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Keywords:

  • functional genomics;
  • gene silencing;
  • Nicotiana benthamiana;
  • Solanaceae;
  • virus-induced gene silencing

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • • 
    Virus-induced gene silencing (VIGS) is a rapid and robust method for determining and studying the function of plant genes or expressed sequence tags (ESTs). However, only a few plant species are amenable to VIGS. There is a need for a systematic study to identify VIGS-efficient plant species and to determine the extent of homology required between the heterologous genes and their endogenous orthologs for silencing.
  • • 
    Two approaches were used. First, the extent of phytoene desaturase (PDS) gene silencing was studied in various Solanaceous plant species using Nicotiana benthamiana NbPDS sequences. In the second approach, PDS sequences from a wide range of plant species were used to silence the PDS gene in N. benthamiana.
  • • 
    The results showed that tobacco rattle virus (TRV)-mediated VIGS can be performed in a wide range of Solanaceous plant species and that heterologous gene sequences from far-related plant species can be used to silence their respective orthologs in the VIGS-efficient plant N. benthamiana. A correlation was not always found between gene silencing efficiency and percentage homology of the heterologous gene sequence with the endogenous gene sequence.
  • • 
    It was concluded that a 21-nucleotide stretch of 100% identity between the heterologous and endogenous gene sequences is not absolutely required for gene silencing.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Gene discovery and expression analyses have resulted in a large number of gene sequences being placed in the public domain (Rhee et al., 2006). Lately, there has been a tremendous increase in the number of publicly available gene sequences, including expressed sequence tag (EST) and genomic sequences from various plant species. However, despite all these gene sequences being available, it is still a daunting task to determine gene function. Therefore, there is a need for the development of novel tools and high-throughput approaches for studying gene function using EST or genomic sequences.

There are various ways to introduce mutations or to down-regulate gene expression in order to study gene function in plants. The four most commonly used methods are chemical mutagenesis, irradiation mutagenesis, insertional mutagenesis and post-transcriptional gene silencing (PTGS). The triggering molecule of PTGS is double-stranded RNA (dsRNA) (Waterhouse et al., 1998), which is cleaved to produce small interfering RNA (siRNA) (Hamilton & Baulcombe, 1999; Hamilton et al., 2002). The siRNA associates with the RNA-induced silencing complex (RISC) and targets homologous RNA for degradation (reviewed in Bartel, 2004). RNA interference (RNAi), is an epigenetic phenomenon that results in sequence-specific degradation of endogenous mRNAs (Waterhouse & Helliwell, 2003; Kerschen et al., 2004; Wang et al., 2005). In the 1990s and the present decade, the transient PTGS system of virus-induced gene silencing (VIGS) by recombinant viruses carrying a fragment of plant sequence has been adapted for studying plant gene function (Baulcombe, 1999; Burch-Smith et al., 2004). Both PTGS approaches, RNAi and VIGS, are becoming powerful tools for studying plant gene function. The generation of stable RNAi plants requires plant transformation and regeneration, which are time consuming, and therefore may not be suitable for high-throughput screens. VIGS, in contrast, can potentially be used as a tool in high-throughput reverse and forward genetic screens (Liu et al., 2002a; Ekengren et al., 2003; Benedito et al., 2004; Anand et al., 2007a). VIGS has been successfully used in several studies to identify the function of plant genes involved in abiotic stress tolerance (Senthil-Kumar & Udayakumar, 2006; Senthil-Kumar et al., 2007), plant defense against pathogens (Liu et al., 2002a,b; Burch-Smith et al., 2004, 2006; Anand et al., 2007a) and in cell developmental processes (reviewed in Burch-Smith et al., 2004; Benedito et al., 2004). Gene identification is rapid using VIGS-mediated forward genetic screens when compared with traditional methods of screening T-DNA, Ethyl methanesulfonate (EMS) and deletion mutants (reviewed in Burch-Smith et al., 2004; Robertson, 2004). The recent development of a tool for avoiding off-target gene silencing has further advanced the use of VIGS (Xu et al., 2006).

The siRNAs (21–28 nucleotides) that are homologous to the gene(s) silenced by PTGS are found in silenced tissues (Hamilton & Baulcombe, 1999; Hamilton et al., 2002). In mammals and Drosophila, 22-nucleotide siRNA molecules are sufficient to induce silencing (Hammond et al., 2000; Bernstein et al., 2001). Thomas et al. (2001) reported that, to achieve PTGS in plants, a dsRNA fragment (silencing inducer) of 300–500 bp with at least one stretch of 23 nucleotides with 100% identity to a targeted transgene mRNA is essential. Several studies have demonstrated that heterologous sequences can be efficiently silenced by VIGS in other species (Benedito et al., 2004; Fofana et al., 2004; Howes & Kumagai, 2005; Senthil-Kumar & Udayakumar, 2006; Anand et al., 2007b).

Unfortunately, VIGS has not yet been optimized for many crop plants. Currently, VIGS can be performed in Nicotiana benthamiana, tomato (Solanum lycopersicum), potato (Solanum tuberosum), Petunia hybrida, pea (Pisum sativum), Arabidopsis thaliana, rice (Oryza sativa), maize (Zea mays), barley (Hordeum vulgare), soybean (Glycine max), cassava (Manihot esculenta), pepper (Capsicum annuum), and wheat (Triticum aestivum) (Benedito et al., 2004; Burch-Smith et al., 2004; Chung et al., 2004; Constantin et al., 2004; Faivre-Rampant et al., 2004; Fofana et al., 2004; Robertson, 2004; Burch-Smith et al., 2006; Ding et al., 2006; Wang et al., 2006; Zhang & Ghabrial, 2006). Various virus-based vectors have been developed, including tobacco rattle virus (TRV), potato virus X (PVX), pea early browning virus (PEBV), tomato golden mosaic virus (TGMV), barley stripe mosaic virus (BSMV), brome mosaic virus (BMV), and cabbage leaf curl virus (CBLCV), which are commonly used for gene silencing studies (reviewed in Benedito et al., 2004; Burch-Smith et al., 2004; Robertson, 2004; Ding et al., 2006). Currently, VIGS cannot be performed in many plant (crop) species because of the lack of a compatible VIGS vector for that particular plant species. Not all viruses are candidates for modification into VIGS vectors because most viruses have suppressors of gene silencing (reviewed in Robertson, 2004). Therefore, a high-throughput method for assessing gene function is not yet available for many crop plants. Fortunately, it has been demonstrated that heterologous gene sequences from both closely and distantly related plant (crop) species can be used to trigger gene silencing in model plants as long as there is minimal nucleotide sequence homology between the gene sequences (Ekengren et al., 2003; Fofana et al., 2004; Ryu et al., 2004; Howes & Kumagai, 2005; Senthil-Kumar & Udayakumar, 2006). For example, a phytoene desaturase (PDS) gene fragment from a monocot (lily; Lilium longiflorum) can cause silencing of endogenous N. benthamiana PDS by VIGS in spite of the remote evolutionary relationship between these two species (Benedito et al., 2004). A drought-induced peanut (Arachis hypogaea) gene, late embryogenic abundant-4 (lea4), was used to efficiently silence its ortholog in tomato (Senthil-Kumar & Udayakumar, 2006). Similarly, a DEAD box helicase gene from Dunaliella salina was used to silence its ortholog in a distantly related species, N. benthamiana (Howes & Kumagai, 2005). Ryu et al. (2004) successfully silenced the endogenous PDS genes of tomato, tobacco (Nicotiana tabacum) and Petunia hybrida using the N. benthamiana PDS gene sequence. However, using heterologous gene sequences it is still not clear how much sequence similarity or identity with the endogenous gene is required for efficient VIGS.

In this study, using TRV-based VIGS vectors, we show that silencing of PDS and the Rubisco small subunit (RBCS) can be achieved in several Solanaceous species using heterologous gene sequences. Similarly, we report silencing of PDS in N. benthamiana using heterologous PDS gene sequences from a wide range of plant species. We also report the relationship between the efficiency of gene silencing and heterologous gene sequence identity with the endogenous gene sequence.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant material and growth conditions

Nicotiana benthamiana L., Nicotiana clevelandii A. Gray, Nicotiana plumbagenifolia L., Nicotiana glutinosa L., Solanum lycopersicum L. (tomato; varieties arka vikas and microtom), Solanum pimpinellifolium L., Solanum peruvianum L., Solanum cheesmaniae (L. Riley) Fosberg and Solanum indicum L. (wild species of egg plant) were used in the experiments. Seeds of the above-mentioned plant species were germinated and seedlings were grown in plastic pots (20 cm diameter) containing potting mixture (Perlite division, Bangalore, India). Plants were grown in growth rooms at 22–25°C with 60% relative humidity and a 12-h photoperiod with light intensity ranging from 300 to 400 µmol m−2 s−1.

For the experiments on silencing of heterologous gene sequences in N. benthamiana, we germinated N. benthamiana seeds in flats using the soil-less potting mixture BM7 (Berger Co., Quebec, Canada). Two-week-old seedlings were transplanted to 10-cm-diameter round pots containing BM7, with one plant per pot. Fertilizer (nitrogen:phosphorus:potassium 20 : 10 : 20) along with a soluble trace element mix (The Scotts Co., Marysville, OH, USA) was applied with water. Glasshouse conditions were kept at 23 ± 3°C and 70% relative humidity with a 16-h extended day with supplemental lighting at 100 µmol m−2 s−1. Three-week-old plants were used in the experiments.

TRV vectors and plasmid construction

The pTRV1 and pTRV2 VIGS vectors (described in Liu et al., 2002a) were obtained from Dr S.P. Dinesh-Kumar (Yale University, USA). RNA was extracted from leaf tissues of different plant species using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and first-strand cDNA was synthesized using oligo(dT)15 primers. The cDNA pool from each species was used to amplify the PDS gene sequence by polymerase chain reaction (PCR) using gene-specific primers that were designed based on the sequence information available from GenBank. Primer sequence information and the fragment sizes of the PCR products used in this study are given in Supplementary Table S1.

The gene fragments amplified by PCR were cloned into the pTRV2 VIGS vector by Gateway cloning according to the manufacturer's recommendations (Invitrogen). Also, the constructs TRV2::NbPDS (Ryu et al., 2004), TRV2::SlPDS (Ryu et al., 2004), TRV2::GFP (Ryu et al., 2004) and TRV2::SlRBCS (Liu et al., 2002a) were used in this study. All the pTRV2 derivatives were confirmed by sequencing. Plasmids were introduced into Agrobacterium tumefaciens strain GV2260 by electroporation.

VIGS

The A. tumefaciens strain GV2260 containing pTRV1 or pTRV2 and its derivatives were grown at 28°C in Luria-Bertani (LB) medium containing appropriate antibiotics. The cells were harvested from cultures grown overnight and re-suspended in the infiltration buffer (10 mm MES, pH 5.5, and 200 µm acetosyringone) to a final absorbance (optical density (OD) at 600 nm) of 1.0, and incubated for 2 h at room temperature in a shaker. For leaf infiltration, each A. tumefaciens strain containing pTRV1 or pTRV2 and its derivatives were mixed in a 1 : 1 ratio in MES buffer (pH 5.5) and infiltrated into lower leaves using a 1-ml needleless syringe (Ratclif et al., 2001; Liu et al., 2002a). Infiltrated plants were maintained under a temperature range of 23–25°C for effective viral infection and spread. Infiltration of 15-d-old tomato plants and 21-d-old N. benthamiana plants induced effective silencing and maintenance of these plants at 25 ± 3°C showed maximum efficiency of gene silencing.

Cloning full-length NbPDS

RNA was extracted from N. benthamiana leaves using Trizol reagent (Invitrogen). RNA samples were treated with RNase-free DNase and a reverse transcription reaction was then performed using a reverse transcriptase–polymerase chain reaction (RT-PCR) kit (Promega, Madison, WI, USA). A FirstChoice RLM-RACE (RNA ligase-mediated rapid amplification of cDNA ends) kit (Ambion Inc., Austin, TX, USA) was used to perform the RACE as per the manufacturer's instruction. The primers used were 5′-GCTTATCTTTGGAGCTCGAGGTCTTC-3′ and 5′-GATAAGACAGCACCTTCCATTGAAGCCAAG-3′. The full-length NbPDS gene (1761 bp) was cloned into the pGEMT vector (Promega) and the sequence was submitted to GenBank (DQ469932).

Real-time quantitative RT-PCR

To quantify endogenous PDS transcript abundances in TRV2::PDS (PDS from different plant species) inoculated plants, real-time quantitative RT-PCR (qRT-PCR) was performed. Total RNA was extracted from silenced and mock-infiltrated plants and first-strand cDNA was synthesized using oligo(dT) primers. qRT-PCR was performed using ABI Prism 7000 (ABI Applied Biosystems, Foster City, CA, USA) using SYBR green (ABI Applied Biosystems). The primers (forward 5′-GCATTT TGATTGATTGCTTTGAACAGA-3′ and reverse 5′- CACAATCGGCATGCAAAGTC-3′) used for quantifying the relative abundances NbPDS transcripts were designed using primer express software version 2.0 (ABI Applied Biosystems). For the relative quantification of gene transcript abundances in silenced and nonsilenced plants, a standard curve method was applied according to the manufacturer's protocol (ABI Applied Biosystems User Bulletin). As a control for silenced and mock-infiltrated plants, a parallel reaction using N. benthamiana elongation factor 1-α (EF2) was performed and the data obtained were used to normalize the data for PDS transcripts. Samples were pooled from three independent experiments (mock & silenced plants) and the real-time PCR was performed twice with three replicates each. Calculations were performed as previously described by Constantin et al. (2004) and the percentage inhibition was determined.

Estimation of chlorophyll content

Chlorophyll was extracted from 100 mg of leaf tissue in an acetone:dimethyl sulphoxide (DMSO) (1 : 1 volume/volume) mix and the supernatant was made up to 1 ml using this mix. The absorbance was recorded at 663 and 645 nm using UV–visible spectrophotometer Model DU800 (Shimadzu Corporation, Kyoto, Japan). Total chlorophyll was estimated as described previously (Hiscox & Israelstam, 1979) and expressed as the percentage reduction relative to the corresponding control (wild-type or mock-infiltrated).

Formulas

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Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Silencing of endogenous PDS and RBCS genes in different plant species by NbPDS and SlRBCS gene sequences

In order to demonstrate that a heterologous gene sequence can be used to silence its orthologs in various plant species, we used N. benthamiana PDS (NbPDS) and tomato RBCS (SlRBCS) gene sequences (Liu et al., 2002a; Ryu et al., 2004) for VIGS in various plant species belonging to the Solanaceae family. All the plant species studied developed photobleaching or yellowing symptoms 2–3 wk after infiltration with TRV2::NbPDS or TRV2::SlRBCS, respectively (Fig. 1a; Supplementary Material Fig. S1a). However, the timing and the extent of photobleaching or yellowing varied among plant species.

image

Figure 1. Silencing of endogenous phytoene desaturase (PDS) in different Solanaceae species by tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) using the Nicotiana benthamiana PDS gene sequence. Fifteen-day-old plants were infiltrated with TRV1 + TRV2::NbPDS. (a) The photobleaching phenotype was photographed at 20 d post infiltration (dpi). (b) The frequency of gene silencing was calculated by counting the number of plants that showed photobleaching symptoms at 28 dpi. (c) The effectiveness of gene silencing was calculated by counting the number of leaves that showed photobleaching on a gene-silenced plant at 28 dpi. (d) The gene silencing efficiency was calculated by measuring the reduction of chlorophyll content in gene-silenced plants at 28 dpi. The values are means of two independent experiments and the bars represent standard error. av, arka vikas; chl, chlorophyll; mock, mock-infiltrated; N., Nicotiana; S., Solanum.

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Transcript abundances  Endogenous PDS transcript abundances in photobleached leaves (third leaf from the top) of TRV2::NbPDS inoculated plants were assessed by RNA dot blot analysis. Endogenous PDS transcript abundances were significantly reduced at 18 d post inoculation in N. benthamiana, N. clevelandii, N. plumbagenifolia, S. lycopersicum, S. peruvianum and S. indicum (data not shown). Plants inoculated with TRV2::00 (mock control) did not show a reduction in endogenous PDS transcripts (data not shown).

Frequency of gene silencing  The gene silencing frequency was calculated by comparing the number of plants that showed photobleaching symptoms with the total number of plants that were inoculated with TRV2::NbPDS (see the Materials and Methods for the formula). Wide variation in silencing frequency was observed among the plant species (Fig. 1b). Nicotiana benthamiana and N. clevelandii showed the highest frequencies (> 90%) of the species studied. Nicotiana plumbagenifolia, S. pimpinellifolium, S. peruvianum and S. cheesmaniae showed silencing frequencies of only 50–60%. The frequency of PDS silencing was lower (< 20%) in N. glutinosa (Fig. 1b). These results indicate that, when heterologous gene sequences are used for VIGS, the silencing frequency can vary for different plant species.

Effectiveness of gene silencing  The effectiveness of gene silencing, in all the plants that showed photobleaching symptoms, was calculated by comparing the number of leaves that showed symptoms with the total number of leaves on the plant (see Materials and Methods for the formula). The effectiveness of gene silencing varied among species and the trend observed was similar to that for the gene silencing frequency (Fig. 1b and Supplementary Material Fig. S1b). Among the different species studied, N. benthamiana, S. lycopersicum, S. pimpinellifolium, S. peruvianum, and S. cheesmaniae showed higher effectiveness for PDS silencing using TRV2::NbPDS. In contrast, N. glutinosa showed less than 40% effectiveness of gene silencing (Fig. 1c). In TRV2::LeRBCS-inoculated plants, the effectiveness of RBCS silencing in various plant species showed a similar trend to that for PDS silencing (Supplementary Material Fig. S1b).

Efficiency of gene silencing  As the PDS and RBCS gene sequences for most of the Solanaceous plant species studied are not known, we could not accurately determine their transcript abundances in silenced plants to measure gene silencing efficiency. Instead, we used chlorophyll content as a measure of silencing efficiency. More chlorophyll degradation indicates higher silencing efficiency and vice versa. The chlorophyll content was assessed in the newly emerged third or fourth leaf, above the infiltrated leaves, that showed the photobleaching phenotype and was compared with the chlorophyll content of leaves of mock-inoculated plants (TRV::00; empty vector) that did not show photobleaching. The greatest reduction (> 90%) in chlorophyll was observed in the PDS gene silenced leaves of N. benthamiana and N. clevelandii (Fig. 1d). The silenced leaves of N. plumbagenifolia and S. peruvianum showed a 70% reduction in chlorophyll content when compared with the leaves of mock-inoculated plants. The other plant species studied showed reductions of less than 60% in chlorophyll content (Fig. 1d). Overall, the trend of chlorophyll reduction in RBCS silenced plants was similar to that in PDS silenced plants, with the exception of S. indicum (Supplementary Material Fig. S1c).

Silencing of the PDS gene in N. benthamiana using heterologous PDS gene sequences from closely and distantly related plant species

VIGS in N. benthamiana has been shown to be highly efficient (Lu et al., 2003; Burch-Smith et al., 2004). To determine whether heterologous gene sequences, from both closely and distantly related plant species, can be used to silence their respective orthologs in N. benthamiana, we cloned PDS sequences from closely related species such as tomato (S. lycopersicum SlPDS), and distantly related species such as pea (Pisum sativum; PsPDS), peanut (Arachis hypogaea; AhPDS), soybean (Glycine max; GmPDS), barrel medic (Medicago truncatula; MtPDS), Arabidopsis (Arabidopsis thaliana; AtPDS), and maize (Zea mays; ZmPDS) into the TRV2 vector and used it to perform VIGS in N. benthamiana. Phenotypic observations indicated that the extent of photobleaching varied from mild to complete bleaching among the different PDS sequences used for silencing (Fig. 2a).

image

Figure 2. Silencing of endogenous phytoene desaturase (PDS) in Nicotiana benthamiana using PDS sequences from different plant species. Twenty-one-day-old plants were infiltrated with Agrobacterium tumefaciens containing TRV1 + TRV2::GFP (mock-infiltrated (mock)) or TRV1 + TRV2::PDS (PDS from different species). (a) Photographs of the photobleached leaves taken at 21 d post inoculation (dpi). (b) The frequency of gene silencing was calculated by counting the number of plants that showed photobleaching symptoms at 28 dpi. (c) The effectiveness of gene silencing was calculated by counting the number of leaves that showed photobleaching on a gene-silenced plant at 28 dpi. (d) The gene silencing efficiency was calculated by measuring the reduction of chlorophyll content in gene-silenced plants at 28 dpi. The values are means of two independent experiments and the bars represent standard error. NbPDS, Nicotiana benthamiana PDS; SlPDS, Solanum lycopersicum PDS; GmPDS, Glycine max PDS; PsPDS, Pisum sativum PDS; AtPDS, Arabidopsis thaliana PDS; MtPDS, Medicago truncatula PDS; ZmPDS, Zea mays PDS; AhPDS, Arachis hypogea PDS.

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Frequency of gene silencing  We determined the frequency of gene silencing in N. benthamiana in a similar manner as for other Solanaceous species (see first part of ‘Results’ section). The silencing frequency was relatively high in most cases when heterologous sequences of PDS were used for VIGS. In all cases, except for the MtPDS sequence, the heterologous PDS sequences caused silencing of endogenous NbPDS in more than 85% of the plants tested (Fig. 2b). The NbPDS and SlPDS sequences caused photobleaching in almost 98% of the plants tested (Fig. 2b). These data clearly demonstrate that heterologous gene sequences can be used to silence their respective orthologs in N. benthamiana.

Effectiveness of gene silencing  The effectiveness of gene silencing (see first part of ‘Results’ section) varied for the different PDS sequences that were used for VIGS (Fig. 2c). The most effective gene silencing was observed when NbPDS and SlPDS sequences were used (Fig. 2c). Almost 40% of the leaves showed photobleaching symptoms when MtPDS and AtPDS sequences were used for VIGS. Only approximately 20% of the leaves showed photobleaching symptoms when PsPDS, AhPDS, GmPDS and ZmPDS sequences were used for VIGS (Fig. 2a).

Efficiency of gene silencing  We calculated the efficiency of gene silencing triggered by heterologous PDS gene sequences in N. benthamaina by two methods. In the first method, we looked at the percentage of chlorophyll reduction in the photobleached leaves, as described in ‘Materials and Methods’ (Fig. 2d). Plants inoculated with TRV2::NbPDS and TRV2::SlPDS showed almost 100% reductions compared with nonsilenced wild-type plants and mock control plants (TRV2::GFP; the GFP sequence does not have any homology to plant DNA and therefore will not cause gene silencing). Plants inoculated with the other clones showed reductions of less than 60% in chlorophyll content (Fig. 2d).

In the second method, we calculated the efficiency of gene silencing by quantifying the abundances of endogenous NbPDS transcripts. qRT-PCR was performed using RNA isolated from the photobleached leaves of silenced plants using the primers designed for the NbPDS gene. As expected, there was a large reduction in NbPDS transcripts in plants inoculated with TRV2::NbPDS and TRV2::LePDS (Table 1). TRV2::AtPDS-inoculated plants showed a reduction of approximately 30% in the abundance of NbPDS transcripts (Table 1). The reduction in endogenous PDS transcripts was only 20% when PDS sequences from garden pea, peanut, soybean, barrel medic and maize were used (Table 1). These results indicate that N. benthamiana could be used to study heterologous gene function. However, the efficiency of PDS gene silencing depends on the extent of homology between the N. benthamiana gene sequence and the heterologous gene sequence that will be used as a gene silencing trigger.

Table 1.  Correlation between phytoene desaturase (PDS) silencing efficiency and percentage nucleotide identity of Nicotiana benthamiana PDS with other heterologous PDS sequences
Heterologous sequences used for VIGSEndogenous transcript inhibition (%)Nucleotide identity with NbPDS (%)Does 21-nucleotide stretch of 100% identity with NbPDS exist?Maximum stretch of nucleotides having 100% identity with NbPDS21-nucleotide stretch with 100% identity containing:*
one nucleotide mismatchtwo nucleotide mismatches
  • VIGS, virus-induced gene silencing.

  • *

    The stretch of nucleotides showing 100% identity when one or two nucleotide mismatches are allowed is shown in parentheses.

Mock  0.5 ± 2.08
NbPDS52.95 ± 4.42100YesAll
SlPDS46.37 ± 3.88 90Yes62
PsPDS18.96 ± 2.57 78Yes31
AhPDS16.75 ± 2.5 72No11Yes (24)
GmPDS21.11 ± 2.64 77No12NoNo
MtPDS20.05 ± 2.6 82No17Yes (23)
AtPDS29.14 ± 2.94 75No14Yes (23)
ZmPDS11.92 ± 2.36 76Yes26

Nucleotide homology between the heterologous gene sequence used for VIGS and the target gene does not always correlate with the gene silencing efficiency

To determine the correlation between the efficiency of gene silencing and the homology of the heterologous gene sequence with the target gene, we compared the overall nucleotide identity (between heterologous PDS gene sequences and the NbPDS sequence) with the percentage of NbPDS transcript reduction in the gene-silenced plants (Supplementary Material Fig. S2; Table 1). SlPDS showed maximum identity to NbPDS among all the heterologous PDS sequences studied. When both SlPDS and NbPDS sequences were used to silence NbPDS, we observed a reduction of approximately 50% in the abundance of endogenous NbPDS transcripts (Table 1). The remaining PDS sequences had approximately 70–80% identity with the NbPDS sequence and they were able to reduce the abundance of endogenous NbPDS transcripts by only 15–30% (Table 1). Overall, these data indicated that there was a correlation (r = 0.89, P < 0.001) between nucleotide homology and endogenous transcript inhibition. We also compared the silencing efficiency determined from the reduction in chlorophyll content with the percentage of nucleotide homology between heterologous PDS gene sequences and NbPDS. A high degree of correlation, with a correlation coefficient of 0.91, was observed between nucleotide homology and gene silencing efficiency. However, when data from NbPDS and SlPDS sequences were not considered, we did not find any correlation between gene silencing efficiency and the per cent nucleotide homology of the other heterologous gene sequences with the target gene (Table 1). For example, the MtPDS sequence had 82% identity with NbPDS but the gene silencing efficiency was only approximately 20%. In contrast, the AtPDS sequence had only 75% identity with the NbPDS sequence but the gene silencing efficiency was approximately 30%.

It has been reported that 21–23 nucleotides with 100% homology between the trigger and the target sequence are required to cause gene silencing during PTGS (Zamore et al., 2000; Voinnet, 2001; Hamilton et al., 2002). We therefore looked for stretches of the various PDS sequences used in this study that had 100% identity with the NbPDS sequence (Supplementary Material Fig. S2). Notably, the GmPDS, AtPDS, MtPDS and AhPDS sequences did not contain a 21-nucleotide stretch of 100% identity with NbPDS. However, the efficiency of gene silencing in NbPDS plants using the above-mentioned sequences ranged from 20 to 30%. It is important to note that the AtPDS, MtPDS and AhPDS sequences had 23–24-bp stretches of continuous homology but with one or two mismatches. Interestingly, although ZmPDS had 76% identity with NbPDS and a 26-nucleotide stretch of 100% identity, the transcript reduction for NbPDS in N. benthamiana inoculated with TRV2::ZmPDS was only c. 10%. In contrast, the GmPDS sequence did not have a 21-nucleotide stretch of 100% identity with NbPDS, even after allowing one or two mismatches, but it was able to reduce the transcript abundance by > 20%. These data suggest that a 21-nucleotide stretch of 100% identity between the trigger and target sequences is not absolutely required for gene silencing. However, the SlPDS sequence that had c. 90% identity with the NbPDS sequence and also had a c. 62-nucleotide stretch of 100% identity with the NbPDS sequence produced the most efficient gene silencing among all the heterologous PDS sequences used in this study.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

In recent years, the PTGS methods of VIGS and RNAi have become popular for studying gene function in plants. In particular, VIGS has gained popularity in plants lacking a high-throughput transformation system. This is mainly because VIGS is a very simple and robust method that does not require stable transformation, and more importantly one can use heterologous gene sequences to silence orthologs in plants that do not have extensive gene sequence information. To date, more than 20 VIGS vectors have been developed, and most of these vectors have a narrow host range (Burch-Smith et al., 2004; Constantin et al., 2004; Fofana et al., 2004). Although VIGS has been widely used, its versatility depends on the efficiency of virus replication and movement in the host, the response of plant species and also the homology of the insert gene (trigger) to the target gene (Baulcombe, 1999; Bartel, 2004; Burch-Smith et al., 2004). Here, we carried out a systematic study to determine the feasibility of using a gene sequence from one plant species to silence its orthologs in various distantly related plant species and also of using gene sequences from various distantly related plant species to silence their orthologs in one plant species.

As host species-specific VIGS vectors are limited, the function of some genes has been studied using heterologous gene sequences in VIGS-efficient species such as N. benthamiana and tomato (Benedito et al., 2004; Senthil-Kumar & Udayakumar, 2006). It has been shown that silencing of the endogenous PDS in the diploid wild species Solanum bulbocastanum and Solanum okadae, in the cultivated tetraploid S. tuberosum and in the distant hexaploid relative Solanum nigrum is possible using the PDS sequence from tomato (Brigneti et al., 2004). The extent of PDS and lea4 gene silencing in tomato by peanut PDS (AhPDS) and lea4 gene sequences was found to be relatively high in one of our previous studies (Senthil-Kumar & Udayakumar, 2006). Although there are many reports demonstrating VIGS using heterologous gene sequences, no systematic study has previously been carried out to determine the frequency, effectiveness and efficiency of VIGS using heterologous gene sequences. In this paper we systematically determined the extent of gene silencing of two different genes in various Solanaceous species using heterologous gene sequences from N. benthamiana or tomato (Fig. 1; Supplementary Material Fig. S1). The extent of gene silencing varied among the plant species tested, and this could be a result of differences in the homology of the heterologous gene sequences used for silencing with the endogenous target gene sequence, and in the ability of the virus (TRV) to replicate and move in the different Solanaceous species studied.

Nicotiana benthamiana is popularly employed as a model plant to study gene function using VIGS. Many laboratories have shown that heterologous gene sequences can be used to silence their respective orthologs in N. benthamiana. For example, in our laboratory we have successfully silenced N. benthamiana PDS using the tomato PDS gene sequence (Ryu et al., 2004). The Prf (gene required for resistance of tomato to Pseudomonas syringae pv. tomato) gene has been successfully silenced in N. benthamiana using the tomato Prf gene sequence in our laboratory as well as in others (Peart et al., 2002; Kang et al., 2004). Several other studies (Burton et al., 2000; Liu et al., 2002a; Lee et al., 2003; Saedler & Baldwin, 2004) have also shown that heterologous gene sequences can be used to silence their orthologs in N. benthamiana. Although there are several such studies, clear understanding of the homology required for silencing is lacking. Here, we carried out a study using PDS sequences from a wide range of plant species to determine the efficiency of endogenous PDS gene silencing in N. benthamiana (Fig. 2).

Understanding the correlation between the sequence identity of trigger vs target sequences and the efficiency of gene silencing is very important when heterologous gene sequences are used for VIGS. Overall, our study showed that variation in the efficiency of gene silencing was correlated with the percentage of nucleotide identity of the heterologous gene sequence and the target sequence (Table 1). In a few cases we observed that a higher percentage of nucleotide identity of the heterologous gene sequence with the target sequence produced a higher efficiency of gene silencing. However, when the difference in the percentage of nucleotide identity was not so great (for example, 75 vs 82%) we did not find a correlation between the gene silencing efficiency and the percentage of nucleotide homology of the heterologous gene sequence with the target gene (Table 1). Thomas et al. (2001) showed that, in assessing the requirement of homology for effective gene silencing, the stretches of 21–23 nucleotides with 100% identity between the trigger sequence and the target gene sequence are more important than the overall percentage of homology. Several other reports (Baulcombe, 2002; Meister & Tuschl, 2004) supported this view. We therefore investigated whether a 21-nucleotide stretch of 100% homology existed between the trigger and target sequences. Surprisingly, in many cases, although there was some degree of silencing, we could not detect a 21-nucleotide stretch of 100% homology between the trigger and target sequences (Table 1). However, AhPDS, MtPDS and AtPDS had a 21-nucleotide stretch of identity with NbPDS, with the exception of one or two mismatches. Interestingly, although GmPDS did not have a 21-nucleotide stretch of identity, even after allowing one or two mismatches, it was c. 20% efficient in silencing NbPDS. In contrast, ZmPDS had 76% nucleotide identity and a 26-nucleotide stretch of 100% identity with NbPDS and was least efficient (c. 11%) in silencing NbPDS. These results suggest that a 21-nucleotide stretch of 100% identity between a trigger sequence and the target sequence is not absolutely required for gene silencing. However, a stretch of more than 21 nucleotides with 100% identity between a trigger sequence and the target sequence is probably required for more efficient gene silencing, as shown by silencing NbPDS using the SlPDS sequence. In addition to adequate homology between the trigger and the target, the extent of viral spread and the effective duration of silencing of the endogenous gene are important parameters that determine the effectiveness of VIGS in many plant systems.

In conclusion, our study clearly shows that gene silencing can occur even with a less than 21-nucleotide stretch of 100% identity between the trigger and target sequences. We have also shown that the overall homology between the heterologous gene sequence and the target gene sequence does not always correlate with the gene silencing efficiency. We speculate that, in addition to the above-mentioned parameters (overall nucleotide homology and a 21-nucleotide stretch of 100% identity), the structure of RNA may play a role in determining the gene silencing efficiency. Nevertheless, our results favor the use of heterologous gene sequences from even distantly related species to silence their respective orthologs in N. benthamiana. We have demonstrated that TRV-mediated VIGS can be used in a wide range of Solanaceous plant species, and this adds to the growing list of plant species that can be used for VIGS-mediated studies.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank all KSM laboratory members for their thoughtful comments during the research program. We also thank Dr Ping Xu for critical reading of the manuscript and C. Ly for figure preparation. MS-K thanks the Council of Scientific and Industrial Research (CSIR), New Delhi for a senior research fellowship award (No. 9/271(86)/2004/EMR-1). He gratefully acknowledges financial support from the Wood Whelan Fellowship of the International Union of Biochemistry and Molecular Biology, the Kirkhouse Trust (Oxford, UK) and the Department of Science and Technology (SR/PF/1183/2005-06, GOI, DST, New Delhi). The authors thank Judy Grider, Kristy Richerson, Narayansamy and Ramanji for plant care during the research period. This work was supported by the Samuel Roberts Noble Foundation, USA and projects of the Department of Crop Physiology, UAS Bangalore.

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  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1 Silencing of endogenous Rubisco small subunit (RBCS) in different Solanaceae species by tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) using the tomato (Solanum lycopersicum) SlRBCS gene sequence.

Fig. S2 Sequence alignment showing nucleotide homology of phytoene desaturase (PDS) gene sequences from various plant species.

Table S1 Primer sequence information and the fragment sizes of the polymerase chain reaction (PCR) products used to clone the phytoene desaturase (PDS) gene fragments in this study.

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