinase activity.KEGG pathway analysis shows that the pathogenesis of TZSV may affect the primary and secondary metabolism of tobacco and the interactions between plants and pathogens [13,14].However, there have been few reports on the study of the two viruses of Orthotospovirus on the same plant.Therefore, the differences in hormone signal transduction, plant defence response and secondary metabolism between the two orthotospoviruses after infection of the same plant are the focus of this study.
Orthotospoviruses are very harmful to crops, but there are still many challenges in effective prevention and control.Disease resistance breeding, phytosanitation, cultivation control strategies, and the use of pesticides and nonbiological or biological inducers to enhance plant resistance to pathogens are of great signi cance to the prevention and control of viruses [15,16].The basis of disease resistance breeding is to analyse the changes in metabolic pathways and differential genes in the process of the plant defence response after orthotospoviruses infect plants to conduct in-depth research on the disease resistance characteristics of host plants.The near-isogenic Sw-7 line and its susceptible recurrent parent material were used for comparative transcriptome and gene expression analyses to identify processes such as host resistance and RNA silencing/antiviral defence as well as key transcription and translation regulatory factor networks of 1244 differential genes and to verify that the PR-5 gene is involved in the resistance process of Sw-7 to TSWV [17].
Through a dynamic transcriptomics study after TSWV infects Arabidopsis thaliana, the results showed that the plant immune defence and protein degradation processes are enhanced after TSWV infects the plant, and photosynthesis and cell wall metabolism are weakened [18].In addition, plant transcription factors play important roles in plant growth and biotic and abiotic stresses.WRKY proteins are a superfamily of transcription factors that participate in many plant processes, including defence responses to biotic and abiotic stresses [19][20][21][22][23][24][25].Therefore, this study used high-throughput sequencing technology to study the defence responses of two orthotospoviruses after infecting peppers and compared them.A comparative transcriptomics analysis method was used to identify the host plant defence-related metabolic processes and the changes in differential genes and transcription factors.The prevention and control of orthotospoviruses provide a reference.

Pepper cultivation and virus preparation
Tomato spotted wilt orthotospovirus (TSWV, GC-1 isolate) and Hippeastrum chlorotic ringspot orthotospovirus (HCRV, HLS1-2 isolate) were isolated from Yunnan Province, China, and stored in our laboratory.Their entire genomes have been sequenced.When in use, the virus was removed from the refrigerator and inoculated by friction on N. benthamiana to activate the virus for subsequent experiments.
The experimental material Capsicum annuum (Solanaceae) used in this study was from Hunan Xiangyan Seed Industry Co., Ltd(Changsha, Hunan province, China).TSWV can systematically infect Xiangyan 11, but HCRV cannot infect Xiangyan 11.The pepper seeds were planted in a plastic pot (10 cm×10 cm×10 cm) with a mixture of soil and humus, and the plants were grown in a greenhouse at 25±2℃, with a constant relative humidity of 65%±5% and a photoperiod of 16:8 (light: dark).The plants were watered twice a week until they grew to the 4-leaf stage, HCRV and TSWV activated viruses were used for mechanical friction inoculation.The control group was treated with inoculation buffer and placed in a greenhouse under the same conditions for growth.After rubbing inoculation with the two viruses, the top three leaf samples were collected (3 biological replicates), frozen in liquid nitrogen immediately, and stored in a -80°C freezer before being prepared for RNA-Seq.Each sample consisted of leaves of 10 plants.The samples were named NC (Xiangyan 11 healthy plants), CK (Xiangyan 11 control group), H (Xiangyan 11 treatment group inoculated with HCRV virus) and T (Xiangyan 11 treatment group inoculated with TSWV virus).There were 3 biological replicates in each group and a total of 12 pepper samples (BioSample accessions: PRJNA665356).

RNA extraction and qRT-PCR experiment
The experimental process of extracting total RNA from 12 pepper leaf samples by the TRIzol method was performed according to the TianGen Biotech manual (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP421).
To determine the time of sample collection, we selected plant leaves with similar plant symptomatic characteristics and growth status in 4 periods (1, 2, 3, and 7 dpi) for qRT-PCR experiments.Using the N gene sequences of HCRV (Accession number: JX833564) and TSWV (Accession number: JN664252) as the reference sequences, Beacon Designer 7.9 (Premier Biosoft International, Palo Alto, CA) was used to design the primers in the qRT-PCR experiment (Table S5), the internal reference gene was UBI3 (Accession number: AY486137.1)[26],the experimental samples were the inoculation samples and CK in 4 periods, and 3 biological replicates were performed for the samples in each period.qRT-PCR was carried out on a qTOWER 2.2 system (Analytik Jena, Jena, Germany) with TB Green Premix Ex Taq II (Takara Biomedical Technology (Beijing) Co., Ltd.).The 2 -ΔΔCT method was used to calculate gene expression [27].We analysed the accumulation of virus genes in the four periods and nally selected the leaves of resistant and susceptible pepper varieties from a speci c period as the follow-up experimental materials.

Transcriptome assembly and differentially expressed genes (DEG) analysis
To ensure data quality, it is necessary to perform quality control and ltering on sequencing data before information analysis.We further rigorously ltered the clean reads obtained through preliminary ltering after sequencing to obtain high-quality clean reads for subsequent information analysis.
The read comparison tool Bowtie2 (2.2.8) was used to compare high-quality clean reads to the ribosomal RNA of the species (mismatch number: 0), remove the reads of the aligned ribosomal RNA, and use the retained data for subsequent analysis.The comparison software TopHat2 (2.1.1)was used to lter out the reads of ribosomal RNA and to compare them to the pepper reference genome (Capsicum Annuum.v.2.0, CM334, Accession number: AYRZ01000000).The known and new mRNAs identi ed and predicted for each sample were statistically summarized.HTSeq[28] was used with a joint model to calculate the FPKM value (fragment kilobase of exon model per million mapped reads) to obtain the gene expression level [29].
edgeR software was used to analyse differences in mRNA expression between groups, and FDR and log2FC were used to screen differential transcripts.The screening conditions were FDR<0.05 and |log2FC|>1.After obtaining the differentially expressed transcripts, GO and KEGG analyses were performed on the differentially expressed transcripts, and Gene Ontology (GO) enrichment analysis was performed for DEG based on the GOenq (1.10.0) and topGO (2.10.0)R packages [30].KOBAS software (Beijing, China) was used to detect the DEG in the KEGG pathways [31].Both annotation analyses used expressed genes (FPKM≥1) as background and corrected p≤0.05.

Quantitative reverse-transcription PCR
To verify the expression of differential genes, the RNA previously extracted was used for quantitative reverse-transcription PCR (qRT-PCR) experiments, and the 2 -ΔΔCT method was used to calculate the expression levels of genes [27].Beacon Designer 7.9 (Premier Biosoft International, Palo Alto, CA) was used to design primers for qRT-PCR experiments (Supplemental Table S1).The instrument used for uorescence quanti cation was a qTOWER 2.2 uorescence quantitative PCR system (Analytik Jena, Jena, Germany).SPSS 26.0 (SPSS Inc., Illinois, USA) was used to perform one-way analysis of variance (ANOVA) on the signi cant difference between means of the qRT-PCR results.Duncan's test was used, and the signi cance level was set to p< 0.05.

Symptoms and qRT-PCR experimental results after virus inoculation
After Xiangyan 11 was inoculated with HCRV and TSWV, the trends of the two viruses in pepper plants were inconsistent.The maximum accumulation of viruses was reached during the 1 dpi period.At 0-1 dpi, HCRV and TSWV both showed increasing trends.
During the period of 1-2 dpi, the accumulations of HCRV and TSWV gradually decreased; thus, 1 dpi was the time point for the maximum accumulation of the two viruses.During the time period of 3-7 dpi, the accumulation of HCRV and TSWV gradually increased.Finally, we chose 1 dpi samples and sent them to Gene Denovo Biotechnology Co., Ltd.(Guangzhou, China) for high-throughput sequencing (Fig. 1).

Transcriptome sequencing results
To obtain the differences in gene expression of Xiangyan 11 inoculated with HCRV and TSWV viruses, we sequenced the leaves of Xiangyan 11 inoculated with the 2 viruses at 1 dpi on an Illumina HiSeq™ 2500 platform and constructed libraries with the sequencing data.In total, 12 cDNA libraries were prepared using poly(A) enrichment from 3 replicates of 4 treatments consisting of samples (NC1, NC2, NC3, CK1, CK2, CK3, T1, T2, T3 and H1, H2, H3). cDNA libraries underwent quality assessment and cleaning, and each sequencing library obtained more than 69 million clean reads (Supplemental Table S2).The Q20 and Q30 values of all libraries exceeded 98.46% and 94.76%, respectively, indicating that these data were suitable for further analysis (Table 1).The original data from the 12 cDNA libraries were saved to the SRA database with accession numbers SRR12726982-SRR12726994.  S3).
Cu inks software was used to reconstruct transcripts based on Tophat2 comparison results and screen the positions of the assembled transcripts on the reference genome for new transcripts.The screening criteria were transcript length ≥ 200 bp and exon number ≥ 1.The number of known mRNAs obtained through screening exceeded 14,669, more than 34,434 new mRNA transcripts were obtained, and the total number of mRNA transcripts exceeded 49,181 (Supplemental Table S4).
We applied stringent ltering criteria to select highly signi cant differentially expressed genes (DEG) with at least two-fold differences between the two conditions by setting a cut-off q-value < 0.01 and log2-fold change > 1 (Fig. 2A).To better understand the diversity of DEG between each treatment, a Venn diagram was generated (Fig. 2B), and 36 DEG existed in all 3 comparison groups.The results showed that the expression levels of genes changed during HCRV and TSWV infection.
In this study, four comparison groups (CK-vs-H, CK-vs-T, T-vs-H and NC-vs-CK) were selected for functional annotation analysis.After obtaining the differentially expressed transcripts of each comparison group, GO (Gene Ontology) functional analysis and KEGG pathway (euKaryotic Orthologous Groups) analysis were performed on the DEG.
Compared with the control group (CK), the H treatment group recorded a total of 2617 DEG belonging to 40 functional groups, and the T treatment group recorded a total of 1587 DEG belonging to 37 functional groups, including biological processes, cell components and molecular functions (Supplemental Figure S1).
To further clarify the response mechanisms of DEG to virus infection of plants, the KEGG database was used to classify and characterize transcriptome DEG into corresponding pathways.Compared with the control group, the H comparison group (CK-vs-H) had 658 enriched DEG in 115 pathways.Among these pathways, the 13 pathways with the most signi cant enrichment (p-value < 0.05) were ribosome, protein processing in the endoplasmic reticulum, carbon xation in photosynthetic organisms, photosynthesisantenna protein, stilbenoid, diarylheptanoid and gingerol biosynthesis, pyruvate metabolism, biosynthesis of amino acids, avonoid biosynthesis, alanine, aspartate and glutamate metabolism, DNA replication, nitrogen metabolism, microbial metabolism in diverse environments, and arginine biosynthesis.In addition, metabolic pathways related to plant immunity, plant-pathogen interactions, plant hormone signal transduction and other pathways were involved in plant immunity DEG (Fig. 3A).Compared with the control group, the T treatment group (CK-vs-T) exhibited 315 enriched DEG in 103 pathways.Among these pathways, the 14 pathways with the most signi cant enrichment (p-value < 0.05) were DNA replication, phenylpropanoid biosynthesis, stilbenoid, diarylheptanoid and gingerol biosynthesis, biosynthesis of secondary metabolites, avonoid biosynthesis, spliceosome, α-linolenic acid metabolism, pyruvate metabolism, metabolic pathways, plant circadian rhythm, amino sugar and nucleotide sugar metabolism, ribo avin metabolism, glutathione metabolism, and protein processing in the endoplasmic reticulum (Fig. 3B).In the T-vs-H comparison group, 251 DEG were enriched in 99 pathways.Among these pathways, the ve pathways with the most signi cant enrichment (p-value < 0.05) were the photosynthesis-antenna protein pathway, ribosome, glutathione metabolism, carotenoid biosynthesis, and monoterpenoid biosynthesis (Fig. 3C).In the NC-vs-CK comparison group, 125 DEG were enriched in 81 pathways.The 10 pathways that were signi cantly enriched (p-value < 0.05) were photosynthesisantenna protein pathways, diterpenoid biosynthesis, pyruvate metabolism, amino sugar and nucleotide sugar metabolism, biosynthesis of unsaturated fatty acids, phenylalanine, tyrosine and tryptophan biosynthesis, tryptophan metabolism, α-linolenic acid metabolism, cutin, suberine and wax biosynthesis, and biosynthesis of secondary metabolites (Fig. 3D).

Identi cation and analysis of plant responses to DEG
To further analyse the immune responses of the two viruses in infected plants, we focused on the plantpathogen interaction pathways, plant hormone signal transduction and phenylpropanoid synthesis pathways enriched by DEG (Fig. 4, Supplemental Table S5).
When a plant is infected by a pathogen, it triggers its own defence response and usually uses two defence mechanisms to resist invasion of the pathogen.In total, 18 genes were differentially expressed in the comparison group of H and control (CK-vs-H), 11 genes were differentially expressed in the comparison group of T and control (CK-vs-T), and 2 genes were differentially expressed in the comparison group of T-vs-H.Therefore, we found that the defensive response of HCRV to Xiangyan 11 was stronger than that of TSWV.
In the XY11-H sample, we found that most DEG were signi cantly upregulated (8 upregulated genes, 2 downregulated genes), including calcium-dependent protein kinase (CDPK), heat shock protein (HSP90), and LRR receptor protein kinase (FLS2), with the highest expression at 2.05.Heat shock proteins play a role in the defence process of ETI, and these genes cause a hypersensitive response (HR) in the process of plant resistance to pathogen infection (Fig. 4A).
In the plant hormone signal transduction pathway, it was found that many hormones were upregulated after infection with TSWV and HCRV.HCRV cannot successfully infect Xiangyan 11, but TSWV can successfully infect Xiangyan 11.After HCRV inoculation with Xiangyan 11, the expression levels of TIR1 and SAUR36 genes in the auxin synthesis pathway were signi cantly downregulated.The upregulated expression of SAPK1 in the abscisic acid (ABA) pathway may affect the synthesis of abscisic acid.In the synthesis of ethylene (ETH), EBF1 downregulates its expression, which relieves the inhibition of the ubiquitinated proteolytic pathway.In the jasmonic acid (JA) synthesis pathway, the two TIFY10A genes in the zinc jasmonic acid domain (JAZ) and the transcription factor MYC2 (bHLH14) are upregulated, and the upregulated expression of JAZ promotes the process of ubiquitinated proteolysis.The upregulated expression of JAZ and MYC2 is conducive to the synthesis of JA and the defence response of plants.The key genes histidine kinase 2 (AHK2) and B-ARR (ARR12) in the cytokinine synthesis pathway (CTK) are both upregulated, and the expression of A-ARR (ARR9) is downregulated.Since the A-ARR gene inhibits the expression of the B-ARR gene, the upregulated expression of the abovementioned genes ultimately increases the accumulation of CTK (Fig. 4B).
The secondary metabolism of plants plays an important role in the defence response of plants, and the synthetic pathway of phenylpropanoids is a common mechanism.The nal product of phenylpropanoid biosynthesis is lignin.The accumulation of lignin on the cell wall forms a physical barrier to pathogenic bacteria.In the phenylpropanoid biosynthesis pathway, the expression levels of caffeic acid methyltransferase (COMT), anti-cinnamic acid 4-monooxygenase (CYP73A), peroxidase (PER) and oxalate O-hydroxycinnamyl transferase (HST) were upregulated after HCRV and TSWV infection.When HCRV infected peppers, acetyl-CoA-benzyl alcohol acetyltransferase (BEAT), cinnamyl alcohol dehydrogenase (CAD), peroxidase (PER), oxalate O-hydroxycinnamyl transferase (HST), and CYP73A were all upregulated, increasing the accumulation of lignin, which is bene cial to the defence response of plants (Fig. 4C).

Changes in transcription factor expression in the plant defence response
Transcription regulation is an important mechanism of eukaryotic gene expression and plays an important role in plant defence responses.Transcription factor family (TF) analysis of DEG in 12 cDNA libraries showed that there were 2350 DEG distributed in 52 transcription factor families, of which C3H (674), bHLH (142), MYB (130), C2H2 (111), ERF (109), MYB-related (103), NAC (77), and WRKY (72), among others, are related to plant defence response (Fig. 5).
We analysed the TFs in the CK-vs-H and CK-vs-T comparison groups.The results showed that TFs such as C3H, MYB, and ARR-B in the CK-vs-H comparison group were upregulated, but WRKY, ERF and LBD TFs were downregulated (Fig. 6A); in the CK-vs-T comparison group, MYB, MYB_related, bHLH, bZIP and other TFs were upregulated, and ERF, WRKY and other TFs were downregulated (Fig. 6B).

qRT-PCR experiment results
To further con rm the gene expression pattern of the differentially expressed transcripts identi ed by RNA-Seq, we selected 10 annotated unigenes for qRT-PCR experiments (Fig. 7).These candidate genes are closely related to the transcription factor family and hormone and phenylpropanoid synthesis.The results showed that the 10 gene expression pro les obtained by qRT-PCR had high similarities with RNA-Seq data.These ndings con rm the reliability of the differential expression analysis data obtained by RNA-Seq and re ect the actual transcriptomic changes in the defence response of peppers after virus infection.

Discussion
Symptoms and transcriptome levels changed after infection of pepper with the two kinds of orthotospoviruses.At 7 dpi, TSWV-infested peppers showed leaf chlorosis and yellowing, followed by leaf deformities and concentric rings.After HCRV friction inoculation of pepper, local cell necrosis appeared on the inoculated leaves.As the plants grew, the new leaves on the upper parts of the inoculated leaves grew normally, and molecular testing revealed that there was no virus present.The resistance difference between TSWV and HCRV after infecting peppers may be related to the molecular structures of the viruses, the transport e ciency of virus particles in plants, the differences in avirulent (Avr) recognized by NLRs (nucleotide-binding leucine-rich repeat receptors), the different strains (TSWV is the American strain, HCRV is the Eurasian strain) and the types of resistance genes in peppers.
The results of comparative transcriptomics studies on peppers infected by TSWV and HCRV showed that a large number of hub genes were upregulated in hormone signal transduction, plant-pathogen interaction and secondary metabolic pathways after the plants were infected with the two viruses.
Previous researchers conducted a comparative transcriptome study after orthotospovirus TZSV was used to infect tobacco, and the KEGG pathway analysis of DEG after infection mainly consisted of multiple processes, such as primary and secondary metabolism, photosynthesis, and plant-pathogen interactions [13].The results of this study are essentially similar to the results of TZSV alone infecting tobacco, but the difference is that the DEG enrichment pathway in this study includes plant hormone signal transduction.The key JA synthesis genes JAZ (TIFY10A and MYC2) in the plant hormone signal transduction pathway were signi cantly upregulated, which ultimately promoted the synthesis of JA.The upregulated expression of JA is conducive to the synthesis of primary and secondary metabolites, such as nicotine and terpene indole alkaloids (TIAs), anthocyanins, artemisinin and other metabolites; as a result, the increase in JA expression is conducive to the disease resistance of plants [33][34][35][36].In addition, studies have found that the nonstructural protein NSs of TSWV inhibits plant MYC-regulated JA signal transduction and enhances its attractiveness to vectors [37].The main purpose of this study was to analyse the DEG in pepper following infection with the two viruses; the transmission vector of the virus was not studied.The interaction between the host and the virus can be evaluated in a follow-up study.
Plant transcription factors play an important role in the defence process of plants.Research results show that the expression levels of transcription factors such as WRKY, MYB, ERF, and MYC (BHLH) are changed after plants are infected by viruses, and these transcription factors participate in the signal transduction of plant hormones, plant-pathogen interactions, the synthesis of secondary metabolites and other pathways and, ultimately, enhance the defence capabilities of host plants [21,24,[38][39][40][41][42][43].The results of this research show that WRKY, MYB, ERF and other TFs underwent major changes compared with the CK after virus inoculation, which is similar to the results of a previous single orthotospovirus infection study.
Orthotospoviruses can complete their own life activities with the help of their host.Plants have evolved two layers of defence mechanisms to prevent their invasion.One is the activation of the antiviral RNA interference (RNAi) defence response, and the other is the recognition of different virus effectors (such as NSs and NSm) through their own cellular innate immune receptors (such as Tsw and Sw-5b) [2,44].In the process of pathogen invasion, plant NLRs recognize speci c pathogen effectors and trigger so-called effector-triggered immunity (ETI).The defence response triggered by Tsw and Sw-5b disease resistance genes belongs to the ETI type [45,46].Previous research has found that the CaHsp25.9 gene has a positive regulatory effect on the tolerance of peppers to heat, salt and drought stress [47].In addition, previous researchers have shown that Hsp70 can improve the heat resistance of peppers [48].This research found that after TSWV and HCRV infect plants, heat shock protein (HSP90) and resistance protein (RPS2) are the hub genes in the ETI pathway in the plant-pathogen interaction pathway.Previous researchers found that SGT1-HSP90 (small glutamine-rich tetratricopeptide repeat-containing protein, SGT1) and the antagonistic genes Rx, RPM1, Mla, N, Prf, Mi, I2, R3a, Lr21 and RPS2 mediated close relationships in plant disease resistance, indicating that R gene-mediated resistance depends on HSP90 [49].Whether there is a mutual dependence between HSP90 and the resistance gene Tsw in pepper remains to be further researched.
The response intensity of Xiangyan 11 in the plant-pathogen interaction pathway after HCRV inoculation was greater than those of TSWV, CDPK, CALM, HSP90, RPS2, and LRR receptor protein kinases.During the defence process of PAMP-triggered immunity (PTI), the calcium ion concentration rapidly ows into the cell wall, which eventually leads to the upregulation of CALM and CDPK.In the process of ETI immunization, the heat shock protein HSP90 is upregulated, which is conducive to the formation of the HR response [50].The predecessors were resistant to two different kinds of Solanaceae plants (Petunia hybrida is resistant to TSWV, while TSWV can infect Nicotiana tabacum systemically).In the analysis of the speci c and shared defence mechanisms triggered by the TSWV virus inoculation, research results show that the defence mechanisms based on reactive oxygen species (ROS) are both present in two species of Solanaceae with different resistance [51].The results of our research did not show that the expression of ROS was upregulated.We found that the hub genes CPK and CALM were upregulated during the process of ROS synthesis.The reason for this phenomenon may be that the synthesis of ROS has not been completed at the initial stage of virus infection.
The results of this study showed that the number of differential genes induced by the two viruses inoculated with Orthotospovirus was signi cantly different after the viruses infected peppers, and signi cantly more genes were differentially induced by HCRV infection than by TSWV infection.We analysed the metabolic pathways of DEG after virus inoculation.The analysis results showed that HCRV and TSWV were signi cantly enriched in the pathways of plant-pathogen interaction, plant hormone signal transduction, and phenylpropanoid synthesis after inoculation.The hub genes of the plant were signi cantly upregulated, ultimately enhancing plant resistance.In addition, we focused on the TFs of pepper.WRKY, MYB, and AP2/ERF, among others, underwent major changes relative to the CK.Therefore, we infer that after HCRV and TSWV infect plants, DEG undergo major changes in the processes of plantpathogen interaction, hormone signal transduction and secondary metabolites and changes in plant TF families, such as WRKY, MYB, and ERF.The selected candidate genes related to plant defence were veri ed by qRT-PCR, and it was found that the qRT-PCR experiment and the RNA-Seq sequencing results correlated well, demonstrating that the RNA-Seq sequencing results were correct and reliable.Gene overexpression and gene knockout experiments on the selected 10 candidate genes should be conducted in a follow-up study to further verify whether the candidate genes play defensive roles in plant defence responses.Through overexpression, gene knockout or gene editing of these candidate genes, the disease resistance of plants is ultimately enhanced, which can be applied to the disease resistance breeding of peppers and other plants.

Conclusion
High-throughput RNA-Seq analysis was used in this study, which allowed us to draw some rules between TSWV, HCRV infection and changes in gene expression in infected peppers.The GO and KEGG pathway enrichment analysis revealed the affected defense response in host plants infected by TSWV and HCRV, and showed how mock-and TSWV-inoculation, HCRV-inoculation triggers different metabolic processes.Our research further analyzes the systemic level changes related to TSWV and HCRV infection, and provides a reference for future exploration of TSWV and HCRV disease resistance breeding.

Figure 1 Expression
Figure 1

Table 1
Quality assessment of the cDNA libraries