JXB Advance Access originally published online on July 25, 2005
Journal of Experimental Botany 2005 56(419):2507-2513; doi:10.1093/jxb/eri244
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RESEARCH PAPER |
Transcript enrichment of Nod factor-elicited early nodulin genes in purified root hair fractions of the model legume Medicago truncatula
Laboratory of Plant Microbe Interactions (LIPM), CNRS-INRA, BP52627, F-31320 Castanet-Tolosan, France
To whom correspondence should be addressed. Fax: +33 5 61 28 50 61. E-mail: fniebel{at}toulouse.inra.fr
Received 18 February 2005; Accepted 6 June 2005
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Abstract |
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This article describes an efficient procedure to study Nod factor-induced gene expression in root hairs of the model legume Medicago truncatula. By developing an improved method of fracturing frozen root hairs, it has been possible to obtain a highly purified root hair fraction from M. truncatula seedlings yielding sufficient RNA for real-time quantitative RT-PCR expression analysis. After Nod factor treatment it was possible to detect up to 100-fold increases of MtENOD11 and pMtENOD11-gus transcript levels in root hair RNA. This corresponds to 5–7-fold higher induction levels than for entire root tissue preparations. Furthermore, the use of these enriched RNA samples has revealed for the first time a very significant induction (30-fold) of the MtENOD40 gene in root hairs in response to Nod factors. It is concluded that the rapid and convenient procedure described here will be particularly useful for detecting tissue-specific low-level gene expression in root hairs responding to Rhizobium Nod factors or other exogenous signals.
Key words: MtENOD11, MtENOD40, real time quantitative RT-PCR, Rhizobium/legume symbiosis, root hair-specific expression
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Plants within the legume family can enter into a symbiotic association with certain soil bacteria collectively referred to as ‘rhizobia’. This symbiosis is a major source of biologically fixed nitrogen and is characterized by the formation on the roots of leguminous plants of a novel nitrogen-fixing organ known as the root nodule. The establishment of this symbiotic interaction involves a specific infection process in which rhizobia enter the plant root via the root epidermis and induce the formation of a nodule structure by reprogramming cortical cell differentiation (for a review see Gage, 2004
).
Epidermal cells in the zone of root hair growth constitute a privileged site of interaction with rhizobia. In these cells, Rhizobium-specific signal molecules known as Nod factors activate a series of early cellular responses, including growth re-orientation of root hairs which precede the infection process (for reviews see Limpens and Bisseling, 2003; Oldroyd and Downie, 2004). In parallel with root hair morphological changes, a limited number of legume genes have been characterized which are specifically activated in the root epidermis in response to Nod factors such as rip1, MtENOD12, and MtENOD11 (Pichon et al., 1992; Cook et al., 1995; Charron et al., 2004).
The relatively small number of genes so far identified as being specifically up-regulated in the root epidermis in response to Nod factors is probably due to the difficulty of detecting tissue-specific gene expression when using whole root tissue preparations. Recent studies of the symbiotic transcriptome have revealed a large collection of genes from the model legume M. truncatula which are potentially implicated during the early stages of the symbiotic interaction (El Yahyaoui et al., 2004; Graham et al., 2004; Mitra et al., 2004). Accessing tissue-specific gene expression data using enriched root-hair cell preparations would provide a powerful means for characterizing the expression of certain of these novel genes.
Whilst methods for root hair isolation have previously been described for Medicago species (Gerhold et al., 1985; Covitz et al., 1998; Ramos and Bisseling, 2003), these are unfortunately not well suited for studying transcription changes elicited by symbiotic Nod factors. The first method described by Gerhold et al. (1985) was developed for protein studies during Rhizobium infection, and requires large quantities of seedlings. Covitz et al. (1998) used the method described by Rohm and Werner (1987) and previously used with success for Vigna (Krause et al., 1994), to prepare RNA from isolated root hairs of untreated M. truncatula roots. Although the paper of Covitz et al. (1998) reports the generation of an ‘enriched’ root hair-specific cDNA library, in fact only one-third of the RNA used for library preparation was purified from root hairs. The remaining RNA was isolated from root tip segments, suggesting that the extraction procedure was probably not very efficient (Covitz et al., 1998). Finally, Ramos and Bisseling (2003) have published a procedure for RNA extraction from root hairs purified from M. truncatula in vitro root cultures. Unfortunately, such A. rhizogenes-transformed root cultures are unable to establish a symbiotic interaction with Rhizobium or to respond to Nod factors (Ramos and Bisseling, 2003).
In this paper, a new procedure for root hair isolation is presented, based on the previously described method of fracturing frozen root hairs (Gerhold et al., 1985), which has been modified and adapted to permit efficient RNA isolation followed by gene expression analysis in root hairs of M. truncatula responding to symbiotic Nod factor signals. Analysis of specific early nodulin gene expression by real time quantitative RT-PCR (qRT-PCR) has revealed a 5–8-fold enrichment of the epidermal-specific MtENOD11 and pMtENOD11-gus marker genes in root hair fractions and also shows for the first time that the MtENOD40 gene (Crespi et al., 1994) is strongly activated in root hairs in response to Nod factors.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Plant material and growth conditions
Both Medicago truncatula Gaertn. cv. Jemalong A17, and the derived transgenic line L416, containing the pMtENOD11-gus fusion (Journet et al., 2001
), were used in these studies. After germination, seedlings with a radicle length of about 1 cm were gently placed on a paper surface (paper support from seed growth pouches, Mega International, Minneapolis, USA) which had been laid on 1.5% agar plates containing a nitrogen-free modified Fahraeus medium (1 mM CaCl2, 0.5 mM MgSO4, 0.7 mM KH2PO4, 0.8 mM Na2HPO4, 50 µM FeEDTA, pH 6.5, including 0.1 mg l–1 of the following microelements: MnSO4, CuSO4, ZnSO4, H3BO3, and Na2MOO4) in square Petri dishes (12x12 cm) (8–10 seedlings per plate). The Petri dishes (slightly inclined from the vertical) were placed in a growth chamber at 25 °C (16 h photoperiod and a light intensity of 70 µE m–2 s–1). After 3 d growth, plants were ready for Nod factor or control (H2O) treatments. At this stage, primary roots were approximately 3–4 cm in length and lateral roots had not yet developed.
Nod factor treatment of seedling roots
Seedling roots growing on the paper support were immersed in 20 ml of either sterile water or a 10–8 M solution of purified S. meliloti Nod factors (Roche et al., 1991), previously diluted in sterile water. After 1 h incubation in a horizontal position, excess liquid was then removed and the plates returned to a vertical orientation and incubated overnight in the growth chamber. After a total of 16–18 h treatment, the seedling roots were harvested either for direct RNA extraction or for the isolation of root hairs using the technique described below.
Root hair isolation method
Prior to root harvesting for root hair isolation, the paper support with 8–10 seedlings was initially transferred onto a sheet of blotting paper (Schleicher & Schuell, Dassel, Germany) in order to remove excess liquid. The root segments with root hairs were then harvested (2–3 cm in length) after removing the root tips (see Fig. 1 in Results), and the 8–10 root segments were combined and transferred to a flat-bottomed polystyrene tube TP30 (Fisher Scientific Labosi, Elancourt, France) previously immersed in liquid nitrogen (note that liquid nitrogen should not be allowed to enter the tube). When the roots freeze a hoar-frost is formed around the root hairs which later on help to visualize the root hair powder at the bottom of the Falcon tube after vortexing. After several min, frozen root portions were transferred to 50 ml Falcon tubes (Becton Dickinson Labware, Le Pont De Claix, France), also previously placed in liquid nitrogen. Fracturing of the root hairs was performed by two cycles of 15 s vortexing at maximum speed, with re-immersion in liquid nitrogen between the cycles. Using this procedure, root hairs are preferentially fractured from the zone containing growing root hairs, whilst most hairs present in the older part of the root remained attached. Gentle tapping of the Falcon tube was necessary to concentrate the fractured root hair powder at the bottom of the tube. It is imperative that tubes containing root hairs are kept frozen throughout the whole process. After removing the remaining root debris with the help of long forceps (roots generally remain intact throughout) an additional 8–10 frozen root segments were then introduced into the same Falcon tube and the root hair fracturing procedure repeated. After processing a total of 50–80 seedling roots in this way, a white powder comprising hoar-frost-covered root hairs should be clearly visible at the bottom of the tube. As described below, RNA extraction from the root hairs can then be performed directly using the Falcon tube containing the root hair powder.
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RNA isolation and cDNA synthesis
Total RNA was extracted from intact roots or isolated root hairs of M. truncatula using the Macherey–Nagel total RNA isolation kit (Hoerdt, France). Intact roots, comprising 8–10 pooled seedling roots (minus the root tips), were first ground in liquid nitrogen before suspension in the RNA extraction buffer. In the case of the fractured root hair-enriched powder in the 50 ml tube (derived from 50–80 seedlings), the powder was directly suspended in RNA extraction buffer added to the tube. After vortexing and a brief centrifugation, extraction buffer with root hairs was then transferred to an Eppendorf tube for further processing. In all cases genomic DNA was removed via on-column DNase treatment following basic kit instructions. The absence of genomic DNA contamination was confirmed by PCR amplification of rip1 (Cook et al., 1995
) sequences using primers positioned on either side of an intron sequence (data not shown). The DNA-free RNA samples were quantified and RNA integrity was checked on a 1% (w/v) agarose gel. First-strand cDNA synthesis with 0.7–1 µg of total RNA was performed with an anchored oligodT (17T+V) and Superscript II reverse transcriptase (Invitrogen, Renfrewshire, UK) following the manufacturer's protocol. 30 ng of an in vitro transcribed nebulin RNA was included in each first strand cDNA reaction in order to control the efficiency of the reverse transcriptase.
Real time quantitative reverse-transcriptase-PCR (qRT-PCR)
qRT-PCR reactions were performed with the Light Cycler Fast Start Reaction Mix MasterPLUS SYBR Green I (Roche, Mannheim, Germany) on a Roche light cycler real time PCR machine according to the manufacturer's instructions. Each qRT-PCR reaction was carried out with 2 µl of a 1/40 or 1/20 (v/v) dilution of the first cDNA strand, with 0.5 µM of each primer in a total reaction volume of 10 µl. The cycling conditions were the following: 95 °C for 9 min followed by 45 cycles of denaturation at 95 °C for 5 s, annealing at 55–60 °C for 10 s, and extension at 72 °C for 10–30 s. At the end of the PCR cycles, PCR amplification specificity was verified by the analysis of a dissociation curve generated by heating the samples from 55–60 °C to 95 °C, as well as by resolving PCR products on 1.5% agarose gels and subsequent purification and sequencing of the corresponding PCR products. Primer sequences as well as the annealing temperatures used to amplify the endogenous MtENOD11, pMtENOD11-gusA, ENOD40, MtGS1a, elongation factor EF1-
, and nebulin transcripts are listed in Table 1. PCR fragments corresponding to each gene were cloned into pGEM-T (Promega, Charbonnières-les-Bains, France), sequenced, and serial dilutions of these plasmids were used to generate a calibration standard curve, where CT (Cycle Threshold) values are plotted as a function of known standard concentrations. The transcript concentration for each sample was calculated based on these standard curves. A negative control reaction without template was always included for each primer combination. Transcript levels for each of the target genes (MtENOD11, pMtENOD11-gus, ENOD40, MtGS1a) were normalized to the endogenous elongation factor EF1- transcript level. For each sample analysed, results represent the mean of values obtained from at least two independent qRT-PCR reactions, and from at least three independent biological experiments.
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Microscopic methods
Root systems or isolated root hairs were observed with a stereomicroscope (Leica Microsystems, Wezler, Germany), a light microscope (Axiophot, Carl Zeiss, Oberkochen Germany) and/or a CCD camera M-1300-HS (Princeton Instruments, Evry, France). Intact root segments or isolated root hairs were stained with methylene blue as previously described (Vasse and Truchet, 1984
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Results |
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A rapid procedure for RNA isolation from Medicago truncatula root hairs responding to Nod factors
To purify large quantities of root hairs from M. truncatula roots responding to Nod factors, the first objective was to establish growth conditions allowing root development with abundant root hairs competent to respond to Nod factors. To facilitate this, use was made of transgenic M. truncatula plants harbouring the pMtENOD11-gus reporter gene, a particularly useful marker to follow specific Nod factor-activated gene expression in the root hair epidermis (Charron et al., 2004
). It was found that root growth and root hair production could be optimized by growing seedlings vertically on a paper support laid on agar in a square Petri dish (Fig. 1A, B; see Materials and methods). The advantage of these growth conditions is that roots grow rapidly, respond well to Nod factor treatment and Rhizobium inoculation, and do not stick to the paper, allowing easy removal of roots and associated hairs.
In addition to the Nod factor-elicited epidermis-specific expression, the pMtENOD11-gus gene is also transcriptionally activated in root cap cells and during the formation of lateral root primordia (Journet et al., 2001). Thus, in order to be able to evaluate Nod factor-specific MtENOD11-related transcript changes in root tissues, young seedlings were used at a developmental stage prior to lateral root primordia formation and the root cap was systematically removed before tissue processing.
In vitro-grown seedlings were tested for root hair isolation using a variety of freezing and fracturing techniques (Gerhold et al., 1985; Ramos and Bisseling, 2003). It was found that virtually pure preparations of root hairs could be obtained by gentle, repeated vortexing of 8–10 frozen M. truncatula seedling roots grouped together in a single 50 ml Falcon tube (Fig. 1C, D; Materials and methods). Root debris could easily be removed after fracturing of root hairs with the help of long forceps. The use of relatively mild vortex conditions resulted in preferentially sectioning at the basal portion of root hairs, and thus allowed the recovery of root hairs which do not appear to be contaminated with other cell types (Fig. 1E, F, G). Vortexing fewer than eight roots together or using younger seedling roots resulted in a high proportion of fragmented root debris which was more difficult to separate from the root hair fraction. Of particular interest, it was found that this method preferentially fractures growing root hairs, corresponding to the root zone which responds to Nod factors (Gage, 2004). It was also observed that many of the root hairs associated with the older region of the root remain attached to the main root, and are therefore eliminated with the root debris (Fig. 1D, F).
Total RNA was then extracted from the fine powder which accumulates at the bottom of the Falcon tube, comprising essentially fractured root hairs (Fig. 1G). As shown in Table 2, an average of 2 µg of total RNA could be extracted from a total of 60–70 plants, representing a yield of approximately 30 ng of root hair RNA per root. Although at least 1 µg of total RNA was reproducibly obtained from 70 seedlings, the variability observed in RNA yields between experiments is probably related to the efficiency of root hair recovery after fracturing.
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Increased transcript levels of nodulin genes in enriched root hair samples
To assess the efficiency of the method described here to generate RNA preparations enriched for root hair-specific transcripts, real time quantitative RT-PCR was used to measure the relative transcript levels of selected M. truncatula genes (Table 1) in isolated root hairs compared with intact root segments. These analyses were performed with RNA extracted from fractured root hairs or from intact root segments (minus the root cap) of plants that had been treated with either 10–8 M Nod factor or control (H2O) solutions. The elongation factor EF1-
gene (Curie et al., 1993), which is expressed at similar levels in different tissues and not induced by Nod factors, was used as an internal control to normalize transcript levels of the target genes listed in Table 1. To evaluate the enrichment of the root hair fractions for epidermal-specific transcripts, use was made of transgenic M. truncatula plants harbouring the Nod factor-activated pMtENOD11-gus gene fusion, thereby allowing both endogenous MtENOD11 and chimeric gus transcript levels to be quantified in the same RNA sample. As shown in Fig. 2A and B, Nod factor treatment resulted in a 26-fold and a 14-fold increase in MtENOD11 and chimeric gus transcript levels, respectively, in intact root segments. Significantly, these Nod factor-dependent increases were substantially higher for both transcripts in purified root hair cell preparations (121-fold and 97-fold, respectively). These results show that the RNA extracted from root hair fractions, prepared using the method described here, is enriched 5–8-fold for specific Nod factor-induced transcripts. As a consequence, the ability to detect specific gene activation in root hairs is considerably enhanced using RNA isolated from root hair fractions, compared with RNA extracted from intact root samples (Fig. 2A, B).
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As further controls, transcript levels of two M. truncatula genes not expected to be induced by Nod factors in root hairs were also analysed. In Medicago, both the glutamine synthetase MtGS1a gene (Carvalho et al., 2000
) and the ENOD40 gene are constitutively expressed in root vascular tissues and, in the case of ENOD40, transcription is also activated in inner cortical and pericycle root tissues following Nod factor treatment (Minami et al., 1996; Fang and Hirsch, 1998). As shown in Fig. 2C, MtGS1a transcripts were predominantly found in RNA preparations from intact root tissues, with 57-fold lower levels in root hair-enriched RNA. As expected, MtGS1a transcript levels were not influenced by Nod factor treatment. These results provide additional evidence that our root hair preparations are not significantly contaminated with inner root tissues. In the case of MtENOD40, these results show that overall transcript levels were not significantly modified following Nod factor treatment in RNA extracted from intact root segments (Fig. 1D). On the other hand, and to our surprise, a very substantial increase (36-fold) in MtENOD40 transcript levels was observed in the root hair-enriched RNA extracts from Nod factor-treated roots. This result was confirmed in three independent biological experiments. Not only does this finding clearly indicate that MtENOD40 is also activated in root hairs during the early stages of the symbiotic association, but it provides an excellent example of a gene whose tissue-specific induction has been overlooked in a global root analysis of gene expression.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this paper, a simple, rapid procedure for isolating highly-enriched root hair fractions of the model legume M. truncatula is presented, and which is applicable to the analysis of gene expression changes elicited in response to Rhizobium Nod factors. This method, based on the fracture of frozen root hairs, preferentially isolates growing root hairs, the privileged site of the symbiotic interaction between rhizobia and the host plant root. An average of 2 µg of total RNA was extracted from root hair fractions derived from 70 seedling roots, which is more than sufficient to perform real time quantitative RT-PCR. Unfortunately, yields for other published protocols are unavailable for comparison.
This study's experiments have demonstrated clearly that transcripts of the epidermal-specific endogenous MtENOD11 gene and the pMtENOD11-gus reporter are both significantly (5-fold and 8-fold) more abundant in RNA from the enriched root hair fraction compared with RNA extracted from intact root segments (Fig. 2A, B), thus validating the enrichment for epidermal-specific transcripts. This is the first time that it has been possible to quantify Nod factor-dependent root hair-specific gene activation precisely. These results reveal firstly that background transcript levels for ENOD11 (and the chimeric construct) are extremely low and, secondly, that the high level of induction (approximately 100-fold) is similar for both the endogenous ENOD11 and the chimeric promoter-gus reporter genes in isolated root hair samples. These data clearly validate the use of the pMtENOD11-gus reporter for evaluating Nod factor-elicited MtENOD11 induction in root hairs (Charron et al., 2004). Differences between the fold-induction of the endogenous MtENOD11 and the chimeric pMtENOD11-gus genes may either be due to the difficulty in quantifying the very low MtENOD11 transcript levels in control tissues, or alternatively to differences in stability between the endogenous and chimeric transcripts.
Although the method described in this paper was developed primarily for purifying root hair-specific RNA, preliminary studies have shown that this method, employing small numbers of seedlings, is also suitable for obtaining root hair-specific protein extracts, thus enlarging the possible applications for this new procedure.
The use of root hair-enriched RNA extracts revealed a novel and unexpected induction of the MtENOD40 gene in the root epidermis following 18 h treatment with Nod factors (Fig. 2D). This suggests that, in addition to a role in nodule organogenesis in inner root tissues, MtENOD40 may also be involved in Nod factor signalling in root hairs. In situ hybridization experiments have previously shown that ENOD40 transcripts are localized in dividing cortical or pericycle cells following rhizobia or Nod factor treatments (Minami et al., 1996; Catoira et al., 2000). Bearing in mind that in situ hybridization is a relatively low sensitivity approach and that root hairs are delicate cells which are often poorly preserved during tissue fixation it is possible that root hair-specific ENOD40 expression was overlooked in these experiments. Interestingly, the use of transgenic alfalfa (M. sativa) expressing a pENOD40-gus gene fusion did reveal a weak, but nevertheless detectable, Nod factor-induced activation of MtENOD40 in root hairs (Fang and Hirsch, 1998). ENOD40 is also constitutively expressed in root vascular tissues (Fang and Hirsch, 1998), resulting in a high background level in untreated intact root samples (Fig. 2D). It is presumed that this high background level and the relatively short period of Nod factor treatment are responsible for the failure to observe detectable induction of MtENOD40 expression in RNA extracted from intact root segments (Fig. 2D). This result underlines the advantage of using cell-specific-RNA preparations to study exogenous signal-induced gene activation in complex plant organs such as roots. From this perspective, it is anticipated that this straightforward non-labour-intensive root hair isolation technique should be extremely useful in determining epidermal-specific expression profiles for new genes uncovered by global transcriptomic analyses, and, in particular, potential regulators such as transcription factors for which induction levels are generally too low to be detected in intact root preparations. This simple procedure is also convenient for generating root hair-specific cDNA libraries corresponding to a variety of growth conditions or treatments, and of course may, in addition, be adapted for use with other plants.
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Acknowledgements |
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We would like to thank Etienne Journet for careful reading of the manuscript and Julie Cullimore for advice concerning the MtGs1a gene, as well as Fabienne Maillet and Jean Dénarié for providing the purified Nod factors. Dorothée Charron kindly supplied the transgenic 416 seeds harboring the pMtENOD11-gus fusion, and Fikri El Yahyaoui provided in vitro transcribed nebulin RNA.
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Footnotes |
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* Present address: Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0116, USA.
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