JXB Advance Access originally published online on April 8, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Journal of Experimental Botany, Vol. 55, No. 399, pp. 983-992, May 1, 2004
© 2004 Oxford University Press
Cell and Molecular Biology, Biochemistry and Molecular Physiology |
RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula
Received 31 October 2003; Accepted 2 February 2004
Laboratory of Molecular Biology, Wageningen University, Dreijenlaan 3, 6703HA Wageningen, The Netherlands
* To whom correspondence should be addressed. Fax: +31 (0)317 483584. E-mail: rene.geurts{at}wur.nl
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
RNA interference (RNAi) is a powerful reverse genetic tool to study gene function. The data presented here show that Agrobacterium rhizogenes-mediated RNAi is a fast and effective tool to study genes involved in root biology. The Arabidopsis gene KOJAK, involved in root hair development, was efficiently knocked down. A. rhizogenes-mediated root transformation is a fast method to generate adventitious, genetically transformed roots. In order to select for co-transformed roots a binary vector was developed that enables selection based on DsRED1 expression, with the additional benefit that chimaeric roots can be discriminated. The identification of chimaeric roots provided the opportunity to examine the extent of systemic spread of the silencing signal in the composite plants of both Arabidopsis and Medicago truncatula. It is shown that RNA silencing does not spread systemically to non-co-transformed (lateral) roots and only inefficiently to the non-transgenic shoot. Furthermore, evidence is presented which shows that RNAi is cell autonomous in the root epidermis.
Key words: A. rhizogenes, Arabidopsis, Medicago truncatula, RNAi, roots, silencing, systemic spreading.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In plants, several reverse genetic techniques are being used to study the function of genes of interest, such as tagged mutants, targeted induced local lesions in genomes (TILLING), co-suppression, and antisense suppression. Recent studies have shown that the formation of double-stranded RNA (dsRNA) can lead to effective and sequence-specific degradation of homologous mRNA in a post-transcriptional fashion. Evidence for the involvement of dsRNA in mediating gene silencing was first discovered in Caenorhabditis elegans, and was termed RNA interference (RNAi) (Fire et al., 1998). Since then it has become clear that dsRNA can effectively suppress gene expression in a wide array of organisms including nematodes, insects, mammals, and plants (Tavernarakis et al., 2000; Elbashir et al., 2001). In retrospect, the phenomenon of RNAi was already known in plants as post-transcriptional gene silencing (PTGS), where the introduction of transgenes (co-suppression) or infection with manipulated viruses (Virus Induced Gene Silencing or VIGS) resulted in post-transcriptional silencing of homologous endogenous genes (Van der Krol et al., 1990; Ratcliff et al., 2001). RNAi is considered to be an ancient and ubiquitous antiviral system of eukaryotic organisms that evolved before the divergence of plant and animals (Sharp, 2001). The process of RNAi can be divided into a few steps. It is initiated by the production of double-stranded RNA, which is recognized and cleaved by a nuclease, named DICER, to produce 21–25 nucleotide small interfering RNAs (siRNAs). In turn, these siRNAs are incorporated into a second enzyme complex, called the RNA-induced silencing complex or RISC, which is responsible for the specific degradation of homologous mRNAs in the cytoplasm (Hammond et al., 2001; Hannon, 2002). Currently RNAi is an important tool in the analysis of gene function in both plants and animals. In C. elegans, in particular, RNAi has been developed to a ‘genomics-scale’ (Fraser et al., 2000; Gönczy et al., 2000; Maeda et al., 2001), and, in Arabidopsis, genome-wide RNAi efforts are underway.
In plants, several different approaches are being used to trigger RNAi in living cells. RNAi can be triggered by generating stable transgenic plants that express RNAs capable of forming a double-stranded hairpin (Waterhouse et al., 1998; Chuang and Meyerowitz, 2000; Wesley et al., 2001). In Nicotiana benthamiana RNAi has been applied using Agrobacterium tumefaciens-mediated transient expression (Johansen and Carrington, 2001) and, in cereals, biolistic delivery of dsRNA to leaf epidermal cells by particle bombardment resulted in interference with the function of endogenous genes at the single cell level (Schweizer et al., 2000). Viruses can also be manipulated to produce dsRNA intermediates of endogenous genes, which will be targeted for degradation after infection of the plant by the virus (Ratcliff et al., 2001).
Most of these studies have focused mainly on targeting genes in the aerial parts of the plant. Recently, it was shown that RNAi can also be used effectively to silence (trans) genes in primary transformed roots of the legumes Medicago truncatula (Medicago) (Limpens et al., 2003) and Lotus japonicus (Kumagai and Kouchi, 2003) by using Agrobacterium rhizogenes-mediated transformation. A. rhizogenes generates adventitious, genetically transformed roots at the site of inoculation in many dicots and can be manipulated to co-transfer the T-DNA of a binary vector that contains the transgene of interest (Chilton et al., 1982). Upon expression of the root locus (rol) genes carried on the Ri T-DNA, roots are formed of which a certain number will be co-transformed with the T-DNA of the binary vector (Nilsson and Olsson, 1997). A. rhizogenes-mediated root transformation has been described for a number of legumes. The transformed roots are morphologically indistinguishable from untransformed roots and, in the case of legumes, they can be nodulated by Rhizobium bacteria and infected by mycorrhizal fungi. A. rhizogenes-mediated transformation offers a fast alternative to generate genetically transformed roots, especially in species where generating stable transgenic lines is very time-consuming. Furthermore, this method has the advantage that root cultures can be clonally propagated without the requirement of additional plant hormones.
It is shown here that A. rhizogenes-mediated RNAi is a fast and effective tool to study the function of genes involved in root biology, not only in legumes but also in Arabidopsis. An Arabidopsis gene, KOJAK (CLSD3) (Favery et al., 2001; Wang et al., 2001), involved in root hair development was efficiently targeted by A. rhizogenes-mediated RNAi. As selection marker for co-transformed roots the gene coding for the fluorescent protein DsRED1, was used as a non-destructive selectable marker. This marker offers the additional advantage to discriminate chimaeric and homogeneously transformed roots and allowed a close examination of the extent of systemic spreading of the silencing signal. RNA silencing did not spread to non-co-transformed (lateral) roots and only with limited efficiency to the non-transgenic shoot of composite plants. Furthermore, evidence is presented which shows that RNA silencing is induced cell autonomously in the root epidermis.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plasmids and vectors
To create pRedRoot the nptII gene and NOS terminator of pBINPLUS (Van Engelen et al., 1995) were removed by a Bsu36I and Bst98I digestion, Klenow treatment and re-ligation. The created plasmid was named pBASIS. The Arabidopsis UBQ10 promoter was PCR amplified using genomic DNA of the accession Columbia as template (primers used: 5'-AAGCTTTGTCCCGACGGTGTTGT-3' and 5'-CCATGGCAAAGATCTGCATCTGTTA-3') and subsequently cloned in frame with the ATG startcodon of DsRED1 (Clontech) in a pGEM-T (Promega) derived vector that contained a NOS terminator. A 2.5 kb HindIII–AscI fragment containing UBQ10::DsRED1-NOS terminator was cloned into pBASIS resulting in pRedRoot.
In order to create inverted repeat constructs, a 1.3 kb fragment of vector pFGC1008 (described at http://ag.arizona.edu/chromatin/fgc1008.html) containing SacI 2927 bp–HindIII 4150 bp (the RNAi box) was PCR amplified and cloned into pBluescriptIISK+ (Stratagene) (primers used: 5'-CACTGACGTAAGGGATGA-3' and 5'-ATGAGCTCTTAATTAAGGATGTGCTGCAAGGCGA-3'). A double CaMV 35S promoter was PCR amplified from vector pMON999 (primers used: 5'-TGATCTCGAGCAAGCTTCTGCAGGTCCAT-3' and 5'-CATGCCATGGAGATCTGCTAGAGTCAGC-3') and cloned XhoI–NcoI in front of this RNAi box to result in the pRNAi vector. Inverted repeats were created in pRNAi by two sequential cloning steps. Target regions are first PCR amplified, and subsequently cloned AscI–SwaI and BamHI–SpeI into the created pRNAi derivative. The resulting inverted repeat construct was inserted KpnI–PacI into the pRedRoot binary vector. Target regions were amplified using the following primer combinations: GFP5 (584 bp) 5'-ATACTAGTGGCGCGCCTTGTTGAATTTAGATGGTGATG-3', 5'-ATGGATCCATTTAAATTTCGAAAGGGCAGATTG-3'; KJK (461 bp) 5'-ATACTAGTGGCGCGCCGTG TGCCAGAAGAAAACC-3', 5'-ATGGATCCATTTAAATGCCAGTAGCCCATCGGAGAAC-3'.
The SpeI–AscI and BamHI–SwaI restriction sites that are included within the primers have been underlined.
Bacterial strains
Agrobacterium strain MSU440 containing the pRi plasmid pRiA4 (Sonti et al., 1995) was used to transform Medicago and Arabidopsis. The binary vectors were introduced into MSU440 by electrotransformation and grown for 2 d at 28 °C under kanamycin selection (50 µg ml–1). Integrity of inverted repeat constructs was checked by mini-prepping and restriction-digestion with HindIII.
Plant material
For Medicago the accession Jemalong A17 and the transgenic line R108-RH2::GFP were used (Hoffmann et al., 1997; Penmetsa and Cook, 1997; Ramos and Bisseling, 2003). R108-RH2::GFP carries the 1.1 kbp promoter region of the pea RH2/DRRG gene (GenBank J03680
[GenBank]
, Chiang and Hadwiger, 1990) in front of GFP5 (Ramos and Bisseling, 2003). For Arabidopsis the accessions Landsberg erecta, Columbia, and Wassilewskija were used. The Gal4 enhancer trap lines J0661 and J0781 are described at http://www.plantsci.cam.ac.uk/Haseloff/IndexCatalogue.html.
Agrobacterium rhizogenes-mediated transformation
Medicago seeds were surface-sterilized by incubating for 10 min in concentrated sulphuric acid, washing six times in sterile water, then 10 min in 4% hypochlorite (commercial bleach), washing seven times in sterile water, and subsequently plated on Färhaeus medium (1 mM MgSO4.7H2O, 0.75 mM KH2PO4, 1 mM Na2HPO4, 15 µM Fe-citrate, 0.75 mM Ca(NO3)2, 0.7 mM CaCl2, 0.35 µM CuSO4.5H2O., 4.69 µM MnSO4.7H2O, 8.46 µM ZnSO4.7H2O, 51.3 µM H3BO3, 4.11 µM Na2MoO4.2H2O, 0.9% Daichin agar (Brunschwig)) containing filter paper. Seeds were vernalized for 1 d at 4 °C and germinated at 25 °C for 24 h in darkness (plates upside down). One-day-old seedlings were transferred to new 9 cm Petri dishes containing Färhaeus medium and a half-round filter paper (5 seedlings per plate), and grown at 21 °C (16/8 h light/darkness) after removal of the seed coat. The Petri dishes were not completely closed by parafilm to enable aeration. The roots of 5-d-old seedlings were removed at the hypocotyl and the wound surface was inoculated with Agrobacterium MSU440 containing the appropriate binary plasmid. The seedlings are co-cultivated with Agrobacterium for 5 d at 21 °C (16/8 h light/darkness) and subsequently transferred to Emergence medium (3 mM MES pH 5.8 containing 2.5 g l–1 KNO3, 0.4 g l–1 MgSO4.7H2O, 0.3 g l–1 NH4H2PO4, 0.2 g l–1 CaCl2.2H2O, 10 mg l–1 MnSO4.4H2O, 5 mg l–1 H3BO3, 1 mg l–1 ZnSO4.7H2O, 1 mg l–1 KI, 0.2 mg l–1 CuSO4.5H2O, 0.1 mg l–1 NaMoO4.2H2O, 0.1 mg l–1 CoCl2.6H2O, 15 mg l–1 FeSO4.7H2O, 20 mg l–1 Na2EDTA, 100 mg l–1 myoinositol, 5 mg l–1 nicotinic acid, 10 mg l–1 pyridoxine HCl, 10 mg l–1 thiamine HCl, 2 mg l–1 glycine, 1% sucrose, 0.9% Daichin agar containing 300 µg ml–1 Cefotaxime (Duchefa)), and covered by a (half-) filter paper. Plants were grown for 6–18 d on Emergence medium. In this period new roots are formed that are potentially co-transformed with the T-DNA of the binary vector.
Transformation of Arabidopsis is done in a similar way with the following differences: Seeds were surface-sterilized by incubating for 5 min in 2% hypochlorite (commercial bleach), washing five times in sterile water, and then vernalized at 4 °C for 3 d. Two-day-old seedlings were used for co-cultivation with the appropriate Agrobacterium MSU440 strain (20 plants per plate). Plants are grown on plates with a filter paper, containing 0.5x Murashige and Skoog (MS) salts (Duchefa), 1% sucrose, and 0.8% (w/v) Daichin agar, for 3 d (21 °C; 16/8 h light/darkness) and subsequently transferred to 0.5x MS plates containing 300 µg ml–1 Cefotaxime (Duchefa).
Nodulation of A. rhizogenes-transformed roots
Three weeks after transformation, composite Medicago plants are starved for nitrate for 3 d (21 °C; 16/8 h light/darkness) on Färhaeus medium (without Ca(NO3)2). Then plants are transferred to agra-perlite (Maasmond-Westland, The Netherlands) saturated with Färhaeus medium (without Ca(NO3)2) and inoculated with 1 ml culture of S. meliloti 2011.pHC60 (OD600 0.1) per plant and grown for 2 weeks (21 °C; 16/8 h light/darkness).
Clonally propagating A. rhizogenes-transformed roots
A. rhizogenes-transformed Arabidopsis roots were excised (
1 cm above the tip) and transferred to 25 ml ARC medium (Czakó et al., 1993) containing 0.05 mg l–1 IAA. After 3 d, the root pieces were transferred to new 25 ml ARC medium without IAA and cultured in the dark at 25 °C for 4 weeks with gentle shaking (100 revolutions min–1).
Microscopy
Imaging of DsRED1 or GFP was done using the Leica MZIII fluorescence stereomicroscope with the appropriate filter settings. Images were processed electronically using Adobe Photoshop 5.5. Spectral imaging of DsRED1 was done according to Gadella et al. (1997). The slit width of the imaging spectrograph was 200 µm and the central wavelength was 550 nm. A 525 nm longpass emission filter was used. Imaging of GFP fluorescence in Medicago line R108-RH2::GFP and in the Arabidopsis J0781 cross-section was performed on a Zeiss LSM 510 confocal laser scanning microsope (Carl-Zeiss); excitation 488 (GFP), 543 nm (propidium iodide/DsRED1); GFP emission was selectively detected by using a 505–530 nm bandpass filter, propidium iodide/DsRED1 emission was detected in another channel using a 560–615 nm bandpass. The root was counter-stained with 0.2 µg ml–1 propidium iodide.
qPCR
Total RNA was extracted from 4-week-old Arabidopsis root cultures according to Pawlowski et al. (1994) followed by DNAseI (Promega) treatment. cDNA was made from 1 µg total RNA using the Taqman Gold RT-PCR kit (Perkin-Elmer Applied Biosystems) in a total volume of 50 µl using the supplied hexamer primers. qPCR reactions were performed in triplicate on 6.5 µl cDNA using the SYBR-GreenR PCR Master kit (Perkin-Elmer Applied Biosystems) (40 cycles of 95 °C for 10 s, 60 °C for 1 min) and real-time detection was performed on the ABI 7700 and analysed using the GeneAmp 5700 SDS software (Perkin-Elmer Applied Biosystems). Optimal amplification efficiencies for the different primer sets were determined by performing qPCR with a concentration range of primers, cDNA, and control plasmids. The specificity of the PCR amplification procedures was checked with a heat dissociation step (from 65–95 °C) at the end of the run and by agarose gel electrophoresis. Results were standardized to the ACTIN2/8 expression levels. Primer combinations used in the qPCR were chosen outside of the regions targeted by the RNAi constructs. The primers used were: AtAct2/8-F: 5'-GGTAACATTGTGCTCAGTGGTGG-3'; AtAct2/8-R: 5'-AACGACCTTAATCTTCATGCTGC-3'; AtKJK-F: 5'-TGAGAGCTCTTGATGGGTTGATGG-3'; AtKJK-R: 5'-TCGGTTTTCTTCTGGCACACGG-3'; GFP4-F: 5'-CTGTCCTTTTACCAGACAACCATTACC-3'; GFP4-R: 5'-CCAGCAGCTGTTAC AAACTCAAGAAG-3'.
Immunoblotting
Proteins were isolated from 4-week-old Arabidopsis J0781 root cultures by grinding 1 g of root tissue in liquid nitrogen and then resuspension in extraction buffer (50 mM TRIS-acetate, pH 7.4, 10 mM potassium-acetate, 1 mM EDTA, 5 mM DTT, 0.5 mM PMSF) followed by two subsequent centrifugation steps: 1000 rpm 15 min and 15 000 rpm 30 min. The supernatant was used for analysis. Protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad). 20 µg protein was separated on a 12.5% SDS-PAGE gel. After electrophoresis, proteins were blotted onto nitrocellulose paper (Schleicher and Schull) and immunostained with 1:1000 diluted rabbit anti-GFP (Molecular Probes) followed by 1:5000 diluted anti-rabbit-HRP and detected using the ECL PlusTM Western Blotting Detection Kit for HRP (Amersham Biosciences) on a Storm 840 (Molecular Dynamics). The blot was stained for total protein using Ponceaux S (Sigma Diagnostics).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
A. rhizogenes-mediated root transformation in Arabidopsis and Medicago using the pRedRoot binary vector
A. rhizogenes-mediated root transformation results in the formation of adventitious roots that are co-transformed with the gene of interest as well as roots lacking this gene. To interpret RNAi experiments it is essential to identify those roots that contain the transgene of interest. Recently it was shown for Medicago that selection on co-transformation can be done using kanamycin resistance (Boisson-Dernier et al., 2001). However, in our laboratory, this selection does not effectively discriminate chimaeric roots (data not shown). Therefore the binary vector pRedRoot was developed (Fig. 1A). The pRedRoot vector provides the possiblity to select transgenic roots based on fluorescence since it contains the gene encoding for the red fluorescent protein, DsRED1 (Matz et al., 1999), under the control of the constitutively expressed UBQ10 promoter of Arabidopsis (Norris et al., 1993). In addition to non-destructive identification of co-transformed roots this vector also allows the detection of chimaeric roots.
|
To test the pRedRoot vector Medicago accessions A17 and R108 as well as Arabidopsis accessions Landsberg erecta, Columbia, and Wassilewskija, were transformed by inoculating freshly cut hypocotyls with the A. rhizogenes strain MSU440 (harbouring pRiA4) containing pRedRoot. The first red fluorescent Medicago roots could be observed approximately 3 weeks after inoculation, whereas in Arabidopsis red fluorescent roots were already formed within 8–10 d. In Fig. 2A a time-lapse experiment is shown, following the accumulation of DsRED1 in Arabidopsis at 1 d intervals. Newly formed roots are visible 4 d after inoculation with A. rhizogenes. However, these roots do not originate from cells expressing DsRED1 and so are not co-transformed. The A. rhizogenes-transformed cells form a callus-like structure from which new adventitious roots are induced. To determine whether the observed red fluorescence was the result of DsRED1 expression, a spectral image of a red fluorescent root versus a control root of Medicago was made using fluorescence spectral imaging microscopy (FSPIM). This showed that the spectrum of DsRED1, with an emission peak at 583 nm, could be clearly distinguished from auto-fluorescence present in Medicago roots (Fig. 3).
|
|
The A. rhizogenes-transformed roots of both Medicago and Arabidopsis have a similar morphology as normal roots. In the case of Medicago, they can be nodulated by the symbiotic partner Sinorhizobium meliloti (Fig. 2B, C). Co-transformation efficiencies varied between experiments, but on average
30% of the newly formed roots in Medicago were co-transformed (ranging from 1–3 co-transformed roots per inoculated seedling), whereas in Arabidopsis efficiencies up to 20% were reached. A. rhizogenes-mediated transformation results in the generation of homogeneously co-transformed roots as well as chimaeric roots. In general, chimaeric roots were observed in about 50% of the cases in Medicago, and only infrequently in Arabidopsis (10%). These results show that pRedRoot is a useful binary vector for A. rhizogenes-mediated transformation to identify transgenic roots as well as co-transformed segments in chimaeric roots.
Silencing of trans GFP in Arabidopsis and Medicago
To test the effectiveness of RNAi in A. rhizogenes-transformed Arabidopsis roots, a GFP transgene was targeted in order to visualize the silencing effects. Therefore, a construct was made containing 584 bp of the coding sequence of GFP5, cloned in both the sense and anti-sense direction separated by a 335 bp spacer, under the control of the 35S promoter in pRNAi (Fig. 1B) and subsequently transferred to pRedRoot resulting in the construct pRR-GFPi. RNA transcribed from this construct produces a hairpin structure, resulting in double-stranded RNA. The pRedRoot vector enabled the identification of co-transformed roots by red fluorescence of DsRED1, while the efficiency of silencing could be determined by quantification of GFP fluorescence.
In Arabidopsis the Gal4 enhancer trap line J0781, expressing GFP (http://www.plantsci.cam.ac.uk/Haseloff/IndexCatalogue.html), was used. J0781 shows strong GFP expression in the root cortex and stele and in the hypocotyl (Fig. 2D, E). Transgenic roots were analysed 12 d after infection with A. rhizogenes carrying pRR-GFPi. In 91% (n=98) of the red fluorescent roots no GFP fluorescence could be detected after co-transformation with pRR-GFPi (Fig. 2E, F), whereas roots (n=63) transformed with the pRedRoot control vector all showed bright GFP fluorescence (Table 1). To verify knock-down of GFP in the A. rhizogenes-transformed roots, GFP mRNA levels were determined by real-time RT-PCR (qPCR) (Fig. 4A). Since Arabidopsis roots are relatively small, independently transformed roots were clonally propagated in order to isolate sufficient RNA material for the qPCR. The results of the qPCR show that GFP mRNA is substantially reduced, at least 10 times, in the pRR-GFPi transformed roots compared with control roots. Since there was still residual GFP mRNA present in the GFP-silenced roots it was determined whether GFP protein could still be detected in these roots. Immunoblotting showed that no GFP protein could be detected in the GFP-silenced roots, whereas in the control roots a high level of GFP protein was detected (Fig. 4B). This correlates well with the absence of detectable GFP fluorescence in these roots.
|
|
Similar results were obtained in Medicago, where a stable transformed line R108-RH2::GFP was used, which contains the 1.1 kb promoter region of the pea RH2 gene in front of GFP (Ramos and Bisseling, 2003). The RH2 promoter is active in root epidermal cells in the zone of the root starting immediately above the root apical meristem and extending into the region containing mature root hairs (Mylona et al., 1994). In line R108-RH2::GFP GFP fluorescence can be detected in the epidermis of young developing roots (Ramos and Bisseling, 2003), which was preserved in A. rhizogenes-transformed roots (Fig. 2K, L). Four weeks after transformation with pRR-GFPi co-transformed roots were analysed. In 86% (n=37) of homogeneously transformed roots no GFP fluorescence could be detected (Table 1). In control plants, transformed with the pRedRoot vector, all transgenic roots (n=12) showed clear GFP fluorescence.
Silencing endogenous genes in Arabidopsis roots
The potential of A. rhizogenes-mediated RNAi with respect to silencing of endogenous genes in Arabidopsis was also investigated. For this purpose the gene KOJAK (KJK/CSLD3) was selected (Favery et al., 2001; Wang et al., 2001). KJK encodes a cellulose synthase-like protein, which is preferentially expressed in trichoblasts (Favery et al., 2001). Root hair formation is initiated in kjk mutants at the correct position but, in general, stops at the bulge stage, resulting in hairless roots. A silencing construct for KJK was made in the pRedRoot vector and pRR-KJKi was transformed by A. rhizogenes-mediated transformation to both Arabidopsis accessions Landsberg erecta and Wassilewskija. The results are summarized in Table 1. Ninety-one per cent (n=58) of the homogeneously transformed roots, as judged by red fluorescence, showed a root hair phenotype. In most roots (62%) root hairs were initiated but failed to elongate, resulting in hairless roots containing small bulges (Fig. 2G). However, in some roots (29%), root hair cells developed more than a bulge and showed some elongation resulting in small root hairs (Fig. 2H). Roots transformed with the pRedRoot control vector all contained normal root hairs. To verify knock down of KJK in the A. rhizogenes-transformed roots, the amount of KJK mRNA was quantified after clonally propagating the co-transformed roots. qPCR showed that KJK mRNA was substantially reduced, by at least eight times, in the pRR-KJKi transformed roots compared with control roots (Fig. 4C). These results show that A. rhizogenes-mediated RNAi is an effective and very fast method to silence endogenous genes in the roots of Arabidopsis. Furthermore, a spectrum of phenotypes can be obtained from RNAi.
Systemic spreading of the silencing signal in roots of Arabidopsis and Medicago
In several plant species it was shown that the induction of RNAi results in systemic spread of the silencing signal (Hamilton et al., 2002). A. rhizogenes-mediated root transformation makes it possible to determine whether the interference signal is systemically transported to non-transformed roots or the non-transgenic shoot of the composite plant. To examine the extent of systemic spread of the silencing signal in A. rhizogenes-transformed roots, the stable GFP expressing Medicago (R108-RH2::GFP) and Arabidopsis (J0781) lines were used. As mentioned above, transformation of these lines with pRR-GFPi efficiently knocked down GFP expression. However, no reduction in GFP fluorescence was detected in non-co-transformed roots on composite plants that also contained silenced roots (Fig. 2E, F).
The use of DsRED1 as the selectable marker enables the identification of chimaeric roots. This provided the possibility to determine the extent of systemic spread of the silencing signal from co-transformed root tissue to non-co-transformed root tissue. Chimaeric roots on Arabidopsis J0781 co-transformed with pRR-GFPi showed knock down of GFP expression in the entire cortex and stele of those particular roots. Figure 2I shows a chimaeric pRR-GFPi transformed J0781 root that appears to be co-transformed only in (part of) the stele as judged by the red fluorescence. However, no GFP fluorescence was detected in both the cortex and the stele of this chimaeric root (Fig. 2J). When such chimaeric pRR-GFPi transformed J0781 roots were clonally propagated, new lateral roots emerged that did not show any DsRED1 fluorescence and regained GFP fluorescence (Fig. 2I, J). This indicates that systemic spread of the silencing signal does take place within the cortex and stele of a single chimaeric A. rhizogenes-transformed root, but not to non-co-transformed lateral roots. Further, the extent of systemic spread of the silencing signal to the epidermis of the root was investigated. Therefore the Medicago R108-RH2::GFP line was used that expresses GFP exclusively in the epidermis (Fig. 2K, L). Upon transformation with pRR-GFPi, chimaeric roots were searched for those that contained transformed epidermal cell files. Figure 2M, N, O shows such a chimaeric root transformed with pRR-GFPi. Approximately half of the epidermal cell files are co-transformed as visualized by red fluorescence. Strikingly, silencing of GFP occurs only in the co-transformed cell files, whereas, in the epidermal cell files lacking DsRED1 expression, GFP fluorescence intensity is as high as in the control roots. Even 6 weeks after transformation, the fluorescence in the non-transformed epidermal cell files was as strong as in the control roots, indicating that RNAi is cell autonomous in the root epidermis.
Systemic spreading of the silencing signal to the shoot
RNAi of a trans 35S::gusA gene in Lotus japonicus indicated some systemic spreading of the silencing signal to the non-transgenic shoots of A. rhizogenes-transformed plants (Kumagai and Kouchi, 2003). To investigate the level of systemic spreading of the silencing signal to the shoot, two additional transgenic Arabidopsis lines were used; one carrying 35S::GFP and a second Gal4 enhancer trap line, J0661, which shows strong GFP fluorescence in root vascular tissue and in the vascular tissue of the cotyledons and leaves. A. rhizogenes-mediated RNAi of GFP in the 35S::GFP transgenic line resulted in 20% of the A. rhizogenes-transformed plants (n=40) showing no, or very low levels, of GFP fluorescence in the shoots 4 weeks after transformation. In 60% of the plants bright GFP fluorescence was visible in some leaves whereas other leaves on the same composite plant lacked GFP fluorescence (Fig. 2P, Q), and even variation within one leaf was observed. 20% of the composite plants did not show any sign of silencing in the shoot, while GFP expression in the co-transformed roots of these composite plants was efficiently knocked down. By contrast with systemic spreading, albeit inefficiently, in the 35S::GFP line, no visible reduction of GFP expression in the vascular tissue of the leaves of enhancer trap line J0661 was observed 4 weeks after transformation (Fig. 2R, S). Red fluorescence due to the DsRED1 protein could occasionally be observed in the vascular tissue of the shoot (Fig. 2R). In the hypocotyl of the A. rhizogenes-transformed enhancer trap line J0781GFP, fluorescence was also not reduced (Fig. 2E).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
It is shown here, by targeting KJK/CSLD3, that endogenous genes can efficiently be silenced in roots of Arabidopsis via RNAi by using Agrobacterium rhizogenes-mediated transformation. A high percentage (91% in the case of KJK) of the homogeneously transformed roots showed phenotypes identical to the described mutant (Wang et al., 2001). Quantification of mRNA levels by qPCR confirmed the knock down of the corresponding gene. However, residual mRNA could still be detected and some variation in the level of expression was also detected between independently transformed roots. This variation in mRNA levels could be an explanation for the observed variation in phenotypes. In these plants 62% of the homogeneously transformed roots initiated root hairs that stopped at the bulge stage without further elongation, but in 29% of the cases some elongation took place resulting in small root hairs. A similar plasticity in phenotypes has been observed for the csld3-1 mutant and is thought to be the result of a reduction in the amount of correctly targeted protein to the membrane, resulting in a reduction of delivery of cellulose polymers to the primary cell wall (Wang et al., 2001). The occurrence of intermediate phenotypes as a result of RNAi has also been reported for stable transformed Arabidopsis plants (Chuang and Meyerowitz, 2000) and can be an additional tool to gain insight into the function of a gene.
Generally, it is thought that a specific mobile silencing signal exists that can travel between cells via plasmodesmata and long distances via the phloem (Palauqui et al., 1997; Voinnet et al., 1998; Jorgensen, 2002; Mlotshwa et al., 2002). For example, in Arabidopsis, biolistic delivery of dsRNA into leaf cells triggered silencing capable of spreading locally and systemically. It was reported that systemic spreading of the silencing signal could be detected 2 weeks after biolistic delivery starting in the veins of non-bombarded leaves and was clearly evident in non-vascular tissues one month after bombardment (Klahre et al., 2002). Strikingly, A. rhizogenes-mediated RNAi of trans GFP in Arabidopsis or Medicago roots showed that systemic transport of the silencing signal does not occur to non-co-transformed roots. Targeting GFP in the Gal4 enhancer trap lines J0661 and J0781 also did not show any systemic spread to the non-transgenic shoot. However, targeting of GFP in a 35S::GFP transgenic line did result in systemic transport of the silencing signal, but the extent of silencing was more limited and greatly variable. Similar results are reported in Lotus japonicus where A. rhizogenes-mediated silencing of a 35S::gusA transgene did not spread to non-co-transformed roots and was limited and variable in the shoots (Kumagai and Kouchi, 2003). The lack of systemic spread of the silencing signal to non-co-transformed roots is in agreement with grafting experiments performed in tobacco, which suggested that silencing is unidirectional from the base to the top of the plant (Palauqui et al., 1996, 1997). The observed variation in spatial patterns of silencing in the shoot of the 35S::GFP line has also been observed in different plant species and for different transgenes under the control of the 35S promoter (Boerjan et al., 1994; Jorgensen et al., 1996; Kunz et al., 1996; Palauqui et al., 1996). The extent of systemic silencing in the shoot could depend on the regulation of the transgene, since no systemic silencing was observed in the shoot of transformed enhancer trap lines J0661 and J0781, which express GFP under the control of an endogenous enhancer element.
The use of DsRED1 as selection marker enabled the selection of chimaeric roots to examine the extent of systemic spread of the silencing signal within root tissue. Arabidopsis line J0781 shows strong GFP expression in the cortex and stele of the root. Chimaeric J0781 roots partly transformed with pRR-GFPi showed silencing of GFP in the entire cortex and vasculatur tissue, indicating that the silencing signal is able to spread systemically in the cortex and stele. Strikingly, lateral roots that formed on these chimaeric roots and were most likely not co-transformed, regained GFP expression. This suggests that within one root system, the silencing signal does not spread to non-co-transformed lateral roots. Formally it cannot be ruled out that co-suppression of the newly introduced transgene is the cause of the chimaeric nature of these roots. However, this seems less likely since it would imply that the hairpin construct is transcriptionally silenced in a cell autonomous way. The absence of systemic spread to the lateral roots is most likely a result of the unidirectional movement of the silencing signal. In contrast with systemic spread in the cortex and vascular tissue in Arabidopsis, no cell-to-cell movement of the silencing signal was observed in the epidermis of Medicago, demonstrating that silencing in the root epidermis is cell autonomous. One explanation for the fact that spreading of the silencing signal is not observed in the epidermis of A. rhizogenes-mediated roots, could be that epidermal cells become symplastically isolated. By dye-coupling experiments in Arabidopsis roots it was shown that cells in the meristem and epidermal cells in the elongation zone are symplastically connected through plasmodesmata, but gradually become symplastically isolated as the epidermal cells differentiate. By the time root hair outgrowth is visible the epidermal cells are symplastically isolated (Duckett et al., 1994). Similarly, it was shown that symplastically isolated stomatal guard cells do not silence systemically (Voinnet et al., 1998). So, the symplastic isolation of cells could cause the immobility of the silencing signal.
RNAi via A. rhizogenes-mediated root transformation is a valuable tool to study genes involved in root development and root–microbe interactions. It is a very fast and efficient system to silence genes in roots. In Arabidopsis, silenced A. rhizogenes-transformed roots can be obtained within 10 d. In particular, for plant species with very time-consuming regeneration times, this methods offers a big advantage. The fact that silencing is triggered cell autonomously in the root epidermis provides the possibility to use inducible and tissue-specific promoters more specifically to regulate RNAi. At the same time it requires a thorough inspection of the chimaeric nature of A. rhizogenes-transformed roots in order to interpret the observed phenotypes correctly. The pRedRoot vector provides this possibility.
|
Acknowledgements |
|---|
The authors thank Mark Kwaaitaal (Biochemistry, Wageningen University) and Jeroen Pouwels (Molecular Biology, Wageningen University) for their help with the immuno-blotting. Toolbox 21 for their work on Arabidopsis line 35S::GFP. Oscar Vorst and Jos Molthoff (Plant Research International, Wageningen) for their assistance with the qPCR. Jan-Willem Borst (Biochemistry, Wageningen University) for assistance with the FSPIM. Arabidopsis line 35S::GFP was kindly provided by Renze Heidstra (Molecular Cell Biology, Utrecht University). EL, TB, and RG are supported by The Netherlands Organization of Scientific Research (NWO), and the EU FP5 program QLG-CT2000-00676.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Boerjan W, Bauw G, Van Montagu M, Inzé D. 1994. Distinct phenotypes generated by overexpression and suppression of S-adenosyl-L-methionine synthetase reveal developmental patterns of gene silencing in tobacco. The Plant Cell 6, 1401–1414.[Abstract]
Boisson-Dernier A, Chabaud M, Garcia F, Bécard G, Rosenberg C, Barker DG. 2001. Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations. Molecular Plant–Microbe Interactions 14, 695–700.[CrossRef]
Chiang CC, Hadwiger LA. 1990. Cloning and characterization of a disease resistance response gene in pea inducible by Fusarium solani. Molecular Plant–Microbe Interactions 3, 78–85.
Chilton MD, Tepfer DA, Petit A, David C, Casse-Delbart F, Tempé J. 1982. Agrobacterium rhizogenes inserts T-DNA into the genome of the host plant root cells. Nature 295, 432–434.[CrossRef]
Chuang C, Meyerowitz M. 2000. Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proceedings of the National Academy of Sciences, USA 97, 4985–4990.
Czakó M, Wilson J, Yu X, Márton L. 1993. Sustained root culture for generation and vegetative propagation of transgenic Arabidopsis thaliana. Plant Cell Reports 12, 603–606.[ISI]
Duckett CM, Oparka KJ. Prior DAM, Dolan L, Roberts K. 1994. Dye-coupling in the root epidermis of Arabidopsis is progressively reduced during development. Development 120, 3247–3255.[Abstract]
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.[CrossRef][Medline]
Favery B, Ryan E, Foreman J, Linstead P, Boudonck K, Steer M, Shaw P, Dolan L. 2001. KOJAK encodes a cellulose synthase-like protein required for root hair cell morphogenesis in Arabidopsis. Genes and Development 15, 79–89.
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811.[CrossRef][Medline]
Fraser AG, Kamath RS, Zipperlen P, Martinez-Campos M, Sohrmann M, Ahringer J. 2000. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408, 325–330.[CrossRef][Medline]
Gadella Jr TW, Vereb Jr G, Hadri AE, Rohrig H, Schmidt J, John M, Schell J, Bisseling T. 1997. Microspectroscopic imaging of nodulation factor-binding sites on living Vicia sativa roots using a novel bioactive fluorescent nodulation factor. Biophysical Journal 72, 1986–1996.
Gönczy P, Echeverri C, Oegema K, et al. 2000. Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 408, 331–336.[CrossRef][Medline]
Hamilton A, Voinet O, Chappell L, Baulcombe D. 2002. Two classes of short interfering RNA in RNA silencing. EMBO Journal 21, 4671–4679.[CrossRef][ISI][Medline]
Hammond SM, Caudy AA, Hannon GJ. 2001. Post-transcriptional gene silencing by double-stranded RNA. Nature Reviews Genetics 2, 110–119.[CrossRef][ISI][Medline]
Hannon GJ. 2002. RNA interference. Nature 418, 244–251.[CrossRef][Medline]
Hoffmann B, Trinh TH, Leung J, Kondorosi A, Kondorosi E. 1997. A new Medicago truncatula line with superior in vitro regeneration, transformation, and symbiotic properties isolated through cell culture selection. Molecular Plant–Microbe Interactions 10, 307–315.[CrossRef]
Johansen LK, Carrington JC. 2001. Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiology 126, 930–938.
Jorgensen RA. 2002. RNA traffics information systemically in plants. Proceedings of the National Academy of Sciences, USA 99, 11561–11563.
Jorgensen RA, Cluster PD, English JJ, Que Q, Napoli CA. 1996. Chalcone synthase co-suppression phenotypes in petunia flowers: comparison of sense vs. antisence constructs and single-copy versus complex T-DNA sequences. Plant Molecular Biology 31, 957–973.[CrossRef][ISI][Medline]
Klahre U, Crété P, Leuenberger SA, Iglesias VA, Meins Jr F. 2002. High molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plants. Proceedings of the National Academy of Sciences, USA 99, 11981–11986.
Kumagai H, Kouchi H. 2003. Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules. Molecular Plant–Microbe Interactions 8, 663–668.
Kunz C, Schöb H, Stam M, Kooter JM, Meins F. 1996. Developmentally regulated silencing and reactivation of tobacco chitinase transgene expression. The Plant Journal 10, 437–450.[CrossRef][ISI]
Limpens E, Franken C, Smit P, Willemse J, Bisseling T, Geurts R. 2003. LysM domain receptor kinases regulating rhizobial Nod factor-induced infection. Science 302, 630–633.
Maeda I, Kohara Y, Yamamoto M, Sugimoto A. 2001. Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi. Current Biology 11, 171–176.[CrossRef][ISI][Medline]
Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA. 1999. Fluorescent proteins from non-bioluminescent Anthozoa species. Nature Biotechnology 17, 969–973.[CrossRef][ISI][Medline]
Mlotshwa S, Voinnet O, Mette MF, Matzke M, Vaucheret H, Wei Ding S, Pruss G, Vance VB. 2002. RNA silencing and the mobile silencing signal. The Plant Cell 14, Supplement, S289–S301.
Mylona P, Moerman M, Yang WC, Gloudemans T, Van de Kerckhove J, van Kammen A, Bisseling T, Franssen HJ. 1994. The root epidermis-specific pea gene RH2 is homologous to a pathogenesis-related gene. Plant Molecular Biology 26, 39–50.[CrossRef][ISI][Medline]
Nilsson O, Olsson O. 1997. Getting to the root: the role of Agrobacterium rhizogenes rol genes in the formation of hairy roots. Physiologia Plantarum 100, 463–473.[CrossRef]
Norris SR, Meyer SE, Callis J. 1993. The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression. Plant Molecular Biology 21, 895–906.[CrossRef][ISI][Medline]
Palauqui J-C, Elmayan T, Dorlhac de Borne F, Crété P, Carles C, Vaucheret H. 1996. Frequencies, timing and spatial patterns of co-suppression of nitrate reductase and nitrite reductase in transgenic tobacco plants. Plant Physiology 112, 1447–1456.[Abstract]
Palauqui J-C, Elmayan T, Pollien JM, Vaucheret H. 1997. Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. EMBO Journal 16, 4738–4745.[CrossRef][ISI][Medline]
Pawlowski K, Kunze R, de Vries S, Bisseling T. 1994. Isolation of total, poly(A) and polysomal RNA from plant tissues. In: Gelvin SB, Schilperoort RA, eds. Plant molecular biology manual. Dordrecht: Kluwer Academic Publishers, 1–13.
Penmetsa RV, Cook DR. 1997. A legume ethylene-insensitive mutant hyperinfected by its rhizobial symbiont. Science 275, 527–530.
Ramos J, Bisseling T. 2003. A method for the isolation of root hairs from the model legume Medicago truncatula. Journal of Experimental Botany 54, 2245–2250.
Ratcliff F, Martin-Hernandez AM, Baulcombe DC. 2001. Tobacco rattle virus as a vector for analysis of gene function by silencing. The Plant Journal 25, 237–245.[CrossRef][ISI][Medline]
Schweizer P, Pokorny J, Schulze-Lefert P, Dudler R. 2000. Double-stranded RNA interferes with gene function at the single-cell level in cereals. The Plant Journal 24, 895–903.[CrossRef][ISI][Medline]
Sharp PA. 2001. RNA interference-2001. Genes and Development 15, 485–490.
Sonti RV, Chiurazzi M, Wong D, Davies CS, Harlow GR, Mount DW, Signer ER. 1995. Arabidopsis mutants deficient in T-DNA integration. Proceedings of the National Academy of Sciences, USA 92, 11786–11790.
Tavernarakis N, Wang SL, Dorovkov M, Ryazanov A, Driscoll M. 2000. Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nature Genetics 24, 180–183.[CrossRef][ISI][Medline]
Van der Krol AR, Mur LA, Beld M, Mol JN, Stuitje AR. 1990. Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. The Plant Cell 2, 291–299.
Van Engelen FA, Molthoff JW, Conner AJ, Nap J-P, Pereira A, Stiekema WJ. 1995. pBINPLUS: an important improved plant transformation vector based in pBIN19. Transgenic Research 4, 288–290.[CrossRef][ISI][Medline]
Voinnet O, Vain P, Angell S, Baulcombe DC. 1998. Systemic spread of sequence-specific transgene RNA degradation in plants is initiated by localized introduction of ectopic promoter-less DNA. Cell 103, 157–167.
Wang X, Cnops G, Vanderhaegen R, De Block S, Van Montagu M, Van Lijsebettens M. 2001. AtCSLD3, a cellulose synthase-like gene important for root hair growth in Arabidopsis. Plant Physiology 126, 575–586.
Waterhouse PM, Graham MW, Wang MB. 1998. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proceedings of the National Academy of Sciences, USA 95, 13959–13964.
Wesley SV, Helliwell CA, Smith NA, et al. 2001. Construct design for efficient, effective and high-throughput gene silencing in plants. The Plant Journal 27, 581–590.[CrossRef][ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. Pang, G. J. Peel, S. B. Sharma, Y. Tang, and R. A. Dixon A transcript profiling approach reveals an epicatechin-specific glucosyltransferase expressed in the seed coat of Medicago truncatula PNAS, September 16, 2008; 105(37): 14210 - 14215. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Kevei, G. Lougnon, P. Mergaert, G. V. Horvath, A. Kereszt, D. Jayaraman, N. Zaman, F. Marcel, K. Regulski, G. B. Kiss, et al. 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase1 Interacts with NORK and Is Crucial for Nodulation in Medicago truncatula PLANT CELL, December 1, 2007; 19(12): 3974 - 3989. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Wan, J. Hontelez, A. Lillo, C. Guarnerio, D. van de Peut, E. Fedorova, T. Bisseling, and H. Franssen Medicago truncatula ENOD40-1 and ENOD40-2 are both involved in nodule initiation and bacteroid development J. Exp. Bot., June 1, 2007; 58(8): 2033 - 2041. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-P. Combier, T. Vernie, F. de Billy, F. El Yahyaoui, R. Mathis, and P. Gamas The MtMMPL1 Early Nodulin Is a Novel Member of the Matrix Metalloendoproteinase Family with a Role in Medicago truncatula Infection by Sinorhizobium meliloti Plant Physiology, June 1, 2007; 144(2): 703 - 716. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Y. Han, Y. S. Kwon, D. C. Yang, Y. R. Jung, and Y. E. Choi Expression and RNA Interference-Induced Silencing of the Dammarenediol Synthase Gene in Panax ginseng Plant Cell Physiol., December 1, 2006; 47(12): 1653 - 1662. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Sunilkumar, L. M. Campbell, L. Puckhaber, R. D. Stipanovic, and K. S. Rathore From the Cover: Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol PNAS, November 28, 2006; 103(48): 18054 - 18059. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-F. Arrighi, A. Barre, B. Ben Amor, A. Bersoult, L. C. Soriano, R. Mirabella, F. de Carvalho-Niebel, E |