Harnessing anthocyanin-rich fruit: a visible reporter for tracing virus-induced gene silencing in pepper fruit
© The Author(s) 2017
Received: 27 August 2016
Accepted: 24 November 2016
Published: 3 January 2017
Virus-induced gene silencing (VIGS) has become a powerful tool for post-genomic technology in plant species. This is important, especially in select plants, such as the pepper plant, that are recalcitrant to Agrobacterium-mediated transformation. Although VIGS in plants has been widely employed as a powerful tool for functional genomics, scattering phenotypic effects by uneven gene silencing has been implemented in order to overcome challenges in experiments with fruit tissues.
We improved the VIGS system based on the tobacco rattle virus (TRV) containing the An2 MYB transcription factor, which is the genetic determinant of purple colored- or anthocyanin-rich pepper. Silencing of endogenous An2 in the anthocyanin-rich pepper with the modified TRV vector for ligation-independent cloning (LIC) lacked purple pigment in its leaves, flowers, and fruits. Infection with TRV–LIC containing a tandem construct of An2 and phytoene desaturase (PDS) resulted in a typical photobleaching event in leaves without the purple pigment, whereas silencing of PDS led to the presence of photobleached and purple-colored leaves. Cosilencing of endogenous An2 and capsaicin synthase in fruits resulted in decreased levels of capsaicin and dihydrocapsaicin as assessed by high performance liquid chromatography analysis coupled with the absence of the purple pigment in fruits.
VIGS with tandem constructs harboring An2 as a visible reporter in anthocyanin-rich pepper plants can facilitate the application of functional genomics in the study of metabolic pathways and fruit biology.
KeywordsVIGS Pepper TRV Reporter system An2 Anthocyanin Capsaicinoid
Over the last decade, more than 90 plant genomes and a large amount of transcriptome data have been available due to revolutionary advances in next generation sequencing technologies (https://en.wikipedia.org/wiki/List_of_sequenced_plant_genomes). Since the advent of the post-genomic era, functional genomics has been increasingly essential in order to identify genes of unknown function. In contrast to the innovative progress in generating sequence data, advances in functional studies of genes are many steps behind. Due to rapid straightforward methods, virus-induced gene silencing (VIGS) has become one of the most widely used tools in plant functional genomics [1, 2].
The pepper is a fleshy fruit-bearing plant and represents a nutritionally and economically important crop as a staple vegetable and food additive. Pepper consumption by humans has increased since it was determined that their fruits synthesize high levels of many health-promoting compounds including capsaicinoids, vitamin C, and carotenoids . Therefore, breeding and biotechnology focused on enhancing the production of these metabolites are in demand although gene repertories regulating the pathways of these compounds are not fully understood. The pepper genome, however, can accelerate the identification of many genes potentially associated with important agronomic traits . Pepper is closely related to the tomato plant, which is a model for fleshy fruit biology and biotechnology. Comparative genomics in Solanaceae accelerated the discovery of genes responsible for many agronomically important traits [4, 5]. Unlike tomato and other Solanaceae plants, pepper is recalcitrant to Agrobacterium-mediated-transformation; thus, VIGS has proven to be a powerful method for determining the function of unknown genes. The TRV system was widely employed for gene silencing in Solanaceae, including pepper plants [6–8]. These previous experiments were limited to vegetative tissues, which led to difficulties in the interpretation of the effects of virus and gene expression in the late stages of fruit development. Therefore, it is essential to develop molecular tools for the functional analysis of gene expression in pepper fruits responsible for biochemical processes of human health-promoting metabolites.
Although VIGS in plants has being widely employed as a powerful tool for functional genomics, scattering phenotypic effects driven by uneven gene silencing has become a problem that must be overcome, especially in fruit tissue. To ameliorate this limitation of VIGS, visual reporter systems were developed using GFP  and DEL-ROS  in GFP overexpressing and anthocyanin-enriched transgenic plants, respectively. Since silenced areas were visually traceable in these systems, dissection and sampling of tissues for subsequent metabolic analysis successfully identified gene functions impacting the metabolites of interest [9–11].
In this study, we improved the VIGS system to monitor gene silencing in pepper fruit. For this purpose, we employed the TRV-LIC VIGS system  for high-throughput cloning and used An2  as a reporter, which is the genetic determinant of purple pigmentation due to the accumulation of anthocyanin in pepper. We successfully examined the cosilencing effects of PDS and capsaicin synthase coupled with An2, and the subsequent phenotypic and metabolic changes in leaves and fruits, respectively. Our results advance reverse genetics methods for fruit traits and ultimately enhance the understanding of the molecular mechanisms of novel genes and regulators in fruit-specific metabolic pathways.
Plant materials and growing conditions
Capsicum annuum cv. NuMex Halloween (hereafter NMH), kindly provided by Prof. Byoung-Cheorl Kang (Seoul National University), was used in this study and maintained in growth chambers. Pungent NMH contained visible amounts of anthocyanin in leaf, stem, flower, and fruit. The plant height was approximately 30 cm. After germination on plates at 30 °C, the seedlings were transferred to plug trays at 25 °C with a 16/8 h light/dark photoperiod until cotyledons were fully expanded (approximately 2 weeks after germination). After agroinfiltration to the abaxial side of the cotyledons, pepper plants were incubated at 16 °C under dark conditions for 1 day. Four-week-post-infiltrated plants were transplanted to pots (130/115 mm) and grown at 20 °C with a 16/8 h light/dark photoperiod. The plants were fertilized by WUXAL according to the manufacturer’s instructions (WUXAL calcium, AGLUKON, Germany) once every 2 months.
List of primer sequences used in this study
Agrobacterium tumefaciens strain GV3101 carrying pTRV1, pTRV2::GFP, pTRV2-LIC::GFP::An2, pTRV2-LIC::PDS::An2, pTRV2-LIC::CS::An2, and was grown overnight at 28 °C in 10 mL YEP medium containing rifampicin (50 µg/mL) and kanamycin (50 µg/mL) as described . The transformed Agrobacterium was harvested by centrifugation at 13,000×g for 15 min at 20 °C, and resuspended in 10 mM MES, 10 mM MgCl2, and 200 µM acetosyringone to a final OD600 nm of 0.7. Cell suspensions were incubated at room temperature with inverting for 4 h. Agrobacterium cultures containing pTRV1 and pTRV2-LIC carrying any GOI were mixed at a 1:1 ratio and infiltrated into the abaxial side of the both cotyledons. Leaves from 4-week-old plants and placenta from fruits from plants 30 days post-anthesis (DPA) were harvested and immediately frozen in liquid nitrogen for qRT-PCR and HPLC. Due to a high frequency of fruit abscission, at least five silenced plants per each construct were grown at 20 °C until harvest.
RNA isolation and quantitative RT-PCR analysis
The total RNA from 100 mg of tissue was extracted using the TRIzol® reagent (Invitrogen, USA). Total RNA (5 µg) was reverse-transcribed with Oligo (dT) primers and Superscript II (Invitrogen), according to the manufacturer’s instructions. Subsequently, qRT-PCR was conducted to analyze gene expression level using the SYBR Green PCR master mix (Invitrogen, USA) and gene-specific primers (Table 1) in the Rotor-Gene 6000 apparatus (QIAGEN, USA), according to the manufacturer’s instructions. All statistical analyses were conducted as described in the manufacturer’s protocol. To normalize the expression levels, the transcript level of CaActin (CA00g80270) was used as a control. Duplicates from 3 biological replicates were used in the qRT-PCR analysis.
Capsaicinoid extraction and HPLC analysis
The placental tissue of each fruit at 30 days-post anthesis was isolated and immediately frozen in liquid nitrogen. For sampling in An2-silenced plants, purple pigment-depleted placenta was carefully collected. Previous method of capsaicinoid extraction  was modified for frozen tissues. Approximately 100 mg of frozen powder from pooled samples of two fruits was extracted with 1.5 mL of an ethyl acetate and acetone mixture (6:4) using TissueLyser II (QIAGEN, USA) at room temperature for 10 min and incubated with shaking at 37 °C for 1 h. After centrifugation at 12,000×g for 5 min at room temperature, 600 µL of the supernatant was transferred to a new tube and evaporated in an Automatic Environmental SpeedVac System AES1010 (Operon, Korea). The extract was dissolved in 500 µL methanol and filtered using an Acrodisc® LC 13-mm syringe filter with a 0.2-µm PVDF membrane (Sigma-Aldrich, USA). Capsaicinoid analysis was performed using the UltiMate® 3000 HPLC (Dionex, USA) including the Inno C-18 column (4.6 mm × 150 mm, YoungJin Biochrom, Korea). A UV detector was operated at 280 nm and the data acquisition was performed with Chromeleon software. Separation of capsaicinoids was achieved under 75% MeOH at a flow rate of 1 mL/min. Each 10 µL aliquot was analyzed with HPLC. The HPLC analyses were performed at NICEM, Seoul National University. Capsaicin and dihydrocapsaicin used as standard compounds were purchased from Sigma-Aldrich (M2028 and M1022, respectively) .
Improvement of VIGS in the pepper plant using a TRV2-LIC system and An2
Silencing and its assessment of endogenous An2 in different tissues
Agrobacterium carrying TRV2::GFP as a control and TRV2::GFP::An2 with TRV1 were infiltrated in the cotyledons of 2-week-old seedlings of NMH to extend the silencing response to meristematic tissues (Fig. 2a). GFP was used for control of gene fragment and the GFP gene fragment is not existed in the pepper genome. Therefore, GFP in the TRV2-LIC would not affect any gene expression or phenotype. The infiltrated plants were grown at 20 °C to maintain silencing for a long period and enhance silencing efficiency. NMH, dwarf, and ornamental pepper cultivars, were characterized by purple leaf pigmentation, flowers, and immature fruits, and harbored orange colored ripe fruits. Fruits were oriented upright, were bullet shaped, and pungent (http://www.chilepepperinstitute.org/cart/product/100/numex_halloween/). Figure 2b demonstrates the phenotypes of the control and An2-silenced peppers in different tissues. An2 silencing using TRV2-LIC systems clearly resulted in anthocyanin deficiency in leaves, flowers, and fruits. In flowers of An2-silenced plants, the absence of the purple pigment was also observed in the stigma and stamen. To examine whether the fruit phenotypes observed in the infiltrated peppers were associated with the down-regulation of An2, qRT-PCR was conducted to compare the gene expression between purple and green fruits. The level of An2 expression was remarkably decreased in TRV2::GFP::An2 compared to the control (Fig. 2c), and the impact of An2 silencing was negligible compared to the control. Therefore, An2 was chosen as a reporter in this VIGS system for NMH by monitoring anthocyanin accumulation. These results also indicate that An2 is the genetic determinant of the A locus .
Validating the cosilencing method in the tandem construct using PDS and An2
Validating cosilencing in fruits using capsaicin synthase and An2
Capsaicinoids are specialized metabolic determinants of pungency and natural vanilloid, which are only found in pepper species. Capsaicin synthase (CS), a homologue of acyltransferase, is the genetic factor of the C or Pun1 locus controlling pungency . Capsaicin synthase catalyzes the last step of the pathway by condensing vanillylamine to 8-methyl-6-nonenoyl-CoA . Capsaicinoids are mostly synthesized in placenta tissue and capsaicin synthases are only expressed during pepper placenta development .
A reporter gene is required in VIGS to visualize the silenced region and monitor the silencing efficiency. PDS has been widely used as a reporter of choice . Since PDS silencing decreased chlorophyll and carotenoid biosynthesis resulting reduced photosynthetic activity, PDS-silenced plant showed growth defect . PDS silencing also caused carotenoid-deficient fruits and affected other metabolisms beyond carotenoid in tomato fruits [10, 23]. Thus, use of PDS as a reporter expect to show poor fruit set and disturb important metabolisms in pepper fruits. Unlike PDS, An2 silencing did not affect other phenotype except anthocyanin pigmentation. Therefore, we choose An2 as a reporter in this study.
Despite the advantages and utility of VIGS in plant functional genomics studies [1, 2], the uneven distribution of silencing in target tissues is a major limitation. Use of a visible reporter would lead to the efficient sampling of silenced tissues from nonsilenced tissues, therefore increasing the sensitivity of subsequent analysis. Several studies have successfully overcome this limitation using transgenic plants of DEL-ROS [1, 10, 11] and GFP . Using the transgenes as visible reporters, cosilencing with the reporter and GOI enables us to identify the precise region where silencing occurs. Ectopic expression of transcription factors, DEL and ROS1 using a ripe-fruit specific promoter resulted in the up-regulation of the anthocyanin biosynthetic genes, leading to anthocyanin accumulation in ripening tomato fruits. Cosilencing the reporter genes with GOI in the transgenic plants facilitated visualization of the silenced region. Fluorescence of the GFP-silenced region in GFP-overproducing plants was clearly decreased after exposure to UV. Therefore, target tissues can be easily collected and the effect of silencing can be more easily identified in systems with higher phenotypic variation compared to the non-collective method. However, these methods are still limited in plant species, which are recalcitrant to Agrobacterium-mediated transformation. In order to extend this concept to most the majority of plant species, we established the endogenous reporter system, An2, under natural variation of anthocyanin-rich pepper. Although we intended to develop this system for fruit genetics, the reporter in NMH can easily be used to monitor the silenced sector of genes in leaves and flowers and facilitate collection of tissues without further expression analysis of GOI, as we revealed that it was possible to cosilence PDS and An2 (Fig. 3). Therefore, our results will be helpful to study genes controlling diverse biological phenomena. Previous studies on CS knockdown by VIGS showed reduced capsaicinoid content in fruits, which is in agreement with the results of our study (Fig. 4) [19, 24], clearly indicating that CS is responsible for capsaicinoid synthesis. CS, originally referred to as Pun1, is believed to catalyze the last step of capsaicinoid biosynthesis although there is no direct evidence that Pun1 has capsaicin synthase activity. Recently, using a protoplast-based assay for de novo capsaicin synthesis and antibodies of the anti-Pun1, which are antagonists of endogenous Pun1 activity, the Pun1 gene and its product were proven to be involved in capsaicin synthesis . These data together with our results reveal that CS or Pun1 primarily controls the final step in capsaicinoid biosynthesis.
VIGS using TRV systems efficiently functioned in vegetative pepper tissues . However, the silencing signal is not well transmitted throughout the whole plant, particularly in reproductive tissues. Silencing of PDS was previously maintained in flowers and tomato fruits and enhanced by low temperature and low humidity . In our study, low temperature was a critical requirement of VIGS in fruits, although fruits were often characterized by evidence of abscission before harvest (data not shown). Further improvement using different environmental conditions and diverse pepper cultivars should be thoroughly examined. In addition, An2 as a transcription factor, should not dramatically affect the developmental or biochemical regulation underlying the function under study, for example, in cases when the GOI is a transcription factor or other master regulator.
Here, we present the utility of the gene silencing approach, which uses an endogenous reporter, An2, in anthocyanin-rich peppers, both by down-regulating PDS in leaves and CS in fruits, ultimately demonstrating the feasibility of this system in metabolic genetics in pepper fruits. Our results demonstrate the power of this tool for addressing the roles of regulatory genes in capsaicinoid biosynthesis and secondary metabolism. The application of this system to examine candidate regulatory genes will be useful and facilitate confirmation of gene functions identified in the model species: tomato and Arabidopsis.
virus-induced gene silencing
tobacco rattle virus
gene of interest
ligation independent cloning
high performance liquid chromatography
The study was conceived and designed by JK, MK, JML, and DC, experiments were performed by JK, ESJ, and JML, and the manuscript was written by JK, JML, and DC. All authors read and approved the final manuscript.
This research was supported by the National Research Foundation of Korea (No. 2014R1A1A1038130) to JML and the Vegetable Breeding Research Center (710001-07) through the Ministry of Agriculture, Food and Rural Affairs of the Korean government to DC. We would like to thank Hyun Jung Kim for technical assistance.
The authors declare that they have no competing interests.
Availability of data and materials
All the data is contained within the manuscript.
Consent for publication
All the authors have approved the manuscript and have made all required statements and declarations.
JML is supported by the National Research Foundation of Korea (No. 2014R1A1A1038130). DC is supported by the Vegetable Breeding Research Center (710001-07) through the Ministry of Agriculture, Food and Rural Affairs of Korea.
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