Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain

Cucumber mosaic virus (CMV) is a destructive pathogen affecting Capsicum annuum (pepper) production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase). Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum ‘Bukang’ cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP). Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic) leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.


Introduction
All viruses are dependent on their host factors for a successful infection of hosts through the virus-host interactions. Physical interactions between viral components and host factors are required for replication, cell-to-cell movement, and systemic movement in viral pathogenesis [1]. Accordingly, host factors are essential components in most steps of virus infection [2,3]. The outcome of such interactions determines host specificity and tissue specificity of virus strains [4,5]. Without the interactions with host factors, viruses are unable to infect; hence, virus-host interactions can be utilized for development of virus-resistant crops [4]. strain containing the bait vector. Yeast co-transformants were incubated in the selection medium lacking tryptophan and leucine (SC-Try, Leu) for 5 d at 30°C. After co-transformation, each colony was streaked on synthetic complete medium (SC) lacking tryptophan, leucine and histidine (SC-Try, Leu, His) and grown for 5 d at 30°C. The pLAM5'-1/pAS2-1 and pTD1-1/pACT2 plasmids (Clontech, Mountain View, CA, USA) were used as a negative control, and pVA3-1/pAS2-1 and pTD1-1/pACT2 were used as a positive control.

β-Galactosidase Filter Lift Assay
To identify interaction between candidate cDNAs and the CMV-P1 RNA1 helicase domain, co-transformed colonies were incubated in the selection synthetic complete liquid medium lacking tryptophan and leucine (SC-Try, Leu) for 3 d at 30°C. After 3 d, cells from each culture were incubated for 4 d at 30°C on synthetic complete medium lacking tryptophan and leucine (SC-Try, Leu) until the diameter of each colony was 0.4-0.7 mm. A 3MM filter (Whatman, Maidstone, Kent, UK) was placed in contact with all of the clones. The filter was then dipped in liquid nitrogen for 15 s and thawed for 1 min at room temperature. After three repeats of this step, the 3MM filter was soaked in Z buffer (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 ÁH 2 O, 10 mM KCl, 1 mM MgSO 4 Á7H 2 O, 39 mM 2-mercaptoethanol, pH 7.0) with X-gal. The filter was then incubated for 8 h at 30°C in the dark and the signal was captured by a digital camera.

PCR Screening
For PCR screening, primers were designed based on the pAD-GAL4-2.1 vector multiple cloning site (MCS) (S1 Table). Using these primers, the DNA fragments were amplified from the yeast clones containing candidate cDNAs by colony PCR. The PCR products were eluted and then cloned using the T-Blunt PCR cloning system (Solgent, Daejeon, South Korea). The ligated DNA fragments were transformed into E. coli strain DH10B and incubated in LB medium containing 50 mg/L kanamycin for selection. To confirm the cloning of PCR fragments, colony PCR was performed using AD vector-specific primers. The clones containing cDNA fragments were incubated in liquid LB medium containing 50 mg/L kanamycin for 1 d at 37°C in a shaking incubator. Plasmids were isolated from cultured cells using the AccuPrep 1 Plasmid Mini Extraction Kit (Bioneer, Daejeon, South Korea) and sequences were determined (NICEM, Seoul National University, Seoul, South Korea).

Sequence Analysis of Candidate Genes
The sequences of candidate genes were determined at the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) and blasted against C. annuum database (http://peppergenome.snu.ac.kr). The candidate gene name, description, and sequence ID are listed in Table 1 and S2 Table. Plasmid Construct for Virus-Induced Gene Silencing (VIGS) The candidate genes were amplified from the C. annuum 'Bukang' cDNA using gene-specific primers designed based on the C. annuum database (http://peppergenome.snu.ac.kr) (S1 Table). A modified ligation-independent cloning system was used for cloning of the inserts into the TRV VIGS vector [22]. All PCR products were purified with the DNA Clean & Con-centrator™-100 (Zymo Research, Irvine, CA, USA). The purified PCR products (15 fmol) were mixed with 5 mM dATP and treated with T4 DNA polymerase (Novagen, Darmstadt, Germany) at 22°C for 30 min. The TRV2-LIC vector was digested with Pst1 and treated with T4 DNA polymerase with dTTP. The treated PCR product and TRV2-LIC vector were mixed in a 5:1 ratio and incubated at 65°C for 2 min and then transferred to 22°C for 10 min. A sample of the mixture (3 μL) was transformed into E. coli DH10B and transformed colonies were selected by colony PCR using LIC primers (S1 Table). Plasmids were extracted from identified colonies (Zymo Research, Irvine, CA, USA). Sequencing analysis was performed at the National Instrumentation Center for Environmental Management (Seoul National University, Seoul, Korea).

Plant Materials and Agrobacterium Infiltration
N. benthamiana plants were grown for 4 weeks at 23°C with a 16-h light/8-h dark cycle. For the VIGS, the TRV VIGS system was used [23,24]. TRV1 or TRV2 derivatives were transformed into Agrobacterium and the resulting strains were incubated in liquid LB medium containing antibiotics (50 mg/L kanamycin and 50 mg/L rifampicin) for 20 h at 30°C. The Agrobacterium cells were harvested and resuspended in infiltration medium (10 mM MgCl 2 , 10mM MES, 200μM acetosyringone), adjusted to 0.4 OD 600 , and incubated at room temperature with shaking for 4 h. Agrobacterium culture containing TRV1 was adjusted to 0.3 OD 600 and incubated as described above. TRV1 and TRV2 or its derivatives were mixed in a 1:1 ratio and infiltrated into N. benthamiana at the four-leaf stage using a 1-mL syringe needle. At 12 d post infiltration (dpi), the silenced plants were used for further experiments.

RNA Extraction and RT-PCR Analysis
Total RNA was extracted from leaves of C. annuum 'Bukang' and silenced N. benthamiana plants using GeneAll R Hybrid-R™ (Gene All Biotechnology, Seoul, South Korea) according to the manufacturer's protocol. First-strand cDNA was synthesized from 4 μg total RNA using M-MLV reverse transcriptase (Promega, Madison, WI, USA) and oligo-(d)T primers (Bioneer, Daejeon, South Korea) according to the manufacturer's protocol. For VIGS, the expression of candidate genes was analyzed by semi-quantitative RT-PCR and real-time PCR using gene-specific primers (S1 Table). For the semi-quantitative RT-PCR, Actin transcript was used as an endogenous control [25]. The real-time PCR was performed using a Lightcycler 1 480 instrument (Roche, Switzerland). Thermal cycling was as follows: denaturing at 95°C for 5 min, followed by 45 cycles of denaturing for 10 s, annealing at 60°C for 20 s and extension at 72°C for 15 s.

Virus Inoculation and Evaluation of Resistance
CMV-P1-GFP inoculum was propagated in N. benthamiana at 23°C with a 16-h light/8-h dark cycle. The silenced plants were inoculated at the four-to-six-leaf stage, and the two oldest leaves were used for carborundum rub-inoculation with virus produced using grinding systemically infected leaves of N. benthamiana in 100 mM potassium phosphate buffer, pH 7.0 (1g tissue; 10 mL buffer). Plants were kept in a growth chamber at 25°C until symptom observation. Nonand mock-inoculated controls were included. For Agrobacterium-mediated inoculation of CMV-P1-GFP, Agrobacterium carrying CMV-P1-GFP was incubated in liquid LB medium containing antibiotics (50 mg/L kanamycin and 50 mg/L rifampicin) for 20 h at 30°C. The Agrobacterium cells were harvested and resuspended in infiltration medium (10 mM MgCl 2 , 10 mM MES, 200 μM acetosyringone), adjusted to 0.4 OD 600 , incubated at room temperature with shaking for 3 h and then infiltrated into N. benthamiana at the four-leaf stage using a 1-mL syringe needle. Virus accumulation was tested using inoculated and upper non-inoculated leaves at 5 and 10 dpi by DAS-ELISA according to the manufacturer's instructions (Agdia, Inc., Elkhart, USA). GFP was visualized using a confocal laser-scanning microscope (LSM 510; Carl Zeiss, Jena, Germany).

Isolation of Candidate Genes Interacting with CMV Helicase Domain
To identify host genes interacting with the P1 Hel domain of CMV-P1, we performed yeast two-hybrid screening analysis. P1 Hel was cloned into a bait vector and a total 100,800 of C. annuum 'Bukang' cDNAs were cloned into prey vector. P1 Hel was co-transformed into YRG-2 yeast strain containing 'Bukang' cDNA (Table 2), and co-transformed yeast cells were grown on the non-selective synthetic complete medium (SC-Leu-Trp) and on the selective medium (SC-Leu-Try-His). When the 156 candidate interacting clones were subjected to a β-galactosidase filter lift assay to confirm interaction, only 82 showed a positive response. These 82 clones interacting with the P1 Hel domain were used for further study (Table 2).

Silencing of the Candidate Genes in N. benthamiana
To test whether the selected genes were required for CMV infection and plant development, the 10 candidate genes were silenced using a TRV-based VIGS system. VIGS was performed with 2 weeks old N. benthamiana plants using previously described protocols and silenced plants were compared with wild-type N. benthamiana. At 12 dpi, VIGS plants showed various developmental phenotypes, such as curved leaves, stunting, and arrested growth, depending on the targeted gene (Fig 2b-2k). Plants infected with TRV::00 (empty vector) were used as negative control (Fig 2l) and Phytoene desaturase (PDS)-silenced plants were used as a silencing control (data not shown). Among 10 candidate genes, VIGS plants for UBI11, ARF1, and ARF showed severe developmental defects at 6 dpi and yellow leaves at 10 dpi (data not shown). Eventually, the VIGS plants of these three lines died at 12 dpi (Fig 2f-2h). TRV::00 plants showed typical TRV symptoms such as curved leaves and slow growth compared to wild type. The VIGS lines for Cysk, FDH, CRT3, AGPase, H3, ARD, and PPM showed similar phenotypes to the TRV::00 control plants (Fig 2). These results demonstrate that VIGS targeting of Cysk, FDH, CRT3, AGPase, H3, and ARD genes does not affect plant growth and that these lines are suitable for studying CMV infectivity in N. benthamiana.
To check expression levels of the targeted genes, semi-quantitative RT-PCR (S2 Table) was performed using gene-specific primers. Infiltrated TRV::00 (empty vector) plants were used a positive control. The mRNA expression levels of Cysk, FDH, CRT3, AGPase, and ARD were significantly reduced in VIGS lines at 12 dpi compared to TRV::00 plants. However, H3 expression was not reduced in H3 VIGS plants (Fig 3a). We also checked for co-silencing of candidate genes in VIGS plants for each gene, and found no effects of the expression of other candidate genes (Fig 3b).

Effects on CMV-P1 Infection of Silencing of the Candidate Genes
To investigate the effects of silencing each candidate gene on CMV-P1 infection, CMV-P1-GFP was inoculated into the upper two leaves of VIGS plants. We monitored GFP florescence of the CMV-P1 virus using a confocal laser-scanning microscope at 5 dpi and 10 dpi (Fig 4). After CMV-P1 infection, the inoculated leaves of the TRV::00 control line showed CysK, Cysteine synthase; AGPase, ADP-glucose pyrophosphorylase; ARF1, ADP-ribosylation factor 1; ARF, ADP-ribosylation factor; H3, Histone-H3; ARD, Acireductone dioxygenase were used. P and N represent positive and negative controls, respectively. Empty is a bait vector alone. SD medium (-LW; lacking tryptophan and leucine and -LWH; lacking tryptophan, leucine, and histidine) was used to select for co-transformation. β-galactosidase (β-Gal) activity assays were performed according to the manufacturer's protocol.   was detected strongly at 10 dpi although no fluorescence was detected in the un-inoculated upper leaves. We also detected GFP signal in the both inoculated and uninoculated leaves of ARD and PMM-silenced plants at 10 dpi. These results indicate that Cysk, AGPase, H3, ARD, and PMM are not main factors for amplification of CMV-P1. The FDH-silenced plants showed weak GFP signal in inoculated leaves, but no GFP signal in uninoculated leaves. In the case of CRT3-silenced plants, there was no CMV-P1-GFP signal in the either the inoculated or the uninoculated, upper leaves at 5 dpi and 10 dpi. To confirm the virus infection in the candidate gene-silenced plants, CMV-P1 coat protein (CP) accumulation was detected by ELISA at 5 and 10 dpi using leaf discs of the inoculated and uninoculated upper leaves in each line. ELISA analysis showed the same trend as the GFPbased analysis (S1 Fig). CP accumulation was not detected in inoculated or un-inoculated leaves of FDH-, CTR3-, ARD-, and PMM-silenced plants at 5 dpi, whereas a reduction of CP accumulation at 10 dpi was observed only in FDH-and CTR3-silenced plants (S1 Fig). To confirm that FDH and CRT3 are necessary for CMV infection, more detailed analysis was performed. Again, CMV-P1-GFP was inoculated into N. benthamiana plants after silencing of FDH and CRT3 genes. After infection, the inoculated leaves of TRV::00 showed strong GFP fluorescence at 11 dpi although weak fluorescence was detected in the uninoculated leaves. However, we did not detect GFP signal at 11 dpi in either inoculated or uninoculated leaves of FDH-and CRT3-silenced plants (S2 Fig). CMV accumulation was also significantly decreased in the FDH-and CRT-silenced plants (Fig 5a). To confirm that the expression levels of the targeted genes were reduced at that time point, real-time PCR was performed using gene-specific primers (S2 Table). Infiltrated TRV::00 (empty vector) plants were used a positive control. The mRNA expression levels of FDH and CRT3 were significantly reduced at 11 dpi in the corresponding VIGS lines compared with TRV::00 plants (Fig 5b). Taken together, these results suggest that FDH and CRT3 are an essential factors for CMV-P1 infection in plants.

Discussion
In this study, we identified novel host factors that interacted with the CMV-P1 helicase domain for CMV infection. We found that N. benthamiana plants silenced for the FDH or CRT3 gene showed decreased infection by CMV-P1, indicating that FDH and CRT3 are required for CMV-P1 infection in the plant.
FDH is one of the most abundant soluble proteins in mitochondria and is found in various organisms such as bacteria, yeast, and plants. FDH catalyzes the oxidation of formate (HCOO-) into CO 2 [28,41]. FDH has a putative mitochondrial signal peptide for targeting to mitochondria [42], although Arabidopsis FDH is localized in both mitochondria and chloroplasts [43,44]. Mitochondria are involved in programmed cell death and the hypersensitive response [45]. In plants, FDH has been reported to function in various stress responses. FDH transcriptional and translational accumulation are induced by stresses such as hypoxia, chilling, drought, dark, wounding, and iron deficiency [46]. In addition, FDH is involved in biotic stress responses. FDH is upregulated by the fungus Colletotrichum lindemuthianum in Phaseolus vulgaris and by Phytophthora citricola in Fagus sylvatica [28,47]. In N. benthamiana, Sonchus yellow net virus and Impatiens necrotic spot virus affect FDH expression [38]. The mitochondrial FDH is up-regulated by both Sonchus Yellow net virus (SYNV) and Impatiens necrotic spot virus (INSV) infection in N. benthamiana [38]. In F. sylyatica seedlings, FDH is induced by infection with the root pathogen P. citricola [47]. These reports indicate that FDH plays a significant role in pathogenicity in plants. In pepper plants silenced for FDH1, bacteria grew rapidly and expression of defense-related genes such as PR1, PR10, and DEF1 was decreased, indicating that FDH1 functions in bacterial disease defense [29]. Indeed, pepper FDH1 transcriptional and translational expression is increased by Xanthomonas campestris pv. vesicatoria. Here, we showed that FDH-silencing in N. benthamiana inhibited infection by CMV-P1 virus (Fig 4, S1 Fig, and Fig 5). Furthermore, we found that pepper FDH directly interacted with helicase domain of CMV-P1 in yeast (Fig 1). These results suggest that FDH has a role in the pathogenesis of viral pathogen as well as bacterial and fungal pathogens.
CRT is a calcium-binding protein in the endoplasmic reticulum (ER) lumen with an established role as a molecular chaperone [48,49]. CRT has been reported to play crucial roles in plants including in reproduction [50,51], tissue regeneration [52,53], abiotic stress responses [54,55], and immunity [31,56,57]. Phylogenetic studies and expression analysis revealed that higher plants contain two distinct groups of CRTs: a CRT1/CRT2 group and a CRT3 group [58]. Arabidopsis CRT1 complements the chaperone functions and calcium storage capacity of mouse CRT, and functions as an alleviator of endoplasmic reticulum (ER) stress [59]. Recently, it was reported CRT2 functions through its N-terminal domain as a self-modulator that can possibly prevent the salicylic acid-mediated runaway defense responses triggered by its C-terminal calcium-buffering activity in response to pathogen invasion [57]. Furthermore, CRT3 is needed for the accumulation of bacterial elongation factor Tu receptor (EFR), a pattern-recognition receptor that is responsible for pathogen-associated molecular pattern-triggered immunity. These findings suggested a role for CRT3 in regulation of plant defense against pathogens [56].
We showed that CRT3 protein directly interacts with helicase domain of CMV-P1 (Fig 1), indicating that CRT3 mediates CMV-P1 in virus infection. In N. tabacum, tobacco mosaic virus movement protein (TMV MP) interacts with CRT for cell-to-cell transport [31]. In Arabidopsis, CRT genes are involved in virus defense pathways. In Arabidopsis, CRT expression is induced after inoculation with Turnip vein clearing virus (TVCV), Oilseed rape mosaic virus (ORMV), Potato virus X (PVX), CMV strain Y, or TuMV [30]. Here, we found that CMV-P1 infection requires CRT3 (Fig 4). These results suggest that CRT genes might be common factors in various virus infection pathways.
In conclusion, we demonstrated that FDH and CRT3 proteins physically interact with the helicase domain of CMV-P1 (Fig 1) and that FDH and CRT3 function in CMV-P1 infection (Figs 4 and 5). These results suggest that FDH and CRT3 mutations or knockouts may provide a new strategy for breeding CMV resistance in crop plants.