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Molecular Plant Advance Access originally published online on June 19, 2009
Molecular Plant 2009 2(5):1084-1094; doi:10.1093/mp/ssp033
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© The Author 2009. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS.

Virus-Induced Gene Silencing in the Culinary Ginger (Zingiber officinale): An Effective Mechanism for Down-Regulating Gene Expression in Tropical Monocots

Tanya Renner, Jennifer Bragga, Heather E. Driscoll, Juliana Cho, Andrew O. Jackson and Chelsea D. Specht1

Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA
a Present address: Genomics and Gene Discovery, USDA, ARS, PWA, WRRC-GGD, 800 Buchanan Street, Albany, CA 94710, USA

1 To whom correspondence should be addressed at 111 Koshland Hall, MC 3102, Berkeley, CA 94720, USA. E-mail cdspecht{at}nature.berkeley.edu, fax 510-642-4995, tel. 510-642-5601.


   Abstract
TOP
 Abstract
INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
 FUNDING
 
Virus-induced gene silencing (VIGS) has been shown to be effective for transient knockdown of gene expression in plants to analyze the effects of specific genes in development and stress-related responses. VIGS is well established for studies of model systems and crops within the Solanaceae, Brassicaceae, Leguminaceae, and Poaceae, but only recently has been applied to plants residing outside these families. Here, we have demonstrated that barley stripe mosaic virus (BSMV) can infect two species within the Zingiberaceae, and that BSMV–VIGS can be applied to specifically down-regulate phytoene desaturase in the culinary ginger Zingiber officinale. These results suggest that extension of BSMV–VIGS to monocots other than cereals has the potential for directed genetic analyses of many important temperate and tropical crop species.

Key Words: Barley stripe mosaic virus • virus-induced gene silencing • VIGS • Zingiber officinale • Monocot

Received for publication March 19, 2009. Accepted for publication April 24, 2009.


   INTRODUCTION
TOP
 Abstract
 INTRODUCTION
RESULTS
 DISCUSSION
 METHODS
 FUNDING
 
Virus-induced gene silencing (VIGS) is a technique that utilizes the RNA interference (RNAi) pathway to down-regulate endogenous gene expression (Dinesh-Kumar et al., 2003; Burch-Smith et al., 2004; Godge et al., 2008). This process begins by abrading leaves with modified viral transcripts that express a plant cDNA sequence of a gene to be targeted for degradation (Kumagai et al., 1995; Ruiz et al., 1998). Once the transcripts begin replicating in vivo, double-stranded RNAs (dsRNAs) are generated by a viral RNA-dependent RNA polymerase, and the dsRNA intermediates are recognized by the plant's defense system and targeted for degradation into small interfering RNAs (siRNAs) by DICER-like enzymes (Benedito et al., 2004; Robertson, 2004). Highly specific silencing of gene expression subsequently occurs as the amplified siRNAs are incorporated into RNA-induced silencing complexes (RISC) that degrade complementary endogenous plant mRNAs (Baulcombe, 2004).

VIGS is a relatively new approach to down-regulate gene expression in plants. The technique was first applied with tobacco mosaic virus (TMV) to interfere with chlorophyll synthesis in Nicotiana tabacum L. (Kumagai et al., 1995). Later potato virus X (PVX–VIGS) was used to silence phytoene desaturase (PDS) in wild-type Nicotiana benthamiana Domin and to express green fluorescence protein (GFP) in transgenic N. benthamiana (Ruiz et al., 1998). However, tobacco rattle virus (TRV) has become the most widely used VIGS vector for members of the Solanaceae and Brassicaceae (Ratcliff et al., 2001; Burch-Smith et al., 2004; Chen et al., 2004; Fu et al., 2005; Burch-Smith et al., 2006; Dong et al., 2007; Godge et al., 2008), and the related pea early browning virus (PEBV) has been applied for developmental analysis of legumes (Constantin et al., 2004, 2008). TRV–VIGS has also recently been used for genetic analyses of the non-model basal eudicots, Papaver somniferum L. (Hileman et al., 2005; Drea et al., 2007), Aquilegia (Gould and Kramer, 2007), and Eschscholzia californica Cham. (Wege et al., 2007). Among the cereal crops, VIGS using barley stripe mosaic virus (BSMV–VIGS) has been applied for barley (Hordeum vulgare L.) (Holzberg et al., 2002; Bruun-Rasmussen et al., 2007) and wheat (Triticum aestivum L.) (Scofield et al., 2005), but application of VIGS for monocots other than cereal grass species has not been described.

Because BSMV–VIGS has been very valuable for analysis of gene function in its natural host Hordeum (Hein et al., 2005; Oikawa et al., 2007; Shen et al., 2007) and in the closely related Triticum (Scofield et al., 2005; Cloutier et al., 2007; Fu et al., 2007; Zhou et al., 2007; Sindhu et al., 2008), we sought to determine whether the technology could be applied to tropical plants of the order Zingiberales. The Zingiberales (tropical gingers and bananas) exhibit a wide range of flower forms, making them an interesting system for investigating the role of specific gene families in the evolution of floral development (Figure 1). The order also exhibits substantial differences in growth habit; hence it is ideal for developmental studies on shoot, rhizome, and root systems. For this purpose, we designed a BSMV–VIGS vector to suppress PDS in the culinary ginger, Zingiber officinale Roscoe, using strategies similar to those successfully applied to barley (Holzberg et al., 2002) and wheat (Tai et al., 2005). Our results suggest wild-type (wt) BSMV is able to establish systemic infections of Z. officinale and Costus spicatus (Jacq.) Sw. We found that in Z. officinale, silencing of endogenous PDS (ZoPDS) results in white striations or fully photobleached leaves in systemically infected plants. We propose using Z. officinale as a model for studying gene function in non-grass monocots.


Figure 1
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  		Figure 1. Photos of the Two Species Used in this Study, Clockwise from Upper Left.

(A) Inflorescence of C. spicatus, showing current and past flowers. One flower opens per day from the conical inflorescence.

(B) Close-up of C. spicatus flower (credit: Madelaine E. Bartlett).

(C) Z. officinale inflorescence with flowers.

(D, E) Z. officinale shoots and rhizomes showing facility for replication of greenhouse experiments. (D) Close-up of the three rhizome sections shown in (E). The growing end of the rhizome is towards the bottom of the photograph, seen as a small protrusion of the rhizome tissue. This protrusion represents a single apical meristem and will grow out as a new vegetative shoot. Lateral thickening at the base will form the rhizome, and roots will develop from axillary meristems along the rhizome.

(E) Three rhizome sections with corresponding shoot and root systems, demonstrating 4 months of growth. Each rhizome section can be separated from adjoining sections and planted as an individual, complete with shoot and root system. Flowering, leafless shoots (C) form directly off the rhizome.

 

   RESULTS
TOP
 Abstract
 INTRODUCTION
 RESULTS
DISCUSSION
 METHODS
 FUNDING
 
BSMV Is Able to Infect Members of the Zingiberales
BSMV has a very broad host range and infects several graminaceous hosts as well as some non-monocot species (Jackson and Lane, 1981). Although there is a single report of Commelina communis L. (Commelinaceae; Commelinales) susceptibility (Jackson and Lane, 1981), extensive studies have not been carried out on monocots belonging to families other than Poaceae, and, to the best of our knowledge, BSMV host range studies with the Zingiberales have not been conducted. Leaves of young Z. officinale shoots were inoculated with extracts of leaves from H. vulgare harboring the wt ND18 strain of BSMV. At 10 d after inoculation, newly emerging leaves developed a lightly striated mosaic phenotype (Figure 2B), and infection was confirmed with a Western blot for viral coat protein (CP) (Figure 3A) and by RT–PCR using primers targeting a 734-nt fragment within ORFs 3 and 4 of RNAβ (Figure 3B). In addition to Z. officinale, we tested the susceptibility of the closely related C. spicatus to BSMV. We were able to confirm the presence of the BSMV in all inoculated plants by Western blotting (Figure 3A) and RT-PCR (Figure 3B) in all C. spicatus-inoculated individuals.


Figure 2
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  		Figure 2. Images of Z. officinale (A–D) and C. spicatus (E–G) demonstrating phenotypes associated with viral infection and gene silencing.

(A) Whole Z. officinale plant with rhizome.

(B) Leaf from BSMV-infected Z. officinale plant showing characteristic yellow stripes.

(C) Leaf from a Z. officinale plant systemically infected with BSMV–ZoPDS. Photobleaching is evident in one sector of the single leaf located directly above the inoculated leaf and can be seen throughout the terminal leaves of plants 30 d post-innoculation.

(D) Terminal leaf of Z. officinale plant with complete photobleaching characteristic of PDS silencing.

(E) Whole C. spicatus stem produced from bulbil in the leaf axil of the parent flowering plant. The rhizome at the base of the shoot has rootlets that developed while this stem was still attached to the parent plant.

(F) Close-up of uninfected C. spicatus leaves.

(G) Close-up of C. spicatus leaf infected with BSMV, showing the infected phenotype of characteristic yellow stripes in a mosaic pattern.

 


Figure 3
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  		Figure 3. Detection of BSMV in gingers.

(A) Western blots showing antibody hybridization to BSMV CP. Detected CP in inoculated lanes indicates viral replication >3 cm from site of abrasion in three different Costus plants (a–c) and in Z. officinale (d). Negative control lanes use leaf material harvested from uninfected C. spicatus (left blot) or Z. officinale (right blot). Positive control lanes on both blots use leaf material from barley (Hordeum) known to be systemically infected with BSMV.

(B) RT–PCR showing presence of viral constructs in plants post inoculation with either wt BSMV or BSMV RNA with the PDS insert. Primers were designed to amplify BSMVβ RNA. Control lanes contain RT–PCR from plants not infected with viral transcripts. (a) C. spicatus, (b) Z. officinale, (c) H. vulgare.

 
Interestingly, new shoots of Z. officinale that developed from growing apices of rhizomes of plants previously infected with BSMV also developed symptoms of the viral infection. These shoots typically emerged 14–20 d post infection and do not appear to be delayed compared with uninfected plants. This observation supports past seed transmission and VIGS studies showing that BSMV is able to infect meristematic tissue of grasses (Jackson and Lane, 1981; Benedito et al., 2004). Our results also indicate that in Z. officinale, BSMV can move systemically from the inoculated leaves of a shoot into the rhizome system and infect new shoots arising from the rhizome. Because of the growth habit of Z. officinale, in which many genetically identical shoots can be generated from the same rhizome, only one shoot may need to be infected to obtain a large number of genotypically identical infected plants bearing terminal flowering shoots.

BSMV Can Elicit VIGS of ZoPDS in Ginger
To determine whether Z. officinale endogenous plant mRNAs can be silenced via a BSMV–VIGS approach, a fragment of the coding region of ZoPDS (GenBank accession number AF049356) was amplified by RT–PCR from Z. officinale mRNA. Once amplified, ZoPDS was sequenced and inserted at the 5’ terminus of the {gamma}b gene to create an infectious BSMV–VIGS vector unable to express the {gamma}b protein (Tai et al., 2005). The ZoPDS fragment is an excellent gene for VIGS assays because it encodes for an enzyme involved in the biosynthesis of carotenoids and, once silenced, PDS is unable to protect chlorophyll from photo-oxidation, resulting in photobleaching due to decreased carotene content (Kumagai et al., 1995; Benedito et al., 2004). Silencing of PDS in H. vulgare (Holzberg et al., 2002) and T. aestivum (Tai et al., 2005) has been shown to reduce levels of carotene content and to result in an obvious photobleached phenotype.

Endogenous gene silencing by BSMV–VIGS was accomplished by inoculating leaves of eight young Z. officinale shoots through leaf abrasion with a combination of BSMV RNA transcripts designated BSMV{gamma}–ZoPDS. This combination consisted of RNA{alpha}, a modified BSMV RNAβ derivative (B7) that is deficient in expression of the coat protein (CP) (Petty and Jackson, 1990), and BSMV RNA{gamma}–ZoPDS transcripts. The RNAβ and RNA{gamma} modifications were introduced previously to enhance VIGS expression in barley and wheat (Holzberg et al., 2002; Tai et al., 2005). The ‘B7’ RNAβ mutant was originally engineered to eliminate CP expression by mutagenesis of the AUG initiation codon of the CP ORF (Petty and Jackson, 1990), and was used by Holzberg et al. (2002) to enhance BSMV–VIGS. Expression of the {gamma}b silencing supressor protein was also disrupted by creation of a BamHI site to eliminate the {gamma}b AUG (Petty et al., 1990) and to provide a site for insertion of cloned DNA fragments (Bragg and Jackson, 2004).

Thirty days post inoculation with BSMV{gamma}–ZoPDS, a silenced PDS photobleached phenotype appeared in the systemic leaves of all eight inoculated plants. Photobleaching was easily visible as partially or fully bleached sectors following the parallel veination along the length of the leaf blades (Figure 2C and 2D). Infected Z. officinale shoots developed varying degrees of photobleaching in new leaves and experienced slowed growth of the infected shoots, with high levels of mortality following complete bleaching of terminal leaves. Of eight plants inoculated that had only a single vegetative shoot, all lost their vegetative shoot apparently due to the death of the shoot apical meristem. Rhizomes remained viable, but did not display any signs of VIGS. wtBSMV-infected plants continued to grow and produce leaves and new shoots with only slight mosaic yellowing. In contrast to the bleaching with BSMV{gamma}–ZoPDS, Z. officinale failed to develop a visible bleached phenotype after infection with BSMV{gamma}–TaPDS (RNA{alpha}, B7 RNAβ, and RNA{gamma}–TaPDS) transcripts, which harbored T. aestivum PDS sequences.

ZoPDS Transcripts Are Specifically Down-Regulated in Photobleached Ginger
RT–PCR analyses revealed a dramatic reduction in the levels of PDS mRNA in the photobleached regions of the systemic leaves of plants inoculated with BSMV{gamma}–ZoPDS (Figure 4). RT–PCR analyses indicated that BSMV{gamma}–TaPDS-inoculated leaves of plants had levels of endogenous PDS mRNAs comparable to those of plants infected with the wild-type BSMV ND18 strain or uninoculated plants (Figure 4). Further comparisons of T. aestivum and Z. officinale PDS sequences show a sequence identity of 77.3%, and varying degrees of identity are illustrated in other genera (Figure 5). A recent VIGS study in H. vulgare shows that cDNA sequences used in viral vectors must have a high percentage of sequence identity to endogenous mRNA for VIGS to be successful (Fu et al., 2007). Our results indicate that BSMV–VIGS is just as sensitive to sequence identity in ginger as in grasses or in non-monocot systems (Burch-Smith et al., 2004; Godge et al., 2008; Scofield and Nelson, 2009).


Figure 4
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  		Figure 4. Agarose Gel with RT–PCR Demonstrating Down-Regulation of PDS in Z. officinale Plants Infected with a BSMV Construct Containing a Z. officinale Endogenous PDS Gene Fragment.

Actin was used as a positive RT–PCR control for RNA extractions from Z. officinale plants infected with BSMV containing no construct (WT), a fragment of the PDS gene from wheat (Ta), or the endogenous Z. officinale PDS fragment (Zo). In all cases, actin is consistently transcribed. Significantly less PDS is transcribed from plants infected with the BSMV vector containing the endogenous PDS gene fragment, indicating successful down-regulation of PDS (ZoPDS+). The negative control reaction lacks reverse transcriptase.

 


Figure 5
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  		Figure 5. Sequence Comparison of the Coding Region of PDS Amplified from Z. officinale (ZoPDS) to T. aestivum, H. vulgare, O. sativa, Z. mays, C. sativus, L. longiflorum, and H. verticillata (GenBank accession numbers EU854153 [GenBank] , DQ270236 [GenBank] , AF049356 [GenBank] , AY062039 [GenBank] , L39266 [GenBank] , AY183118 [GenBank] , AY500378 [GenBank] , AY639658 [GenBank] ) utilizing the program GeneDoc version 2.06.02 (Nicholas et al., 1997).

Nucleotides shaded by black boxes with white lettering (100% conserved), gray shading with white lettering (80% or greater conserved), gray shading with black lettering (60% or greater conserved), no shading with black lettering (less than 60% conserved).

 

   DISCUSSION
TOP
 Abstract
 INTRODUCTION
 RESULTS
 DISCUSSION
METHODS
 FUNDING
 
The monocot order Zingiberales (tropical gingers) contains approximately 2500 species that form specialized pollination relationships via alterations in floral form. Members of the order comprise a major component of both tropical and subtropical ecosystems and include crop plants (e.g. banana, plantain, ginger), sources of traditional medicines and spices (e.g. cardamom, turmeric, galanga), and horticulturally important ornamentals (e.g. Heliconia, Bird-of-Paradise, Canna). Detailed studies of two families, Costaceae and Zingiberaceae, indicate that specialized relationships with animal pollinators have led to increased rates of diversification (i.e. rapid radiations) in bird-pollinated and bee-pollinated lineages (Specht, 2005, 2006). Species within these two families thus represent ideal evolutionary models for comparative morphology and developmental genetic studies.

Of the tropical gingers, Zingiber officinale is the most extensively described and widely cultivated for use of its aromatic rhizomes in cooking and home remedies. Extracts from Z. officinale have been shown to have pharmacological activities, and may be effective in inhibiting a variety of illnesses, including the promotion of tumors, inflammation, and emesis (Kawai et al., 1994; Katiyar et al., 1996; Penna et al., 2003). Zingiber officinale and related species are easily cultivated vegetatively from rhizome cuttings or vegetative bulbils. This characteristic obviates the need for seed production for successful reproduction and permits rapid generation of multiple genetically identical individuals. In addition, gingers are herbaceous perennials with fast growth rates and short times to maturity (6–7 months seed to seed), making them realistically amenable to gene silencing and subsequent phenotyping. In order to further understand and add to research of the Zingiberales, we demonstrate that virus-induced gene-silencing (VIGS) using a cereal virus, barley stripe mosaic virus (BSMV), can be used effectively to study gene function in Z. officinale.

BSMV Infection of Zingiberales
While H. vulgare (subfamily Pooideae: tribe Triticeae) is the natural host for BSMV, systemic viral infection with BSMV has been demonstrated for most subfamilies and many tribes of grasses (Jackson and Lane, 1981). BSMV–VIGS has been successfully demonstrated for barley and wheat (Pooideae: Triticeae), and is proving to be extremely valuable for analysis of genes affecting morphogenesis and disease resistance (Hein et al., 2005; Scofield and Nelson, 2009). Here, we demonstrate that systemic BSMV infections of distantly related monocots, such as gingers, occur after inoculation with both wild-type and engineered ND18 strain transcripts. Plants infected with wt BSMV developed a mild yellow mosaic on the normally bright green leaves, but did not show obvious reductions in overall plant growth or in the timing of transition from vegetative to reproductive phase. In the absence of intentional leaf abrasion, BSMV does not appear to be easily transmitted to surrounding plants in the greenhouse. This is an important practical feature for gene silencing in the Zingiberales, since many plants become too large for growth in chambers and require high levels of humidity that are difficult to maintain if plants are grown in isolation.

In each of the tested species, BSMV moved from the initial sites of infection to developing leaves above the site of inoculation. Additionally, in Zingiber, we observed the movement of the virus downward through the infected shoot and into the rhizome (underground stem), where it ultimately infected new shoots developing from the rhizome tip (see Figure 1). The recently reported stability of BSMV–VIGS (Bruun-Rasmussen et al., 2007) demonstrates the potential to control gene expression for a considerable period of time during plant development. If Z. officinale BSMV persists over several vegetative and flowering cycles, this presents the opportunity to create a large number of ginger shoots with down-regulated gene expression from inoculation of a single leaf. After a cluster is infected, we should be able grow individual shoots separately to test the effects of gene down-regulation under a variety of environmental conditions.

BSMV–VIGS Is Effective in Zingiber officinale
We have shown that BSMV–VIGS is an efficient method for inducing the down-regulation of the expression of specific target genes in Z. officinale. A modified BSMV{gamma} RNA containing a partial sequence of the Z. officinale PDS gene was able to effect the down-regulation of endogenous PDS and cause visible photobleaching of leaf and stem tissue. The extent of PDS silencing in Z. officinale after BSMV–VIGS inoculation is similar to that found in studies of VIGS in other monocots (Holzberg et al., 2002; Tai et al., 2005). However, Z. officinale shoots with photobleached leaves showed high rates of mortality, and rhizomes were not able to produce new shoots or develop inflorescences for over 50 d after inoculation. The vegetative shoots showing down-regulated PDS eventually died, and failed to regenerate photosynthetic tissue, suggesting complete gene silencing in the shoot apical meristem. It is therefore likely that gene silencing will be an effective means to elucidate the functions of genes involved in developmental and biochemical pathways. Future analyses with other marker genes that do not lead to photo-oxidation are planned to determine the duration of gene down-regulation, as well as to test the physical movement of gene silencing throughout the plant.

VIGS has become a widely used technique, most commonly applied to eudicot plants using TRV-derived vectors and Agrobacterium-mediated transfer into host cells. Despite reports of a host range for TRV that includes monocots (see TEC Release October 2005: www.pbltechnology.com), we were unable to infect Zingiberales using Agrobacterium-mediated infiltration of TRV in several attempts using various published delivery methods. The use of a native monocot virus to affect VIGS in phylogenetically distant monocot taxa presents an effective means of transferring VIGS technology to a wide range of crop species, model organisms, and non-model species within the monocots. The transfer of this technology provides a high-throughput means for assaying the function of a large number of genes recently identified and sequenced through EST databases and genome sequencing projects being developed for a number of diverse grasses (Z. mays, O. sativa, H. vulgare, Triticum spp., Sorghum spp., Panicum virgatum L., Brachypodium distachyon (L.) P. Beauv., Saccharum officinarum L.) and non-grass monocots (Musa acuminata Colla, Asparagus officinalis L., Phalaenopsis spp., Ananas comosus (L.) Merr., Allium cepa L.). The simple topical application method for introduction of the virus increases the ease with which BSMV can be used to assess gene function across monocots. We are currently testing the efficacy of the virus in infecting species of Allium (Alliaceae; Asparagales), Hippeastrum (Amaryllidaceae; Asparagales), Iris (Iridaceae; Asparagales), Acorus (Acoraceae; Acorales), and Chamaedorea (Arecaceae; Arecales).

In addition to studying host gene function, VIGS has the potential to provide a useful method for assaying host factors involved in viral pathogenicity (Zhu and Dinesh-Kumar, 2008). Biochemical assays have been used to identify various host translation initiation factors associated with viral replication proteins (Quadt et al., 1993). VIGS provides an additional means for testing the function of candidate host factors in viral pathogenicity, providing a high-throughput mechanism for screening potential new host factors and testing for the effects of candidate host factors on pathogenesis. The recent spread of the vector-borne banana virus, banana bunchy top virus (BBTV), has resulted in the spread of banana bunchy top disease and the subsequent failure of banana crops in Hawaii (Conant, 1992) and across the South Pacific and Southeast Asia (Dale, 1987). BSMV–VIGS in banana (Musa acuminata: Musaceae; Zingiberales) could provide a reverse genetics approach to help elucidate host mechanisms involved in viral pathogenicity.

BSMV–VIGS as a Tool for Studying Gene Function in the Zingiberales
BSMV–VIGS is likely to be effective in other members of the Zingiberales that are susceptible to BSMV infection. This should enable targeted studies for identifying gene function to be carried out in this ecologically and evolutionarily important group of tropical crops and ornamentals. The ginger family, Zingiberaceae, includes species such as turmeric (Curcuma longa L.), galanga (Kaempferia galanga L.), cardamom (Elettaria cardamomum Maton), and ginger root (Z. officinale), all of which have uses as spices and medicinals. Most rhizomes of Zingiberaceae species accumulate high levels of pharmacologically active metabolites derived from the phenylpropanoid pathway. Several of these, gingerols in Zingiber and curcuminoids in Curcuma, have been isolated and characterized, but little is known about their biosynthesis. Recent biochemical studies have started to identify enzymes involved in the biosynthetic pathways (Ramirez-Ahumada et al., 2006; Kita et al., 2008); however, nothing is known about the genetic network involved in biosynthesis. Our developed BSMV–VIGS tool could be used to functionally analyze ESTs believed to be associated with the biosynthesis of these important compounds.

Our interest in developing BSMV–VIGS in Zingiberales extends to floral developmental evolution. We are interested in dissecting the genetic networks leading to development of the diverse floral forms found across the order, particularly floral forms involved in the attraction of distinct pollinators. Unlike grasses, Zingiberales are ‘petaloid monocots’, having floral organs comprising sepals, petals, stamens, and carpels. The formation of the staminodes and the labellum may be a question of organ identity, with these structures functionally homologous to petals yet sharing positional homology with stamens. A group of transcription factors, many of which belong to the MADS-box family, are involved in floral organ identity in several model plant systems (Saedler et al., 2001; Theissen, 2001). VIGS has been successfully used to study MADS-box gene function in a variety of eudicots (Schwartz-Sommer et al., 1990; Liu et al., 2004; Hileman et al., 2005; Drea et al., 2007; Gould and Kramer, 2007) and future studies using VIGS may allow us to determine how these organ identity genes influence floral form throughout the Zingiberales (e.g. Gao et al., 2006).


   METHODS
TOP
 Abstract
 INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
FUNDING
 
Testing Infection with wtBSMV
Hordeum vulgare leaves infected for 5–6 d with the BSMV ND18 strain were ground in a mixture of 50 mL of 10 mM sodium phosphate buffer, pH 7.0 and 1% Celite Analytical Filter Aid (World Minerals). The extract was used to inoculate the leaves of young (~2 weeks after transplantation) vegetative shoots of Z. officinale (eight plants) and C. spicatus (four plants). Plants selected for inoculation had a maximum of two leaves, both of which were inoculated and subsequently grown in ambient light under shading conditions in a whitewash-coated greenhouse maintained at 85% humidity. wtBSMV infection of the emerging leaves was indicated by visual observation (Figure 2B) and confirmed by RT–PCR of the RNAβ subunit (Figure 3). At 14 d post-inoculation (DPI), total RNA was extracted from ~0.5 g of leaf tissue using Purelink Plant Reagent (Invitrogen) protocol described by the supplier and the RNA was used for cDNA synthesis. For this purpose, 1.5 µg of the total RNA extract was used to synthesize cDNA using the viral RNA-specific primer BSMV3’ 5'-TGG TCT TCC CTT GGG GGA CCG AAG CT-3'. PCR was performed using the forward primer TGB3 EcoRI 5'-GCG AAT TCC ATG GCA ATG CCT CAT CCC C-3’ and BSMV3’ as the reverse primer with iTaq polymerase (BioRad Laboratories) and 5% DMSO with the following thermocycling protocol: 95°C for 3 min, 35 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s and a single 7-min final extension cycle at 72°C.

PDS Amplification
Total RNA was isolated from uninfected Z. officinale leaves with the Purelink Plant Reagent (Invitrogen) and subsequently used for cDNA synthesis using the iScript cDNA Synthesis Kit (BioRad Laboratories). To amplify a 466-bp product of Z. officinale PDS from the leaf RNA, forward and reverse primers (PDS-F 5'-CTT ATG TTG ARG CYC AAG ATG G-3’ and PDS-R 5'-GTG TTC TTS AGT TTT CKR TCA AAC-3', respectively) were designed from a conserved region (Figure 4) in Hydrilla verticillata L. f. Royle, Lilium longiflorum Thunb., Crocus sativus L., Zea mays L., Oryza sativa L., H. vulgare, and T. aestivum (GenBank accession numbers AY639658 [GenBank] , AY500378 [GenBank] , AY183118 [GenBank] , L39266 [GenBank] , AF049356 [GenBank] , AY062039 [GenBank] , and DQ270236 [GenBank] , respectively). PCR was carried out utilizing the iProof polymerase kit (BioRad Laboratories) with 0.05 mg mL–1 BSA at 98°C for 4 min, 35 cycles at 98°C for 10 s, 62°C for 30 s, and 72°C for 30 s, and a single 7-min final extension cycle at 72°C.

Creating a ZoPDS–VIGS Construct
To apply BSMV–VIGS to Z. officinale, existing full-length cDNA plasmids derived from the ND18 strain (Petty et al., 1989) were used to generate RNAs for the infection mixture. These included the wt RNA{alpha} plasmid and a modified BSMVβ plasmid (B7), containing a mutation in the CP start codon (Petty and Jackson, 1990). The B7 plasmid RNA was included in the infection mixture because Holzberg et al. (2002) have indicated that disruption of CP synthesis enhances the persistence of VIGS. The infection mixture also contained transcripts from the BSMV{gamma}–ZoPDS plasmid, which is similar to the BSMV{gamma}–TaPDS described for wheat VIGS by Tai et al. (2005). Both plasmids were derived from BSMV RNA{gamma}–{gamma}bBamHI, which has an introduced BamHI site that alters the start codon of the {gamma}b ORF and blocks expression of the {gamma}b protein (Petty et al., 1990; Bragg and Jackson, 2004). PDS cDNA amplified from Z. officinale was then digested with BamHI and inserted non-directionally into the BamHI site of BSMV RNA{gamma}–{gamma}bBamHI to produce the BSMV{gamma}–ZoPDS plasmid. Orientation of ZoPDS in the BSMV{gamma}–ZoPDS transcript used for the infection mixture was determined to be in the forward direction via sequencing.

The BSMV plasmids were prepared separately for in vitro transcription reactions by linearization with Mlu I ({alpha} and {gamma} plasmids) or Spe I (β plasmid), and synthesized in vitro in reactions containing ~500 ng of plasmid DNA and bacteriophage T7 RNA polymerase (Petty et al., 1989). After synthesis, the RNAs were combined and extracted with phenol/chloroform, ethanol precipitated, and re-suspended in 50 µl of 50 mM glycine, 30 mM sodium phosphate monobasic, 1% bentonite (Sigma), and 1% Celite (Petty et al., 1989). The RNAs were mixed and applied to the plant by directly rubbing the mixture on the leaves of each plant. The plants were grown as described above before leaf symptoms were evaluated at various times after inoculation. The BSMV{gamma}–TaPDS construct containing the T. aestivum PDS gene was substituted for BSMV{gamma}–ZoPDS in some experiments to evaluate its effectiveness for VIGS in Z. officinale. The T. aestivum and Z. officinale PDS sequences have 77.3% sequence identity as determined by the program Geneious v3.7 (Drummond et al., 2007; available at www.geneious.com/). The nucleotide sequence for the ZoPDS gene was submitted to GenBank (accession number EU854153).

Quantifying PDS Down-Regulation
Total RNA was isolated from uninfected control Z. officinale leaves, and leaves infected with BSMV{gamma}–TaPDS and BSMV{gamma}–ZoPDS. Tissue (~0.5 g) was ground in the presence of the Purelink Plant Reagent (Invitrogen) and extracted using the recommended procedures, and the extracts were subjected to DNase treatment (RQ1 RNase-Free DNase, Promega). The DNase-treated RNAs were subsequently used for total cDNA synthesis (iScript cDNA Synthesis Kit, BioRad Laboratories) and RT–PCR. PDS RNAs were amplified using the same primers as those used to amplify ZoPDS from cDNA, and the resulting products were analyzed by agarose gel electrophoresis. Forward and reverse primers for Actin (5'-GAT GGA TCC TCC AAT CCA GAC ACT GTA-3’ and 5'-GTA TTG TGT TGG ACT CTG GTG ATG GTG T-3', respectively) were used as controls during cDNA amplification with iProof polymerase (Biorad Laboratories) and 50 mg ml–1 of BSA.


   FUNDING
TOP
 Abstract
 INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
 FUNDING
 
This research was partially funded by the UC Berkeley Committee on Research and the College of Natural Resources.


   Acknowledgements
 
We thank V.F. Irish, S.P. Dinesh-Kumar, E. Kramer, and A. Litt for early advice on VIGS in non-model plants and Doug Dahlbeck for his assistance and persistence in early attempts at infecting gingers with TRV. Tessa Burch-Smith provided additional insights for growing gingers once infected by the virus. The Specht Lab and especially Madelaine Bartlett, Chodon Sass, Ana Almeida, Kali Lader, Irene Liao, Julie Huston, and Candice Cherk contributed ideas, advice, and physical labor to assist in developing virus-infected gingers. No conflict of interest declared.

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