C3HC4-Type RING Finger Protein NbZFP1 Is Involved in Growth and Fruit Development in Nicotiana benthamiana

C3HC4-type RING finger proteins constitute a large family in the plant kingdom and play important roles in various physiological processes of plant life. In this study, a C3HC4-type zinc finger gene was isolated from Nicotiana benthamiana. Sequence analysis indicated that the gene encodes a 24-kDa protein with 191 amino acids containing one typical C3HC4-type zinc finger domain; this gene was named NbZFP1. Transient expression of pGDG-NbZFP1 demonstrated that NbZFP1 was localized to the chloroplast, especially in the chloroplasts of cells surrounding leaf stomata. Virus-induced gene silencing (VIGS) analysis indicated that silencing of NbZFP1 hampered fruit development, although the height of the plants was normal. An overexpression construct was then designed and transferred into Nicotiana benthamiana, and PCR and Southern blot showed that the NbZFP1 gene was successfully integrated into the Nicotiana benthamiana genome. The transgenic lines showed typical compactness, with a short internode length and sturdy stems. This is the first report describing the function of a C3HC4-type RING finger protein in tobacco.


Introduction
Zinc finger proteins are one of the most abundant types of transcription factors in eukaryotic genomes. Zinc fingers are structurally composed of multiple cysteines and/or histidines, and zinc ions play an important role in the stability of the protein itself. The RING domain of RING finger proteins, which are members of the zinc finger family, was first identified as a DNA-binding motif in the transcription factor TFIIIA from Xenopus laevis [1]. In addition to DNA, RING domains also bind RNA, protein, or lipid substrates. The RING motif is relatively small and consists of four pairs of ligands that bind two ions [2]. C3HC4-type RING finger proteins are involved in numerous cellular processes, including transcription, signal transduction, and recombination. Functions attributed to the RING domain itself include protein-protein interactions and ubiquitination. Most RING finger proteins are E3 ubiquitin ligases that mediate the transfer of ubiquitin to target proteins and play important roles in diverse aspects of cellular regulation in plants [3].
In our previous study, we used Hrip1, which can result in the formation of necrotic lesions that mimic a typical hypersensitive response and apoptosis-related events including DNA laddering [12], as a bait protein, yeast two hybrid system was performed and five candidate interacting proteins were screened. During the process of further verification about the interaction between Hrip1 and candidate proteins, interestingly, one of the five candidate interacting proteins, which is involved in fruit development in Nicotiana benthamiana, in the meanwhile, after blast in NCBI and sol genomics network, there is no relevant report about this gene. In the present study, we emphasized the gene which is involved in fruit development in Nicotiana benthamiana, a pair of primers were designed according to the results of yeast two hybrid and sol genomics network information and C3HC4-type RING finger gene was obtained from a tobacco cDNA, this new gene was named NbZFP1. Prokaryotic expression of NbZFP1 in vitro and subcellular localization of NbZFP1 in tobacco were performed. The function of NbZFP1 was analyzed by tobacco rattle virus (TRV) based on Virus-induced gene silencing system and over expression in tobacco. This research provides a foundation of the molecular mechanisms of C3HC4-type RING finger proteins in tobacco.

Plasmids and Bacterial Strains
The prokaryotic expression vector pGEX-6P-2 was purchased from Amersham Biosciences (Pittsburgh, pennsylvania state, USA). pGEX-6P-2 contains the coding region for glutathione Stransferase (GST). The plant expression vector pBI121, the subcellular localization vector pGDG, and Agrobacterium GV3101 were derived from lab stocks. E. coli strain BL21 (DE3) pLysS was purchased from TransGen Biotech Inc. (China). The VIGS system vectors were a gift from Yule Liu, Tsinghua University.

Plant Cultivation
Tobacco seeds were germinated on 1/2 MS medium in a growth chamber that was maintained at 25uC with 12 h of light and 12 h of darkness. Following germination, the seedlings were transferred to an autoclaved soil mix containing 1:3 (v/v) highnutrient soil and vermiculite in 867.567.5-cm pots. One plant per pot was kept in the growth chamber at 25uC with 50% humidity and 16 h of light. The plants were watered on alternate days.

NbZFP1 Cloning and Construction of the Prokaryotic Expression Plasmid
Total RNA of Nicotiana benthamiana was extracted using a plant tissue RNA extraction reagent (TransGen Biotech, Beijing, China), and mRNA was used to synthesize first-strand cDNA.
NbZFP1 was amplified from first-strand cDNA of Nicotiana benthamiana via polymerase chain reaction (PCR) using the sense primer P1 (59-CGGGATCCATGTCACTTTCTGGTCGT-39) and the antisense primer P2 (59-GCGTCGACTCAGGTTTT-CAGCCCTGT-39). The PCR thermocycling protocol was as follows: 95uC for 5 min, followed by 30 cycles of 95uC for 30 s, 60uC for 30 s, and 72uC for 1.5 min, with a final extension of 72uC for 10 min. The PCR product was visualized by ethidium bromide-containing agarose gel electrophoresis (1% agarose, 100 V for 20 min) and subsequent UV transillumination. The PCR product was then purified and digested with BamHI and SalI, inserted into the pGEX-6P-2 vector, and transformed into BL21(DE3) pLysS. Positive colonies were selected on Luria-Bertani (LB) agar plates containing ampicillin (100 mg/ml) and screened by direct colony PCR. The extracted plasmid, designated pGEX-6p-2-NbZFP1, was subjected to DNA sequencing by Beijing Genomics Institution, Beijing, China.

Expression and Purification of the Recombinant Protein
The cells transformed with pGEX-6p-2-NbZFP1 were cultured in LB medium containing ampicillin (100 mg/ml) at 37uC with shaking for eight hours. Isopropyl b-D-thiogalactoside (IPTG) was then added to a final concentration of 0.2 mM to induce expression at 16uC for 8 h. The culture transformed with the empty pGEX-6P-2 vector was used as a control. The bacteria were pelleted at 5000 g for 20 min at 4uC. The pellets were resuspended in buffer I (50 mM Tris and 200 mM NaCl, pH 8.0), and the cells were broken by sonication. After adequate sonication, the broken cells were pelleted at 12000 rpm for 1 h at 4uC, and the supernatant was collected. At this point, the samples containing pGEX-6p-2-NbZFP1 and empty pGEX-6P-2 were ready to be purified. Because pGEX-6P-2 contains the coding region for glutathione S-transferase (GST), we used GST affinity purification technology to purify GST-NbZFP1 and GST alone in Figure 1. Phylogenetic analysis of the relationships between NbZFP1 and C3HC4-type RING finger genes in other species. We selected twenty-five typical C3HC4-type RING finger genes with high similarity from different species, and we analyzed the similarity of their RING finger domains and full-length sequences with NbZFP1. Phylogenetic trees were generated by the maximum likelihood (ML) method using MEGA4. Bootstrap values from 1000 replicates are indicated at each branch. (a) A maximum likelihood (ML) tree of NbZFP1 and genes from other species was constructed based on RING finger domain sequences. (b) A maximum likelihood (ML) tree of NbZFP1 and genes from other species was constructed based on full-length nucleotide sequences. doi:10.1371/journal.pone.0099352.g001 the first step. After desalination, ion exchange chromatography was applied in the next purification step [13]. Both samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

Subcellular Location of the NbZFP1 Protein
The plasmid pGEX-6P-2-NbZFP1 was used as a template to amplify NbZFP1 by PCR with specific primers containing BamHI and SalI restriction enzyme sites. The product was digested with BamHI and SalI and cloned into pGDG that was cut with BamHI and SalI. After successful construction of pGDG-NbZFP1, we transformed the plasmid into Agrobacterium GV3101 using the freeze-thaw method [14]. Agrobacterium GV3101 harboring pGDG-NbZFP1 was grown in culture until the optical density of the culture reached 1.0 at 600 nm. The bacteria were pelleted at 5000 g for 15 min at room temperature. The pellets were resuspended in buffer (10 mM MES, 10 mM MgCl 2 , 200 mM acetosyringone, pH 5.6). Bacterial suspensions were then maintained at room temperature for 2-3 h. Infiltrations were performed by gently inserting a 1-ml disposable syringe into the abaxial surface of fully expanded Nicotiana benthamiana leaves that were approximately 2.5 cm wide at the mid-leaf and slowly depressing the plunger [15]. Following agroinfiltration, the plants were maintained in a growth chamber at 25uC with a 16/8 h light/dark photoperiod. The leaves were examined by microscopy between 40 h and 90 h post-infiltration.

VIGS Technique for Silencing NbZFP1 in N. benthamiana
The VIGS system includes the pTRV1 and pTRV2 vectors. Two adaptors with PstI restriction sites were inserted into the pTRV2 vector. This vector allows the insertion of gene silencing fragments by ligation-independent cloning (LIC) [16]. The NbZFP1 C3HC4-type RING finger domain gene, referred to as NbZFP1-A1, was amplified with the primers 59-CGACGACAA-GACCCTTGCTGTGTTTGTCAGGAA-39 and 59-GAGGA-GAAGAGCCCTTCAGGTTTTCAGCCCTGT-39. A version of the NbZFP1 gene lacking the C3HC4-type RING finger domain, referred to as NbZFP1-A2, was amplified with the primers 59-CGACGACAAGACCCTATGTCACTTTCTGGTCGT-39 and 59-GAGGAGAAGAGCCCTTGGCTCAACATCCA-CAGG-39. The PCR products were purified with polyethylene glycol/MgCl 2 to remove any nonspecific PCR products and primers. A total of 50 ng of purified PCR product was treated with T4 DNA polymerase (New England Biolabs) in 16reaction buffer containing 5 mM dATP and dithiothreitol at 22uC for 30 min followed by 20 min of inactivation of T4 DNA polymerase at 70uC. The TRV2-LIC vector was then digested with PstI and similarly treated with T4 DNA polymerase; however, dTTP replaced dATP [17]. A total of 50 ng of treated PCR product and TRV2-LIC vector were mixed and incubated at 65uC for 2 min and subsequently at 22uC for 10 min. Then, 6 ml of the mixture was transformed into E. coli DH5a competent cells, and the transformants were tested by colony PCR.
Sequence-validated pTRV2-NbZFP1-A1 and pTRV2-NbZFP1-A2 plasmids were each introduced into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method [14]. Overnight cultures were grown at 28uC in the appropriate antibiotic selection medium. On the following day, the cultures were spun down, and the cells were resuspended in infiltration medium (10 mM MES, 10 mM MgCl 2 , 200 mM acetosyringone, pH 5.6), adjusted to an OD 600 of 1, and incubated at room temperature for 3 h. Agrobacterium cultures containing pTRV1 and pTRV2 were mixed at a 1:1 ratio and used to infiltrate plants at the 4-leaf stage using a 1-ml needle-less syringe [18]. Total RNA was extracted from the leaves or flowers of wild-type and VIGS N. benthamiana plants using the RNeasy plant mini kit (Qiagen). First-strand cDNA was synthesized using 1 mg of total RNA, gene-specific primers, and SuperScript reverse transcriptase (Invitrogen) according to the manufacturer's protocol. The expression level of NbZFP1 was monitored by real-time PCR, and the Actin gene was amplified as a quantitative control. qPCR was conducted with iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and an iCycler (Bio-Rad) according to the manufacturer's instructions.

Tobacco Transformation
Primer 5.0 was used to design the sense primer, P1 (59-CGGGATCCATGTCACTTTCTGGTCGT-39), and the antisense primer, P2 (59-GCGTCGACTCAGGTTTT-CAGCCCTGT-39). The primers were then used to amplify the ORF of NbZFP1 by polymerase chain reaction (PCR). Primers P1 and P2 contained BamHI and SalI restriction sites. The PCR products and pBI121 vector were double digested, adapter ligation was performed, and the ligation products were then transformed into DH5a cells. Positive colonies were selected on Luria-Bertani (LB) agar plates containing kanamycin (50 mg/ml) and screened by direct colony PCR. The fusion vector pBI121-NbZFP1 was transformed into Agrobacterium GV3101. Agrobacterium GV3101 harboring pBI121-NbZFP1 was used to transform N. benthamiana according to the method proposed by Horsch et al [19], and the transformed plants were then transferred to soil for seed setting. T 1 progeny plants from the seeds of independent T 0 transformants were grown on 1/2 MS medium with kanamycin selection, and green plantlets with roots were transferred to soil.
PCR analysis of T 0 plants was performed for the detection of putative transgenic tobacco plants. Genomic DNA from fresh, fully expanded tobacco leaves was used for the PCR analysis. pBI121 harbors a gus gene; thus, amplification of a specific fragment of the gus gene was performed by adjusting the PCR conditions for 30 cycles. The primers forward-F (59-ATGGTCCGTCCTGTAGAAACC-39) and primers reverse-R (59-GACTGCCTCTTCGCTGTACAG-39) were used for amplification of the specific gus fragment. After positive identification of transgenic plants, we performed Southern blotting to determine the NbZFP1 copy numbers according to the protocol provided with the DIG High Prime DNA Labeling and Detection Starter Kit II (Amersham Biosciences).
30 Single copy T 1 progeny plants were grown on 1/2 MS medium containing kanamycin, and 30 wild progeny plants were grown without antibiotic as a control. Green plantlets with roots were transferred to pots containing soil. The height of the plants were measured beginning at the four-leaf stage in 10-day intervals. The internode lengths of wild-type and transgenic tobacco were measured at the sixty day after four-leaf stage.

Statistical Analysis
The data were analyzed separately for each experiment with SPSS 16.0 software. The means were compared using Tukey's HSD test. The significance of the different between the means of wild-type and over-expression tobacco in the same growth condition were calculated by chi square test using SPSS 16.0 software. *** indicate significance at the 0.001 of confidence level.

NbZFP1 Cloning
Using a pair of specific primers, the gene we have designated as NbZFP1 was cloned from N. benthamiana by RT-PCR. The length of the full open reading frame (ORF: GenBank accession number KJ169550) for this gene is 576bp.

Phylogenic Analysis
To define the relationships between NbZFP1 and C3HC4-type RING finger genes in other species, phylogenetic analyses were performed with RING finger domain sequences and full-length sequences [20]. The maximum likelihood (ML) tree [21] of the C3HC4-type RING finger domain-containing motif suggested that C3HC4-type RING finger genes are highly conserved in 26 species. In addition, NbZFP1 is more closely related to the C3HC4-type RING finger genes of Solanum tuberosum, Lycopersicon esculentum, and Solanum lycopersicum than those of other species (Figure 1a). The tree of full-length sequences of C3HC4-type RING finger genes showed that the similarities between NbZFP1 and the C3HC4-type RING finger genes in the other 25 species are not high (Figure 1b).

Purification and Detection of Recombinant NbZFP1 Protein
NbZFP1 was cloned into the prokaryotic expression vector pGEX-6p-2, and the resulting recombinant plasmid, pGEX-6p-2- NbZFP1, was confirmed by colony PCR and DNA sequencing. The recombinant plasmid was then transformed into E. coli BL21 (DE3) to express the recombinant protein. The expressed protein was soluble in E. coli and was purified with a GSTrap HP column followed by a HiTrap desalting column and a HiTrap Q HP column (Figure 2a). The molecular weight of the GST tag is 26 kDa, and SDS-PAGE analysis showed a single band corresponding to the purified recombinant protein with an approximate molecular mass of 50 kDa (Figure 2b). This result indicated that the NbZFP1 protein was expressed in E. coli.

Subcellular Localization of the NbZFP1 Protein
To confirm the subcellular localization of the NbZFP1 protein, we constructed a recombinant vector, pGDG-NbZFP1, for transient expression. The empty pGDG vector was used as a negative control. Because the Rac1 protein was shown to be localized to the plasma membrane [22], the pGDG-Rac1 vector was constructed for expressing Rac1-GFP as a positive control. Agrobacterium GV3101 containing pGDG, pGDG-Rac1, or pGDG-NbZFP1 was used to infiltrate healthy tobacco plants. Laser scanning confocal micrographs showed that GFP alone localized throughout the whole cell, including the plasma membrane, nucleus, and cytoplasm (Figure 3a and Figure 3b). The fusion protein Rac1-GFP was localized to cell membranes only (Figure 3c and Figure 3d). The protein of interest, NbZFP1-GFP, was located in the chloroplast, especially in the chloroplasts of cells surrounding leaf stomata (Figure 3e-h).

Developmental Phenotypes Revealed by Silencing NbZFP1
TRV-mediated VIGS is an effective tool for assessing the functions of genes in the reproductive tissues of plants [23]. Silencing of phytoene desaturase (PDS) leads to the inhibition of carotenoid synthesis, causing the plants to exhibit a photobleached phenotype [24]. Thus, we assessed the gene silencing efficiency of our TRV-VIGS clones by suppressing the expression of the PDS gene in N. benthamiana. The PDS suppression phenotype was visible at 10 days post-infiltration in the upper leaves of the plant and persisted indefinitely. This result indicated that the TRV-VIGS system could be successfully used to induce silencing of other desirable endogenous plant genes [18].
We then cloned two different fragments of NbZFP1 gene separately into the TRV2-LIC vector for silencing. The first of these fragments was obtained from the NbZFP1 C3HC4-type RING finger domain gene NbZFP1-A1, while the other fragment was obtained from NbZFP1-A2, which is a version of NbZFP1 that lacks the C3HC4-type RING finger domain. We infiltrated plants at the four-leaf stage with a 1:1 mixture of TRV1 and the TRV2-LIC-NbZFP1 fragment for each of the clones, and we monitored the infiltrated plants throughout their entire lifespan. We then determined the degree of silencing of NbZFP1 fragments by qPCR. The results revealed a greater than 90% reduction in transcript levels in the silenced plants (Figure 4a). Silencing NbZFP1 resulted in fruits that were smaller than those of the TRV-infected control (Figure 4c), although no other phenotypic defects were observed (Figure 4b).

Generation of NbZFP1 Transgenic Tobacco Plants
The plant expression vector pBI121 containing NbZFP1 was transformed into tobacco via the Agrobacterium-mediated method [19]. Nine independent kanamycin-resistant NbZFP1 transgenic tobacco lines were obtained. These transgenic tobacco plants were verified by PCR, and eight transgenic lines were positive (Figure 5a). We randomly selected 3 transgenic plants and performed a Southern blotting experiment, which showed that NbZFP1 was successfully integrated into all 3 transgenic tobacco plants and two of the lines had a single copy of NbZFP1 (Figure 5b).

Phenotypic Effect of NbZFP1 in Transgenic Lines
We selected T 0 plants with a single copy of NbZFP1 and obtained their seeds. These seeds were then sown on 1/2 MS medium with kanamycin selection. Wild type plants were grown on 1/2 MS medium without antibiotic. 30 wild-type and transgenic plantlets with roots were transferred to soil, respectively. The height of the plants were measured 10 days after the plants had reached the four-leaf stage, the internode lengths of wild-type and transgenic tobacco were measured at the sixty day after four-leaf stage. We found that transgenic lines were shorter than wild type lines after the four-leaf stage, and the regenerated plants were compact with short internodes and sturdy stems (Figure 6b, Figure 6c, Figure 6d). However, the phenotype of transgenic fruits was normal (Figure 6a).

Discussion
C3HC4-type RING finger proteins play different roles in diverse physiological processes. Many C3HC4-type RING finger genes in plants have been shown to play important roles in the regulation of growth and development [25]. The C3HC4-type RING finger proteins can be divided into three types according to their role. Because the specificity of the ubiquitin proteasome pathway is determined by E3 ligases, the various functions of C3HC4-type RING finger proteins may be explained by the fact that they act as E3 ligases with different targets in diverse physiological processes. Indeed, C3HC4-type RING finger proteins can regulate many cellular processes, including homeostasis, development, cell division, growth, hormone responses, and stress responses [26]. Some C3HC4-type RING finger genes function as cis-elements and play key roles in the transcriptional regulation of genes controlling various biological processes, including abiotic stress responses [27]. In addition, C3HC4-type RING finger genes also participate hormonal regulation. Phytohormones are the most important signaling molecules in plants, and the concentration of a given phytohormone influences plant growth and development. For example, auxin regulates cell processes and promotes cell elongation. GAs are a large family of plant hormones, some of which are bioactive growth regulators that control seed germination, stem elongation, and flowering [28].
In this study, we provide a look at the C3HC4-type zinc finger protein of tobacco. Sequence analysis indicated that the gene encodes a 24-kDa protein with 203 amino acids, and we refer to this gene as NbZFP1. The results of VIGS analysis show that the C3HC4-type zinc finger protein had a clear effect on the process of tobacco seed pod development, although the height of the plants subjected to VIGS treatment was normal. When NbZFP1 was transformed into tobacco, we found that the fruit phenotype of T 1 transgenic lines containing a single copy of NbZFP1 was normal, although these lines were characterized by compactness, short internodes, and sturdy stems. We next examined the subcellular localization of NbZFP1, and we found that NbZFP1 was localized in the chloroplast, especially in the chloroplasts of cells surrounding leaf stoma. We therefore hypothesized that NbZFP1 has a role in stomatal movements. It is known that stomatal opening and closing is mediated by ABA-triggered changes in ion fluxes in guard cells [29,30,31]. ABA is considered to be an important hormone, as it plays a critical role in the response of plants to various stresses. ABA is not only a stress signal but is also required to fine-tune growth and development under non-stress conditions. The physiological processes controlled under these conditions include the regulation of growth, stomatal aperture, hydraulic conductivity, and seed dormancy [32,33,34]. ABA negatively influences the size of guard cells and the internode length, and it also acts together with other phytohormones, such as brassinosteroids, gibberellic acid, and auxin, in the regulation of plant growth and development [35,36,37]. The characteristics of the transgenic lines are consistent with the function of zinc finger proteins in other plants. For example, the EPF zinc finger protein family of Petunia hybrida and the SUPERMAN or NNT zinc finger protein of Arabidopsis thaliana are both involved in regulating the process of reproductive development [38,39,40] The rice zinc finger protein PROG1 is directly involved in plant growth regulation and domestication [41]. NbZFP1 might be regulate the concentration of the phytohormone abscisic acid (ABA) through a specific mechanism, as ABA negatively regulates the internode length, or might act together with other phytohormones related to auxin signal transduction or the auxin/cytokinin signal transduction process, consequently affecting the growth and development of tobacco. However, it is currently unclear how NbZFP1 influences fruit development. Ma et al. [25] found that among 29 C3HC4-type RING finger genes in rice, 5 genes were preferentially expressed in reproductive tissues or organs. Commonly, a high level of expression or preferential expression in tissues or organs suggests that the highly/preferentially expressed gene may play an important role there. Thus, these 5 rice genes may play an important role at the reproductive stage.
Overall, C3HC4-type RING finger genes have been shown to play important roles in the regulation of growth and development in plants; however, these genes undoubtedly have many functions that have not yet been discovered. The current study provides preliminary data that requires further validation and establishes a basis for further studies. Much work remains to be carried out in the future.