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This work was supported by the Bioenergy Science Center. The BioEnergy Science Center is a US Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science.
Switchgrass (Panicum virgatum L.) is a C4 perennial grass and has been identified as a potential bioenergy crop for cellulosic ethanol because of its rapid growth rate, nutrient use efficiency and widespread distribution throughout North America. The improvement of bioenergy feedstocks is needed to make cellulosic ethanol economically feasible, and genetic engineering of switchgrass is a promising approach towards this goal. A crucial component of creating transgenic switchgrass is having the capability of transforming the explants with DNA sequences of interest using vector constructs. However, there are limited options with the monocot plant vectors currently available. With this in mind, a versatile set of Gateway-compatible destination vectors (termed pANIC) was constructed to be used in monocot plants for transgenic crop improvement. The pANIC vectors can be used for transgene overexpression or RNAi-mediated gene suppression. The pANIC vector set includes vectors that can be utilized for particle bombardment or Agrobacterium-mediated transformation. All the vectors contain (i) a Gateway cassette for overexpression or silencing of the target sequence, (ii) a plant selection cassette and (iii) a visual reporter cassette. The pANIC vector set was functionally validated in switchgrass and rice and allows for high-throughput screening of sequences of interest in other monocot species as well.
The development of next-generation sequencing technologies has rapidly increased the amount of nucleotide-based information for many plant species (Varshney et al., 2009), but the generation and study of gain-of-function or loss-of-function mutants is still a requirement for validating gene functionality in higher plants. Various methods exist for elucidating the function of genetic elements in model plant species and applied crop species including virus-induced gene silencing (VIGS) (Ding et al., 2007; Scofield and Nelson, 2009), transient-induced gene silencing (Douchkov et al., 2005; Marzin et al., 2008), transient overexpression assays (Bargmann and Birnbaum, 2009; Saxena et al., 2011), and insertional mutagenesis and activation tagging systems (Pogorelko et al., 2008; Qu et al., 2008; Tadege et al., 2008). However, the stable expression of transgenes and the suppression of native genes in planta are the two most common methods for thoroughly understanding the biological role or function of the genes being studied. These methods typically require cloning a sequence of interest into a vector backbone and introducing the DNA into the plant using a variety of transformation methods.
Agrobacterium-mediated transformation and particle bombardment are the most widely used methods for transforming plants. Agrobacterium-mediated transformation was originally developed for dicotyledonous species, although transformation efficiencies have improved over time for monocotyledonous species using this method (for a review, see Shrawat and Lörz, 2006). Many studies have compared these two methods alongside each other, with Agrobacterium-mediated transformation resulting in transgenic plants with lower copy number, lower frequency of transgene silencing, and higher stable expression (Dai et al., 2001; Travella et al., 2005). However, Lowe et al. (2009) demonstrated that decreasing the amount of DNA used for particle bombardment can significantly increase the number of single-copy events in maize, although it can negatively affect the transformation frequency. Both methods remain relevant to monocot transformation, and each offers its own advantages and disadvantages.
Many commonly used vector backbones exist for plant transformation methods (for a review, see Komori et al., 2007). Current vector sets have enhanced or replaced older vector backbones (McCormac et al., 1997; Goderis et al., 2002; Thomson et al., 2002), and more advanced plant vectors include reporter gene or epitope tag fusions for tracking gene expression, visualizing protein subcellular localization and other genetic studies (Earley et al., 2006; Chakrabarty et al., 2007; Coutu et al., 2007; Nakagawa et al., 2007), Gateway-compatible sites for rapid gene cloning (Curtis and Grossniklaus, 2003; Miki and Shimamoto, 2004; Tzfira et al., 2005), and multisite Gateway compatibility for multi-sequence delivery capabilities (Karimi et al., 2007). Gateway technology takes advantage of the bacteriophage lambda site-specific recombination system, facilitating the exchange of DNA fragments from an ‘entry’ vector to a ‘destination’ vector in a rapid and efficient manner (Hartley et al., 2000). Available plant transformation vector sets constitute ‘destination’ vectors and contain the attR1 and attR2 recombination sites flanking (i) the chloramphenicol resistance gene (Cmr) and (ii) a negative selectable marker gene (ccdB). Overexpression vectors contain the attR1-Cmr-ccdB-attR2 cassette, and RNAi-mediated suppression vectors contain the attR1-Cmr-ccdB-attR2 cassette downstream of an inverted repeat of itself, resulting in a hairpin loop of the target sequence after recombination and transcription. In either case, these Gateway-compatible cassettes are located downstream of an appropriate promoter. The Gateway system allows for rapid recombinational cloning and ease of screening for positive clones.
Many of the available plant vector sets are tailored for functional analysis in dicot species and utilize only a limited number of constitutive promoters to control expression. For instance, the cauliflower mosaic virus (CaMV) 35S and 2x35S promoters have been commonly used in vectors to drive expression and/or suppression of genes of interest (Curtis and Grossniklaus, 2003; Earley et al., 2006; Nakagawa et al., 2007). However, the expression of the CaMV 35S promoter is typically lower in monocots than dicots (Himmelbach et al., 2007; Mazarei et al., 2008; Mann et al., 2011a), and native monocot promoters are more commonly used to transform monocot species (Miki and Shimamoto, 2004; Himmelbach et al., 2007). While the vector sets above are designed for a variety of applications, few of them are suited for high-throughput forward genetics studies in monocots. Himmelbach et al. (2007) constructed a Gateway-compatible vector set for monocots with a wide range of flexibility for replacing elements and cassettes, and the ZmUbi1 promoter driving the target gene cassette showed the highest levels of overexpression; however, the ZmUbi1 promoter also drives the selection cassette (hph), and repeated use of the same promoter for two different transgenes within a single construct can lead to transcriptional gene silencing (De Wilde et al., 2000; Butaye et al., 2005). Novel promoters with strong constitutive expression in monocots and efficient selection cassettes are needed to improve the efficiency of genetic engineering in monocots, as these tools can affect the expression level of the transgene and the transformation efficiency (Prakash et al., 2008). Recent monocot promoters have been isolated and characterized (Sivamani and Qu, 2006; Mann et al., 2011a), but not yet incorporated into and functionally validated in vectors for high-throughput reverse genetic studies in monocots. In this study, we describe the construction and functional validation of a Gateway-compatible vector set that facilitates the optimization of monocot transformation methods and subsequent applications for high-throughput production of stable transgenics in switchgrass and other monocot species.
Results and discussion
The pANIC vectors (the vectors are named after Panicum virgatum, the scientific name for switchgrass) are a versatile set of Gateway-compatible destination vectors for use in overexpression or RNAi-mediated suppression of sequences of interest in higher plant species, particularly monocot species. A preliminary description of the pANIC vector set has been published, but with no performance data (Mann et al., 2011b). All pANIC vectors contain three cassettes (Figure 1). These include (i) a Gateway-compatible cassette for overexpression or suppression of the target gene, (ii) a plant selectable marker cassette for conferring resistance (containing either the bar or hph gene) to the transformed plant, and (iii) a visual reporter gene cassette (GUSPlusTM or pporRFP) for optimization of the transformation process, visual tracking of transgenic events and rapid identification of transgenic plants. In addition, all combinations of vector components were also constructed with biolistic and binary T-DNA backbones for flexibility with different plant transformation methods.
The pANIC vector cassettes
To allow for high levels of transgene overexpression and sufficient levels of RNAi-mediated suppression of endogenous genes, the ZmUbi1 promoter was utilized to drive expression of the Gateway-compatible cassettes. The ZmUbi1 promoter has been used extensively for expression in a wide variety of monocot plant transformation systems and is a constitutively expressed promoter that regulates gene expression in most tissue types during most stages of plant development (i.e. ubiquitous expression), although some discrepancies have been reported (Cornejo et al., 1993). A previously described Gateway-compatible cassette for overexpression (Earley et al., 2006) was utilized, which contains the AcV5 epitope tag for protein fusions downstream of the Gateway-compatible attR2 recombination site and has been functionally validated in plants. The Gateway-compatible cassette for RNAi-mediated suppression has been previously described (Miki and Shimamoto, 2004) and is widely used for rice genetic studies. This cassette contains inverted repeats of the Gateway-compatible arrangement (attR1-CmR-ccdB-attR2) with an Escherichia coli-derived gusA linker sequence, creating a hairpin-loop structure that, upon transcription, can be recognized by the RNAi-pathway and employed for targeted gene suppression within the plant. For these RNAi Gateway-compatible systems, regions of the targeted gene (we have used 200–500 bp for switchgrass) can be generated and recombined from entry clones into the destination vector in a high-throughput manner.
Selectable marker cassettes
For transformation in monocots, hygromycin B and bialaphos are the two most commonly used selection agents, although a variety of alternatives have been implemented in previous studies (Li et al., 2003; LaFayette et al., 2005; Ochiai-Fukuda et al., 2006). The bialaphos acetyltransferase (bar) gene, conferring resistance to bialaphos, was used in initial switchgrass transformation efforts (Richards et al., 2001; Somleva et al., 2002). However, the hygromycin B phosphotransferase (hph) gene has been successfully implemented in more recent studies to confer hygromycin B resistance to the transformed tissue (Burris et al., 2009; Xi et al., 2009; Li and Qu, 2011). Therefore, pANIC vectors were designed and constructed with either the bar or hph plant selectable marker gene to give options to the researcher for the preferred application. Both genes were placed under the transcriptional control of the rice actin 1 gene (OsAct1) promoter (McElroy et al., 1990), although pANIC 10A and 12A contain the switchgrass ubiquitin 2 (PvUbi2) promoter in place of OsAct1 (described in more detail below) (Figure 1). In the binary T-DNA vectors, the selection cassette was inserted near the left border (LB) to decrease false-positive stable insertions and increase the insertion rate of the gene of interest cassette, because transfer of the T-DNA is initiated at the right border (RB).
Visual reporter cassettes
The use of chemical selection agents during transformation allows for positive selection of tissue stably transformed with the DNA of interest. In addition, reporter genes have been shown to significantly enhance the selection process, allowing for rapid identification of stable events, decreasing the number of non-transformed regenerated escapes and reducing the amount of downstream gene-silencing phenomena (Stewart, 2001; Nishizawa et al., 2006; Saika and Toki, 2009). Cotransformation systems can be utilized for introduction of a vector containing a reporter cassette simultaneously with a vector containing the gene of interest, although the actual rate of cotransformation for both constructs can vary. Vectors designed with the reporter gene cassette on the same backbone as the gene of interest cassette could allow earlier identification of stable events for the gene of interest and eliminate issues previously observed with cotransformation.
To accommodate a variety of reporter systems, the pANIC vectors were constructed with GUSPlus™, an enhanced derivative of uidA, or the pporRFP gene isolated from the coral Porites porites (Alieva et al., 2008), which produces pporRFP, a DsRed-type fluorescent protein. The pporRFP reporter gene was previously validated for switchgrass genetic transformation (Burris et al., 2009), and red fluorescent proteins (RFPs) are advantageous when compared with the green fluorescent protein (GFP) for fluorescence in plants as a result of the lower level of autofluorescence in the red wavelengths (Jach et al., 2001; Dietrich and Maiss, 2002; Nishizawa et al., 2006). The GUSPlus™ and pporRFP genes are expressed under the control of the switchgrass ubiquitin 1 gene (PvUbi1) promoter that has been isolated and characterized for strong constitutive expression in switchgrass and rice (Mann et al., 2011a).
Biolistic and binary backbones
Both biolistic and binary T-DNA vector backbones were constructed for the pANIC vector set. All pANIC vectors were originally cloned into the pCR4 backbone for biolistic bombardment (the pANIC 5A-E and 7A-E series), containing the ampicillin (bla) and kanamycin (nptII) resistance genes for bacterial selection. To facilitate Agrobacterium-mediated transformation, the pCR4 backbone was replaced with the pPZP201BK binary T-DNA backbone (Covert et al., 2001) to create an alternate set of binary vectors (the pANIC 6A-E and 8A-E series). The pPZP201BK backbone contains the nptII gene for bacterial selection, the ColE1 replication of origin for high copy number replication in E. coli, the pVS1 origin for low copy number replication in Agrobacterium sp., the pBR322 bom site for conjugational transfer, the rep and sta regions from pVS1 for vector stability in Agrobacterium in the absence of selection pressure, and the LB and right border (RB) for Agrobacterium-mediated plant transformation (Hajdukiewicz et al., 1994).
Functional evaluation of the pANIC vectors
To validate the functionality of the Gateway-compatible overexpression cassette, the gusA coding region (GUS) was recombined into the Gateway-compatible cassette of pCR4B-ZmUbi1-GW, the precursor to all overexpression pANIC vectors (see Materials and Methods) under the transcriptional regulation of the ZmUbi1 promoter and tested for expression via particle bombardment into callus cultures from the switchgrass genotype Alamo 2 along with the pAHC25 positive control vector (Christensen and Quail, 1996) and a negative control (gold particles alone) (Figure 2). The quantitative measurements of the fluorometric assay display similar levels of expression for the ZmUbi1 promoter in the pANIC vector backbone and the vector of origin (pAHC25). An additional vector was tested for expression levels of GUS under the control of the CaMV 35S promoter. GUS expression levels under the regulation of the CaMV 35S promoter were significantly lower than those observed under the regulation of the ZmUbi1 promoter similar to previous observations (Himmelbach et al., 2007; Mazarei et al., 2008).
To test the functionality of the Gateway-compatible RNAi cassette in the pANIC vectors, sequence fragments of approximately 400 bp from the switchgrass cinnamoyl CoA reductase1 (PvCCR1) and cinnamate-4-hydroxylase1 (PvC4H1) genes were cloned and recombined into pANIC 4B, a previously cloned precursor to the pANIC 8B vector that contained the rubi3 promoter in place of the PvUbi1 promoter for the reporter cassette (Sivamani and Qu, 2006; Mann et al., 2011a). Following switchgrass stable transformation as previously described (Xi et al., 2009), the regenerated transgenic plants were tested for gene expression levels using qRT-PCR (Figure 3). These results demonstrate that the lignin biosynthetic genes PvCCR1 and PvC4H1 were down-regulated significantly in the transgenic lines when compared with the wild-type controls, validating that the Gateway-compatible RNAi cassette of the pANIC vector set is effective for suppressing gene expression in switchgrass.
To test the functionality of the hph selection cassette under the control of the OsAct1 promoter, rice (Oryza sativa L. japonica) cv. Taipei 309 (TP309) embryogenic callus cultures were transformed with pANIC 7A using particle bombardment. After 3 weeks, stably transformed and hygromycin-resistant rice calli had grown rapidly as compared to the wild-type controls (Figure 4), and at least 33 independent lines of stably transformed rice calli were isolated from the four initial plates that were bombarded. These results demonstrate that the hygromycin selection cassette in the pANIC vector set is functional and that the OsAct1 promoter is a highly effective promoter for driving hygromycin B resistance in rice transformation. Functional hygromycin selection was also observed while using the pANIC 6A vector for Agrobacterium-mediated transformation of switchgrass, and actively growing transgenic calli could be observed on hygromycin selection (hygromycin B 40–60 mg/L) using the pporRFP gene as a visual marker (data not shown). Two additional pANIC vectors have been constructed for overexpression (pANIC 10A) and RNAi-mediated suppression (pANIC 12A) with the PvUbi2 promoter (Mann et al., 2011a) driving the hygromycin B selectable marker gene, as some data suggest that the promoter driving the expression of the selectable marker gene can significantly affect the transformation efficiency and recovery of transformants through the selection process (Prakash et al., 2008; Vogel and Hill, 2008). Both pANIC 10A and pANIC 12A vectors have been functionally validated for gene of interest overexpression/suppression, RFP-expression and hygromycin B selection using similar methods as mentioned above and are available as part of the vector set (data not shown, diagram of vectors displayed in Figure 1).
The reporter cassettes were validated in switchgrass and rice callus using biolistic bombardment assays. The pANIC 7B vector was bombarded into embryogenic rice ‘TP309’ and switchgrass genotype ST1 callus cultures followed by histochemical staining and visual observation for expression. GUSPlus™ expression was observed in rice and switchgrass calli (Figure 5), validating the functionality of the PvUbi1:GUSPlus™ reporter cassette in the pANIC vectors. To test the functionality of the PvUbi1:pporRFP reporter cassette, the pANIC 7A vector was bombarded into embryogenic rice TP309 and switchgrass ST1 callus cultures. After 10 days, callus cultures were viewed for detection of fluorescence under a microscope. pporRFP foci were observed in numerous callus pieces for rice and switchgrass (Figure 6). Additionally, real-time tracking of stable transgenic events during different growth stages throughout the tissue culture and regeneration process can be observed using pporRFP expression (Figure 7). This demonstrates the dual selection capabilities (i.e. antibiotic resistance and visual marker) of the pANIC vector set and the advantage this can offer for optimized stable transformation of monocot species.
The pANIC series is a versatile set of vectors that has been constructed for stable transformation using Agrobacterium-mediated or particle bombardment methods in monocot species and has been validated in rice and switchgrass. This vector set contains Gateway-compatible components for overexpression or RNAi-mediated suppression analyses, as well as multiple combinations of selection marker cassettes and visual reporter cassettes. The Gateway-compatible cassettes allow for convenient insertion of any open reading frame or other target sequence of interest through rapid recombinational cloning. These combined elements provide the researcher with the ability to select the features that are pertinent to their specific transformation system and allow high-throughput production of stable transgenics and functional genomics studies in a particular monocot species. The antibiotic selection (hph) in tandem with the fluorescence marker (pporRFP) works as a definitive tool for rapid screening and real-time tracking of positive transformation events. This dual selection capability of the pANIC vector set offers an advantage previously unavailable in monocot vector sets. The RFP visualization method also provides a reduction in the amount of time and labour invested in the culturing of tissue during the selection process and no damage or loss of putative transgenic callus tissue or plant organs during the screening process.
The functional validation of the pANIC vectors in rice and switchgrass demonstrates the versatility of these vectors and their components, and these data suggest that the pANIC vector set is a valuable tool for the elucidation of candidate DNA sequence functionality in a broad range of monocot transformation systems. This vector set is freely available for non-commercial research purposes.
The Gateway-compatible overexpression cassette (attR1-CmR-ccdB-attR2) coupled with an AcV5 epitope and the OCS terminator was PCR amplified from plasmid pEarleyGate 304 (Earley et al., 2006) and cloned into the pCR4B-TOPO backbone (Invitrogen, Carlsbad, CA, USA), creating pCR4B-GW. The ZmUbi1 promoter was amplified from the pAHC25 plasmid (Christensen and Quail, 1996) using PCR primers supplemented with PstI and EcoRI restriction sites. The PCR product was digested with PstI and EcoRI and pCR4B-GW was digested with NsiI and treated with Antarctic Phosphatase (New England Biolabs, Ipswich, MA). The ZmUbi1 fragment and linearized pCR4B-GW plasmid were ligated together using T4 DNA Ligase (Fisher Scientific, Pittsburgh, PA), creating pCR4B-ZmUbi1-GW.
For the Gateway-compatible RNAi hairpin-loop cassette, the plasmid pANDA (Miki and Shimamoto, 2004) was used as a template, although the entire Gateway-compatible hairpin-loop cassette could not be released using restriction endonucleases. Therefore, it was reconstituted as follows: The NOS terminator from pCAMBIA 1305.2 (http://www.cambia.org/daisy/cambia/585.html) was PCR amplified and cloned between the SacI and EcoRI sites of pMCS03 (a modified pSmart HC-Kan, Lucigen Corp., Middleton, WI) to create pMCS03A. The plasmid pANDA was digested with HindIII and SacI, and the GUS linker and sense-strand Gateway cassette (attR1-CmR-ccdB-attR2) region were ligated between the same sites in pMCS03A to create pMCS03B. The Zmubi1 promoter and antisense-strand Gateway cassette (attR1-CmR-ccdB-attR2) region from pANDA was released with HindIII and cloned into the HindIII site of pMCS03B to create pMCS03C. The resulting Gateway-compatible RNAi hairpin-loop cassette was subsequently released from pMCS03C by digestion with PmeI and AscI and cloned in place of the Gateway-compatible overexpression cassette in pCR4B-ZmUbi1-GW, creating pCR4B-ZmUbi1-GW-RNAi.
For resistance to hygromycin B or bialaphos, the hph and bar genes were PCR amplified from pMDC99 and pMDC123, respectively (Curtis and Grossniklaus, 2003), and cloned into the pCR4B-TOPO backbone, creating pCR4B-bar and pCR4B-hph. The OsAct1 promoter was excised from pCOR113 (McElroy et al., 1991) using Acc65I, filled in with DNA Polymerase I Large (Klenow) Fragment (Promega Corporation, Madison, WI), digested with HindIII and gel purified with the QIAquick Gel Extraction kit (Qiagen, Valencia, CA). pCR4B-bar and pCR4B-hph were digested with PmeI and HindIII and gel purified, followed by ligation with the OsAct1 promoter fragment. The resulting plasmids, pCR4B-OsAct1-bar and pCR4B-OsAct1-hph, were digested with PstI and gel purified. The plasmids pCR4B-ZmUbi1-GW and pCR4B-ZmUbi1-GW-RNAi were digested with SbfI, treated with Antarctic Phosphatase [New England Biolabs (NEB)] and ligated with the gel-extracted OsAct1-hph and OsAct1-bar cassettes, resulting in pCR4B-GW-hph, pCR4B-GW-bar, pCR4B-GW-RNAi-hph and pCR4B-GW-RNAi-bar, respectively.
For the reporter cassettes, a recently isolated switchgrass ubiquitin promoter (PvUbi1) (Mann et al., 2011a) was PCR amplified and cloned directly upstream of the pporRFP coding region (Alieva et al., 2008) or the GUSPlus™ coding region with the rice glycine-rich protein signal peptide sequence (GRP) from pCAMBIA 1305.2 (http://www.cambia.org/daisy/cambia/585.html). These reporter cassettes (PvUbi1-pporRFP and PvUbi1-GUSPlus) were digested with ScaI and EcoRI followed by blunt ending using T4 DNA polymerase (Fisher Scientific). The pCR4B-GW-hph, pCR4B-GW-bar, pCR4B-GW-RNAi-hph and pCR4B-GW-RNAi-bar backbones were digested with PmeI and treated with Antarctic Phosphatase (NEB). These fragments were blunt-end ligated to each other, creating pANIC 5A-E (overexpression) and pANIC 7A-E (RNAi-mediated suppression) for particle bombardment. For construction of pANIC 6A-E and pANIC 8A-E, the pCR4B-TOPO backbone was removed from pANIC 5A-E and pANIC 7A-E by digestion with SbfI and AscI. The binary backbone T-DNA plasmid pPZP201BK (Covert et al., 2001) was digested with PstI and AscI and ligated to the pANIC 5A-E and pANIC 7A-E inserts, resulting in pANIC 6A-E and pANIC 8A-E. Following cloning, all resulting plasmids were transformed into One Shot®ccdB Survival™ 2 T1 Phage-Resistant (T1R) chemically competent E. coli cells (Invitrogen) for propagation and plasmid maintenance. All amplified regions and plasmids were sequence verified at the University of Tennessee Molecular Biology Resource Facility.
Clonase reactions into destination vectors
Sequences of interest were cloned into pCR8/GW/TOPO per the manufacturer’s instructions (Invitrogen) and recombined into destination vectors using Gateway® LR Clonase® II enzyme mix (Invitrogen). The resulting expression vectors produced from the recombination reactions were transformed into E. coli strain DH5α using standard heat shock method.
Plant material and growth conditions
Switchgrass cv. Alamo genotypes Alamo 2 and ST1 were maintained and cultured as previously described (Alexandrova et al., 1996; Mann et al., 2011a). ST1 was provided by Zeng-Yu Wang from the Noble Foundation (Xi et al., 2009). Seeds of rice cv. Taipei 309 were provided by the USDA National Plant Germplasm System and maintained and cultured as previously described (Mann et al., 2011a).
DNA particle bombardment and stable transformation of switchgrass and rice callus
Transient expression assays were performed using biolistic transformation of rice and switchgrass embryogenic callus cultures as previously described (Mann et al., 2011a). For stable transformation of rice, DNA particle bombardment was performed using three-month-old rice callus cultures and 150 ng of plasmid DNA per bombardment. Rice callus cultures were incubated for 6 h pre- and 18 h post-bombardment on 0.6 MNB osmotic medium (Chen et al., 1998). Each vector was used to complete three replicate bombardments, and each replicate consisted of 50 bombarded calli. Rice callus cultures were selected on MNBH50 containing hygromycin B (Calbiochem®, Merck KGaA, Darmstadt, Germany) as described (Chen et al., 1998) to ensure independent events were recovered. For stable transformation of switchgrass, Agrobacterium-mediated transformation was performed using previously published protocols (Burris et al., 2009; Xi et al., 2009).
Isolation of PvCCR1 and PvC4H1 fragments for RNAi-mediated suppression
Sequence fragments of approximately 400 bp were selected for PvCCR1 (from Genbank accession number GQ450304.1) (Escamilla- Treviño et al., 2010) and PvC4H1 (Hui Shen and Richard Dixon, pers. commun). The primer pairs used for cloning the RNAi fragments are CCR1RNAi.F (5′-AACGGGCAGACCGTGTGC-3′), CCR1RNAi.R (5′-TGCTCCGCCACCGCCTTG-3′), C4H1RNAi.F (5′-CACCCAACGATCCAGTCC-3′) and C4H1RNAi.R (5′-GGTGTCGACCTTGTCCTG-3′). Sequence fragments were cloned into pCR8/GW/TOPO (Invitrogen) and sequence confirmed.
Quantitative reverse transcriptase-PCR
RNA isolation and reverse transcription were performed as previously described (Shen et al., 2011a). Briefly, 3 μg of switchgrass total RNA was isolated using the RNeasy Mini kit (Qiagen), treated with DNase (Applied Biosystems, Ambion, Austin, TX) and used for reverse transcription with a reverse transcript III kit (Invitrogen) according to the manufacturer’s protocol. The cDNA samples were diluted 20-fold, and 2 μL of the diluted cDNA samples was used as templates for qRT-PCR assays. qRT-PCR and data analysis were as previously described (Karlen et al., 2007). The qRT-PCR primers are CCR1qRT.F (5′-GCGTCGTGGCTCGTCAA-3′), CCR1qRT.R (5′-TCGGGTCATCTGGGTTCCT-3′), C4H1qRT.F (5′-GGGCAGTTCAGCAACCAGAT-3′) and C4H1qRT.R (5′-CGCGTTTCCGGGACTCTAG-3′).
Quantification of GUS activity
Fluorometric assays to quantify GUS activity were performed with 4-methylumbelliferyl-β-d-glucuronide (MUG) (Sigma-Aldrich, St. Louis, MO) (Jefferson et al., 1987). Proteins were extracted from calli using GUS extraction buffer consisting of 50 mm sodium phosphate pH 7.0, 10 mm Na2EDTA, 0.1% sodium lauryl sacosyl, 0.1% Triton X-100, and 10 mmβ-mercaptoethanol (Sigma). Frozen calli and ice-cold buffer were combined at a 1 : 1 ratio (w/v) and ground in a chilled mortar and pestle. The homogenate was centrifuged for 10 min at 12 000 g at 4 °C, and the supernatant was removed and centrifuged for an additional 5 min. Clarified extracts were stored at −80 °C prior to analysis. To measure GUS activity, 50 μL of extract was mixed with 450 μL of GUS extraction buffer with 1 mm MUG. An aliquot of 100 μL was removed immediately and added into 1.9 mL stop buffer (0.2 m sodium carbonate) as a control sample. The rest of the mixture was incubated at 37°C. At timed intervals, 100 μL was withdrawn and the enzymatic reaction terminated in stop buffer. GUS activity was determined using microtiter plates and a Synergy HT multi-detection microplate reader (Bio-Tek Instruments Inc., Winooski, VT). A standard curve was prepared with 4-methyllumbelliferone (MU) (Sigma). Protein concentration was determined by the dye-binding method (Bradford, 1972) using a Bradford assay reagent kit (Pierce, Rockford, IL) with bovine serum albumin (Sigma) as a standard. GUS activity is expressed as nmol of MU per min per mg of protein.
Red fluorescent plants and callus tissues were observed using an Olympus SZX12 stereo zoom fluorescent microscope. Filter sets were used for RFP (535/30 nm excitation, 600/50 nm emission) and GFP (475/30 nm excitation, 535/40 nm emission) (Chroma Technology Corporation, Rockingham, VT). Digital imaging was performed using an Olympus QColor 3 CCD camera integrated with QCapture 2.56 imaging software (QImaging Corp., Surrey, BC, Canada). While exposure times varied depending on the intensity of the fluorescent expression in each plant tissue, all fluorescent and bright field images were obtained using identical exposure times for the transgenic and control samples.
All the pANIC vectors described above have been donated to the Arabidopsis Biological Resource Center (ABRC) and are freely available for non-commercial research purposes through ordering online at http://abrc.osu.edu.
The authors gratefully acknowledge Dr Zeng-Yu Wang (Noble Foundation) for providing the switchgrass ST1 plants for tissue culture and transformation. We further thank Dr Mark Curtis for providing plasmids pMDC99 and pMDC123, Dr Keith Earley for providing plasmid pEarleyGate304, Dr Ko Shimamoto for providing plasmid pANDA, Dr Janice Zale and Jason Burris for providing plasmids pAHC25 and pCOR113, and Dr Mikhail Matz for providing the pporRFP gene.
DGJM participated in the design, construction and validation of the vectors and drafted the manuscript. PRL participated in the design and construction of the vectors and contributed to manuscript revision. LLA participated in the design of the vectors. ZRK, MM, HB, MCH, CRP and HS participated in experimental design and validation of the vectors. RAD, WAP and CNS were overall study directors and supervisors, participated in the study design, helped to revise the manuscript and obtained the funding. All authors read and approved the final version.