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Plant Science

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Overexpression of a pathogenesis-related gene NbHIN1 confers resistance toTobacco Mosaic Virus in Nicotiana benthamiana by potentially activating thejasmonic acid signaling pathway

Haoran Penga, Yundan Pua, Xue Yanga, Gentu Wua, Ling Qinga, Lisong Mab,c,⁎, Xianchao Suna,⁎⁎

a College of Plant Protection, Southwest University, Chongqing 400716, Chinab College of Plant Protection, Hebei Agriculture University, Baoding 071001, Chinac Division of Plant Science, Research School of Biology, The Australian National University, ACT, Acton, 2601, Australia

A R T I C L E I N F O

Keywords:Nicotiana benthamianaHIN1Tobacco mosaic virusResistanceRNA-SeqNbRAB

A B S T R A C T

Harpin proteins secreted by plant-pathogenic gram-negative bacteria induce diverse plant defenses againstdifferent pathogens. Harpin-induced 1 (HIN1) gene highly induced in tobacco after application of Harpin proteinis involved in a common plant defense pathway. However, the role of HIN1 against Tobacco mosaic virus (TMV)remains unknown. In this study, we functionally characterized the Nicotiana benthamiana HIN1 (NbHIN1) geneand generated the transgenic tobacco overexpressing the NbHIN1 gene. In a subcellular localization experiment,we found that NbHIN1 localized in the plasma membrane and cytosol. Overexpression of NbHIN1 did not lead toobserved phenotype compared to wild type tobacco plant. However, the NbHIN1 overexpressing tobacco plantexhibited significantly enhanced resistance to TMV infection. Moreover, RNA-sequencing revealed the tran-scriptomic profiling of NbHIN1 overexpression and highlighted the primary effects on the genes in the processesrelated to biosynthesis of amino acids, plant-pathogen interaction and RNA transport. We also found thatoverexpression of NbHIN1 highly induced the expression of NbRAB11, suggesting that jasmonic acid signalingpathway might be involved in TMV resistance. Taken together, for the first time we demonstrated that over-expressing a pathogenesis-related gene NbHIN1 in N. benthamiana significantly enhances the TMV resistance,providing a potential mechanism that will enable us to engineer tobacco with improved TMV resistance in thefuture.

1. Introduction

Plants are continuously threatened by different kinds of biotic orabiotic stresses. However, all current plant species have been successfulin counteracting these unfavorable environmental factors via tran-scriptional and post transcriptional regulations including gene expres-sion and physiological dynamics [1–3]. In the incompatible plant-pa-thogen interactions, a localized cell death response is often observedaround the infected site. This local phenomenon, known as the hy-persensitive response (HR), benefits the host plant for preventing fur-ther spread of the pathogen from the invasion site. Plant-pathogenicgram-negative bacteria secret Harpin proteins into the extracellularspace of host plant to function as pathogen independent HR elicitors[4]. In the early 1980s, the independent isolation of hrp (for hy-persensitive reaction and pathogenicity) mutants from Pseudomonassyringae pv. Phaseolicola [5] and pv. syringae [6] was reported by using

mutational approach. The hrp genes encode the structural proteins,effectors and Harpin proteins that play durable roles in pathogenicbacteria either contributing to pathogenicity as virulence factors oreliciting hypersensitive reactions on resistant or non-host plants [7].Furthermore, genetic and biochemical studies have demonstrated thatHarpin proteins are components of Type III secretion systems, reg-ulatory proteins, proteinaceous elicitors of the hypersensitive reaction,and enzymes required for synthesis of periplasmic glucans.

Harpin elicits HR in non-host plant tobacco and over two decadesago tobacco genes induced by Harpin have been cloned and named asHIN (Harpin-induced) gene [8]. Two classes of HIN genes were iden-tified in Nicotiana tabacum including NtHIN1 (4 clones) and NtHIN2(116 clones) and the functional characterization of NtHIN1 in detailsrevealed a role in plant-bacteria interaction [8]. Similar genes havebeen found in tomato and Arabidopsis thaliana. HIN1 shows a sequencesimilarity with the A. thaliana NDR1 gene (non-race-specific disease

https://doi.org/10.1016/j.plantsci.2019.02.018Received 3 December 2018; Received in revised form 18 January 2019; Accepted 25 February 2019

⁎ Corresponding author at: College of Plant Protection, Hebei Agriculture University, Baoding 071001, China.⁎⁎ Corresponding author at: College of Plant Protection, Southwest University, Chongqing 400716, China.E-mail addresses: Lisong.ma@anu.edu.au (L. Ma), sunxianchao@163.com (X. Sun).

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resistance gene) [9]. Database searches for HIN1 and NDR1-relatedsequences reveal that in A. thaliana these genes belong to a large NHL(NDR1/HIN1-Like) family with at least 29 members [10]. It has beendocumented that some members of NHL gene family play a role inplant-pathogen interaction. Mutation of NDR1 gene in A. thalianacompromises the resistance to both bacterial and fungal pathogens [9].NHL2 overexpressing Arabidopsis plants exhibit elevated levels of PR-1expression and light-dependent ‘speck disease-like’ symptoms in theleaves of transgenic plants [10]. NHL10 is up-regulated in the hy-persensitive response to Cucumber mosaic virus infection and is specifi-cally induced in an incompatible plant-bacteria interaction [11].NHL25 and NHL3 transcripts also accumulate specifically during in-fection with avirulent bacterial pathogen strains [12]. Overexpressionof NHL3 in Arabidopsis plants results in increased resistance to P. syr-ingae pv. tomato DC3000 [13]. However, the biochemical function ofthe HIN1 gene against plant virus remains to be determined.

Advances in next-generation sequencing (NGS) technologies aretransforming biology research [14]. The large-scale study of the tran-scriptome with RNA-seq approach has opened the opportunity to un-derstand a wide variety of response of plants in response to diversetreatments or stresses. RNA-Seq (deep-sequencing of cDNA) showssignificant advantages such as sensitive, resolution and comprehensive.It is becoming a popular tool for identifying and quantifying gene ex-pression at a genome scale level [15–17]. It enables genome-wide ex-pression studies on the cellular responses and pathways of microbeaffected by different treatment via differential gene expression profiling[18–20]. The release of whole genomic sequences of tobacco offers afoundation to study the sequencing-based transcriptome (RNA-Seq),which hold the potentials to uncover the key factors in response ofdifferent stresses [21].

Tobacco mosaic virus (TMV) infects all tobacco species as well asmany other plants worldwide and causes severe losses in tobacco pro-duction. To date no effective chemical treatments were found to protecttobacco plants from TMV virus infection [22]. To find a strategy totackle TMV disease and understand the role of NbHIN1 gene in responseto TMV infection, in this study we isolated the NbHIN1 gene from to-bacco and characterized its function by exploring sequence comparison,subcellular localization and overexpression in tobacco for anti-TMVactivity. Furthermore, we exploited RNA-Seq to analyze the tran-scriptome of transgenic plants carrying NbHIN1.

2. Materials and methods

2.1. Plant materials and bacterial and yeast strains

Tobacco seeds (N. benthamiana) were surface sterilized for 3min in75% ethanol, rinsed with sterile water for five times, and then germi-nated in 1/2 MS medium in a growth chamber maintained at 25℃ (14 hlight/10 h dark). Following germination, seedlings were transferred toplantlets filled with autoclaved soil consisting of 1:1 (v/v) high-nutrientsoil and vermiculite in pots and then cultured in a growth chamber at25℃ with 50% humidity (14 h light/10 h dark).

The yeast-two hybrid series vector pGBKT7 and pGADT7 were takenfrom laboratory stocks that were purchased from Clontech. The pCFP-AtRop10 and pSPDK661 (TMV-GFP) were gifts from Yule Liu (TsinghuaUniversity, Beijing, China) [23]. The plant expression vector pFGC5941was a gift from Zhiliang Zheng (City University of New York, New York,USA) [24]. The plant expression vector pCV-dsRFP-N1 and BiFC seriesvector pCV-nYFP-C1 and pCV-cYFP-C1 were gifts from Jianping Chen(Zhejiang Academy of Agricultural Sciences, Zhejiang, China) [25]. ThepEASY-T5 vector and Escherichia coli Trans5α were purchased fromTransGen Biotech (China) and was grown in LB at 37℃. Agrobacteriumtumefaciens EHA105 was grown in Kana medium supplemented with Rifat 28℃, 200 rpm in an orbital shaker and harvested at log phase ofgrowth (OD600= 1.0) for infiltration. Yeast AH109 (Saccharomycescerevisiae) was grown in PDA or SD medium at 30℃.

2.2. RNA extraction and cDNA synthesis

The leaves of plants were grounded into powder in liquid nitrogenusing mortars. RNA was extracted using an Eastep® Super Total RNAExtraction Kit (Promega, LS1040, Beijing, China). The RNA sample wasthen reverse transcribed with a PrimeScriptTM RT reagent Kit (TaKaRa,RR037 A, Shiga, Japan) in a 10 μL reaction.

2.3. Cloning and protein bioinformatics analysis

Based on the full-length sequence of tobacco HIN1 in NCBI database(AF212183), the coding sequence of the HIN1 gene was cloned withgene-specific primer set (Supplementary Table S1). Other plants HIN1sequences used for comparison were retrieved from the GenBank da-tabase. MEGA 6.0 was employed to phylogenetic analyses of the nu-cleotide sequences with the Maximum-likelihood method [26]. Thereliability of the trees was assessed using 1000 bootstrap replicates. Thecomputation of various physical and chemical parameters for NbHIN1protein were calculated using ExPASy-ProtParam tool. The transmem-brane helices in proteins were predicted using TMHMM Server v.2.0[27]. Protein subcellular localization was predicted using PSORT Pre-diction [28].

2.4. Vector construction

For localization, the open reading frame of NbHIN1 was insertedinto the BamHI and SalI restriction sites of pCV-dsRFP-N1. The obtainedplasmid or empty control plasmid was transformed into Agrobacteriumtumefaciens strain EHA105. For yeast two-hybrid, the open readingframe of NbHIN1 were cloned into AD vector of the pGADT7 using therestriction sites of NdeI and EcoRI. The open reading frame of NbRAB11gene were cloned into BD vector of pGBKT7 using the restriction sites ofNdeI and EcoRI.

For BiFC, the open reading frame of NbHIN1 was cloned into thevector of pCV-nYFP-C1 using the restriction sites of SmaI and BamHI.The open reading frame of NbRAB11 gene was cloned into the pCV-cYFP-C1 using the restriction sites of SmaI and BamHI.

2.5. Agroinfiltration of N. benthamiana leaves and confocal microscopy

NbHIN1 Agrobacterium tumefaciens strain EHA105 carrying thepCFP-AtRop10 plasmid serves as a plasma membrane marker control.Agrobacteria-mediated transient expression was performed followingthe methods described previously [29]. For localization, infiltrated N.benthamiana leaves were harvested 36 h after infiltration and leaf discswere visualized using a LSM780 confocal laser scanning microscopeequipped with a 40*/1.2 water-immersion objective (Zeiss, Germany).Excitation of RFP was done at 543 nm with a HeNe laser. The590–620 nm filter captured emission. Excitation of GFP was done at488 nm with an Ar-ion laser and emission was captured with a505–530 nm pass filter. Images were scanned eight times.

2.6. Generation of transgenic plants

The coding sequence of NbHIN1 with a downstream in-frame His-Tag was cloned using Nco I and BamHI restriction sites. The specificprimers used in this study are listed in supplementary Table S1. Afterdigestion with NcoI and BamHI, the NbHIN1 sequence was inserted intopFGC5941 under the control of the CaMV35S promoter. The obtainedrecombinant plasmid pFGC5941-NbHIN1 was transformed intoAgrobacterium tumefaciens strain EHA 105. Generation of transgenic N.benthamiana was performed following the leaf disk method [30].Transgenic plants were selected using phosphinothricin (5mg·L−1) andconfirmed using PCR and western blot. Positive plants were propagatedasexually in MS medium and then transferred to soil for seeds. Plantsgrowth at the 6-leaf stage were ready for virus inoculation. WT tobacco

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plants were also cultivated in the same growing condition, propagatedand transplanted at the same time with the transgenic tobacco plantsand served as controls for TMV inoculation and other analyses.

2.7. PCR and western blot

Standard PCR was performed with the primer set of 35S andNbHIN1 (see supplementary Table S1) and WT tobacco plants served asthe negative control. The rapid DNA extraction was performed by usingTIANcombi DNA Lyse&Det PCR Kit (TIANGEN, KG203, Beijing, China).The amplifications were performed at 94℃ for 3min, followed by 35cycles of denaturation at 94℃ for 30 s, 56℃ for 30 s and elongation at72℃ for 1min, 72℃ for 5min. PCR product was electrophoresed on 1%agarose gel and visualized by Bio-Rad gel imager. Protein samples wereextracted with Laemmli buffer [31] and subjected to electrophoresis onSDS–PAGE gel followed by western blot assays using anti-His antibodiesand were detected using ECL western blotting substrate.

2.8. TMV inoculation, ELISA and quantitative RT-PCR

For TMV inoculation, two approaches were employed. One wasperformed following the agroinfiltration of tobacco leaves method de-scribed previously [23] and infiltrated region was observed after 4 daysafter infiltration. Another approach was to apply 100 μL extracts ofTMV-GFP infected leaves to every leaf by rubbing and pictures weretaken under UV light after 2 days. Each experiment was repeated threetimes with at least three independent plants per time. Crude extractsprepared from leaves of symptomatic plants were applied to directdouble antibody sandwich (DAS)-ELISA for detection of the TMV. Allthe anti-bodies are polyclonal rabbit serum provided by Bingsheng Qiu(Institute of Microbiology, Chinese Academy of Sciences, China).Quantitative Real-time quantitative PCR (qPCR) was performed using aCFX Touch Real-time PCR machine (Bio-Rad) and QuantinovaTM SYBRGreen PCR Kit (QIAGEN, Germany) to determine the relative expressionlevels of target genes. Gene specific primers were designed according tothe coding sequences of each gene using Primer 5.0 software. Quanti-fication of the relative changes in gene transcript levels was performedusing the 2−△△CT method [32].

2.9. RNA-Seq

Quality of total RNAs was verified on an Agilent 2100 Bioanalyzerand based on the rRNA ratio 25S/18S, RNA integrity number, and theabsence of smear. The amplified fragments were sequenced usingIllumina HiSeqTM 2500 by Gene Denovo Co. (Guangzhou, China). Denovo assembly and differentially expressed gene analysis were de-scribed previously [33].

2.10. Plant treatment with exogenous hormones

N. benthamiana plants at the 6-leaf stage were sprayed with 0.1mmol•L−1 MeJA (Sigma-Aldrich). Control plants were sprayed withsterile water. Samples were collected at 1-hour interval for up to 12 hand immediately frozen in liquid nitrogen and stored at -80℃ until usefor RNA isolation.

2.11. Yeast two-hybrid assay

Y2H assay was performed according to the protocol described pre-viously [34]. Briefly, the yeast strain was co-transformed with bait andprey plasmid combinations using lithium-acetate and polyethyleneglycol 3350. Transformants harboring both bait and prey plasmids wereselected on plates containing minimal medium lacking Leu and Trp (SD-WL). Empty prey vector pGBKT7 or pGADT7 used as bait or prey servedas controls. One colony per combination was picked from SD-WL platesto inoculate 1mL SD-WL culture. After 36 h growth, cells were collected

by centrifugation and resuspended in 25 μL 0.9% NaCl from OD600= 1to OD600= 0.00001 and spotted on SD-WL and SD-AHWL plates sup-plementing with 40 μg/mL X-α-Gal (Clontech, Mountain View, USA)and 200 ng·mL−1 Aureobasidin A (Clontech, Mountain View, USA).After 3 days incubation, the plates were checked for growth and pho-tographed.

2.12. Bimolecular fluorescence complementation (BiFC)

BiFC assay was performed according to the method described pre-viously [34]. Briefly, the leaves of 4- to 5-week-old N. benthamianaplants were infiltrated with Agrobacteria containing the correspondingconstructs at an absorbance density of 0.5. Leaf discs 48 h after in-filtration were imaged. Confocal microscopically analysis was per-formed with the LSM710 (Zeiss, Germany).

2.13. Statistical analysis

All experiments and data presented here involved at least threerepeats. The data are presented as means and standard deviations. Thestatistical analysis was performed with SPSS software (version 17.0)using Student’s t-test.

3. Results

3.1. Phylogenic and localization analysis of NbHIN1

The coding sequence of NbHIN1 was cloned and assigned the newGenBank number KU195817. The NbHIN1 gene was predicted to en-code a protein with 229 amino acid residues. To determine the physicaland chemical properties of this protein, bioinformatics tools were used.ExPASy-ProtParam analysis indicated that it has a predicted molecularmass of 26.2 kDa and includes a conserved domain LEA-14 (101–203aa). PSORT Prediction analysis showed that the maximum likelihood oflocalization in the plasma membrane is 60%. Furthermore, BLASTanalysis showed that NbHIN1 shared approximately 90% similaritywith HIN1 from other solanaceae plants. Phylogenetic tree analysisshowed that NbHIN1 was clustered into the same subgroup withCapsicum annuum HIN1 (CaHIN1). However, it has no analogy with thatof monocotyledons such as rice and sorghum (Fig. 1).

Confocal microscopic observation showed that the control CFPprotein localized in plasma membrane (Fig. 2). Interestingly, NbHIN1-RFP signals were clearly visualized in the plasma membrane as evi-denced by the overlapped localization with the CFP signals and in cy-tosol (Fig. 2). Based on these observations, we concluded that NbHIN1localized in the plasma membrane and cytosol.

3.2. Overexpression of NbHIN1 enhances the resistance to TMV

To investigate the role of NbHIN1 in the resistance against TMV, wegenerated the transgenic tobacco NbHIN1-OE under the control of the35S promoter (Fig. 3A). PCR analysis showed that the T-DNA insertionwas present in the tested 10 independent transgenic tobacco lines andNbHIN1 fusion protein with His tag was detected in the transgenic line1, 3, 4, 7, 8 and 10 as well, but transgenic line 1 and 3 accumulatedmore amount of protein compared to other tested lines and line 1 and 3were selected for further experiment (Fig. 3 B & C). No visible pheno-type was observed in the NbHIN1-OE transgenic line 3 compared towildtype (WT) plants (Fig. 3D).

To examine the response of NbHIN1-OE-3 line to TMV, we in-filtrated the transgenic line 3 and WT tobacco plant leaves withAgrobacterium tumefaciens carrying the TMV-GFP. Surprisingly, novisible GFP signals were observed in the infiltrated leaves of NbHIN1-OE transgenic line at 4 and 5 dpi whereas pronounced green fluores-cence signals were visualized in the leaves of WT plant. At 6 and 7 dpi,green fluorescence signals can be detected in the young leaves of WT

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plants due to systemic spread of the virus. However, in the infiltratedleaves of NbHIN1-OE line the GFP signals slightly expanded (Fig. 4A).ELISA assay on the inoculated leaves showed that the accumulations ofTMV in WT plants were significantly higher than that in NbHIN1-OE atthese tested days, which are consistent with the microscopic observa-tions (Fig. 4B). To further validate these findings viral supernatant wasapplied to leaves by rubbing. Interestingly, the macroscopic greenfluorescent spots appeared at 2 dpi both in the inoculated leaves oftransgenic and WT plants, but more spots were clearly visualized in theWT plants. At 3 dpi, the green fluorescent signals increasingly accu-mulated compared to that at 2 dpi, but the level of signals present intransgenic plant was much lower than that in WT plants. At 4 dpi, theGFP signals in WT plants fully expanded to the young leaves while inNbHIN1-OE transgenic line limited and weak signals were observed inthe young leaves (Fig. 4C). Furthermore, the number of TMV-GFP

transcripts in inoculated leaves of overexpression and WT lines wasquantified by qPCR and Fig. 4D showed that the expression level of GFPin WT plants was significantly higher than that in the NbHIN1 over-expression line. Taken together, these findings strongly indicated thatoverexpressing NbHIN1 in N. benthamiana significantly enhanced theresistance against TMV.

3.3. Transcriptomic analysis of the genes that were affected by NbHIN1overexpression

It has been proved that overexpressing NbHIN1 largely increasedTMV resistance. To further dissect the molecular mechanism underlyingthe increased resistance against TMV in NbHIN1-OE line, we applied theRNA-Seq approach to assess the genome-wide expression profiles ofNbHIN1-OE line and WT plant, respectively. Total RNA was extracted

Fig. 1. Phylogenetic analysis of the NbHIN1 proteinamong NHL (NDR1/HIN1-Like) family proteins fromother plants. The numbers on the branches representedthe values of Bootstrap test. Ca: Capsicum annuum, Nt:Nicotiana tabacum, St: Solanum tuberosum, Sl: Solanumlycopersicon, At: Arabidopsis thaliana, Os: Oryza sativa,Sb: Sorghum bicolor, Pt: Populus trichocarpa, Cs: Cucumissativus, Gm: Glycine max, Car: Coffea arabica, Fv:Fragaria vesca subsp. Vesca, Pp: Prunus persica.

Fig. 2. NbHIN1 localizes in the plasma membrane and cytosol. Co-transient expression of NbHIN-dsRFP and plasma membrane maker AtRop10-CFP in the epidermalcells of N. benthamiana leaves by agroinfiltration. RFP and CFP fluorescence were visualized using confocal imaging 72 h after infiltration and were depicted in redand blue, respectively. NbHIN1 was clearly visualized in the plasma membrane as evidenced by the co-localization with the plasma membrane marker AtRop10-CFPand in cytosol. The white scale bars represented 20 μm.

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Fig. 3. Generation and validation of transgenicN. benthamiana plants stably expressingNbHIN1. (A) Schematic diagram of the35S::NbHIN1-His fusion construct. (B) PCRanalysis to determine the T-DNA insertion inthe representative transgenic plants. WT to-bacco plants served as the negative control.“+” indicated the positive control and “-” re-presented the negative water control. (C)Immunoblot of NbHIN1-His fusion protein ac-cumulating in the NbHIN1-expressing tobaccolines. Protein extracts from leaves were sub-jected to immunoblot and probed with an anti-HIS antibody (α-HIS) and protein sizes wereindicated on the left. (D) NbHIN1 over-expressing tobacco line (NbHIN-OE-3) did notdisplay any visible phenotypes in comparisonto WT plant. Representative pictures weretaken 3-week after planting.

Fig. 4. NbHIN1-overexpresing N. benthamiana plants exhibit significantly increased resistance to TMV. (A) The green fluorescent signals representing the viralreplication in NbHIN1-OE and WT tobacco plants that were inoculated with Agrobacterium tumefaciens carrying TMV-GFP construct were imaged at 4-, 5-, 6- and 7-daypost inoculation (dpi). (B) Detection of TMV virus content by indirect-ELISA. (C) The green fluorescent signals in NbHIN1-OE and WT tobacco plants inoculated withTMV-GFP virus extracts by rubbing were visualized at 2-, 3- and 4-day post inoculation (dpi). (D) qPCR analysis showing the transcript levels of TMV-GFP in NbHIN1-OE and WT plants. Expression levels were represented as the fold change and normalized to actin gene. The results are mean values (± SD) from three independentexperiments. The statistical analyses were performed using Student’s t-test (∗0.01< P < 0.05, ∗∗0.001< P < 0.01, ∗∗∗P < 0.001).

Table 1Summary of the RNA-seq reads.

Sample group T-1 T-2 WT-1 WT-2

Raw reads 6710428500 6362495400 7699279500 7103187600Clean reads 6580729382 6245392012 7546242685 6975422942GC content (%) 44.88 44.91 44.72 44.54Uniquely mapped reads 34712632 (79.96%) 32275203 (80.28%) 39584542 (79.87%) 37068869 (80.66%)Multiple mapped reads 2233966 (5.15%) 2098560 (5.22%) 2500564 (5.05%) 2277714 (4.96%)

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from two independent NbHIN1-OE line 1 and 3 plants (T-1 and T-2) andtwo WT (WT1 and WT2) Nicotiana benthamiana plants, respectively. 43million and 48 million clean reads number in total were obtained fromthe two libraries of NbHIN1 and WT plants, respectively. After qualitycontrol filtering and trimming adaptor sequencing, around 32 millionclean reads from every library were mapped to N. benthamiana genome.The summary of RNA-seq reads was showed in Table 1. Sequence readshave been deposited in the NCBI Sequence Read Archive (SRA) underaccession number PRJNA450205.

The transcriptome of NbHIN1-OE and WT plant represented highreproducibility among biological replicates, as indicated by the Pearsoncorrelation coefficient is 0.9945 and 0.9987, respectively, indicatingthat the RNA-seq data was accurate and reproducible. The volcano plotsof the differentially expressed genes (DEGs) demonstrated that 102genes in N. benthamiana were differentially expressed after NbHIN1overexpression compared to those in WT plant, in which 48 genes wereup-regulated and 54 genes were down-regulated with more than two-fold changes (Fig. 5). In particular, we list top 10 down and up-regu-lated genes in Table 2.

To elucidate the biological function or pathways that DEGs in-volved, gene ontology (GO) enrichment analyses were conducted. GOanalysis revealed that GO terms were classified into three main classes:biological process, cellular component and molecular function in-cluding 21 sub-categories (Fig. 6), Among the biological process class,metabolic process (21.4%) and cellular process (21.2%) were the twodominant groups, while in the molecular function class, binding(51.3%) was the major group. In addition, KEGG annotation revealedthat all annotated DEGs were classified into 23 categories and the top20 abundant biochemical pathways with numbers of assigned geneswere shown in Supplementary Fig. S1. To validate the RNA-Seq results,we selected two highly upregulated genes: NbRAB11 (Ras-related pro-tein Rab11) (Niben101Scf35013g00007) and NbPARN (poly(A)-specificribonuclease) (Niben101Scf05961g02014) from DGEs for qRT-PCRanalysis. qPCR showed that the expression patterns (8.05-fold and 4.07-fold) of these two genes were consistent with those in RNA-Seq, sug-gesting that our RNA-Seq data is accurate and reliable (Fig. 7).

3.4. NbHIN1 regulates NbRAB11 expression and exogenous MeJAtreatment induces their expression

Overexpression of NbHIN1 highly regulates the expression ofNbRAB11 based on our RNA-seq analysis. To assess whether exogenousMeJA affects the expression of NbHIN1 and NbRAB11, we treated theplant with 0.1 mmol•L−1 MeJA and quantified the gene expression byqRT-PCR. The results showed that the expression of two genes afterMeJA treatment was significantly increased at different time points.However, they displayed a similar expression pattern in 5–12 h andpeaked in 9 h after treatment (Fig. 8). These findings indicated thatoverexpressing NbHIN1 could activate the JA-mediated signalingpathway through inducing the NbRAB11 gene expression. To furtherunderstand the function of NbHIN1 and NbRAB11, we examinedwhether there was a physical interaction between them using yeasttwo-hybrid analysis and bimolecular fluorescence complementationassay. However, no direct interaction was observed either in Y2H or inBiFC assay (Supplementary Fig. S2).

4. Discussion

HIN1 is a class of protein produced during the process of hy-persensitivity induced by Harpin protein in non-host plants. In thisstudy, we descried the cloning, molecular and functional character-ization of NbHIN1 in response to TMV infection. Using transient tobaccoexpression system, we found that NbHIN1 localized in the plasmamembrane and cytosol. Stable expression of NbHIN1 in tobacco plantresulted in significantly enhanced resistance of tobacco against TMVinfection. Moreover, RNA-sequencing revealed that overexpression ofNbHIN1 highly induced the expression of NbRAB11, suggesting thatjasmonic acid signaling pathway might be involved in TMV resistance.This is the first example of study on overexpression of NbHIN1 geneconferring TMV resistance.

Previous studies showed that several members of the NHL familyhave the cell membrane subcellular localization [13,35]. However, weobserved that NbHIN1 localized both in the membrane and in cytosol(Fig. 2). The observed cell membrane localization of NbHIN1 is con-sistent with the predicted highest likelihood of protein localization bybioinformatics tool. It has been reported that some members in the NHLfamily reside in the endoplasmic reticulum due to sarcolipin-like se-quences present in the protein [37]. These findings suggest that thesubcellular localization of HIN1 protein is diverse. The ArabidopsisNDR1 protein with cell membrane localization was found to function inan amplification of the initial event following pathogen perception[13,36]. Because NbHIN1 exhibits sequence similarity to NDR1, its cellmembrane localization suggested that it might function as an elicitor ofHR reaction following pathogen perception.

Our studies showed that the isolated ORF of HIN1 from N. ben-thamiana encodes a protein containing 229 amino acid residues with apredicted and conserved LEA-14 domain, which is also present in otherHIN1 genes [37]. Previous studies have demonstrated that the plantgene family LEA-14 expressed in the plant both in response to pathogenand drought, suggesting a common molecular mechanism between theplant response to biotic and abiotic stresses [38,39]. The existence ofthis conserved domain in NbHIN1 strongly indicated that it might beinvolved in plant defense response. It has been reported that HIN1 isrequired in the acquired resistance of plant in response to fungal andbacterial infection. For example, overexpression of rice OsHIN1 inducesresistance to rice blast [40]. In previous study, it was found that thetranscription level of HIN1 was significantly increased after TMV in-fection [41], which suggested that HIN1 as an important componentparticipates in a rapid resistance reaction after TMV infection. We ob-served that overexpression of NbHIN1 can largely inhibit tobacco mo-saic virus infection both at RNA and protein levels (Fig. 4). AlthoughHIN1 serves as a marker gene for cell death, transgenic N. benthamianaline overexpressing NbHIN1 grew normally and did not display any

Fig. 5. Volcano plots of the differentially expressed genes. The transcriptome ofNbHIN1-overexpressing plants was compared to wild-type plants. Differentiallyexpressed genes were represented with red dots (up-regulated) or green dots(down-regulated), and others (no differential difference) indicated with blackdots. The x-axis specified the fold-changes (FC) and the y-axis specified thenegative logarithm to the base 10 of the FDR values as statistical significance.

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visible cell death symptoms (Fig. 3) and same observation has beenreported in the transgenic soybean overexpressing GmHIN1 line [42].

We found that the NbRAB11 is highly differentially expressed genebased on our transcriptome analysis. The RAB protein family representsthe largest member of the Ras superfamily of monomeric G proteins,also referred to as small ATPases [43]. It has been documented thatRAB is involved in resistance against biotic and abiotic stresses. Forexample, in salt stressed tomato plants, expression of RAB11 was re-pressed and expression of RAB2 was induced as shown by microarrayanalysis of total RNA [44]. Similarly, transgenic tobacco plants over-expressing Prosopis juliflora RAB7 displayed resistance to high-salt stress[45] and ectopic expression of OsRAB7 enhanced tolerance of peanutplants to several abiotic stresses. Furthermore, in rice, several lines ofevidence suggest that OsRAB11 acts with OsOPR8 to positively regulateJA-mediated signaling through activation of the JA biosynthetic

pathway [46]. cis-12-oxo-phytodienoic acid reductase (OPR) is a keyenzyme in JA accumulation in response to environmental stress[46,47]. The expression of OPR is tissue-specific and induced by avariety of abiotic and biotic stresses, including wounding, infection,and signaling molecules [48–50]. In this study, overexpression ofNbHIN1 highly regulated the NbRAB11 expression and resulted in sig-nificantly increased resistance to TMV, suggesting that jasmonic acid-mediated signaling pathway might be involved in TMV resistance.Moreover, it has been reported that salicylic acid and jasmonic acid areessential for systemic resistance against TMV in N. benthamiana [51].Based on these findings, we hypothesize NbHIN1 is able to induceNbRAB11 and there is an OPR-Like gene in N. benthamiana that couldact with NbRAB11 to regulate the JA signaling pathway.

Based on transcriptomic profiling of NbHIN1 overexpression line,we found that the PARN gene is significantly up-regulated (Fig. 7).

Table 2Top 10 down and up-regulated DEGs in response to overexpression of NbHIN1.

Gene Log2 fold change Seq.Descripition

Niben101Scf01795g04035 −13.1635 BnaA05g32900D [Brassica napus]Niben101Scf03015g06015 −13.0810 Ribulose bisphosphate carboxylase small chain 8B, chloroplasticNiben101Ctg12716g00002 −12.0768 Filament-like plant proteinNiben101Scf01795g04036 −11.9549 Peptidyl-prolyl cis-trans isomerase DNiben101Scf01795g04037 −11.3581 Filament-like plant protein 3Niben101Scf09116g02014 −10.4041 Alanine-tRNA ligaseNiben101Scf01534g02026 −9.6970 Annexin D3Niben101Scf04528g13017 −8.1137 Histone H2ANiben101Scf01795g04026 −4.7435 Glutamyl-tRNA(Gln) amidotransferase subunit ANiben101Scf03830g02001 −4.4594 BZIP family transcription factor [Medicago truncatula]Niben101Scf05146g03008 10.7690 CASP-like protein 4D1Niben101Scf02348g10009 9.2877 Amino acid permease 6Niben101Scf02015g04019 9.0768 Curved DNA-binding proteinNiben101Scf08020g06001 8.1056 late embryogenesis abundant hydroxyproline-rich glycoprotein [Arabidopsis thaliana]Niben101Scf01740g17007 6.0000 ATP-dependent RNA helicase ded1Niben101Scf17372g01008 4.2854 Receptor-like protein kinaseNiben101Scf01150g08001 4.1260 nodulin MtN21 /EamA-like transporter family proteinNiben101Scf02110g00028 2.6871 Pectinacetylesterase family proteinNiben101Scf35013g00007 2.5575 Ras-related protein Rab11Niben101Scf03600g02026 2.5564 Sister chromatid cohesion 1 protein 2

Fig. 6. GO annotation and functional classification of all deferentially expressed genes. Gene ontology terms are classified as biological process, cellular componentand molecular functions. The x-axis legend showed a description of the 21 functional categories and the y-axis indicated the number of genes in a specific functioncluster.

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Previous studies reported that PARN is involved in the processing ofcertain non-coding RNAs [52], such as the processing of snoRNA andsmall Cajal RNA (scaRNA), and it is also responsible for cellular DNAdamage [53,54] and antiviral infections. Studies have found that zincfinger antiviral proteins can selectively recruit PARN proteins, therebyexcising the poly(A) tail of viral mRNA and degrading RNA itself fromthe 3′-5′ direction through the exonuclease complex to inhibit HIV-1infection [55]. We can speculate that overexpression of NbHIN1strongly induced the expression of PARN which might result in thedegradation of TMV mRNA and thereby inhibit viral replication. Ourresults indicated that NbHIN1 and NbRAB11 were up-regulated at 5 hand 2 h after MeJA treatment, respectively, and the expression patternsof these two genes at 5–12 h was similar (Fig. 8), indicating that thesetwo genes have a certain correlation in response to jasmonic acid andmay be involved in the same pathway. However, there is no direct in-teraction between NbRAB11 and NbHIN1and the specific relationshipbetween them requires further study. Related studies have shown thatTaHIN1 rapidly responded to strip rust infection and was strongly in-duced after treatment with salicylic acid and jasmonic acid [56]. Thesefindings suggested that overexpression of NbHIN1 activates certain

hormone pathways to gain resistance.

5. Conclusion

Taken together, our findings indicate NbHIN1 has a function in thedefense response against TMV. The information presented here under-lines the importance of understanding the molecular function ofNbHIN1 in TMV resistance. We demonstrate for the first time thatoverexpressing a pathogenesis-related gene NbHIN1 in N. benthamianasignificantly enhances the TMV resistance by potentially activatingjasmonic acid signaling pathway. All in all, these findings will expandour understanding towards the function of HIN1 and will provide in-valuable resources for engineering the tobacco plant against TMV in thefuture.

Author contributions

XS and HP designed the experiment. HP, YP, XY and GW performedthe experiment. HP, XS, LQ and LM wrote the manuscript. All authorsread and approved the final manuscript.

Fig. 7. Comparison of NbRAB11 and NbPARN expression levels between RNA-Seq and qPCR. Error bars represent the standard deviations of qRT-PCR signals (n=3).

Fig. 8. Relative expression of NbHIN1 and NbRAB11 in N. benthamiana leaves after MeJA treatment within 12 h. Expression of NbHIN1 and NbRAB11 were de-termined by quantitative reverse-transcripts polymerase chain reaction and normalized to N. benthamaian actin gene. Values represent means ± standard error (SE)from three independent experiments. The statistical analyses were performed using Student’s t-test (∗0.01< P < 0.05, ∗∗0.001< P < 0.01, ∗∗∗P < 0.001). “CK”indicated the plant without MeJA treatment.

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Funding

This study was partly supported by the National Natural ScienceFoundation of China (30900937, 31670148) and the FundamentalResearch Funds for the Central Universities (XDJK2016A009,XDJK2017C015).

Conflict of interest statement

The authors declare that the research was conducted in the absenceof any commercial or financial relationships that could be construed asa potential conflict of interest.

Acknowledgement

We would like to thank Dr. Yule Liu (Tsinghua University, Beijing,China) for providing the pSPDK661 vector, Dr. Zhiliang Zheng (CityUniversity of New York, New York, USA) for providing the plant ex-pression vector pFGC5941 vector, Dr. Jianping Chen (ZhejiangAcademy of Agricultural Sciences, Zhejiang, China) for providing ex-pression vector pCV-dsRFP-N1 and BiFC series vector pCV-nYFP-C1 andpCV-cYFP-C1and Dr. Bingsheng Qiu (Institute of Microbiology, ChineseAcademy of Sciences, China) for providing anti-bodies.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in theonline version, at doi:https://doi.org/10.1016/j.plantsci.2019.02.018.

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