Silencing of the Chalcone Synthase Gene by a Virus Vector Derived from the Cucumber Mosaic Virus in Petunia

Abstract

Virus-induced gene silencing (VIGS) systems are widely used to downregulate target host genes in plants. The Cucumber mosaic virus (CMV), which has a broad host range, has been used as a VIGS vector system in research. In this study, a VIGS vector system derived from the pepo strain of CMV (CMV pepo) was tested for its use in functional analysis of a target gene in Petunia. The CMV pepo caused systemic infections without severe viral symptoms in petunia plants. A vector containing a short fragment (57 bp) of the chalcone synthase gene (PhCHS-A) was constructed (PhCHSA-CMVpepo) and petunia plants were mechanically inoculated with it. Corollas were white in plants infected with PhCHSA-CMVpepo, but were purple (original color) in plants infected with wild-type CMV pepo. In white corollas, the mRNA level of PhCHS-A was downregulated compared to that in purple corollas. These results indicate that the CMV pepo vector system is useful for VIGS research in flowers of the genera Petunia.

Introduction

Loss of function approaches have been useful for reverse genetic studies. These studies have utilized knock-down genes, as in T-DNA insertion mutagenesis, recombination, and transposon-tagging, or downregulation of gene expression using RNA interference (RNAi) or virus-induced gene silencing (VIGS). Gene silencing is one of the most extensively applied techniques for functional characterization of different target genes in many plant species including horticultural crops and is responsible for crop improvement (Pandey et al., 2015). This gene silencing process, through mRNA degradation and translational inhibition in RNAi or VIGS, is called post-transcriptional gene silencing (PTGS: [Al-Kaff et al., 1998; Napoli et al., 1990]). Post-transcriptional gene silencing has been shown to function as an endogenous defense mechanism against viruses by directly targeting the replicative form of the virus (Voinnet, 2001). In VIGS systems, defense mechanisms are used to downregulate target host mRNAs by introducing fragments of target host genes into a modified viral genome. In contrast to agrobacterium-mediated plant transformation, the VIGS process does not require plant regeneration from tissue cultures: therefore, VIGS phenotypes can be observed in a relatively short time. Furthermore, VIGS can reveal the mature phenotypes of embryo-lethal disruptions to gene functions. The first report of VIGS was presented in 1995, when the tobacco mosaic virus (TMV) vector system was used to knock-down a gene involved in the carotenoid biosynthesis pathway by the expression of a phytoene desaturase (PDS) gene fragment (Kumagai et al., 1995). This first VIGS study demonstrated the potential of virus vector systems for rapidly inducing downregulation phenotypes through the expression of part of a target gene, and made VIGS a common tool for the rapid assessment of gene functions in plants. These vector systems have been developed using many plant viruses, such as potato virus X (Angell and Baulcombe, 1997), tomato golden mosaic virus (Kjemtrup et al., 1998), tobacco rattle virus (Ratcliff et al., 2001), tomato yellow leaf curl China virus (Tao and Zhou, 2004), apple latent spherical virus (Igarashi et al., 2009), cucumber mosaic virus (CMV) strain Y (Otagaki et al., 2006), and bean pod mottle virus (Zhang and Ghabrial, 2006). Moreover, some other viruses have been developed for monocotyledonous (Holzberg et al., 2002; Hsieh et al., 2013; Liu et al., 2016).

Silencing of genes related to color pigmentation, such as the PDS and chalcone synthase (CHS) genes, provide visual indicators for the location and extent of gene silencing in infected plants (Chen et al., 2004; Kumagai et al., 1995; Ratcliff et al., 2001). When PDS, which is involved in carotenoid biosynthesis, was downregulated, photosynthetic tissues such as leaves turned white due to chlorophyll degradation (known as photobleaching). On the other hand, CHS, which is involved in the biosynthesis of anthocyanins, functions as an indicator of gene silencing in floral tissues. Plants which originally had red, purple, or blue flowers, produced white flowers when CHS gene expression was downregulated by VIGS (Broderick and Jones, 2014; Chen et al., 2004).

The Cucumber mosaic virus (CMV), which belongs to the genus Cucumovirus (family Bromoviridae) has been reported to infect over 1200 species in over 100 families of plants. Thus, CMV has an extremely broad host rage. (Mochizuki and Ohki, 2012). The virus has a single-stranded RNA genome divided into three segments (RNA1, RNA2, and RNA3; Fig. 1A), is classified into subgroups IA, IB, and II by phylogenetic analysis, based on its genome sequences (Roossinck, 2002). Generally, subgroup I strains are more virulent than subgroup II strains, and disease symptoms are also different among strains. The differences among strains in the ability to produce systemic infection in host plant species is possibly related to genetic differences between the strains. The pepo strain of CMV (CMV pepo) was originally obtained from Cucurbita pepo in Japan (Ozaki et al., 1973) and has been widely used for basic viral research using tobacco plants. Several virus strains could not infect shoot apical meristem (SAM), but CMV pepo could infect shoot apical meristems cells (Mochizuki and Ohki, 2004, 2005). Furthermore, the vector system derived from CMV pepo has been improved to allow the analysis of gene function in a model plant (Mochizuki et al., 2009). However, the CMV pepo system has been not used in various horticultural crops.

Fig. 1

Structure of the CMV pepo vector system. Structure of CMV pepo vectors. A 57 bp DNA fragment derived from PhCHS-A (black-box) was inserted into the back of a capsid protein (CP) gene.

Petunia was used as plant material in this research because it is a good model organism for floricultural studies and is a typical solanaceous crop (Gerats and Vandenbussche, 2005). Additionally, petunia plants are a high economically important crop in horticultural markets. In this study, as a first step toward the development of a functional analysis of target host genes in horticulture crops using the CMV pepo system, we investigated the availability of the CMV pepo vector system in petunia plants. In addition, we discuss the effect of a relatively short target gene insert for gene silencing.

Materials and Methods

Plant materials and growth condition

Petunia × hybrida (cultivar ‘Picobella Blue’) that has purple corollas was used in this study. The seeds were sown in plug trays on soil mix (Sakata Seed Co., Yokohama, Japan) and grown under florescent grow lights. After 3–4 weeks, plants were transplanted into 9 cm pots and moved to growth chambers (Nippon Medical and Chemical Instruments Co., Osaka, Japan). Plants were grown under florescent grow lights at approximately 200 μmol·m⁻²·s⁻¹ with a 16 h light and 8 h dark cycle with temperature at the 23°C. Plants were fertilized with 1000-fold diluted Hyponex Solution (Type: 6-10-5; Hyponex Japan, Osaka, Japan).

Plasmid construction

The plasmids, pCP1II, pCP2II, and pCP3TP2, contained one tripartite component of the CMV pepo strain genomic RNA (RNA1, RNA2, and RNA3, respectively) (Mochizuki et al., 2009). These plasmids were procured from Dr. Mochizuki, Osaka Prefecture University. For gene-silencing experiments, a 57 bp fragment, corresponding to the nucleotide sequences of the PhCHS-A gene was introduced into the pCP3TP2 vector (Fig. 1B) by the modified inverse PCR method using a KOD-Plus-Mutagenesis Kit (Toyobo. Co., Osaka, Japan; [Hashimoto et al., 2001]). We designed primers containing a part of pCP3TP2 vector, containing fragments corresponding to nucleotide sequences of PhCHS-A (underlined) to be inserted in the forward primer: 5'-TTGTTTATTTTGTTTCTTGGGGTTGAATTTATTTCCGTGTTTTCCAGAACC-3' and reverse primer: 5'-TTACACTTCAATTCATATAAGCCCATCAGACTGGGAGCACCCCAGACG-3', and carried out PCR reactions with these primers and the pCP3TP2 vector under following conditions: 2 min at 94°C, 10 cycles of 10 s at 98°C, 4 min at 68°C. After the inverse PCR reactions, DpnI restriction enzymes were added to the PCR reaction mixture and it was incubated at 37°C for 1 h. Then, part of the mixture was used for the ligation reaction and the self-ligated PCR products (vectors) were used for the transformation of E. coli cells (strain DH5α). The self-ligated PCR products were sequenced in order to check that the pCP3TP2 vector contained the fragments corresponding to the nucleotide sequences of PhCHS-A. The resulting vector was named pCP3TP2:PhCHSA.

Inoculation methods

A mechanical inoculation for leaves of plants was performed as described in a previous study (Mochizuki et al., 2009). Petunia plants at the four to six true leaf stages were used for mechanically inoculation. Leaves were mechanically inoculated with RNAs transcribed in vitro from the CMV vector plasmids after linearization with appropriate restriction endonucleases or phosphate buffer (mock control). In vitro transcription was performed using the T7 RiboMAX^TM large-scale RNA-production system (Promega, Madison, WI, USA). The virus that was propagated using RNAs transcribed in vitro from the original CMV vector plasmids was termed wild-type CMV pepo. For the virus extraction of wild-type CMV pepo, leaves infected with wild-type CMV pepo were ground in 0.1 M phosphate buffer (pH 7.2). The virus extract solution was centrifuged at 10,000 × g for 3 min at 4°C and the supernatant was used for inoculation. Then, 10 μL of mixture of RNA1, RNA2, and RNA3 derived from the CMV vector, phosphate buffer or wild-type CMV pepo was placed on the adaxial side of leaves which carborundom powder was dusted onto. The solution was softly spread with gloved fingers. Two to four leaves were inoculated and these leaves were then rinsed with distilled water. Inoculated plants were grown in a plant growth chamber maintained at 23°C with a 16 h light/8 h dark cycle.

RNA extraction and RNA analysis of CMV pepo

Total RNA was purified from leaves and corollas of Petunia plants using TRIzol reagent (Life Technologies Japan Ltd., Tokyo, Japan). First strand cDNA from total RNAs was produced using PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s protocol. Fragments of pCP3TP2 and pCP3TP2:PhCHSA vectors of CMV pepo were amplified by PCR using 5'-ATATGATCTTTCGGCGATGC-3' in the CP gene and 5'-TTTAGCCGTAAGCTGGATGG-3' in the 3'-untraslated region (UTR) as forward and reverse primers, respectively. PCR was performed with Ex Taq DNA polymerase (Takara Bio Inc.) under the following conditions: 30 s at 95°C, 30 cycles of 5 s at 95°C, 60 s at 68°C.

cDNA Cloning and expression analysis

Full-length cDNA of PhCHS-A was generated by PCR with Ex Taq and 5'-ATGGTGACAGTCGAGGAGTA-3' and 5'-ACTTAAGTAGCAACACTGTGGAGG-3' as forward and reverse primers. Expression analyses of the PhCHS-A gene in infected Petunia petals were carried out by semiquantitative reverse transcriptase-PCR (RT-PCR) using forward primer 5'-CCTGCAATTTTGGACCAAGT-3' and reverse primer 5'-GCCCAAATCCAAAAAGAACA-3'. PCR was performed with Ex Taq DNA polymerase under following conditions: 30 s at 95°C, 25 cycles of 5 s at 95°C, 60 s at 68°C. The Petunia ubiquitin (PhUbq) gene was used as the control and primers used for PhUbq were identical to those described in a previous study (Nishijima et al., 2011).

Results and Discussion

Inoculation of wild type CMV pepo

To confirm if the CMV pepo vector system was viable, we tested the infection using viral RNA of wild-type CMV pepo in petunia plants. Petunia plants at the four to six true leaf stages were mechanically inoculated. Inoculated leaves and upper leaves in infected plants did not show obvious abnormalities or symptoms growing: they grew slightly slower without withered leaves (Fig. 2A). However, some infected plants showed mild leaf mosaic symptoms. Seven days after inoculation, we succeeded in detecting a PCR fragment, corresponding to a length of 286 bp, using specific primers for RNA3 of CMV pepo from inoculated and upper leaves (Fig. 2B). Furthermore, the PCR fragment was detected in corollas. Therefore, this study is the first to describe a systemic infection in petunia caused by the CMV pepo strain and indicates the viability of the CMV pepo vector system in petunia plants. Previous studies reported that the CMV pepo strain was able to induce systemic infections in many plant species, such as tobacco, tomato, pumpkin, spinach, and corn, but unable to induce infection in radish (Ozaki et al., 1973).

Fig. 2

Petunia plants inoculated with wild-type CMV pepo. (A) a and b: Control petunia plants (Mock). c and d: Petunia plants infected with wild-type CMV pepo. (B) Detection of a PCR fragment corresponding to 286 bp derived from RNA3 in CMV pepo. M: molecular marker; n: No template; a and b: Non-infected petunia plants; WP: pCP3TP2 plasmid DNA; c and d: Petunia plants infected with wild-type CMV pepo.

Phenotype and transcript levels of PhCHS-A in the corolla

The CMV pepo vector system is a characteristic method for vector construction, because the system does not require the cDNA clone preparation, e.g., PhCHS-A, for the insertion of DNA fragments in advance of vector construction. If the sequence of the target gene is obtained from a database, the inverse PCR can be carried out, followed by custom DNA primer synthesis with the target gene sequence. In Petunia, the whole-genome sequences and assemblies of inbred derivatives of two wild parents, P. axillaris and P. inflata, were previously reported (Bombarely et al., 2016). Therefore, we obtained the sequence of PhCHS-A from a database (Sol Genomics Network; https://solgenomics.net/) and constructed the pCP3TP2:PhCHSA vector using the modified inverse PCR method. The pCP3TP2:PhCHSA vector contained a 57 bp DNA fragment corresponding to nucleotide positions 1177–1233 of the PhCHS-A gene encoding a part of the second exon of the gen that was amplified through inverse PCR as described above. RNAs transcribed from the CMV vector plasmids (pCP1II, pCP2II, and pCP3TP2:PhCHSA) for PhCHS-A silencing were mechanically inoculated in the leaves of petunia plants. We extracted RNA from the upper leaves of plants 7 to 10 days after inoculation and checked the infected plants using RT-PCR. All inoculated plants were infected with the virus (PhCHSA-CMVpepo) derived from vectors designated pCP1II, pCP2II, and pCP3TP2:PhCHSA (data not shown). The first white-corollas phenotype was observed 70 days after inoculation, and 92% (11/12) of the plants showed this phenotype (Fig. 3A). By contrast, plants in the control sample or those inoculated with wild-type CMV pepo had the purple corollas phenotype. One plant in which leaves were infected with PhCHSA-CMVpepo did not have the white corollas phenotype for unknown reasons. It may have been caused by reduced infectivity because corollas of the plant were not infected with virus as confirmed by RT-PCR (data not shown). Using a TMV-based VIGS vector, an optimized method such as plant stage for inoculation, cultivars, and environmental growing conditions, increased the reliability of VIGS in petunia (Broderick and Jones, 2014). As a result, further research is needed in order to optimize inoculation techniques and growing conditions for the CMV pepo vector system.

Fig. 3

Expression analysis of PhCHS-A and corollas of Petunia plants. (A) Corolla of Petunia infected by PhCHSA-CMVpepo (a): wild-type plant (mock); (b): Petunia plant infected with wild type CMV pepo; (c), (d), and (e): Petunia plants infected with PhCHSA-CMVpepo. Bar = 10 mm. (B) Semiquantative PCR analysis of PhCHS-A and PhUbq was carried out according to the procedures explained in the Materials and Methods section. M: molecular marker; Mc: wild-type Petunia plant (mock); Mp: pCP3TP2 plasmid DNA containing a fragment of PhCHS-A; WT: Petunia plant infected with wild-type CMV pepo; ch1, ch2, and ch3: Petunia plants infected with PhCHSA-CMVpepo.

To confirm PhCHSA-CMVpepo virus multiplication, total RNAs isolated from corollas of infected plants were used for RT-PCR using specific primers for CMV pepo (Fig. 4). A PCR fragment corresponding to the expected length of 343 bp was observed, so it is likely that the multiplication of PhCHSA-CMVpepo occurred in the white corollas of petunia plants. We investigated the expression levels of PhCHS-A mRNA in the white corollas of plants infected with PhCHSA-CMVpepo using semiquantitative PCR and their total RNA. The levels of PhCHS-A mRNA were decreased in corollas of infected plants compared to those of non-infected and wild-type CMVpepo-infected corollas (Fig. 3B). These results indicate that the CMV pepo vector system can be successfully used as a VIGS vector in petunia plants.

Fig. 4

Detection of PCR fragments corresponding to 286 bp derived from RNA3 in wild-type CMV pepo and 343 bp from a pCP3TP2 vector containing a fragment of PhCHS-A in PhCHSA-CMVpepo. M: molecular marker; WP: pCP3TP2 plasmid DNA; MP: pCP3TP2 plasmid DNA containing a fragment of PhCHS-A; WT1 and WT2: Petunia plants infected with wild type CMV pepo; ch1, ch2, and ch3: Petunia plants infected with PhCHSA-CMVpepo.

It was reported that the insertion length of a foreign gene affected the stability of the insert fragment in the VIGS vector and silencing of the target gene (Hsieh et al., 2013; Igarashi et al., 2009; Yamagishi et al., 2015). Insert fragments of short length were more stable compared to long ones. However, long insert fragments have a strong possibility of silencing the target gene because they may contain an effective position in the target sequence for silencing. As a preliminary test, we found that the insertion of a 500 or 150 bp green fluorescent protein (GFP) fragment into RNA3 of CMV pepo resulted in loss of infectivity (data not shown). Additionally, a 30 bp fragment of PhCHS-A inserted into RNA3 of CMV pepo had no effect to produce white corollas. These results indicate that insert size influences the infectivity of the CMV pepo vector and that insertion length and position of the target gene should be considered when using the CMV pepo vector system. Previous studies reported that virus vectors carrying about 200 bp of the target gene efficiently induced stable VIGS (Igarashi et al., 2009). A ~200 bp cDNA fragment of CHS was used for downregulation of the CHS gene in some studies, while a 194 bp fragment of the CHS-J mRNA corresponding to nucleotide positions 654–847 was used for VIGS studies using vectors derived from TRV in petunia plants (Chen et al., 2004). In soybean, a 244 bp fragment of the CHS7 gene was cloned into an RNA 2 vector derived from CMV strain Y and used for VIGS studies (Nagamatsu et al., 2007). However, in this study, the 57 bp nucleotide fragment corresponding to the sequence positions 1177–1233 of PhCHS-A gene, produced an adequate effect for the production of white corollas and the downregulation of the PhCHS-A mRNA (Fig. 3). It is conceivable that its target position, which is in the second exon of the PhCHS-A genome, is important for PhCHS-A mRNA silencing. In previous studies, white corollas in transgenic and non-transgenic varieties of petunia with suppression of PhCHS-A mRNA levels were associated with mRNA degradation. In the former transgenic petunia, overexpression of the sense PhCHS-A gene caused the production of white corollas, and this was the first example of RNA-silencing induced by a sense transgene. The silencing of both the PhCHS-A transgene and endogenous PhCHS-A gene was induced in transgenic petunia, an event termed cosuppression (Napoli et al., 1990). Moreover, non-transgenic varieties of petunia, with bicolor flowers, such as star-type and picotee-type flowers exhibited a similar PhCHS-A gene silencing and cosuppression system to that in corollas (Jorgensen, 1995; Morita et al., 2012). The production of PhCHS-A short interfering RNAs (siRNAs) was observed in both transgenic and non-transgenic petunia subjected to PhCHS-A RNA silencing (De Paoli et al., 2009; Kasai et al., 2013). The siRNAs production was confined to the second exon and siRNAs were commonly detected in both silencing systems. The gene silencing process by VIGS is similar to that of RNAi (Chicas and Macino, 2001). During replication of RNA viruses, double stranded RNAs (dsRNAs) are produced as replication intermediates. These dsRNAs are recognized by an RNAse-III-like enzyme, namely Dicer-like endonuclease, and cleaved into siRNAs (Pandey et al., 2015). Single-stranded siRNAs are incorporated into the RNA-induced Silencing Complex (RISC) involving Argonaute and recognize and guide homology-dependent degradation of target RNA. These results, together with our results, suggest that the biosynthetic processes of siRNAs and the subsequent production of secondary siRNAs in the second exon are important for PhCHS-A silencing. Therefore, the VIGS mechanism depends on the availability of specific transcript regions in target genes to be processed into siRNAs. Some reports have examined the gene silencing of PDS and GFP by short nucleotide fragments, but did not investigate the minimum target sequence for gene silencing (Hirai et al., 2008; Tasaki et al., 2016; Thomas et al., 2001). Further studies should focus on the production of siRNA with respect to specific sequences or structural features, using deep sequencing of small RNAs in many gene silencing processes.

In conclusion, our study demonstrated that the CMV pepo vector could act as an inducer for the downregulation of endogenous genes in petunia. Furthermore, the CMV pepo vector system was efficiently used to construct a plasmid with a part of a target gene from a host plant. In addition, the CMV pepo strain induced systemic infections in petunia plants without severe viral symptoms. These results suggest that the CMV pepo vector meets the conditions for a VIGS vector and represents a useful tool for VIGS research.

Acknowledgements

We thank Dr. Takaaki Nishijima for the analysis of the PhUbq gene expression in Petunia plants. We also thank Mrs. Hiroko Uegaki and Mrs. Fumino Inamoto for technical assistance.

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