An endogenous artificial microRNA system for unraveling the function of root endosymbioses related genes in Medicago truncatula
- Emanuel A Devers†1, 2,
- Julia Teply†1,
- Armin Reinert1,
- Nicole Gaude1 and
- Franziska Krajinski1Email author
© Devers et al.; licensee BioMed Central Ltd. 2013
Received: 11 February 2013
Accepted: 10 May 2013
Published: 16 May 2013
Legumes have the unique capacity to undergo two important root endosymbioses: the root nodule symbiosis and the arbuscular mycorrhizal symbiosis. Medicago truncatula is widely used to unravel the functions of genes during these root symbioses. Here we describe the development of an artificial microRNA (amiR)-mediated gene silencing system for M. truncatula roots.
The endogenous microRNA (miR) mtr-miR159b was selected as a backbone molecule for driving amiR expression. Heterologous expression of mtr-miR159b-amiR constructs in tobacco showed that the backbone is functional and mediates an efficient gene silencing. amiR-mediated silencing of a visible marker was also effective after root transformation of M. truncatula constitutively expressing the visible marker. Most importantly, we applied the novel amiR system to shed light on the function of a putative transcription factor, MtErf1, which was strongly induced in arbuscule-containing cells during mycorrhizal symbiosis. MtPt4 promoter driven amiR-silencing led to strongly decreased transcript levels and deformed, non-fully truncated arbuscules indicating that MtErf1 is required for arbuscule development.
The endogenous amiR system demonstrated here presents a novel and highly efficient tool to unravel gene functions during root endosymbioses.
In the past decades, legumes have been established as important model systems to discover the molecular and physiological background of the root nodule and arbuscular mycorrhizal symbiosis. Analysis of gene function during root endosymbioses requires reverse genetics approaches based on expression perturbation experiments. In the past, RNA interference (RNAi) or virus induced gene silencing (VIGS) has been widely applied to produce plant knock-down mutants. Both systems exploit endogenous posttranscriptional gene silencing (PTGS) pathways of eukaryotes [1–7].
An efficient VIGS system has not yet been established for M. truncatula, hence RNAi approaches have been widely applied to elucidate gene functions in Agrobacterium rhizogenes transformed roots. However, previous knock-down approaches in this system using RNAi constructs often did not lead to consistent results due off-target effects of RNAi approaches. RNAi is based on a hairpin construct with short inverted sequence fragments of the gene of interest separated by an intron and is processed via the IR-PTGS pathway. The expressed RNA folds into a perfect matched double strand and is processed by DCL4 to short interfering RNAs (siRNAs). However, in some cases the approach is limited by inefficient knock down of the target gene in legumes due to unknown causes . Additionally, the RNAi approach leads to heterogeneous accumulation of siRNA products, derived from the expressed hairpin which can lead to unspecific downregulation of related genes (off-targets), especially in large gene families with high sequence similarity . Also, a mechanism called transitivity leads to an amplification and spreading of the siRNA species, yielding secondary siRNAs independent of the primary siRNA signal . These secondary siRNAs cover sequence information outside of the designed RNAi construct, thus enhancing off-target effects. There is precedent for artificial miRNAs to be more specific as RNAi constructs [11, 12], here we suggest artificial miRNAs as an alternative tool for gene knock down approaches. However, we do not provide a direct comparison of both approaches with regard to efficiency and target specificity.
Analyzing gene functions by gene knock out approaches in A. rhizogenes transformed root systems is also hampered by a high variability within the experimental system with independent transformation events being present in a root system after A. rhizogenes transformation. Hence, to facilitate investigating gene functions in non-uniformly transformed root systems, a strong expression strength of the gene knock down constructs is required. However, the widely applied 35S promoter for driving knock down constructs mediates a rather weak expression strength in M. truncatula roots , with particularly weak expression in arbuscule-containing cells of mycorrhizal roots . We therefore developed a vector series with three different promoters for knock down construct expression, either the 35S promoter or the ubiquitin 3 promoter of Arabidopsis thaliana or the MtPt4 promoter of M. truncatula. The latter is mediating a particular strong expression in arbuscule-containing cells .
Arbuscules are intracellular fungal structures formed in the plant’s inner cortical cell layers. The development of arbuscules requires a profound reprogramming of the root cell , and a wide number of genes which are specifically expressed in arbuscule containing cells have been identified [17, 18]. However, an analysis of the precise role of these genes during arbuscule development and function is often hampered by the previously mentioned inconveniences regarding expression perturbation experiments in mycorrhizal M. truncatula roots.
Here we demonstrate that mtr-miR159b is effectively processed from its precursor molecule and thus represents a highly suitable backbone for the expression of amiRs in M. truncatula. Efficient target gene knock-down could be validated by an amiR against a visible marker in an heterologous system and in M. truncatula. Additionally, we used the MtPt4 promoter, which mediates a strong expression in mycorrhizal roots  for driving the expression of an amiR against a previously identified putative transcription factor (MtErf1). Knock-down of MtErf1 expression resulted in reduced expression of levels of Rhizophagus irregularis genes indicating reduced mycorrhizal colonization. Moreover, MtErf1 seemed to be required for arbuscule development, since only truncated, non-fully branched arbuscules were present in roots with amiR-silenced MtErf1 expression.
Results and discussion
miR159b represents a suitable backbone for artificial microRNA (amiR) expression in M. truncatula
A vector system for expression perturbation experiments by A. rhizogenesmediated root transformation
For reverse genetic approaches in M. truncatula roots, we developed a vector series (pRed), where a constitutively expressed dsRED gene allows the easy detection of transformed roots (Additional file 1: Figure S2). We have developed expression vectors (pRed-Exp) and vectors for RNAi (pRed-RNAi). Both types of vectors are available with three different promoters for expression of the gene or RNAi construct, namely the 2×35S promoter, the ubi3 promoter of Arabidopsis thaliana and the MtPt4 promoter of M. truncatula.
The miR159b-mediated amiR expression mediates strong silencing in tobacco leaves
The miR159b backbone driven amiR constructs lead to efficient knock-down in M. truncatularoots
MtPt4 promoter driven amiR silencing of MtErf1 points to a role of this TF in arbuscule-development
Finally we wanted to confirm that amiR-mediated gene silencing also works efficiently in arbuscule-containing cells. Since the 35S promoter seems to be only weakly active in arbuscule-containing cells , we used the MtPt4 promoter of M. truncatula, to enable a strong and specific expression of the amiRs. MtPt4 encodes for a phosphate transporter, which is strongly induced in arbuscule-containing cells [15, 25].
The endogenous amiR-mediated gene silencing system presented here provides a useful tool to investigate the function of genes involved in root endosymbioses. In addition, we showed that the MtPt4 promoter provides a strong expression of amiR constructs in mycorrhizal roots. AmiR-silencing of a putative transcription factor MrErf1 indicated a putative function of this gene during arbuscule development, since only defective arbuscules were observed in roots with reduced MtErf1 expression due to amiR-mediated gene silencing.
The following plants were used in this study: Nicotiana benthaminana cv. TW16, Medicago truncatula cv. Jemalong (A17) and Medicago truncatula cv. 2HA stably transformed with pKDSR(I) (see vectors and cloning procedures for vector details). Bacterial strains include Escherichia coli TOP10 (Invitrogen) or DH5α for cloning purposes, Agrobacterium rhizogenes strain ARqua1  for Medicago root transformations and Agrobacterium tumefaciens strain GV2260  for the leaf infiltration assay. The fungus Rhizophagus irregularis (strain BB-E, provided by Agrauxine, France) was propagated and used for mycorrhizal colonization studies as described in .
Medicago truncatulagrowth, transformation and inoculation
Medicago truncatula seeds were scarified using concentrated H2SO4 and subsequently sterilized with HClO. The seeds were laid on a water agar plate and kept at 4°C in the dark overnight to synchronize the germination. Afterwards, the seeds were transferred to room temperature but remained in the dark for two days. M. truncatula root transformation was carried out with a modified protocol according to . Seedlings were subsequently transferred to a fresh water agar plate, kept in the dark and incubated at room temperature for a further two days. Finally the seedlings were transferred into vertical square plates (amiRdsRED) or jars (amiRMtErf1) containing Fahraeus medium plus 25 μg/ml kanamycin. The plates and jars were kept in a phytotron for three weeks to allow the growth of transformed roots. The phytotron conditions were: 200 μE·m-2·s-1, 16 h/8 h day-night cycle, 22°C and 65% humidity. After three weeks the plants expressing the amiR- and control construct indicated by a DsRed fluorescence, were potted in a soil mix of quartz sand (0.6–1.2 mm)/expanded clay/vermiculite/inoculum mix (7:1:1 [v/v]). All potted plants were fertilized twice a week with 20 μM Pi Hoagland’s solution .
M. truncatula Jemalong, genotype 2HA was used for plant transformation with the vector pKDSR(I) (see vectors) .
Leaf infiltration assays
Nicotiana benthamiana plants were grown for 4 weeks in a phytotron and used for the infiltration prior to the flowering phase. The experiment was repeated on two individual plants and six infiltrations each. Prior to infiltration, the plants were watered for 3h until the soil was water saturated. The leaves were infiltrated either with Agrobacterium tumefaciens (GV2260) containing pRed-35Spro::amiR-dsred or empty pRed-Expr. The bacteria were harvested, washed with AS-media (10 mM MES, 10 mM MgCl2, pH 5.6), resuspended to an OD600 of 0.8 with AS-media containing 150 μM acetosyringone and incubated for 3 h at room temperature on a shaker (50 rpm). Using a syringe, 500 μl of the bacterial suspension was infiltrated into the abaxial side of each leaf. Infiltration boarders were marked with a permanent marker. The plants were placed into a phytotron and analyzed after two and three days. After analysis, the marked leaf areas were excised, frozen in liquid nitrogen and stored at −80°C for further protein and RNA extraction.
Staining and determination of fungal structures
Fungal structures were visualized with wheat germ agglutinin (WGA) conjugated with Alexa Fluor 488 (Invitrogen). In short, the approx. 1 cm long root sections were submerged for 5 min in 90°C hot 10% KOH [w/v] and washed five times with phosphate buffered saline (PBS) buffer (pH 7.4). The roots were then incubated overnight in PBS buffer (pH 7.4) containing 0.01% WGA Alexa Fluor 488 [w/v].
The DsRed fluorescence of tobacco leaves and the roots of chimeric M. truncatula plants were monitored using a stereo-fluorescence microscope Leica M165 FC with a DsRed filter system (Leica 10447227). The exposure time for fluorescence was 500 ms and the gain setting was 2.0×. Overlay images were produced using Leica LAS-AF Version 2.8.1.
Confocal Images of WGA-Alexa Fluor stained arbuscules were collected on a Leica TCS-SP5 confocal microscope (Leica Microsystems, Exton, PA USA) using a 63× water immersion objective NA 1.2, zoom 1.6. Alexa Fluor was excited at 488 nm and emitted light was collected from 505 to 582 nm. Optical sections were acquired at 0.3–0.5 μm intervals. Images were processed using ImageJ software (Wayne Rasband, National Institutes of Health, USA; http://imagej.nih.gov/ij).
Mapping of small RNA and degradome reads to miRNA precursors and design of artificial miRNAs
All M. truncatula precursor sequences belonging to the families of miR159 and miR319, were gathered and analyzed for small RNA and degradome read location as well as abundance. Small RNA and degradome reads were previously mapped to the Medicago 3.0 genome . The information of the precise location of the reads and their abundance was manually annotated to the appropriate precursor. The 21 nt long artificial miRNA sequences against dsRED and MtErf1 were designed using the web miRNA designer WMD3 (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi) following the instructions given on the website. Only green flagged sequences were chosen and checked against their target sequence and the Medicago genome v3.5 using psRNAtarget (http://plantgrn.noble.org/psRNATarget/). The sequence showing no or the least possible off-targets was chosen for further construction of the artificial miRNA.
Vectors and cloning procedure
The vector pKDSR(I) was created by converting the unique AscI of pRedroot  to a PacI site, in a way that the gene with its promoter and terminator was flanked by two PacI sites. Likewise, a KpnI site of pK7GWIWG2(I)  was converted into a PacI site. The PacI flanked cassette was excised from pRedroot and ligated into the novel PacI site of pK7GWIWG2(I). The resulting vector was named pKDSR(I).
For the construction of the pRed-amiR vectors, the final amplification product of the overlapping PCR was first cloned into pCR2.1 (Invitrogen). The PCR product included parts of the pBluescript II SK (+) multiple cloning site. The amiR precursor molecule was removed from the pCR2.1 vector by using the SpeI and MluI restriction sites and ligated into the appropriate sites of pRed vectors. Final pRed-amiR constructs were sequenced and used for root transformation (pRed-35Spro::amiR-dsred, pRed-MtPt4pro::amiR-MtErf1) as described recently . Additionally, for tobacco leaf infiltration assays, the amiR-dsred precursor molecule was inserted via NotI and KpnI sites into pORE-E4 .
Overlapping PCR using the miR159b backbone to design an artificial miRNA
The miR159b backbone was synthesized by gene synthesis (MWG) and an additional MluI site was added to the 3′ end of the miR precursor. This molecule was cloned into the SpeI and PstI restriction site of pBluescript II SK (+). The resulting pB159b vector represents the template for the overlapping PCR to generate amiR precursor molecules. For this purpose four different primers are designed according to .
Primer I: GTX1…X21AAATTGGACACGCGTct (X1-X21 are the designed amiR sequence).
Primer II: TTY1…Y21ACAAAAAGATCAAGGC (Y1-Y21 are the amiR sequence in reverse complement orientation).
Primer III: TTZ1…Z21TCTAAAAGGAGGTGATAG (Z1-Z21 are the amiR sequence in reverse complement orientation, with the exception that Z11 and Z12 have to be modified to not pair (also no G:U non-Watson-Crick pairing) to position X10 and X11, respectively. Also Z21 has to be changed to not pair to X1.
Primer IV: GAN1…N21 AATTAGGTTactagt (N1-N21 are reverse complement of Z1-Z21). Primer sequences used to create amiRDsRED and amiRMtErf1 are given in the Additional file 2: Table S1.
Three independent PCRs were performed with pB159b as template and the following three primer combinations (1) primer A + primer I, (2) primer II + primer III, (3) primer B + primer IV. The PCR products were loaded into a single well of an 2% agarose gel followed by gel purification. The resulting mixture of products was used as a template for a final PCR using primer A + primer B. The single PCR product was subcloned into pCR2.1 (Invitrogen) and sequenced to check for a correct amiR precursor sequence and a stem-loop folding identical to mtr-miR159b.
RNA extraction, RT-PCR and quantitative RT-PCR
Total RNA was extracted from liquid nitrogen frozen and ground tissue using the miRVana miRNA extraction Kit (Ambion) and Plant Isolation Aid step (Ambion) according to the manufacturer’s instructions. All PCRs were carried out as described earlier in [23, 38].
Protein extraction and Western blotting
Frozen plant tissue was ground and proteins were extracted with rigorous vortexing in 4 ml of homogenization buffer per gram fresh weight. The homogenization buffer consisted of 100 mM HEPES pH 7.5, 10% glycerol, 5 mM DTT, cOmplete ULTRA tablet – EDTA free (Roche) proteinase inhibitor cocktail. The extract was centrifuged at 14000 g for 15 min (4°C) and the supernatant was collected and aliquots were frozen at −20°C. The protein concentration was determined using QuickStart Bradford Protein Assay (Biorad) with the provided γ-globulin standard following the manufacturer’s instructions using the microtiter plate protocol.
For the western blot analysis an equal amount of 15 μg of protein from extracts of tobacco plants and 10 μg of proteins from Medicago root extracts were resolved on separately on 1 mm 12% SDS-PAGE mini-gels, respectively, and subsequently transferred onto Immobilon-P PVDF membranes (Millipore) by semi-dry blotting according to Immobilon-P transfer membrane user guide. Detection of DsRed was carried out with primary antibodys using 0.4 μg/ml (1:1250) rabbit anti-RFP tag antibody (GenScript) mixed with rabbit anti-RubisCO activase (Agrisera) in a 1:10000 dilution, the latter serving to detect RubisCO activase as a loading control. The secondary goat anti-rabbit antibody conjugated with alkaline phosphatase (Abcam) was used as a 1:10000 dilution. The protein bands were visualized using NBT/BCIP (Roche). The specific proteins were identified by comparison to a pre-stained protein size marker (Thermo scientific).
To test for difference between plant genotype and treatment, data were analyzed by Student’s t-test for pairwise comparisons using the Sigmaplot software package (Systat, Germany).
The Max Planck Society supported this work. We thank Igor Kryvoruchko for constructing the pKDsRed vector. We thank Ursula Krause for providing the anti-RubisCO activase antibody and Derek Nedveck for critical reading of the manuscript.
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