Overexpression of a pathogenesis-related gene NbHIN1 confers resistance to Tobacco Mosaic Virus in Nicotiana benthamiana by potentially activating the jasmonic acid signaling pathway

Plant Science 283 (2019) 147–156

Contents lists available at ScienceDirect

Plant Science journal homepage: www.elsevier.com/locate/plantsci

Overexpression of a pathogenesis-related gene NbHIN1 confers resistance to Tobacco Mosaic Virus in Nicotiana benthamiana by potentially activating the jasmonic acid signaling pathway ⁎

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

T

⁎⁎

a

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

A R T I C LE I N FO

A B S T R A C T

Keywords: Nicotiana benthamiana HIN1 Tobacco mosaic virus Resistance RNA-Seq NbRAB

Harpin proteins secreted by plant-pathogenic gram-negative bacteria induce diverse plant defenses against different pathogens. Harpin-induced 1 (HIN1) gene highly induced in tobacco after application of Harpin protein is 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) gene and 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 to observed phenotype compared to wild type tobacco plant. However, the NbHIN1 overexpressing tobacco plant exhibited significantly enhanced resistance to TMV infection. Moreover, RNA-sequencing revealed the transcriptomic profiling of NbHIN1 overexpression and highlighted the primary effects on the genes in the processes related to biosynthesis of amino acids, plant-pathogen interaction and RNA transport. We also found that overexpression of NbHIN1 highly induced the expression of NbRAB11, suggesting that jasmonic acid signaling pathway might be involved in TMV resistance. Taken together, for the first time we demonstrated that overexpressing 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 the future.

1. Introduction Plants are continuously threatened by different kinds of biotic or abiotic stresses. However, all current plant species have been successful in counteracting these unfavorable environmental factors via transcriptional and post transcriptional regulations including gene expression and physiological dynamics [1–3]. In the incompatible plant-pathogen interactions, a localized cell death response is often observed around the infected site. This local phenomenon, known as the hypersensitive response (HR), benefits the host plant for preventing further spread of the pathogen from the invasion site. Plant-pathogenic gram-negative bacteria secret Harpin proteins into the extracellular space of host plant to function as pathogen independent HR elicitors [4]. In the early 1980s, the independent isolation of hrp (for hypersensitive reaction and pathogenicity) mutants from Pseudomonas syringae 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 pathogenic bacteria either contributing to pathogenicity as virulence factors or eliciting hypersensitive reactions on resistant or non-host plants [7]. Furthermore, genetic and biochemical studies have demonstrated that Harpin proteins are components of Type III secretion systems, regulatory 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 decades ago tobacco genes induced by Harpin have been cloned and named as HIN (Harpin-induced) gene [8]. Two classes of HIN genes were identified in Nicotiana tabacum including NtHIN1 (4 clones) and NtHIN2 (116 clones) and the functional characterization of NtHIN1 in details revealed a role in plant-bacteria interaction [8]. Similar genes have been found in tomato and Arabidopsis thaliana. HIN1 shows a sequence similarity with the A. thaliana NDR1 gene (non-race-specific disease

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: [email protected] (L. Ma), [email protected] (X. Sun).

⁎⁎

https://doi.org/10.1016/j.plantsci.2019.02.018 Received 3 December 2018; Received in revised form 18 January 2019; Accepted 25 February 2019 Available online 13 March 2019 0168-9452/ © 2019 Elsevier B.V. All rights reserved.

Plant Science 283 (2019) 147–156

H. Peng, et al.

2.2. RNA extraction and cDNA synthesis

resistance gene) [9]. Database searches for HIN1 and NDR1-related sequences reveal that in A. thaliana these genes belong to a large NHL (NDR1/HIN1-Like) family with at least 29 members [10]. It has been documented that some members of NHL gene family play a role in plant-pathogen interaction. Mutation of NDR1 gene in A. thaliana compromises the resistance to both bacterial and fungal pathogens [9]. NHL2 overexpressing Arabidopsis plants exhibit elevated levels of PR-1 expression and light-dependent ‘speck disease-like’ symptoms in the leaves of transgenic plants [10]. NHL10 is up-regulated in the hypersensitive response to Cucumber mosaic virus infection and is specifically induced in an incompatible plant-bacteria interaction [11]. NHL25 and NHL3 transcripts also accumulate specifically during infection with avirulent bacterial pathogen strains [12]. Overexpression of NHL3 in Arabidopsis plants results in increased resistance to P. syringae pv. tomato DC3000 [13]. However, the biochemical function of the HIN1 gene against plant virus remains to be determined. Advances in next-generation sequencing (NGS) technologies are transforming biology research [14]. The large-scale study of the transcriptome with RNA-seq approach has opened the opportunity to understand a wide variety of response of plants in response to diverse treatments or stresses. RNA-Seq (deep-sequencing of cDNA) shows significant advantages such as sensitive, resolution and comprehensive. It is becoming a popular tool for identifying and quantifying gene expression at a genome scale level [15–17]. It enables genome-wide expression studies on the cellular responses and pathways of microbe affected by different treatment via differential gene expression profiling [18–20]. The release of whole genomic sequences of tobacco offers a foundation to study the sequencing-based transcriptome (RNA-Seq), which hold the potentials to uncover the key factors in response of different stresses [21]. Tobacco mosaic virus (TMV) infects all tobacco species as well as many other plants worldwide and causes severe losses in tobacco production. To date no effective chemical treatments were found to protect tobacco plants from TMV virus infection [22]. To find a strategy to tackle TMV disease and understand the role of NbHIN1 gene in response to TMV infection, in this study we isolated the NbHIN1 gene from tobacco and characterized its function by exploring sequence comparison, subcellular localization and overexpression in tobacco for anti-TMV activity. Furthermore, we exploited RNA-Seq to analyze the transcriptome of transgenic plants carrying NbHIN1.

The leaves of plants were grounded into powder in liquid nitrogen using mortars. RNA was extracted using an Eastep® Super Total RNA Extraction Kit (Promega, LS1040, Beijing, China). The RNA sample was then 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 with gene-specific primer set (Supplementary Table S1). Other plants HIN1 sequences used for comparison were retrieved from the GenBank database. MEGA 6.0 was employed to phylogenetic analyses of the nucleotide sequences with the Maximum-likelihood method [26]. The reliability of the trees was assessed using 1000 bootstrap replicates. The computation of various physical and chemical parameters for NbHIN1 protein were calculated using ExPASy-ProtParam tool. The transmembrane helices in proteins were predicted using TMHMM Server v.2.0 [27]. Protein subcellular localization was predicted using PSORT Prediction [28]. 2.4. Vector construction For localization, the open reading frame of NbHIN1 was inserted into the BamHI and SalI restriction sites of pCV-dsRFP-N1. The obtained plasmid or empty control plasmid was transformed into Agrobacterium tumefaciens strain EHA105. For yeast two-hybrid, the open reading frame of NbHIN1 were cloned into AD vector of the pGADT7 using the restriction sites of NdeI and EcoRI. The open reading frame of NbRAB11 gene were cloned into BD vector of pGBKT7 using the restriction sites of NdeI and EcoRI. For BiFC, the open reading frame of NbHIN1 was cloned into the vector of pCV-nYFP-C1 using the restriction sites of SmaI and BamHI. The open reading frame of NbRAB11 gene was cloned into the pCVcYFP-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 the pCFP-AtRop10 plasmid serves as a plasma membrane marker control. Agrobacteria-mediated transient expression was performed following the methods described previously [29]. For localization, infiltrated N. benthamiana leaves were harvested 36 h after infiltration and leaf discs were visualized using a LSM780 confocal laser scanning microscope equipped with a 40*/1.2 water-immersion objective (Zeiss, Germany). Excitation of RFP was done at 543 nm with a HeNe laser. The 590–620 nm filter captured emission. Excitation of GFP was done at 488 nm with an Ar-ion laser and emission was captured with a 505–530 nm pass filter. Images were scanned eight times.

2. Materials and methods 2.1. Plant materials and bacterial and yeast strains Tobacco seeds (N. benthamiana) were surface sterilized for 3 min in 75% ethanol, rinsed with sterile water for five times, and then germinated in 1/2 MS medium in a growth chamber maintained at 25℃ (14 h light/10 h dark). Following germination, seedlings were transferred to plantlets filled with autoclaved soil consisting of 1:1 (v/v) high-nutrient soil and vermiculite in pots and then cultured in a growth chamber at 25℃ with 50% humidity (14 h light/10 h dark). The yeast-two hybrid series vector pGBKT7 and pGADT7 were taken from laboratory stocks that were purchased from Clontech. The pCFPAtRop10 and pSPDK661 (TMV-GFP) were gifts from Yule Liu (Tsinghua University, Beijing, China) [23]. The plant expression vector pFGC5941 was a gift from Zhiliang Zheng (City University of New York, New York, USA) [24]. The plant expression vector pCV-dsRFP-N1 and BiFC series vector pCV-nYFP-C1 and pCV-cYFP-C1 were gifts from Jianping Chen (Zhejiang Academy of Agricultural Sciences, Zhejiang, China) [25]. The pEASY-T5 vector and Escherichia coli Trans5α were purchased from TransGen Biotech (China) and was grown in LB at 37℃. Agrobacterium tumefaciens EHA105 was grown in Kana medium supplemented with Rif at 28℃, 200 rpm in an orbital shaker and harvested at log phase of growth (OD600 = 1.0) for infiltration. Yeast AH109 (Saccharomyces cerevisiae) was grown in PDA or SD medium at 30℃.

2.6. Generation of transgenic plants The coding sequence of NbHIN1 with a downstream in-frame HisTag was cloned using Nco I and BamHI restriction sites. The specific primers used in this study are listed in supplementary Table S1. After digestion with NcoI and BamHI, the NbHIN1 sequence was inserted into pFGC5941 under the control of the CaMV35S promoter. The obtained recombinant plasmid pFGC5941-NbHIN1 was transformed into Agrobacterium tumefaciens strain EHA 105. Generation of transgenic N. benthamiana was performed following the leaf disk method [30]. Transgenic plants were selected using phosphinothricin (5 mg·L−1) and confirmed using PCR and western blot. Positive plants were propagated asexually in MS medium and then transferred to soil for seeds. Plants growth at the 6-leaf stage were ready for virus inoculation. WT tobacco 148

Plant Science 283 (2019) 147–156

H. Peng, et al.

by centrifugation and resuspended in 25 μL 0.9% NaCl from OD600 = 1 to OD600 = 0.00001 and spotted on SD-WL and SD-AHWL plates supplementing 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 photographed.

plants were also cultivated in the same growing condition, propagated and transplanted at the same time with the transgenic tobacco plants and 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 and NbHIN1 (see supplementary Table S1) and WT tobacco plants served as the negative control. The rapid DNA extraction was performed by using TIANcombi DNA Lyse&Det PCR Kit (TIANGEN, KG203, Beijing, China). The amplifications were performed at 94℃ for 3 min, followed by 35 cycles of denaturation at 94℃ for 30 s, 56℃ for 30 s and elongation at 72℃ for 1 min, 72℃ for 5 min. PCR product was electrophoresed on 1% agarose gel and visualized by Bio-Rad gel imager. Protein samples were extracted with Laemmli buffer [31] and subjected to electrophoresis on SDS–PAGE gel followed by western blot assays using anti-His antibodies and were detected using ECL western blotting substrate.

2.12. Bimolecular fluorescence complementation (BiFC) BiFC assay was performed according to the method described previously [34]. Briefly, the leaves of 4- to 5-week-old N. benthamiana plants were infiltrated with Agrobacteria containing the corresponding constructs at an absorbance density of 0.5. Leaf discs 48 h after infiltration were imaged. Confocal microscopically analysis was performed with the LSM710 (Zeiss, Germany). 2.13. Statistical analysis

2.8. TMV inoculation, ELISA and quantitative RT-PCR

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

For TMV inoculation, two approaches were employed. One was performed following the agroinfiltration of tobacco leaves method described previously [23] and infiltrated region was observed after 4 days after infiltration. Another approach was to apply 100 μL extracts of TMV-GFP infected leaves to every leaf by rubbing and pictures were taken under UV light after 2 days. Each experiment was repeated three times with at least three independent plants per time. Crude extracts prepared from leaves of symptomatic plants were applied to direct double antibody sandwich (DAS)-ELISA for detection of the TMV. All the 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 a CFX Touch Real-time PCR machine (Bio-Rad) and QuantinovaTM SYBR Green PCR Kit (QIAGEN, Germany) to determine the relative expression levels of target genes. Gene specific primers were designed according to the coding sequences of each gene using Primer 5.0 software. Quantification of the relative changes in gene transcript levels was performed using the 2−△△CT method [32].

3. Results 3.1. Phylogenic and localization analysis of NbHIN1 The coding sequence of NbHIN1 was cloned and assigned the new GenBank number KU195817. The NbHIN1 gene was predicted to encode a protein with 229 amino acid residues. To determine the physical and chemical properties of this protein, bioinformatics tools were used. ExPASy-ProtParam analysis indicated that it has a predicted molecular mass of 26.2 kDa and includes a conserved domain LEA-14 (101–203 aa). PSORT Prediction analysis showed that the maximum likelihood of localization in the plasma membrane is 60%. Furthermore, BLAST analysis showed that NbHIN1 shared approximately 90% similarity with HIN1 from other solanaceae plants. Phylogenetic tree analysis showed that NbHIN1 was clustered into the same subgroup with Capsicum annuum HIN1 (CaHIN1). However, it has no analogy with that of monocotyledons such as rice and sorghum (Fig. 1). Confocal microscopic observation showed that the control CFP protein localized in plasma membrane (Fig. 2). Interestingly, NbHIN1RFP signals were clearly visualized in the plasma membrane as evidenced by the overlapped localization with the CFP signals and in cytosol (Fig. 2). Based on these observations, we concluded that NbHIN1 localized in the plasma membrane and cytosol.

2.9. RNA-Seq Quality of total RNAs was verified on an Agilent 2100 Bioanalyzer and based on the rRNA ratio 25S/18S, RNA integrity number, and the absence of smear. The amplified fragments were sequenced using Illumina HiSeqTM 2500 by Gene Denovo Co. (Guangzhou, China). De novo assembly and differentially expressed gene analysis were described previously [33].

3.2. Overexpression of NbHIN1 enhances the resistance to TMV 2.10. Plant treatment with exogenous hormones To investigate the role of NbHIN1 in the resistance against TMV, we generated the transgenic tobacco NbHIN1-OE under the control of the 35S promoter (Fig. 3A). PCR analysis showed that the T-DNA insertion was present in the tested 10 independent transgenic tobacco lines and NbHIN1 fusion protein with His tag was detected in the transgenic line 1, 3, 4, 7, 8 and 10 as well, but transgenic line 1 and 3 accumulated more amount of protein compared to other tested lines and line 1 and 3 were selected for further experiment (Fig. 3 B & C). No visible phenotype was observed in the NbHIN1-OE transgenic line 3 compared to wildtype (WT) plants (Fig. 3D). To examine the response of NbHIN1-OE-3 line to TMV, we infiltrated the transgenic line 3 and WT tobacco plant leaves with Agrobacterium tumefaciens carrying the TMV-GFP. Surprisingly, no visible GFP signals were observed in the infiltrated leaves of NbHIN1OE transgenic line at 4 and 5 dpi whereas pronounced green fluorescence 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

N. benthamiana plants at the 6-leaf stage were sprayed with 0.1 mmol•L−1 MeJA (Sigma-Aldrich). Control plants were sprayed with sterile water. Samples were collected at 1-hour interval for up to 12 h and immediately frozen in liquid nitrogen and stored at -80℃ until use for RNA isolation. 2.11. Yeast two-hybrid assay Y2H assay was performed according to the protocol described previously [34]. Briefly, the yeast strain was co-transformed with bait and prey plasmid combinations using lithium-acetate and polyethylene glycol 3350. Transformants harboring both bait and prey plasmids were selected on plates containing minimal medium lacking Leu and Trp (SDWL). Empty prey vector pGBKT7 or pGADT7 used as bait or prey served as controls. One colony per combination was picked from SD-WL plates to inoculate 1 mL SD-WL culture. After 36 h growth, cells were collected 149

Plant Science 283 (2019) 147–156

H. Peng, et al.

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

transcripts in inoculated leaves of overexpression and WT lines was quantified by qPCR and Fig. 4D showed that the expression level of GFP in WT plants was significantly higher than that in the NbHIN1 overexpression line. Taken together, these findings strongly indicated that overexpressing NbHIN1 in N. benthamiana significantly enhanced the resistance against TMV.

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

3.3. Transcriptomic analysis of the genes that were affected by NbHIN1 overexpression It has been proved that overexpressing NbHIN1 largely increased TMV resistance. To further dissect the molecular mechanism underlying the increased resistance against TMV in NbHIN1-OE line, we applied the RNA-Seq approach to assess the genome-wide expression profiles of NbHIN1-OE line and WT plant, respectively. Total RNA was extracted

Fig. 2. NbHIN1 localizes in the plasma membrane and cytosol. Co-transient expression of NbHIN-dsRFP and plasma membrane maker AtRop10-CFP in the epidermal cells of N. benthamiana leaves by agroinfiltration. RFP and CFP fluorescence were visualized using confocal imaging 72 h after infiltration and were depicted in red and blue, respectively. NbHIN1 was clearly visualized in the plasma membrane as evidenced by the co-localization with the plasma membrane marker AtRop10-CFP and in cytosol. The white scale bars represented 20 μm. 150

Plant Science 283 (2019) 147–156

H. Peng, et al.

Fig. 3. Generation and validation of transgenic N. benthamiana plants stably expressing NbHIN1. (A) Schematic diagram of the 35S::NbHIN1-His fusion construct. (B) PCR analysis to determine the T-DNA insertion in the representative transgenic plants. WT tobacco plants served as the negative control. “+” indicated the positive control and “-” represented the negative water control. (C) Immunoblot of NbHIN1-His fusion protein accumulating in the NbHIN1-expressing tobacco lines. Protein extracts from leaves were subjected to immunoblot and probed with an antiHIS antibody (α-HIS) and protein sizes were indicated on the left. (D) NbHIN1 overexpressing tobacco line (NbHIN-OE-3) did not display any visible phenotypes in comparison to WT plant. Representative pictures were taken 3-week after planting.

Fig. 4. NbHIN1-overexpresing N. benthamiana plants exhibit significantly increased resistance to TMV. (A) The green fluorescent signals representing the viral replication 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-day post inoculation (dpi). (B) Detection of TMV virus content by indirect-ELISA. (C) The green fluorescent signals in NbHIN1-OE and WT tobacco plants inoculated with TMV-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 NbHIN1OE and WT plants. Expression levels were represented as the fold change and normalized to actin gene. The results are mean values ( ± SD) 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). Table 1 Summary of the RNA-seq reads. Sample group

T-1

T-2

WT-1

WT-2

Raw reads Clean reads GC content (%) Uniquely mapped reads Multiple mapped reads

6710428500 6580729382 44.88 34712632 (79.96%) 2233966 (5.15%)

6362495400 6245392012 44.91 32275203 (80.28%) 2098560 (5.22%)

7699279500 7546242685 44.72 39584542 (79.87%) 2500564 (5.05%)

7103187600 6975422942 44.54 37068869 (80.66%) 2277714 (4.96%)

151

Plant Science 283 (2019) 147–156

H. Peng, et al.

3.4. NbHIN1 regulates NbRAB11 expression and exogenous MeJA treatment induces their expression Overexpression of NbHIN1 highly regulates the expression of NbRAB11 based on our RNA-seq analysis. To assess whether exogenous MeJA affects the expression of NbHIN1 and NbRAB11, we treated the plant with 0.1 mmol•L−1 MeJA and quantified the gene expression by qRT-PCR. The results showed that the expression of two genes after MeJA treatment was significantly increased at different time points. However, they displayed a similar expression pattern in 5–12 h and peaked in 9 h after treatment (Fig. 8). These findings indicated that overexpressing NbHIN1 could activate the JA-mediated signaling pathway through inducing the NbRAB11 gene expression. To further understand the function of NbHIN1 and NbRAB11, we examined whether there was a physical interaction between them using yeast two-hybrid analysis and bimolecular fluorescence complementation assay. However, no direct interaction was observed either in Y2H or in BiFC assay (Supplementary Fig. S2). 4. Discussion HIN1 is a class of protein produced during the process of hypersensitivity induced by Harpin protein in non-host plants. In this study, we descried the cloning, molecular and functional characterization of NbHIN1 in response to TMV infection. Using transient tobacco expression system, we found that NbHIN1 localized in the plasma membrane and cytosol. Stable expression of NbHIN1 in tobacco plant resulted in significantly enhanced resistance of tobacco against TMV infection. Moreover, RNA-sequencing revealed that overexpression of NbHIN1 highly induced the expression of NbRAB11, suggesting that jasmonic acid signaling pathway might be involved in TMV resistance. This is the first example of study on overexpression of NbHIN1 gene conferring TMV resistance. Previous studies showed that several members of the NHL family have the cell membrane subcellular localization [13,35]. However, we observed that NbHIN1 localized both in the membrane and in cytosol (Fig. 2). The observed cell membrane localization of NbHIN1 is consistent with the predicted highest likelihood of protein localization by bioinformatics tool. It has been reported that some members in the NHL family reside in the endoplasmic reticulum due to sarcolipin-like sequences present in the protein [37]. These findings suggest that the subcellular localization of HIN1 protein is diverse. The Arabidopsis NDR1 protein with cell membrane localization was found to function in an amplification of the initial event following pathogen perception [13,36]. Because NbHIN1 exhibits sequence similarity to NDR1, its cell membrane localization suggested that it might function as an elicitor of HR reaction following pathogen perception. Our studies showed that the isolated ORF of HIN1 from N. benthamiana encodes a protein containing 229 amino acid residues with a predicted and conserved LEA-14 domain, which is also present in other HIN1 genes [37]. Previous studies have demonstrated that the plant gene family LEA-14 expressed in the plant both in response to pathogen and drought, suggesting a common molecular mechanism between the plant response to biotic and abiotic stresses [38,39]. The existence of this conserved domain in NbHIN1 strongly indicated that it might be involved in plant defense response. It has been reported that HIN1 is required in the acquired resistance of plant in response to fungal and bacterial infection. For example, overexpression of rice OsHIN1 induces resistance to rice blast [40]. In previous study, it was found that the transcription level of HIN1 was significantly increased after TMV infection [41], which suggested that HIN1 as an important component participates in a rapid resistance reaction after TMV infection. We observed that overexpression of NbHIN1 can largely inhibit tobacco mosaic virus infection both at RNA and protein levels (Fig. 4). Although HIN1 serves as a marker gene for cell death, transgenic N. benthamiana line overexpressing NbHIN1 grew normally and did not display any

Fig. 5. Volcano plots of the differentially expressed genes. The transcriptome of NbHIN1-overexpressing plants was compared to wild-type plants. Differentially expressed genes were represented with red dots (up-regulated) or green dots (down-regulated), and others (no differential difference) indicated with black dots. The x-axis specified the fold-changes (FC) and the y-axis specified the negative logarithm to the base 10 of the FDR values as statistical significance.

from two independent NbHIN1-OE line 1 and 3 plants (T-1 and T-2) and two WT (WT1 and WT2) Nicotiana benthamiana plants, respectively. 43 million and 48 million clean reads number in total were obtained from the two libraries of NbHIN1 and WT plants, respectively. After quality control filtering and trimming adaptor sequencing, around 32 million clean reads from every library were mapped to N. benthamiana genome. The summary of RNA-seq reads was showed in Table 1. Sequence reads have been deposited in the NCBI Sequence Read Archive (SRA) under accession number PRJNA450205. The transcriptome of NbHIN1-OE and WT plant represented high reproducibility among biological replicates, as indicated by the Pearson correlation coefficient is 0.9945 and 0.9987, respectively, indicating that the RNA-seq data was accurate and reproducible. The volcano plots of the differentially expressed genes (DEGs) demonstrated that 102 genes in N. benthamiana were differentially expressed after NbHIN1 overexpression compared to those in WT plant, in which 48 genes were up-regulated and 54 genes were down-regulated with more than twofold changes (Fig. 5). In particular, we list top 10 down and up-regulated genes in Table 2. To elucidate the biological function or pathways that DEGs involved, gene ontology (GO) enrichment analyses were conducted. GO analysis revealed that GO terms were classified into three main classes: biological process, cellular component and molecular function including 21 sub-categories (Fig. 6), Among the biological process class, metabolic process (21.4%) and cellular process (21.2%) were the two dominant groups, while in the molecular function class, binding (51.3%) was the major group. In addition, KEGG annotation revealed that all annotated DEGs were classified into 23 categories and the top 20 abundant biochemical pathways with numbers of assigned genes were shown in Supplementary Fig. S1. To validate the RNA-Seq results, we selected two highly upregulated genes: NbRAB11 (Ras-related protein Rab11) (Niben101Scf35013g00007) and NbPARN (poly(A)-specific ribonuclease) (Niben101Scf05961g02014) from DGEs for qRT-PCR analysis. qPCR showed that the expression patterns (8.05-fold and 4.07fold) of these two genes were consistent with those in RNA-Seq, suggesting that our RNA-Seq data is accurate and reliable (Fig. 7).

152

Plant Science 283 (2019) 147–156

H. Peng, et al.

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

Log2 fold change

Seq.Descripition

Niben101Scf01795g04035 Niben101Scf03015g06015 Niben101Ctg12716g00002 Niben101Scf01795g04036 Niben101Scf01795g04037 Niben101Scf09116g02014 Niben101Scf01534g02026 Niben101Scf04528g13017 Niben101Scf01795g04026 Niben101Scf03830g02001 Niben101Scf05146g03008 Niben101Scf02348g10009 Niben101Scf02015g04019 Niben101Scf08020g06001 Niben101Scf01740g17007 Niben101Scf17372g01008 Niben101Scf01150g08001 Niben101Scf02110g00028 Niben101Scf35013g00007 Niben101Scf03600g02026

−13.1635 −13.0810 −12.0768 −11.9549 −11.3581 −10.4041 −9.6970 −8.1137 −4.7435 −4.4594 10.7690 9.2877 9.0768 8.1056 6.0000 4.2854 4.1260 2.6871 2.5575 2.5564

BnaA05g32900D [Brassica napus] Ribulose bisphosphate carboxylase small chain 8B, chloroplastic Filament-like plant protein Peptidyl-prolyl cis-trans isomerase D Filament-like plant protein 3 Alanine-tRNA ligase Annexin D3 Histone H2A Glutamyl-tRNA(Gln) amidotransferase subunit A BZIP family transcription factor [Medicago truncatula] CASP-like protein 4D1 Amino acid permease 6 Curved DNA-binding protein late embryogenesis abundant hydroxyproline-rich glycoprotein [Arabidopsis thaliana] ATP-dependent RNA helicase ded1 Receptor-like protein kinase nodulin MtN21 /EamA-like transporter family protein Pectinacetylesterase family protein Ras-related protein Rab11 Sister chromatid cohesion 1 protein 2

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

visible cell death symptoms (Fig. 3) and same observation has been reported in the transgenic soybean overexpressing GmHIN1 line [42]. We found that the NbRAB11 is highly differentially expressed gene based on our transcriptome analysis. The RAB protein family represents the largest member of the Ras superfamily of monomeric G proteins, also referred to as small ATPases [43]. It has been documented that RAB is involved in resistance against biotic and abiotic stresses. For example, in salt stressed tomato plants, expression of RAB11 was repressed and expression of RAB2 was induced as shown by microarray analysis of total RNA [44]. Similarly, transgenic tobacco plants overexpressing Prosopis juliflora RAB7 displayed resistance to high-salt stress [45] and ectopic expression of OsRAB7 enhanced tolerance of peanut plants to several abiotic stresses. Furthermore, in rice, several lines of evidence suggest that OsRAB11 acts with OsOPR8 to positively regulate JA-mediated signaling through activation of the JA biosynthetic

Fig. 6. GO annotation and functional classification of all deferentially expressed genes. Gene ontology terms are classified as biological process, cellular component and 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 function cluster. 153

Plant Science 283 (2019) 147–156

H. Peng, et al.

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 determined 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.

hormone pathways to gain resistance.

Previous studies reported that PARN is involved in the processing of certain non-coding RNAs [52], such as the processing of snoRNA and small Cajal RNA (scaRNA), and it is also responsible for cellular DNA damage [53,54] and antiviral infections. Studies have found that zinc finger antiviral proteins can selectively recruit PARN proteins, thereby excising the poly(A) tail of viral mRNA and degrading RNA itself from the 3′-5′ direction through the exonuclease complex to inhibit HIV-1 infection [55]. We can speculate that overexpression of NbHIN1 strongly induced the expression of PARN which might result in the degradation of TMV mRNA and thereby inhibit viral replication. Our results indicated that NbHIN1 and NbRAB11 were up-regulated at 5 h and 2 h after MeJA treatment, respectively, and the expression patterns of these two genes at 5–12 h was similar (Fig. 8), indicating that these two genes have a certain correlation in response to jasmonic acid and may be involved in the same pathway. However, there is no direct interaction between NbRAB11 and NbHIN1and the specific relationship between them requires further study. Related studies have shown that TaHIN1 rapidly responded to strip rust infection and was strongly induced after treatment with salicylic acid and jasmonic acid [56]. These findings suggested that overexpression of NbHIN1 activates certain

5. Conclusion Taken together, our findings indicate NbHIN1 has a function in the defense response against TMV. The information presented here underlines the importance of understanding the molecular function of NbHIN1 in TMV resistance. We demonstrate for the first time that overexpressing a pathogenesis-related gene NbHIN1 in N. benthamiana significantly enhances the TMV resistance by potentially activating jasmonic acid signaling pathway. All in all, these findings will expand our understanding towards the function of HIN1 and will provide invaluable resources for engineering the tobacco plant against TMV in the future. Author contributions XS and HP designed the experiment. HP, YP, XY and GW performed the experiment. HP, XS, LQ and LM wrote the manuscript. All authors read and approved the final manuscript. 154

Plant Science 283 (2019) 147–156

H. Peng, et al.

Funding

[17] I. Nookaew, M. Papini, N. Pornputtapong, G. Scalcinati, L. Fagerberg, M. Uhlén, J. Nielsen, A comprehensive comparison of RNA-Seq-based transcriptome analysis from reads to differential gene expression and cross-comparison with microarrays: a case study in Saccharomyces cerevisiae, Nucleic Acids Res. 40 (2012) 10084–10097. [18] A. Heo, H.J. Jang, J.S. Sung, W. Park, Global transcriptome and physiological responses of acinetobacter oleivorans DR1 exposed to distinct classes of antibiotics, Plos One 9 (2014) e110215. [19] N. Qin, X. Tan, Y. Jiao, L. Liu, W. Zhao, S. Yang, A. Jia, RNA-Seq-based transcriptome analysis of methicillin-resistant Staphylococcus aureus biofilm inhibition by ursolic acid and resveratrol, Sci. Rep. 4 (2014) 5467. [20] C.F. Le, R. Gudimella, R. Razali, R. Manikam, S.D. Sekaran, Transcriptome analysis of Streptococcus pneumoniae treated with the designed antimicrobial peptides, DM3, Sci. Rep. 6 (2016) 26828. [21] N. Sierro, J.N. Battey, S. Ouadi, N. Bakaher, L. Bovet, A. Willig, S. Goepfert, M.C. Peitsch, N.V. Ivanov, The tobacco genome sequence and its comparison with those of tomato and potato, Nat. Commun. 5 (2014) 3833. [22] R. Liu, R.A. Vaishnav, A.M. Roberts, R.P. Friedland, Humans have antibodies against a plant virus: evidence from Tobacco mosaic virus, Plos One 8 (2013) e60621. [23] Y. Liu, M. Schiff, R. Marathe, S.P. Dinesh‐Kumar, Tobacco Rar1, EDS1 and NPR1/ NIM1 like genes are required for N‐mediated resistance to Tobacco mosaic virus, Plant J. 30 (2002) 415–429. [24] Z. Xin, A. Wang, G. Yang, P. Gao, Z.L. Zheng, The Arabidopsis A4 subfamily of lectin receptor kinases negatively regulates abscisic acid response in seed germination, Plant Physiol. 149 (2009) 434–444. [25] Y. Lu, F. Yan, W. Guo, H. Zheng, L. Lin, J. Peng, M.J. Adams, J. Chen, Garlic virus X 11‐kDa protein granules move within the cytoplasm and traffic a host protein normally found in the nucleolus, Mol. Plant Pathol. 12 (2011) 666–676. [26] K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, S. Kumar, MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods, Mol. Biol. Evol. 28 (2011) 2731–2739. [27] A. Krogh, B. Larsson, V.H. Gunnar, E.L. Sonnhammer, Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes, J. Mol. Biol. 305 (2001) 567–580. [28] P. Benkert, M. Biasini, T. Schwede, Toward the estimation of the absolute quality of individual protein structure models, Bioinformatics 27 (2010) 343–350. [29] L. Ma, E. Lukasik, F. Gawehns, F.L. Takken, The use of agroinfiltration for transient expression of plant resistance and fungal effector proteins in Nicotiana benthamiana leaves, Plant Fungal Pathogens, Springer, 2012, pp. pp. 61–74. [30] R.B. Horsch, J.E. Fry, N.L. Hoffmann, D. Eichholtz, S.G. Rogers, R.T. Fraley, A simple and general method for transferring genes into plants, Biol. Sci. 227 (1985) 1229–1231. [31] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680. [32] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2 −ΔΔCT method, Methods 25 (2001) 402–408. [33] K. Liu, S. Feng, Y. Pan, J. Zhong, Y. Chen, C. Yuan, H. Li, Transcriptome analysis and identification of genes associated with floral transition and flower development in sugar apple (Annona squamosa L.), Front. Plant Sci. 7 (2016) 1695. [34] L. Ma, M. Djavaheri, H. Wang, N.J. Larkan, P. Haddadi, E. Beynon, G. Gropp, M.H. Borhan, Leptosphaeria maculans effector protein AvrLm1 modulates plant immunity by enhancing MAP kinase 9 phosphorylation, iScience 3 (2018) 177–191. [35] P. Coppinger, P.P. Repetti, B. Day, D. Dahlbeck, A. Mehlert, B.J. Staskawicz, Overexpression of the plasma membrane‐localized NDR1 protein results in enhanced bacterial disease resistance in Arabidopsis thaliana, Plant J. 40 (2004) 225–237. [36] C. Knepper, E.A. Savory, B. Day, The role of NDR1 in pathogen perception and plant defense signaling, Plant Signal. Behav. 6 (2011) 1114–1116. [37] S.B. Lee, B.K. Ham, J.M. Park, Y.J. Kim, K.H. Paek, BnNHL18A shows a localization change by stress-inducing chemical treatments, Biochem. Biophys. Res. Commun. 339 (2006) 399–406. [38] F.D. Ciccarelli, P. Bork, The WHy domain mediates the response to desiccation in plants and bacteria, Bioinformatics 21 (2004) 1304–1307. [39] K. Goyal, L.J. Walton, A. Tunnacliffe, LEA proteins prevent protein aggregation due to water stress, Biochem. J. 388 (2005) 151–157. [40] R. Rakwal, G.K. Agrawal, S. Tamogami, H. Iwahashi, Transcriptional profiling of OsHin1 in rice plants: a potential role in defense/stress and development, Plant Sci. 166 (2004) 997–1005. [41] S. Guo, S.M. Wong, Deep sequencing analysis reveals a TMV mutant with a poly(A) tract reduces host defense responses in Nicotiana benthamiana, Virus Res. 239 (2017) 126–135. [42] A. Maldonado, R. Youssef, M. Mcdonald, E. Brewer, H. Beard, B. Matthews, Overexpression of four Arabidopsis thaliana NHL genes in soybean (Glycine max) roots and their effect on resistance to the soybean cyst nematode (Heterodera glycines), Physiol. Mol. Plant Pathol. 86 (2014) 1–10. [43] H. Stenmark, V.M. Olkkonen, The rab GTPase family, Genome Biol. 2 (2001) reviews3007.3001-3007.3007. [44] S. Zhou, S. Wei, B. Boone, S. Levy, Microarray analysis of genes affected by salt stress in tomato, Afr. J. Environ. Sci. Technol. 1 (2007) 14–26. [45] S. George, A. Parida, Over-expression of a Rab family GTPase from phreatophyte Prosopis juliflora confers tolerance to salt stress on transgenic tobacco, Mol. Biol. Rep. 38 (2011) 1669–1674. [46] C. Li, A.L. Schilmiller, G. Liu, G.I. Lee, S. Jayanty, C. Sageman, J. Vrebalov, J.J. Giovannoni, K. Yagi, Y. Kobayashi, G.A. Howe, Role of β-oxidation in jasmonate biosynthesis and systemic wound signaling in tomato, Plant Cell. 17 (2005)

This study was partly supported by the National Natural Science Foundation of China (30900937, 31670148) and the Fundamental Research Funds for the Central Universities (XDJK2016A009, XDJK2017C015). Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a 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 (City University of New York, New York, USA) for providing the plant expression vector pFGC5941 vector, Dr. Jianping Chen (Zhejiang Academy of Agricultural Sciences, Zhejiang, China) for providing expression vector pCV-dsRFP-N1 and BiFC series vector pCV-nYFP-C1 and pCV-cYFP-C1and Dr. Bingsheng Qiu (Institute of Microbiology, Chinese Academy of Sciences, China) for providing anti-bodies. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.plantsci.2019.02.018. References [1] J. Zhu, Salt and drought stress signal transduction in plants, Annu. Rev. Plant Biol. 53 (2002) 247–273. [2] J.D. Jones, J.L. Dangl, The plant immune system, Nature 444 (2006) 323. [3] K. Yamaguchi-Shinozaki, K. Shinozaki, Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses, Annu. Rev. Plant Biol. 57 (2006) 781–803. [4] M.S. Choi, W. Kim, C. Lee, C.S. Oh, Harpins, multifunctional proteins secreted by gram-negative plant-pathogenic bacteria, Mol. Plant Microbe Interact. 26 (2013) 1115–1122. [5] P.B. Lindgren, R.C. Peet, N.J. Panopoulos, Gene cluster of Pseudomonas syringae pv. "phaseolicola" controls pathogenicity of bean plants and hypersensitivity of nonhost plants, J. Bacteriol. 168 (1986) 512–522. [6] F. Niepold, D. Anderson, D. Mills, Cloning determinants of pathogenesis from Pseudomonas syringae pathovar syringae, Proc. Natl. Acad. Sci. U. S. A. 82 (1985) 406–410. [7] R.N. Goodman, A.J. Novacky, The Hypersensitive Reaction in Plants to Pathogens: a Resistance Phenomenon, American Phytopathological Society (APS), 1994. [8] S. Gopalan, W. Wei, S.Y. He, hrp gene‐dependent induction of hin1: a plant gene activated rapidly by both harpins and the avrPto gene‐mediated signal, Plant J. 10 (1996) 591–600. [9] K.S. Century, A.D. Shapiro, P.P. Repetti, D. Dahlbeck, E. Holub, B.J. Staskawicz, NDR1, a pathogen-induced component required for Arabidopsis disease resistance, Science 278 (1997) 1963–1965. [10] P. Dörmann, S. Gopalan, S.Y. He, C. Benning, A gene family in Arabidopsis thaliana with sequence similarity to NDR1 and HIN1§, Plant Physiol. Biochem. 38 (2000) 789–796. [11] M.S. Zheng, H. Takahashi, A. Miyazaki, H. Hamamoto, J. Shah, I. Yamaguchi, T. Kusano, Up-regulation of Arabidopsis thaliana NHL10 in the hypersensitive response to Cucumber mosaic virus infection and in senescing leaves is controlled by signalling pathways that differ in salicylate involvement, Planta 218 (2004) 740–750. [12] A. Varet, J. Parker, P. Tornero, N. Nass, T. Nürnberger, J.L. Dangl, D. Scheel, J. Lee, NHL25 and NHL3, two NDR1/HIN1-like genes in Arabidopsis thaliana with potential role(s) in plant defense, Mol. Plant Microbe Interact. 15 (2002) 608–616. [13] A. Varet, B. Hause, G. Hause, D. Scheel, J. Lee, The Arabidopsis NHL3 gene encodes a plasma membrane protein and its overexpression correlates with increased resistance to Pseudomonas syringae pv. tomato DC3000, Plant Physiol. 132 (2003) 2023–2033. [14] M.L. Metzker, Sequencing technologies—the next generation, Nat. Rev. Genet. 11 (2010) 31. [15] U. Nagalakshmi, Z. Wang, K. Waern, C. Shou, D. Raha, M. Gerstein, M. Snyder, The transcriptional landscape of the yeast genome defined by RNA sequencing, Science 320 (2008) 1344–1349. [16] Z. Wang, M. Gerstein, M. Snyder, RNA-Seq: a revolutionary tool for transcriptomics, Nat. Rev. Genet. 10 (2009) 57.

155

Plant Science 283 (2019) 147–156

H. Peng, et al.

971–986. [47] M.J. Hong, Y. mi Lee, Y.S. Son, C.H. Im, Y.B. Yi, Y.G. Rim, J.D. Bahk, J.B. Heo, Rice Rab11 is required for JA-mediated defense signaling, Biochem. Biophys. Res. Commun. 434 (2013) 797–802. [48] C. Biesgen, E. Weiler, Structure and regulation of OPR1 and OPR2, two closely related genes encoding 12-oxophytodienoic acid-10, 11-reductases from Arabidopsis thaliana, Planta 208 (1999) 155–165. [49] A. Stintzi, B. John, The Arabidopsis male-sterile mutant, opr3, lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis, Proc. Natl. Acad. Sci. 97 (2000) 10625–10630. [50] A. Stintzi, H. Weber, P. Reymond, J. Browse, E.E. Farmer, Plant defense in the absence of jasmonic acid: the role of cyclopentenones, Proc. Natl. Acad. Sci. 98 (2001) 12837–12842. [51] F. Zhu, D.H. Xi, S. Yuan, F. Xu, D.W. Zhang, H.-H. Lin, Salicylic acid and jasmonic acid are essential for systemic resistance against Tobacco mosaic virus in Nicotiana benthamiana, Mol. Plant Microbe Interact. 27 (2014) 567–577. [52] H. Berndt, C. Harnisch, C. Rammelt, N. Stöhr, A. Zirkel, J.C. Dohm,

[53]

[54]

[55]

[56]

156

H. Himmelbauer, J.P. Tavanez, S. Hüttelmaier, E. Wahle, Maturation of mammalian H/ACA box snoRNAs: PAPD5-dependent adenylation and PARN-dependent trimming, Rna 18 (2012) 958–972. H.C. Reinhardt, P. Hasskamp, I. Schmedding, S. Morandell, M.Av. Vugt, X. Wang, R. Linding, S.-E. Ong, D. Weaver, S.A. Carr, M.B. Yaffe, DNA damage activates a spatially distinct late cytoplasmic cell-cycle checkpoint network controlled by MK2mediated RNA stabilization, Mol. Cell 40 (2010) 34–49. M.A. Cevher, X. Zhang, S. Fernandez, S. Kim, J. Baquero, P. Nilsson, S. Lee, A. Virtanen, F.E. Kleiman, Nuclear deadenylation/polyadenylation factors regulate 3′ processing in response to DNA damage, EMBO J. 29 (2010) 1674–1687. Y. Zhu, G. Chen, F. Lv, X. Wang, X. Ji, Y. Xu, J. Sun, L. Wu, Y.-T. Zheng, G. Gao, Zinc-finger antiviral protein inhibits HIV-1 infection by selectively targeting multiply spliced viral mRNAs for degradation, Proc. Natl. Acad. Sci. 108 (2011) 15834–15839. L. Deng, X. Wang, X. Liu, G. Cai, C. Tang, G. Wei, L. Huang, Z. Kang, Isolation and expression analysis of a TaHin1 gene induced by stripe rust fungus in wheat, Sci. Agric. Sin. 43 (2010) 1977–1984.