Virus-induced gene silencing in Nicotiana benthamiana using a DEAD-box helicase sequence derived from Dunaliella salina

Abstract

Viral vectors can be used to introduce sequences that cause gene silencing in plants. In this study, a sequence from the salt-tolerant green alga Dunaliella salina was used to silence a DEAD box helicase gene in Nicotiana benthamiana, a plant related to tobacco. Phenotypic changes due to silencing were observed, and changes in expression of the DEAD box helicase gene were quantified using real-time PCR. The results suggest that the gene is involved in critical RNA processing functions. More generally, it was demonstrated that although N. benthamiana is only distantly related to D. salina, gene silencing could be induced in N. benthamiana using a sequence from D. salina; this indicates that it may be possible to use gene silencing in N. benthamiana to characterize genes from a wide variety of organisms.

Introduction

Researchers have found that plants have a built-in mechanism of post-transcriptional gene silencing (Robertson 2004). Transformation with a homologous transgene causes expression of both the transgene and the similar endogenous gene to be suppressed (van der Krol et al. 1990). This effect has also been observed in response to plant viruses containing homologous genes, implicating gene silencing as a possible defense against viruses (Al-Kaff et al. 1998).

Viral vectors have been developed to induce gene silencing in plants (Kumagai et al., 1995). These vectors consist of modified plant viruses that can be designed to carry foreign sequences to be transcribed along with the viral genes in the infected plant (Busto and Kumagai, 2004). If the vector is engineered to contain part of a plant gene in the antisense orientation, then it will produce complementary mRNA that will bind to the corresponding mRNA in the cytoplasm of the plant cell, forming double-stranded RNA. The presence of double-stranded RNA triggers the gene silencing mechanism of the plant, causing the production of small interfering RNA molecules that target their specific sequence for destruction (Waterhouse and Helliwell, 2002; Hamilton and Baulcombe, 1999). The selective destruction of the mRNA molecules effectively silences expression of the gene.

Viral vectors have become an important tool in plant genetics research. Kumagai et al. demonstrated that virus-induced gene silencing could be used to disable steps in a carotenoid biosynthetic pathway (1995). It was also shown that the pathway could be re-routed with a viral vector to produce a non-native carotenoid (Kumagai et al., 1998). Recombinant protein products have been produced in plants by transfection with viral vectors carrying heterologous genes (Kumagai et al., 2000). Since expression or silencing of a gene begins soon after inoculation, it is much faster to use viral vectors for such studies rather than developing transgenic or "knockout" plants. Functional genomics research benefits from this convenience, which allows large numbers of genes to be quickly characterized without directly manipulating the genetic makeup of the plants.

In this work, a virus-induced gene silencing experiment was conducted with a partial gene sequence obtained from a cDNA library for Dunaliella salina, a species of marine algae. The sequence was found to be similar to a gene in higher plants encoding a DEAD box RNA helicase. The DEAD box helicases are a family of proteins involved in such functions as ribosome assembly, translation initiation, and RNA splicing (Roberts et al., 2004). It was therefore expected that successful silencing of this DEAD box helicase gene in a higher plant would disrupt important regulatory pathways and affect the health of the plant significantly. Dunaliella salina is only remotely related to higher plants, but it was hypothesized that the D. salina sequence could be used to induce silencing of the DEAD box helicase gene in Nicotiana benthamiana, a relative of tobacco, despite their phylogenetic distance. This would demonstrate that it is possible to indirectly characterize genes from organisms such as D. salina, which cannot be easily manipulated genetically, by investigating the effects of using those genes to induce silencing in a higher plant species.

Dunaliella salina is a unicellular green alga of considerable scientific and commercial interest. It is able to withstand extreme salt concentrations, and one of its responses to stressful conditions is the production of carotenoids. It is cultivated commercially as a source of β-carotene, a precursor of vitamin A and an antioxidant that can be used in food and nutrition supplements (Chidambara Murthy et al., 2004). Studying the function of genes in D. salina could provide valuable information about the molecular mechanisms of halotolerance and carotenoid production. Genes from D. salina have previously been cloned and expressed in Escherichia coli (Premkumar et al., 2003), and very recently there has been some success in producing transgenic D. salina by bombardment with gold micro-particles (Lu et al., 2004). Developing a system for expressing or silencing D. salina genes in N. benthamiana by using viral vectors would provide a convenient alternative that would facilitate rapid gene characterization and allow comparisons to be made between the D. salina genome and that of higher plants. This work explores the potential of such a system by employing viral vectors in the study of an uncharacterized D. salina gene.
Figure 1.  The TTOSA1 APE pBAD #5 viral vector contains bacterial plasmid pBR322, genes from tobacco mosaic virus and an insert that codes for green fluorescent protein.

Figure 1. The TTOSA1 APE pBAD #5 viral vector contains bacterial plasmid pBR322, genes from tobacco mosaic virus and an insert that codes for green fluorescent protein.

Materials and Methods

Sequence identification

The uncharacterized cDNA clone SJ5013 was searched against GenBank using NCBI BLAST and against a D. salina stress-responsive EST database using PipeOnline 2.0 (stress-genomics.org). A translated BLAST query showed similarity to an RNA helicase protein in Arabidopsis thaliana (At2g35340). A match found in the D. salina EST database (contig2001Jun082218_1540) also showed similarity to this protein. A 459 bp segment from SJ5013 representing the highly conserved region was used as the DEAD box helicase sequence for silencing experiments. The alignment between this sequence and the A. thaliana sequence is shown below:

SJ5013: 1 GDHIALMNIFDGWAESNFSTQWCYENYVQVSHSVRTLSRYATVR 132

GDHIAL+ ++ W E+NFSTQWCYENY+Q VR++ R +R

At2g35340: 913 GDHIALLKVYSSWKETNFSTQWCYENYIQ----VRSMKRARDIR 952

Construction of viral vector

A stock of C600 E. coli cells containing the plasmid pBlue ARF #6 was cultured in a shake flask with 30 mL of luria-bertani medium and 30 μL of ampicillin (100 μg/mL), incubated at 37oC and agitated at 230 rpm for 12-16 hours. Plasmid DNA was purified from the culture using the Qiagen Midi Prep plasmid purification kit (a modified alkaline lysis procedure). The pBlue ARF #6 plasmid contains the DEAD box helicase sequence as well as portions of a gene encoding an adenosine ribosylation factor.

The DEAD box helicase sequence was amplified from the pBlue ARF #6 plasmid by PCR using the following primers:

5' ATC CTA GGA GAC CAC ATC GCA CTC ATG AAC ATC 3'

5' ATC TCG AGG GAG CAT GGC AAA GAG GTT GAA AGC 3'

These primers incorporate a XhoI and AvrII restriction site on either end of the sequence to facilitate ligation into the viral vector. The PCR reaction was run with each primer at a concentration of 0.5 μM, and Vent DNA polymerase (New England Biolabs, Beverly, MA) was used. An Eppendorf Mastercycler was used for the PCR reaction, which began at 97ºC for 2 minutes, followed by 5 cycles at 97ºC (1 min), 60ºC (1 min), 72ºC (2 min); then 20 cycles of 94ºC (1 min), 60ºC (1 min), 72ºC (2 min); and a final 7 minutes at 72ºC.

The 471 bp DEAD box helicase PCR product (the DBH insert) was purified on a 1% agarose gel using a Qiagen MiniElute Gel Extraction kit (Qiagen, Valencia, CA). It was then digested sequentially with XhoI and AvrII restriction endonucleases (New England Biolabs).

The viral vector used for the experiment was TTOSA1 APE pBAD #5 (Figure 1). The vector was isolated as plasmid DNA from a C600 cell culture as described above. It was digested sequentially with XhoI and AvrII restriction endonucleases to remove the insert encoding green fluorescent protein. It was then purified by excision from a 1% low-melt agarose gel. A ligation reaction was carried out using T4 DNA ligase (New England Biolabs) with an approximately 3:1 ratio of DBH insert to viral vector; it was run for 35 minutes at 25oC before 1 μL of the solution was used to transform 200 μL of C600 competent cells. Transformed cells were plated on ampicillin-selective media, and colonies were screened using restriction enzymes.

Host plant

N. benthamiana is a member of the tobacco family and is susceptible to viral vectors derived from tobacco mosaic virus. Seeds were sown 2-3 weeks prior to transfection. Seedlings were kept moist by misting, and larger plants were watered daily. Plants were grown indoors under artificial lighting.
Figure 2.  The green fluorescent protein insert was replaced with the DEAD box helicase insert to create the viral vector TTOSA1 DBH #1. This vector was used to silence the DEAD box helicase gene.

Figure 2. The green fluorescent protein insert was replaced with the DEAD box helicase insert to create the viral vector TTOSA1 DBH #1. This vector was used to silence the DEAD box helicase gene.

Transfection of plants

The constructed viral vector, TTOSA1 DBH #1 (Figure 2), was linearized by digestion with KpnI. The Ambion mMessage mMachine SP6 in vitro transcription kit (Ambion, Austin, TX) generated mRNA from the linearized TTOSA1 DBH #1 vector. The reaction solution was combined with 9 volumes of FES before being mechanically applied to the leaves of plants. Two leaves were inoculated per plant, and 50 μL was used for each leaf. Simultaneous inoculations were performed using the unmodified TTOSA1 APE pBAD #5 vector as a control. Uninfected plants were also kept and raised alongside the experimental plants for comparison.

Confirmation of silencing with Real-time PCR

Four days after inoculation, 0.15-0.2 g samples of leaf tissue from both uninfected plants and plants transfected with TTOSA1 DBH #1 were frozen in liquid nitrogen and homogenized using a mortar and pestle that had been baked overnight at 240oC. RNA was recovered from the tissue by extraction with TRIzol Reagent (Invitrogen, Carlsbad, CA). RNA cleanup was performed with the Qiagen RNeasy Plant Mini Kit. Samples were treated with DNase I (Invitrogen) before a BioRad iScript kit (BioRad, Hercules, CA) was used to synthesize cDNA.

Real-time PCR reactions were set up using BioRad iQ SYBR Green Supermix. The following primers were used for the amplification of the DEAD box helicase sequence:

5' GCG TCT TCG ACA TCT TTC AA 3' (upstream)

5' CAA TGG TTC TGA CGA TGA GG 3' (downstream)

Reactions were also run with primers for a selected reference gene, the small subunit of rubisco:

5' ACA AGA AGA AGT ACG AGA CTC TCT 3' (upstream)

5' CGA ACA TAG GTA AGC TTC CAC ATG 3' (downstream)

Duplicate real-time PCR reactions were set up for each of the following conditions: wild-type cDNA with DBH primers, wild-type cDNA with rubisco primers, transfected plant cDNA with DBH primers, and transfected plant cDNA with rubisco primers. A series of reactions containing successive 10-fold dilutions of the transfected plant cDNA was also set up to generate a standard curve. Reactions were run on a BioRad iCycler real-time PCR machine.

Results

Four days after inoculation, all plants began showing signs of successful viral infection, such as slight crinkling and wilting in the stem and leaves. After seven days, plants transfected with TTOSA1 DBH #1 showed discoloration of the leaves and stunted growth compared to plants transfected with TTOSA1 APE pBAD #5. Plants transfected with TTOSA1 APE pBAD #5 expressed green fluorescent protein and were observed to fluoresce under a handheld ultraviolet light, but did not show the same disease-like symptoms. After 13 days, plants transfected with TTOSA1 DBH #1 showed heavy necrosis on the leaves and appeared to be in much worse health than the other plants (Figure 3).

Figure 3.  Nicotiana benthamiana plants 13 days after inoculation. Left: Plant transfected with TTOSA1 APE pBAD #5 shows signs of viral infection and expresses green fluorescent protein. Middle: Plant transfected with TTOSA1 DBH #1 shows heavy necrosis, discoloration, and stunted growth. Right: Healthy, uninfected plant.

Figure 3. Nicotiana benthamiana plants 13 days after inoculation. Left: Plant transfected with TTOSA1 APE pBAD #5 shows signs of viral infection and expresses green fluorescent protein. Middle: Plant transfected with TTOSA1 DBH #1 shows heavy necrosis, discoloration, and stunted growth. Right: Healthy, uninfected plant.

Real-time PCR results indicated that expression levels of the DEAD box helicase gene were much lower in plants transfected with TTOSA1 DBH #1 (Figure 4). The dilution series, however, did not yield a suitable standard curve to determine amplification efficiency, and melt curve analysis showed that non-selective binding was occurring with the DBH primers. An approximation calculation was made using the Ct method:

Wild-type Ct = 40.32 – 20.63 = 19.69

Transfected plant Ct = 45.62 – 19.93 = 25.69

Ct = 19.69 – 25.69 = -6

2-6 = 64-fold decrease in DEAD box helicase expression in transfected plants

Discussion

The DEAD box helicase sequence studied in this work is highly conserved among distantly related species. This suggests that it is associated with vital cellular functions, and its putative RNA-binding ability implies a likely role in RNA processing. Further study of this gene may help to elucidate mechanisms of gene expression and regulation in many different organisms. It is also possible that the D. salina gene has particular characteristics related to halotolerance, which would be of potential interest to those studying the mechanisms of stress tolerance or to those attempting to optimize productivity in D. salina bioreactors. Virus-induced gene silencing is one way to approach the characterization of this gene.

Figure 4. In real-time PCR, amplification is tracked by a fluorescent dye that binds to double-stranded DNA. For each run, the PCR cycle number at which the total fluorescence surpasses a threshold value (horizontal dashed line) is the Ct value for that run. If there is less cDNA for the gene in the sample, then it will take longer for the sequence to amplify, and the Ct value will be higher. The curves shown here represent a) Rubisco small subunit in transfected plants b) Rubisco small subunit in wild-type plants c) DEAD box helicase in wild-type plants d) DEAD box helicase in transfected plants. In transfected plants, expression of rubisco does not appear to be affected, but expression of the DEAD box helicase is reduced significantly.

Figure 4. In real-time PCR, amplification is tracked by a fluorescent dye that binds to double-stranded DNA. For each run, the PCR cycle number at which the total fluorescence surpasses a threshold value (horizontal dashed line) is the Ct value for that run. If there is less cDNA for the gene in the sample, then it will take longer for the sequence to amplify, and the Ct value will be higher. The curves shown here represent a) Rubisco small subunit in transfected plants b) Rubisco small subunit in wild-type plants c) DEAD box helicase in wild-type plants d) DEAD box helicase in transfected plants. In transfected plants, expression of rubisco does not appear to be affected, but expression of the DEAD box helicase is reduced significantly.

The phenotypic changes observed in the plants transfected with TTOSA1 DBH #1, along with the real-time PCR results, made it evident that gene silencing had occurred. The DEAD box helicase gene that was silenced is itself involved in pathways that affect gene regulation, so it is not surprising that silencing it significantly affected the growth and development of the plants. It is possible, however, that some of the disease-like symptoms observed were in fact caused by a pathogen able to take advantage of the weakened immunity of the plants. Further studies should be performed to show that the results can be replicated. Nevertheless, the phenotypic changes observed in this experiment were observed consistently among the four separate plants transfected with TTOSA1 DBH #1.

These results have demonstrated that it is possible to use a sequence from Dunaliella salina to induce silencing in Nicotiana benthamiana. This means that it may be possible to perform similar experiments with other sequences from D. salina or from other uncharacterized organisms. Large-scale functional genomics studies may be able to take advantage of this opportunity to explore the functions of genes from organisms that cannot be easily manipulated in the laboratory. Aided by the speed and convenience of viral vectors, gene silencing experiments like this could lead to many interesting and important discoveries in a relatively short amount of time.

Acknowledgements

Support for this work was provided through NSF grant 0243600 and the University of Hawaii Sea Grant College Program. The authors would also like to acknowledge Ms. Jennifer Busto for guidance in conducting the real-time PCR experiments, as well as the Greenwood Molecular Biology Facility for use of the real-time PCR thermocycler. NCBI BLAST and PipeOnline 2.0 (stress-genomics.org) were used for sequence indentification and alignment.


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