Methods for transient assay of gene function in floral tissues
- Yongjin Shang†1, 2, 3,
- Kathy E Schwinn†1,
- Michael J Bennett1,
- Donald A Hunter1,
- Toni L Waugh1, 2,
- Nilangani N Pathirana1, 3,
- David A Brummell1,
- Paula E Jameson3, 4 and
- Kevin M Davies1Email author
© Shang et al; licensee BioMed Central Ltd. 2007
Received: 18 September 2006
Accepted: 08 January 2007
Published: 08 January 2007
There is considerable interest in rapid assays or screening systems for assigning gene function. However, analysis of gene function in the flowers of some species is restricted due to the difficulty of producing stably transformed transgenic plants. As a result, experimental approaches based on transient gene expression assays are frequently used. Biolistics has long been used for transient over-expression of genes of interest, but has not been exploited for gene silencing studies. Agrobacterium-infiltration has also been used, but the focus primarily has been on the transient transformation of leaf tissue.
Two constructs, one expressing an inverted repeat of the Antirrhinum majus (Antirrhinum) chalcone synthase gene (CHS) and the other an inverted repeat of the Antirrhinum transcription factor gene Rosea1, were shown to effectively induce CHS and Rosea1 gene silencing, respectively, when introduced biolistically into petal tissue of Antirrhinum flowers developing in vitro. A high-throughput vector expressing the Antirrhinum CHS gene attached to an inverted repeat of the nos terminator was also shown to be effective. Silencing spread systemically to create large zones of petal tissue lacking pigmentation, with transmission of the silenced state spreading both laterally within the affected epidermal cell layer and into lower cell layers, including the epidermis of the other petal surface. Transient Agrobacterium-mediated transformation of petal tissue of tobacco and petunia flowers in situ or detached was also achieved, using expression of the reporter genes GUS and GFP to visualise transgene expression.
We demonstrate the feasibility of using biolistics-based transient RNAi, and transient transformation of petal tissue via Agrobacterium infiltration to study gene function in petals. We have also produced a vector for high throughput gene silencing studies, incorporating the option of using T-A cloning to insert the gene sequence of interest. These techniques should allow analysis of gene function in a much broader range of flower species.
The proliferation of DNA sequences from EST and genome studies has driven an increasing interest in rapid assay systems as alternatives to stable transgenics for establishing gene function. Transient over-expression of gene sequences using biolistics (particle bombardment) is now well established for functional assays. In particular, it has been extensively applied in studies on plant pigmentation, using flower petals or developing maize seeds. However, this technique is limited in the range of tissues and biological systems to which it can be applied. Most notably, unless an obvious change in phenotype occurs, it is difficult to obtain a sufficient quantity of transformed cells to enable molecular or biochemical analysis of the impact of the transgene.
More recently, the use of Agrobacterium tumefaciens infiltration (agroinfiltration) for transient assays has become established for processes such as assigning gene function [e.g. [1–5]], promoter element analysis  and inducible gene studies . The majority of results have been obtained using Nicotiana benthamiana, which is particularly suited to this method. However, the agroinfiltration transient assay system has recently been optimized for other species, including Lactuca sativa (lettuce), L. serriola (wild lettuce), Solanum lycopersicum (tomato) and some cultivars of Arabidopsis thaliana (Arabidopsis) . Vegetative tissues have typically been used for agroinfiltration, although tomato fruit  and hairy root cultures (using A. rhizogenes) have also been used .
The development of RNA-interference (RNAi) gene silencing methods, based on the triggering of sequence-specific RNA degradation in a similar manner to antisense  or sense suppression [11, 12] but with higher efficiency, has allowed the improvement of transient assay systems for loss of gene function [13–16]. Most of the RNAi systems have been with virus-induced gene silencing (VIGS), initially in N. benthamiana  and subsequently in other species as new viral vectors have been developed, for example for Arabidopsis , Hordeum vulgare (barley, ), Pisum sativum (pea, ), Glycine max (soyabean, ) and tomato . However, for each target plant species a suitable virus vector must be identified, and even when a suitable viral species is known, its use may be limited by local biosecurity regulations.
Agroinfiltration has also been used as a delivery system for transient RNAi [3, 9]. However, as with VIGS, agroinfiltration requires that the host is amenable to infection by the pathogen, and without the induction of tissue necrosis. Biolistic delivery offers an alternative delivery system that avoids the need for a pathogen and allows use of simple vectors lacking T-DNA or virus sequences. Other developments of agroinfiltration or RNAi technology include the suppression of gene silencing to allow higher levels of transgene expression , and the use of novel vector structures for higher throughput, such as the use of Gateway cloning [e.g. ] or vectors with an inverted repeat of the transcript termination sequence rather than the target gene sequence .
Antirrhinum majus (Antirrhinum) and petunia (most commonly Petunia hybrida and Petunia 'Mitchell') are classic model systems, with growing EST and genomics resources. Both species have been used in studies of floral organ development, floral scent production, self-incompatibility and the biosynthesis and regulation of production of anthocyanin pigments in flowers (see reviews of Schwarz-Sommer et al. ; Gerats and Vandenbussche ). For Antirrhinum, although Agrobacterium-mediated systems are available , production of stable transgenics remains a difficult process. Thus, we were interested in establishing additional transient assay systems for these model species.
We report here the establishment of agroinfiltration for petunia and N. tabacum (tobacco) floral tissues, and the use of biolistics for transient RNAi in Antirrhinum. These systems have been applied to flowers in situ and flower buds cultured in vitro. In addition, we have developed and tested a new high-throughput vector for RNAi assays.
Results and discussion
Assay of gene function in flowers using transient RNAi
To enable the use of a sealed chamber biolistic apparatus, detached flower buds of Antirrhinum were used. We had previously determined that Antirrhinum buds could successfully develop in vitro, with buds 5–10 mm in length developing relatively normal pigmentation and expanding to open flowers, although the youngest buds did not always reach the normal size and developed more slowly.
Although excised flower buds and a sealed chamber 'gene gun' were used in this study, it is assumed that the chamber-less guns would allow transient RNAi experiments with petals in situ.
An improved vector for assay of gene function using RNAi
Transient gene expression in petals using agroinfiltration
Agroinfiltration (using strain LBA4404) of Antirrhinum petals was unsuccessful (data not shown). Both lack of Agrobacterium infection and induced necrosis in target tissues have been noted as problems when developing agroinfiltration protocols, and the identification of Agrobacterium strains more compatible with the host plant has been successful for some species . There are no previous reports on the use of floral tissue for transient assays with agroinfiltration, so it is not known whether lack of success with transient infiltration can be correlated to the infectability of species with Agrobacterium when generating stably transformed plants. Antirrhinum is readily infected by A. tumefaciens or A. rhizogenes ; however, regeneration of plantlets from transformed tissues is difficult.
Biolistics-based transient RNAi in floral tissues was demonstrated for the classic model species Antirrhinum, and agroinfiltration methods for transient gene expression were successfully established for floral tissues of two other model species, tobacco (N. tabacum) and petunia. To our knowledge, this is the first report describing the application of these techniques to floral tissue. These methods should allow analysis of gene function in a broader range of flower species. Furthermore, a construct was developed for high-throughput RNAi silencing and successfully tested in petals of Antirrhinum.
The 'Mitchell' petunia line (sometimes referred to as W115) was obtained from the University of Auckland, New Zealand, and is Petunia axillaris × (P. axillaris × P. hybrida) . Antirrhinum line 603 and wild type lines H75A and JI522 were obtained as seed from Prof Cathie Martin and Rosemary Carpenter of the John Innes Centre, Norwich (UK). The tobacco line used was N. tabacum cv Samsun.
pDAH1 vector construction
The plasmid DAH1 (Figure 3) was derived from pGEM5Zf (In Vitro Technologies, Auckland, New Zealand). Linker1F and Linker1R (5'-NotI-PstI-MfeI-NotI-3') were inserted into the SphI/NsiI site of pGEM5Zf, destroying the SphI and NsiI sites, to produce pAF. A PstI-EcoRI fragment containing the CaMV 35S promoter from pART7 , Linker2F and Linker2R (BamHI/SacI) and the nos terminator were then ligated into the PstI/MfeI sites of pAF to produce pAO. A second nos terminator fragment (with 97 bp of the 3'-end of the GFP sequence) was PCR-amplified with the primers BglII-SGFP-NOS and XbaI-EcoRI-ASNOS, digested with BglII and XbaI and cloned in the antisense orientation into the XbaI/BamHI sites of pAO to make the nos hairpin vector pAP. A multiple cloning site was then created by inserting Linker3F (Figure 3A, top strand) and Linker3R (Figure 3A, lower strand) into pAP digested with XbaI and EcoRI (destroying the upstream XbaI site) to produce pDAH1. The oligonucleotide sequences used were:
Primer BglII-SGFP-NOS 5'-CGCAGATCTCCACATGGTCCTTCTTGA-3'
The binary vectors used were pBINm-gfp5-ER [38, 39] and p27IGUS, a vector based on pART27 but containing a GUS reporter gene with an intron (IGUS; ). IGUS was PCR-amplified from pMOG410 and cloned into pART7 which had been digested with KpnI and SmaI. The cassette containing 35S:IGUS:OCS was released by NotI digestion and ligated into NotI-digested pART27.
Hairpin vectors for dsRNA
cDNA encoding the ORF of CHS was PCR-amplified from a pool of first strand cDNA derived from floral RNA (isolated from Antirrhinum wild type line H75A) using FastStart Taq polymerase (Roche Applied Science) and gene-specific primers. pPN187 was constructed based on the vector pRNA69 , containing the CaMV 35S promoter, multiple cloning sites (separated by the Yabby5 intron) for inserting sense and antisense sequences for the gene of interest, and the ocs terminator. The CHS ORF was ligated in a sense orientation into the XhoI site and in an antisense orientation using ClaI/XbaI sites. pPN283 was constructed by ligating the PCR-amplified CHS ORF into pDAH1 using T-A cloning. pPN107 was made by ligating the ORF of Rosea1 into pRNA69 in a sense orientation using the XhoI/BclI sites and in an antisense orientation using the ClaI/XbaI sites.
In vitro culture of Antirrhinum floral buds
Whole buds (3–10 mm in length; minus sepals) were surface sterilized for 10 minutes using 10% (v/v) bleach containing 1–2 drops of Tween20/100 mL. Buds were then rinsed three times with sterile water and maintained on medium #2 (1/2 × MS macro salts/L; 1 × MS micro salt/L; 1 × MS iron/L; 1 × LS vitamins/L; 3% sucrose (w/v)/7.5% agar (w/v) during and after particle bombardment. The cultured buds were placed under artificial lights (16 h photoperiod) at 25°C after bombardment.
Particle bombardment of floral buds
Particle bombardment used a helium-driven particle inflow gun based on Vain et al. , but modified by the addition of a high speed, direct current solenoid valve for accurate valve opening times down to 8 ms. The bombardment conditions were a solenoid valve opening time of 30 ms, a pressure setting of 400 kPa, a shooting distance of 13 cm, and a partial vacuum of approximately -95 kPa. Preparation of the DNA/gold suspension was essentially as in Schwinn et al. , with the gold in 50 μL water prior to precipitation of plasmid DNA onto the gold particles. A final DNA concentration (for each construct of interest) of 2 μg DNA per mg of 1.0 μm gold particles was used. Each bombardment used 5 μL of DNA/gold suspension. Buds were bombarded six times and then cultured. Transformation was monitored by including an internal control vector, pRT99GFP, which was co-precipitated onto the gold particles with the construct of interest (at one-fifth the concentration). Also, pRT99GFP alone (at the same concentration) was used for control bombardment experiments.
Agrobacterium infiltration of floral tissue
Attached tobacco flowers were infiltrated with A. tumefaciens strain LBA4404 harbouring either p27IGUS or pBINm-gfp5-ER. The LBA4404 cells were cultured in 10 ml LB broth with antibiotic overnight, pelleted and re-suspended in medium #1003 (AB media salts + NaH2PO4 240 mg/L+ glucose 10 g/L + MES 14.693 g/L) supplemented with 100 μM acetosyringone, and cultured for 4 h. The cells were then pelleted and re-suspended to a concentration of A600 = 0.5 in 1% (w/v) glucose solution (pH 5.3) supplemented with 100 μM acetosyringon. Flower buds or opened flowers were pierced with a needle and infiltrated with the A. tumefaciens culture using a syringe. When using detached flowers, the flowers were cut into half across the middle of the tube, agroinfiltrated using a vacuum chamber, blotted with Whatman paper and cultured in petri-dishes containing moistened Whatman paper. The agroinfiltrated, detached flowers were cultured at 25°C under artificial lights (16 h photoperiod) for 2 to 2.5 days before examining reporter gene activity.
Reporter gene assays
1.5 to 3 days following agroinfiltration, flowers were histochemically assayed for GUS activity. Flower samples were incubated for 12 to 48 h at 37°C in X-gluc staining buffer (5-bromo-4-chloro-3-indoyl-β-D glucuronide dissolved in dimethyl formamide then diluted to 0.5 mg/L X-gluc in 50 mM phosphate buffer (pH 7.0) containing 1% (v/v) Triton X-100), and then placed in 70% (v/v) ethanol to remove the chlorophyll and preserve the sample. In some instances a brown colour developed due to tissue necrosis, and this was removed by treatment in a 5% acetic acid/ethanol (v/v) solution at 70°C for 30 min. To enable penetration of the GUS substrate into the petals they were either cut into pieces or wounds were made in the petals.
Light microscopy used an Olympus BH2 microscope and fluorescent microscopy used an Olympus SZX microscope. Images were recorded using a Leica DC 50 digital camera.
We thank Ian King for skilled care of the plants, Prof. Cathie Martin and Rosemary Carpenter for the Antirrhinum lines, Dr Jim Haseloff for pBIN mGFP5-ER, Drs Tony Lough and Kim Richardson for pRNA69, Dr Simon Coupe for pRT99GFP, Daniel Park for editorial comments and help in formatting the manuscript, and Tony Corbett for help in preparing the figures. This work was supported by the Marsden Fund Council from Government funding administered by the Royal Society of New Zealand (contract CRO101: YS, MJB, NNP, PEJ), the New Zealand Foundation for Research, Science and Technology (contract C02X0202 New Products & Technologies for the VHFN Industries: DAH, DAB; contract C02X0203 Knowledge and Economic Benefit from Sustainable PGT: KES, KMD, TLW), and a Top Achiever Doctoral Scholarship (NNP), a Bright Future Scheme administered by the Tertiary Education Commission.
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