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AP2-ERFTranscriptionFactorsMediateNodFactor–DependentMt ENOD11 Activation in Root Hairs via a Novelcis-Regulatory Motif W

Andry Andriankaja,a,1,2 Aurelien Boisson-Dernier,a,1,3 Lisa Frances,a Laurent Sauviac,a Alain Jauneau,b

David G. Barker,a and Fernanda de Carvalho-Niebela,4

a Laboratory of Plant Microbe Interactions, Centre National de la Recherche Scientifique–Institut National de la Recherche

Agronomique, BP 52627, Castanet-Tolosan Cedex, Franceb Institut Federatif de Recherche 40 Pole de Biotechnologie Vegetale, BP17 Auzeville, 31326 Castanet-Tolosan, France

Rhizobium Nod factors (NFs) are specific lipochitooligosaccharides that activate host legume signaling pathways essential for

initiating the nitrogen-fixing symbiotic association. This study describes the characterization of cis-regulatory elements and

trans-interacting factors that regulate NF-dependent and epidermis-specific gene transcription in Medicago truncatula.

Detailed analysis of the Mt ENOD11 promoter using deletion, mutation, and gain-of-function constructs has led to the

identification of an NF-responsive regulatory unit (the NF box) sufficient to direct NF-elicited expression in root hairs. NF box–

mediated expression requires a major GCC-like motif, which is also essential for the binding of root hair–specific nuclear

factors. Yeast one-hybrid screening has identified three closely related AP2/ERF transcription factors (ERN1 to ERN3) that are

able to bind specifically to the NF box. ERN1 is identical to an ERF-like factor identified recently. Expression analysis has

revealed that ERN1 and ERN2 genes are upregulated in root hairs following NF treatment and that this activation requires a

functional NFP gene. Transient expression assays in Nicotiana benthamiana have further shown that nucleus-targeted ERN1

and ERN2 factors activate NF box–containing reporters, whereas ERN3 represses ERN1/ERN2-dependent transcription

activation. A model is proposed for the fine-tuning of NF-elicited gene transcription in root hairs involving the interplay between

repressor and activator ERN factors.

INTRODUCTION

Legumes have the unique capacity to establish symbiotic asso-

ciations with nitrogen-fixing soil bacteria known as rhizobia,

thereby facilitating growth in nitrogen-limiting soils. This endo-

symbiotic interaction leads to the formation of specialized root

organs known as nodules, within which rhizobia reduce atmo-

spheric nitrogen for the benefit of the host plant. Rhizobial

infection in temperate legumes such as Medicago and Lotus is

usually initiated in root hairs and is concomitant with nodule

organogenesis in the root cortex. Rhizobial attachment to the tip

of a growing root hair induces curling around the entrapped

bacteria, which subsequently enter the plant cell via a plasma

membrane invagination and the formation of an intracellular

tubular structure called the infection thread. Bacteria divide

within the infection thread, which progresses through the outer

root cortex toward the newly formed nodule primordium, within

which bacteria are released into host cells and subsequently

differentiate into nitrogen-fixing bacteroids (Timmers et al., 1999;

Gage, 2004).

The success of this symbiotic interaction requires specific and

reciprocal signaling between the two partners. In response to

flavonoids secreted by host roots, rhizobia produce specific

lipochitooligosaccharide signal molecules called Nod factors

(NFs) that play a pivotal role in recognition and controlled host

root infection (D’Haeze and Holsters, 2002). Purified NFs are

sufficient to induce many of the early plant symbiotic responses

normally observed during preinfection stages of the interaction.

These include rapid ion fluxes, membrane depolarization, spe-

cific calcium oscillations, root hair morphogenetic changes, and

specific activation of early nodulin genes (Geurts et al., 2005;

Oldroyd et al., 2005; Stacey et al., 2006).

The specificity and subnanomolar activity of NFs argue that

receptors mediate NF signal transduction pathways in host roots.

Based on rhizobial genetic studies, it has been proposed that an

initial signaling receptor pathway is required for early preinfection

responses, followed by a second entry receptor pathway that is

necessary for bacterial root infection and involving a different

specificity for NF recognition (Ardourel et al., 1994). Genetic

studies conducted with the model legumes Lotus japonicus and

Medicago truncatula have led to the identification and cloning of

host genes essential for early NF perception/transduction (Geurts

1 These authors contributed equally to this work.2 Current address: The Samuel Roberts Noble Foundation, 2510 SamNoble Parkway, Ardmore, OK 73401.3 Current address: Division of Biological Sciences, University ofCalifornia, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0116.4 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Fernanda deCarvalho-Niebel ([email protected]).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.107.052944

The Plant Cell, Vol. 19: 2866–2885, September 2007, www.plantcell.org ª 2007 American Society of Plant Biologists

et al., 2005; Oldroyd et al., 2005; Stacey et al., 2006). These

studies have revealed that NFs are likely to be perceived via LysM

domain–containing receptor kinases such as L. japonicus NFR1/

NFR5 and M. truncatula NFP (Madsen et al., 2003; Radutoiu et al.,

2003; Arrighi et al., 2006). Following perception, NF signal trans-

duction requires a number of essential genes encoding putative

ion channels (DMI1/CASTOR/POLUX) (Ane et al., 2004; Imaizumi-

Anraku et al., 2005), LRR receptor–like kinases (DMI2/SYMRK/

NORK) (Endre et al., 2002; Stracke et al., 2002), and nucleoporins

(NUP85 and NUP133) (Kanamori et al., 2006; Saito et al., 2007), all

acting upstream of the NF-induced calcium spiking response in

root hairs (Miwa et al., 2006). This specific calcium signal is

potentially decoded by the calcium- and calmodulin-dependent

kinase DMI3 (Levy et al., 2004; Mitra et al., 2004a), which is

required for subsequent signaling steps that lead to the tran-

scriptional activation of early nodulin genes (Charron et al., 2004;

Mitra et al., 2004b).

Although significant progress has been made in deciphering

the primary signal perception/transduction components of NF

signaling, little is known about the mechanisms leading to

transcriptional gene activation in response to NFs. Genetic

studies have identified two GRAS-type transcription regulators,

NSP1 and NSP2, lying downstream of DMI3 and required for

subsequent NF-induced gene expression in both Medicago and

Lotus (Kalo et al., 2005; Smit et al., 2005; Heckmann et al., 2006;

Murakami et al., 2007). Although a direct transcriptional regula-

tory function has not yet been demonstrated for these factors, it

is proposed that they could be coactivators of other DNA binding

transcription factors (Udvardi and Scheible, 2005). Most re-

cently, a genetic approach has identified an ERF transcription

factor named ERN (for ERF Required for Nodulation) that is

necessary for NF-elicited gene activation and could potentially

be involved in the direct transcriptional activation of early nodulin

genes (Middleton et al., 2007).

In addition to genetic approaches, the direct identification of

NF-responsive cis-regulatory elements and trans-acting factors

should also contribute to elucidating the molecular mechanisms

regulating NF-elicited gene expression. To this end, we have

made use of the well-characterized M. truncatula Mt ENOD11

gene as a model to determine the molecular mechanisms of

NF-dependent gene regulation (Charron et al., 2004). Mt ENOD11,

which encodes an atypical repetitive Pro-rich protein, is a widely

used marker gene for both the early preinfection and subsequent

infection stages of the symbiotic association (Journet et al.,

2001). During preinfection stages, Mt ENOD11, along with Mt

ENOD12 and rip1, are the earliest M. truncatula genes charac-

terized to date that are specifically activated in root hairs in

response to NFs (Journet et al., 1994; Cook et al., 1995; Charron

et al., 2004). NF-elicited Mt ENOD11 transcriptional activation in

the root epidermis is both rapid and strong and is dependent on

the genetically defined NFP/DMI/NSP NF signaling pathway

(Catoira et al., 2000; Ben-Amor et al., 2003; Oldroyd and Long,

2003; Charron et al., 2004; Mitra et al., 2004b; Sauviac et al.,

2005).

In a previous article, we showed that the 411-bp Mt ENOD11

promoter sequence directly upstream of the start codon is suf-

ficient to confer both preinfection (NF-mediated) and infection-

related expression (Boisson-Dernier et al., 2005). By contrast,

the �257-bp truncated promoter is only capable of driving

infection-related expression. This finding suggested that the Mt

ENOD11 regulatory sequences required for early NF-elicited

activation lie within the �411 to �257 promoter region and can

be dissociated from the infection-responsive regulatory region.

In this study, we have performed a detailed analysis of the �411

to �257 Mt ENOD11 promoter region using deletion, point

mutation, and gain-of-function approaches. This has led to the

definition of a novel NF-responsive regulatory unit called the NF

box and associated regulatory motifs that are necessary and

sufficient to confer NF-induced activation in root hairs. Yeast

one-hybrid screening of root hair–specific cDNA libraries has

identified three NF box binding AP2/ERF transcription factors,

including the recently described ERN factor essential for nodu-

lation (Middleton et al., 2007). These transcription factors, named

ERN1, ERN2, and ERN3, are highly expressed in root hairs and

upregulated by NFs only in the presence of a functional NFP-

dependent signaling pathway. Importantly, ERN1 and ERN2 act

as transcriptional activators, while ERN3 acts as a putative

repressor of NF box–containing target reporters in transiently

expressing Nicotiana benthamiana cells. These results led us to

propose that ERN factors are key players in the transcriptional

regulation of NF-activated early nodulin genes operating via a

complex regulatory mechanism involving both repressor and

activator factors.

RESULTS

The 2411 to 2358 Mt ENOD11 Promoter Region Mediates

NF-Elicited Root Hair Expression

In order to characterize cis-regulatory sequences required for

NF-elicited expression in root hairs, we performed a detailed

functional analysis of the �411 to �257 Mt ENOD11 promoter

region fused to the b-glucuronidase (GUS) reporter gene in

transgenic M. truncatula roots. Because NF-elicited gene acti-

vation is restricted to a specific zone of the root epidermis

(Journet et al., 2001), a quantitative measurement of GUS activity

in entire root extracts frequently underestimates tissue-specific

GUS expression in root hairs. For this reason, each construct

was analyzed by both fluorimetric and histochemical GUS

assays using a large number of independent transgenic roots

as described (see Methods) (Boisson-Dernier et al., 2005). In

transgenic roots carrying a 2.3-kb Mt ENOD11 promoter fused to

the GUS reporter gene (PMt ENOD11-GUS), histochemical GUS

activity is totally absent in root hairs of control water-treated

roots, whereas a basal activity level is observed in lateral root

primordia (Journet et al., 2001; Charron et al., 2004; Boisson-

Dernier et al., 2005; Sauviac et al., 2005). This nonsymbiotic

expression, detected by fluorimetric assays, has proved useful

as an internal control in this study.

Figure 1A illustrates the results of fluorimetric assays per-

formed on a series of 59 promoter deletions between positions

�411 and�257 of the Mt ENOD11 promoter. While GUS activity

corresponding to basal expression in nonepidermal root tissues

was similar for all deletions studied, the capacity to drive

NF-elicited expression was dramatically reduced in the �367E11

NF Regulation of Mt ENOD11 by ERF Factors 2867

Figure 1. Identification of a 54-bp Nod Factor–Responsive Element (NFE) within the Mt ENOD11 Promoter Sufficient to Drive Root Hair–Specific

Expression.

GUS activity driven by Mt ENOD11 59 promoter deletions and gain-of-function constructs fused to the GUS reporter gene were analyzed in transgenic

M. truncatula roots.

(A) Schematic representation of Mt ENOD11 59 promoter deletion fusions. Numbers above the promoters indicate the positions of 59 deletions relative

to the translation initiation codon ATG. Fluorimetric GUS activities were quantified in root extracts following either water (white bars) or NF (10�9 M) (gray

bars) treatment. The percentage values refer to the proportion of NF-induced GUS activity (i.e., after subtraction of the water control value) of a given

construct in relation to the�411E11 reference construct. Asterisks indicate statistically significant differences (P < 0.05, Student’s t test) compared with

the reference �411E11 construct (see Supplemental Table 1 online).

(B) Fluorimetric analyses of reporter GUS activities driven by the gain-of-function 4xNFE construct and the control P35S min construct following NF

treatments (10�9 M). The asterisk indicates a statistically significant difference (P < 0.05, Student’s t test) compared with the reference P35S min

construct.

Error bars represent 6SD of mean activity values derived from three independent experiments performed with n number of individual roots.

2868 The Plant Cell

deletion. This indicates that sequences between�411 and�367

are important for NF-elicited gene activation in root hairs.

Furthermore, the fact that the �411E11 promoter deletion

lacking sequences between �357 and �258 was still capable

of driving NF-elicited gene activation (data not shown) suggests

that this region is not essential for the NF-elicited response.

To determine whether the�411 to�357 Mt ENOD11 promoter

region can, by itself, direct NF-elicited epidermal expression in a

heterologous context, a gain-of-function construct comprising a

tetramer of this sequence named NFE (for Nod Factor–Responsive

Element) fused to the �47-bp cauliflower mosaic virus (CaMV)

35S minimal promoter (here named P35S min) (Pontier et al.,

2001) was analyzed in transgenic M. truncatula roots. A tetramer

of NFE was used in this gain-of-function approach, since multimers

have been shown to confer expression to minimal promoters

more efficiently than monomers, probably by compensating for

the absence of general positive regulatory elements (Wu et al.,

1998; Pontier et al., 2001; Rushton et al., 2002). In water-treated

samples, transgenic roots carrying the 4xNFE construct exhib-

ited a higher basal GUS activity than roots carrying the P35S min

construct, suggesting that sequences within 4xNFE can act as

general positive transcriptional enhancers. Following NF appli-

cation, control roots with the P35S min construct showed no

induction of GUS activity in the root epidermis, whereas roots

carrying 4xNFE exhibited clear NF-elicited GUS activity in root

hairs (Figure 1B), although at lower levels than the reference

�411E11 construct (Figure 1A). This gain-of-function promoter

analysis demonstrates that the �411 to �357 NFE sequence is

sufficient to confer NF-elicited root hair–specific expression.

NF-Mediated Gene Activation Requires a Conserved

GCC-Like Motif

In order to identify putative cis elements within the NFE region

that could be involved in NF-regulated gene expression, we

searched for conserved sequences within NFE that are common

to other NF-responsive gene promoters. Comparison between

NFE and 59 upstream sequences of two other epidermal

NF-activated genes, Mt ENOD12 (Journet et al., 1994) and Mt

ENOD9 (D. Barker, unpublished data), allowed us to identify

NFE-homologous regions within both promoters (Figure 2A).

Promoter deletion analysis further revealed that truncated�534-

bp Mt ENOD12 and�552-bp Mt ENOD9 promoters including the

NFE-homologous regions were able to direct NF-responsive

reporter expression, whereas deletions lacking the NFE-homologous

region (�323 bp for Mt ENOD12 and �525 bp for Mt ENOD9)

were no longer responsive to NFs (data not shown). As shown

in Figure 2A, a striking feature of NFE and NFE-homologous

regions is the presence of a perfectly conserved GCAGGCC

motif, which is reminiscent of, but not identical to, GCC box

motifs (AGCCGCC) (Ohme-Takagi and Shinshi, 1995). This con-

served GCC-like motif is located within a particularly AT-rich

region, and in the case of NFE, it is flanked by consensus binding

sites for CAAT binding and DOF transcription factors (Kusnetsov

et al., 1999; Yanagisawa and Schmidt, 1999). Interestingly, DOF

and CAAT box sites are also found in the vicinity of the GCC-like

motif in the NFE-homologous sequences of both the Mt ENOD12

and Mt ENOD9 promoters. In addition, an HD-ZIP–like binding

site overlapping the CAAT box motif and a conserved W box

binding site for WRKY transcription factors can be identified

within the Mt ENOD11 NFE (Figure 2A).

To investigate the importance of the conserved GCC-like motif

for NF-elicited gene activation, we deleted the entire motif from

within the �411E11 promoter region to create the DGCC con-

struct (Figure 2B). As a control, a block deletion of an upstream

G(A/T)-rich sequence, overlapping the W box motif, was also

generated (DG[A/T]). As shown in Figure 2B, block deletion of the

GCC-like motif alone resulted in a significant reduction in the

proportion of transgenic roots exhibiting epidermal NF-elicited

histochemical GUS activity, whereas deletion of the upstream

G(A/T)-rich sequence did not alter NF-induced GUS activity.

Quantitative fluorimetric assays confirmed the drastic effect of

deleting the GCC-like motif in relation to NF-elicited GUS acti-

vation, whereas nonsymbiotic expression in control roots re-

mained unaltered (Figure 2B; see Supplemental Table 1 online).

These results indicate that within NFE, the GCC-like motif and/or

its flanking sequences, including the potential CAAT box and

HD-ZIP–like motif, are important for NF-elicited gene activation.

In order to address the importance of individual cis elements

within the NFE region, base substitutions were introduced at

sites overlapping or in the proximity of the GCC-like motif. For

this purpose, we generated �411E11 derivative constructs mu-

tated in the central GGCC core (GCcore) as well as in the HD-ZIP–

like/CAAT box and DOF sites (see Methods) (Figure 2C). These

experiments confirmed that, as for the block deletion, the spe-

cific mutation of the GCcore (mGCcore) results in a major reduction

(>10 fold) in NF-elicited GUS activity, similar to the levels for

control roots (Figure 2C; see Supplemental Table 1 online).

Although less striking than with the GCcore mutation, a significant

threefold to fivefold reduction of the NF-mediated response was

also observed in roots transformed with the mDOF and mHDZ

constructs, suggesting that these flanking sequences also par-

tially influence the efficiency of the NF-dependent response.

Importantly, none of these point mutations modified the non-

symbiotic GUS activity levels in roots (Figure 2C; see Supple-

mental Table 1 online).

Taken together, these results underline the importance of the

GCC-like GCAGGCC motif with its GCcore sequence for NF-

elicited expression in root hairs. This central GC-rich sequence

may function in cooperation with neighboring cis elements,

which together form a fully functional regulatory unit. This

particular combination of cis motifs, therefore, could constitute

a promoter signature for NF-elicited gene expression in root

hairs.

The NF Box Defines a Novel Regulatory Unit Sufficient for

NF-Elicited Gene Activation

To further evaluate to what extent these motifs are sufficient to

direct NF-elicited expression in root hairs, additional gain-of-

function experiments were performed. For this, tetramers of two

promoter fragments of ;30 bp in length derived from either the

59 or the 39 end of NFE were fused to the P35Smin-GUS reporter

(Figure 3A). In agreement with the deletion and point mutation


Figure 2. Functional Analyses of Conserved cis Motifs Involved in NF-Elicited Expression.

(A) Partial sequence alignment of NFE and NFE-like sequences within the Mt ENOD11, -9, and -12 promoters. Asterisks indicate conserved nucleotides

at the given positions. The fully conserved 8-bp GCC-like motif is shaded. Putative W box (GTCA), HD-ZIP–like (AAATAATTG), CAAT box (CAAT or

ATTG), and DOF (TAAAG or CTTTA) sequences are indicated by double underlining, underlining, boldface lettering, and boxing, respectively.

(B) GUS activity of the �411E11 reference and derived constructs block-deleted within the conserved GCC-like and/or G(A/T)-rich motifs of NFE. The

relative strength of root hair–specific GUS activity was visually screened in individual NF-treated roots (see Methods) and is represented as the

percentage of roots exhibiting clear-cut GUS activity in the root hairs. GUS activity levels were also measured by fluorimetric assays in protein extracts

from either water-treated (white bars) or NF-treated (gray bars) roots. The percentage values refer to NF-elicited GUS activities (i.e., after subtraction of

the water control value) relative to that obtained with the �411E11 reference construct.

(C) Quantitative analysis of GUS activity driven by the �411E11 construct and its mutated derivatives. Substituted nucleotides are shown in italics and

underlined. The percentage values refer to NF-induced GUS activities relative to the reference construct.

In (B) and (C), error bars represent 6SD and asterisks indicate significant differences compared with the reference construct (P < 0.05, Student’s t test)

(see Supplemental Table 1 online). n represents the number of individual transgenic roots analyzed.

2870 The Plant Cell

experiments, NF-elicited GUS activity was only observed in roots

carrying the 4xNFE39 construct, which includes the GCC-like,

HD-ZIP–like/CAAT, and DOF motifs (Figure 3B). Interestingly, a

similar level of GUS reporter activity was observed with either the

4xNFE or the 4xNFE39 construct, indicating that NFE39 contains

all of the regulatory elements required for the root hair–specific

NF response (cf. Figures 1B and 3B). Furthermore, following

Sinorhizobium meliloti inoculation, the 4xNFE39 construct driving

the P35Smin-GUS reporter was activated during preinfection

(NF-dependent) symbiotic stages (data not shown). However, as

expected, this construct was unable to drive infection-related

expression in either root tissues or the infection zone of the

nodule (Boisson-Dernier et al., 2005) (see Supplemental Figure

1 online). Nonsymbiotic expression in lateral root primordia was

also absent in roots transformed with this construct (data not

shown). Thus, we conclude that the 30-bp NFE39 sequence,

renamed NF box, constitutes a novel regulatory unit sufficient to

confer NF-dependent root hair–specific expression.

A Yeast One-Hybrid Screen Identifies NF Box

Binding Proteins

Initial experiments to determine whether nuclear proteins can

bind specifically to the NF box regulatory unit were performed

using the gel shift technique. Since experiments with extracts

from whole roots had not been conclusive, we increased the

sensitivity of the gel shift assay using protein extracts from

isolated root hairs. As shown in Figure 4A, a weak but reproduc-

ible NF box–specific retardation signal was observed in these

assays, with a slightly enhanced level in NF-treated root hair

samples. More importantly, competition studies showed that this

retardation signal was dependent on the presence of the GCcore

motif (Figure 4A), thus confirming the importance of this motif

within the NF box for the specific binding of potential trans-acting

factors present in M. truncatula root hairs.

In order to identify the root hair transcription factors mediating

NF-elicited gene activation, a yeast one-hybrid screen was

Figure 3. The NF Box Confers an NF-Specific Response in the Root Epidermis.

(A) Schematic representation of the NFE sequence and associated cis motifs. The sequences NFE59 and NFE39 (NF box) were used to generate the new

gain-of-function constructs tested in (B).

(B) Histochemical and quantitative analyses of GUS activity directed by gain-of-function 4xNFE59 and 4xNFE39 (4xNF box) constructs. Data are

represented as mean values obtained by the analysis of n individual roots from three independent experiments. Error bars represent 6SD. The asterisk

indicates a significant difference compared with the reference construct (P < 0.05, Student’s t test) (see Supplemental Table 1 online). Bars ¼ 200 mm.


Figure 4. Isolation of NF Box Binding Proteins.

(A) Band shift experiments using an NF box–containing DNA probe with root hair protein extracts from 6-h water- or NF-treated roots. In competitor lane

reactions, 50 to 100 molar excesses of cold NF box or mNF box probes were included.

(B) Schematic representation of the one-hybrid strategy used to screen cDNA libraries from NF-treated root hairs using a HIS3 reporter gene under the

control of a minimal HIS3 promoter fused to a tetramer of the NF box and expressed in the yeast YM4271 reporter strain. The number of positive clones

corresponding to each of the three cDNA classes (NFbB1, NFbB2, and NFbB3) isolated after two rounds of screening and able to grow on 5 mM 3-AT is

indicated.

(C) Confirmation of the interaction of NFbB1, NfbB2, and NFbB3 with the NF box by retransformation of yeast YM4271 containing the tetramer NF box-

HIS3 reporter with the isolated plasmids expressing GAL4-NFbB fusions and control GAL4 plasmids. Yeast cells were grown on synthetic dropout (SD)

Leu�His� þ 5 mM 3-AT medium.

(D) Binding specificity of NFbB proteins. YM4721 reporter strains carrying tetramer NF box, tetramer NFE, and trimer p53 cis (p53bs) sequences were

transformed with plasmids expressing the mouse GAL4-p53 factor that interacts with the p53 binding site, the NFbBs GAL4-NFbBs proteins, and the

water control (�). Yeast growth was examined under either nonselective SD Leu� conditions (�3-AT) or selective SD Leu�His� conditions on medium

supplemented with 5 mM 3-AT (þ3-AT).

2872 The Plant Cell

performed using the NF box as bait. A YM4271 yeast reporter

strain containing a HISTIDINE3 (HIS3) gene under the control of a

chimeric promoter comprising a tetramer of the NF box se-

quence fused to the HIS3 minimal promoter was used to screen

GAL4-fused cDNA libraries derived from NF-elicited root hairs

(see Methods) (Figure 4B). This NF box reporter yeast strain is

unable to grow on selective medium (without His and supple-

mented with 5 mM 3-aminotriazole [3-AT]), thus allowing efficient

screening. After initially screening 12 3 106 yeast transformants,

67 candidates were selected for a subsequent screening on

more stringent selective medium, followed by retransformation

of yeast with isolated plasmid DNAs. This resulted in 27 con-

firmed positive clones that could be classified into three closely

related groups designated NFbB1 (5 clones), NFbB2 (15 clones),

and NFbB3 (7 clones) for NF box binding proteins (Figure 4B).

To examine the specificity of the interactions of NFbB1,

NFbB2, and NFbB3 with the NF box bait sequence in yeast,

the corresponding plasmids were used to transform individual

yeast strains harboring HIS3 reporters fused to either the tetramer

NF box or the NFE tetramer. As a negative control, we made use

of a trimer of the unrelated p53 human binding site (Matchmaker;

Clontech) (Figures 4C and 4D). Significant yeast growth was only

detected when the three NFbB clones were expressed in the

presence of the NF box or NFE HIS3 reporters, but not with the

p53 HIS3 reporter (Figure 4D). This shows that reporter yeast

growth is dependent on the direct interaction of NFbB1, NFbB2,

and NFbB3 with NF box–containing target sequences.

NF Box–Interacting Factors Belong to the ERF Transcription

Factor Family

Sequence analysis revealed that all positive NFbB clones corre-

sponded to in-frame GAL4AD fusions. NFbB2 and NFbB3

cDNAs are 1098 and 885 bp in length and contain open reading

frames of 313 and 238 amino acids, respectively. Positive NFbB1

cDNA clones (857 bp) are all 59 truncated at approximately the

60-bp position and encode a protein of 248 amino acids missing

the 20 N-terminal residues. This indicates that the 59 truncated

NFbB1 is perfectly able to bind to NF box–containing target

sequences in yeast.

Subsequent BLAST analysis revealed that NFbB1, -2, and -3

all belong to the large family of AP2/ERF transcription factors

(Riechmann and Meyerowitz, 1998). Furthermore, it turns out

that NFbB1 is identical to the symbiosis-associated ERF-like

transcription factor called ERN that was recently characterized

by Middleton et al. (2007). For simplicity, therefore, we have

renamed our three AP2/ERF transcription factors ERN1 (NFbB1),

ERN2 (NFbB2), and ERN3 (NFbB3). ERN1, -2, and -3 all contain

the highly conserved AP2/ERF domain putatively involved in

DNA binding (Wessler, 2005) (Figure 5). ERN1 and ERN2 are the

most closely related and contain, in addition to the conserved

AP2/ERF domain, an identical 22–amino acid stretch at the C

terminus that is absent in the ERN3 sequence (Figure 5A).

Phylogenetic analysis of the conserved ERN AP2/ERF domain

in relation to all known Arabidopsis thaliana family members

(http://datf.cbi.pku.edu.cn) shows that the three ERNs, together

with Arabidopsis RAP2.11, form a distinct group within the ERF

family, as reported for ERN1 (data not shown) (Middleton et al.,

2007). A very high level of sequence conservation (;80 to 90%)

is observed between the AP2/ERF DNA binding domains of all

three ERNs and RAP2.11, while there is only ;50% conservation

between ERN AP2/ERF DNA binding domains and other ERF

proteins such as At ERF1 (Figure 5B).

Importantly, the critical amino acid residues within the AP2/

ERF DNA binding domain of the Arabidopsis At ERF1 factor

required for binding to its target DNA GCC box are only partially

conserved in ERN and RAP2.11 AP2/ERF DNA binding domains

(Figure 5B) (Allen et al., 1998). Therefore, we predict that these

differences are related to the specific binding of ERNs to the

novel NF box GCcore motif.

NF-Elicited Upregulation of ERN Genes in M. truncatula

Root Hairs

To study ERN gene expression in M. truncatula root hairs,

quantitative RT-PCR analysis was performed on RNA isolated

from purified root hairs (Sauviac et al., 2005). As shown in Figure

6A, all three genes are constitutively expressed in untreated

roots and root hairs. In all cases, transcript levels are ;10-fold

higher in root hairs compared with whole root samples. Following

NF treatment, both ERN1 and ERN2 genes are upregulated in

root hairs and in entire roots, while the level of ERN3 transcripts is

not significantly modified. On the other hand, no NF induction of

these genes was detected for the nodulation-defective nfp-2

mutant (Figure 6B). This shows that, as for their target gene Mt

ENOD11, NF-elicited activation of the ERN genes is dependent

on the NFP locus, an essential component of NF perception and

signaling in root hairs (Ben Amor et al., 2003; Arrighi et al., 2006).

To determine whether ERN genes are expressed at later

stages of the nodulation process, quantitative RT-PCR analysis

was performed on RNA extracted from roots and isolated nod-

ules of aeroponically grown plants. Similar ERN1 transcript levels

were observed in noninoculated root samples from plants grown

for 3 d (Figure 6B) and 10 d (Figure 6C) in aeroponic conditions,

while ERN2 and ERN3 showed significantly enhanced transcript

levels in root samples of 10-d aeroponically grown plants. This

upregulation of ERN2 and ERN3 may be associated with the

development of lateral roots, which are not yet visible in 3-d

aeroponically grown plants. During nodulation, the ERN1 tran-

script level is higher in young 4-d-old nodules compared with

roots, whereas ERN2 and ERN3 transcript levels are lower in

nodules compared with roots. However, for the moment, we

cannot rule out that a localized low-level induction in nodules

may be masked by the high basal level of ERN2 and ERN3

expression in roots. Finally, ERN genes display a similar expres-

sion profile in different M. truncatula organs, for which transcript

levels were mainly detected in root and leaf tissues (see Sup-

plemental Figure 2 online).

Nuclear Localization of ERNs in N. benthamiana Cells

To examine the subcellular localization of the ERN transcription

factors, yellow fluorescent protein (YFP) N-terminally fused ERN

proteins under the control of the 35S promoter were analyzed in

N. benthamiana leaves. As expected, controls expressing a 35S-

YFP construct showed fluorescence labeling in both cytoplasmic


strands and nucleoplasm (Figures 7A and 7B). Although ERNs do

not have a typical nuclear localization signal, our experiments

clearly show that YFP-ERN fusion proteins are targeted to the

nucleus of transformed cells (Figures 7C to 7H). This is also the

case for other AP2/ERF proteins (Lee et al., 2004; Krizek and

Sulli, 2006). In addition to the homogeneous nucleoplasm local-

ization observed for all three fusions, the YFP-ERN1 fusion

protein was also frequently associated with subnuclear particles

(Figure 7D). These subnuclear particles were very dynamic, with

an appearance that often varied with time and with a variable

frequency between experiments.

ERN3 Negatively Modulates ERN1/ERN2 trans-Activation of

a Target NF Box Reporter in N. benthamiana Cells

To determine whether ERN1, -2, and -3 proteins are capable of

trans-activating Mt ENOD11 in plant cells, transient expression

assays in N. benthamiana were performed. Leaves were coinfil-

trated with either target 4xNF box or control P35Smin-GUS

reporters in combination with effectors comprising 3HA-tagged

ERN proteins under the control of the 35S promoter (Figure 8A).

As shown in Figures 8B and 8C, ERN1 and ERN2 effectors

specifically trans-activated the target 4xNF box reporter but not

the control P35Smin-GUS reporter. Importantly, ERN1 and

ERN2 effectors deleted for their respective AP2/ERF DNA bind-

ing domains (ERN1D and ERN2D, respectively) were no longer

able to induce the target 4xNF box GUS reporter, indicating that

specific DNA binding is required for trans-activation (Figure 8D).

By striking contrast, the ERN3 effector was unable to activate the

target 4xNF box reporter, despite the accumulation of significant

quantities of the tagged ERN3 protein (Figure 8C; see Supple-

mental Figure 3 online).

To address the question of whether ERN3 can modulate the

trans-activation properties of the ERN1/ERN2 transcriptional

activators, a combination of effector proteins was expressed in

N. benthamiana using the target 4xNF box reporter. Coexpres-

sion of both ERN1 and ERN2 resulted in similar GUS activity

levels to those observed when expressing ERN1 alone (Figure

8E). However, coexpression of ERN3 with either ERN1 or ERN2

led to a significant reduction in GUS activity levels compared with

those obtained with ERN1 or ERN2 (Figure 8E; see Supplemental

Figure 4 online). Strikingly, the ERN3 protein lacking its AP2/ERF

Figure 5. NF Box Binding Proteins Named ERN1, ERN2, and ERN3 Belong to the ERF Transcription Factor Family.

Comparison of the deduced amino acid sequences of the three entire ERN1, ERN2, and ERN3 proteins (A) and the corresponding AP2/ERF DNA

binding domains aligned by ClustalW (Thompson et al., 1997) with those of the related Arabidopsis RAP2.11 proteins (B). Identical amino acid residues

are shaded gray. The conserved AP2/ERF DNA binding domain in (A) is underlined, and the 20–amino acid stretch conserved in ERN1 and ERN2 is

double underlined. In (B), the conserved AP2/ERF DNA binding domain is indicated. The black wavy bar and arrows represent predicted a-helix and

b-sheet regions, respectively, within the AP2/ERF domain of At ERF1 (Allen et al., 1998). Asterisks correspond to conserved amino acid residues

required for DNA binding to GCC box motifs.

2874 The Plant Cell

DNA binding domain (ERN3D) was equally capable of attenuat-

ing the transcriptional activation of the target 4xNF box reporter

by ERN1 (Figure 8E; see Supplemental Figure 4 online). This

suggests that ERN3-mediated repression is an active mecha-

nism that does not absolutely require the DNA binding domain.

DISCUSSION

NF-Elicited Gene Activation in Root Hairs Requires a

Regulatory Unit with a Novel GCC-Like Motif

Mt ENOD11 is a well-characterized molecular marker that has

been widely used to study NF signaling in the root epidermis

(Charron et al., 2004; Miwa et al., 2006; Sun et al., 2006; Marsh

et al., 2007). In this study, we performed a detailed analysis of

the Mt ENOD11 promoter in order to elucidate the molecular

mechanisms of NF-regulated gene expression. A gain-of-function

approach has shown that a 33-bp promoter sequence that we

call the NF box is sufficient to direct specific NF-mediated

expression, which is elicited during the early preinfection stage of

the S. meliloti association. On the other hand, the NF box alone is

not sufficient to confer Mt ENOD11 expression during the later

stage of rhizobial infection, which we have previously shown to

depend upon a proximal 257-bp promoter region lying down-

stream of the NF box (Boisson-Dernier et al., 2005). We propose

that NF box–mediated gene expression is activated through

initial NF perception (via the NFP/DMI/NSP signaling pathway)

and that the infection-related expression requires a second

distinct signaling pathway that is presumably activated at a later

stage before bacterial entry. The NF box sequence is also found

Figure 6. The ERN1, ERN2, and ERN3 Genes Are Upregulated in NF-Treated M. truncatula Root Tissues.

(A) Quantitative RT-PCR for ERN1, ERN2, and ERN3 transcripts in total RNA samples extracted from 3-d in vitro grown wild-type intact roots or isolated

root hairs treated with either water (white bars) or 10�8 M NFs (gray bars) for 6 h.

(B) Quantitative RT-PCR for ERN transcripts in total RNA samples extracted from 3-d aeroponically grown wild-type (A17) or nfp-2 mutant roots treated

with either water (white bars) or 10�9 M NFs (gray bars) for 6 h.

(C) Quantitative RT-PCR for ERN transcripts in total RNA samples extracted from nitrogen-starved 10-d aeroponically grown wild-type roots (N0) or

isolated nodules harvested at 4 d (N4), 10 d (N10), or 14 d (N14) after inoculation.

Values represent averages of either two or three independent biological experiments after normalization against EF1a transcript levels (see Methods).

Multiplication factors above the shaded bars refer to NF:water expression ratios. Error bars represent 6SD.


in the promoters of other NF-responsive M. truncatula genes

such as Mt ENOD12 and Mt ENOD9, which also encode putative

Pro-rich cell wall–associated proteins presumably involved in

root hair cell wall modification during preinfection stages (Pichon

et al., 1992; Journet et al., 2001). It should be noted that the NF

box is not present in the 200-bp promoter region of the pea

(Pisum sativum) Ps ENOD12B gene that Vijn et al. (1995) have

reported to confer NF-elicited expression in dividing cortical cells

of the nodule primordia. We suggest that the NF box may be a

regulatory unit specifically associated with early NF signaling in

the root epidermis. The NF box is also distinct from the recently

identified conserved Root Hair Element comprising the strictly

conserved CACG motif, which is essential for root hair–specific

activation of genes encoding angiosperm cell wall–related pro-

teins (Kim et al., 2006). We thus propose that the NF box acts

through an alternative regulatory pathway and therefore may be

of interest for engineering novel inducible synthetic promoters for

legume-centric research and technology development (Rushton

et al., 2002).

Block deletion and point mutation analysis has revealed that

a GCC-like (GCAGGCC) motif within the NF box is essential for

NF-dependent expression in root hairs and for binding of root

hair–specific nuclear protein factors. This motif is reminiscent

of, although not identical to, the well-characterized GCC box

(GCCGCC) recognized by ERF transcription factors. Despite the

lack of the GCC box consensus sequence, a number of essential

nucleotides for specific protein binding, indicated in boldface in

the GCC box core (GCCGCC) (Hao et al., 1998), can be located

to identical relative positions within the NF box (GCAGGCC).

Additional studies will now be required to determine the DNA

binding specificities of NF box–interacting ERN factors (dis-

cussed in more detail below) for this novel variant of the classical

GCC box.

Mutations within the CAAT box and DOF-like motifs flanking

or partially overlapping the GCC-like motif significantly reduce

NF-responsive gene activation, but not as dramatically as muta-

tions within the GCC-like motif. These flanking or overlapping

nucleotides can contribute to the binding affinity of transcription

factors to the central GC-rich region, as reported previously for

other factors (Tournier et al., 2003; Xue and Loveridge, 2004).

Alternatively, the GCC-like motif may act in cooperation with

neighboring cis motifs to specify expression, as has been

reported for a number of plant genes (Buttner and Singh, 1997;

Riechmann and Meyerowitz, 1998; Diaz et al., 2002; Brown et al.,

2003; Yamamoto et al., 2006). The future characterization of the

core cis-regulatory elements specifically recognized by ERN

factors should make it possible to scan the M. truncatula genome

to discover NF-dependent regulons.

Novel ERF Factors Interacting with the NF Box

Yeast one-hybrid screening using the NF box sequence as bait

resulted in the identification of three closely related AP2/ERF

transcription factors specifically interacting with this NF-responsive

sequence. Interestingly, one of these NF box–interacting ERF

factors is identical to the ERN transcription factor very recently

identified by a genetic approach (Middleton et al., 2007). The

ERN deletion mutant bit1-1 is defective in root infection and

nodulation as well as in NF-elicited expression of Mt ENOD11 in

root hairs. The three NF box–interacting factors, now named

ERN1 (previously named ERN), ERN2, and ERN3, all contain the

typical AP2/ERF DNA binding domain and belong to the same

phylogenetically related group V of the ERF subfamily that

includes Arabidopsis RAP2.11 (Okamuro et al., 1997; Nakano

et al., 2006; Middleton et al., 2007). We have shown here that

Figure 7. Nuclear Localization of ERN1-, ERN2-, and ERN3-YFP Fusion

Proteins.

Confocal images of epidermal N. benthamiana cells expressing control

YFP ([A] and [B]), YFP-ERN1 ([C] and [D], YFP-ERN2 ([E] and [F]), and

YFP-ERN3 ([G] and [H]) fusion proteins under the control of the 35S

promoter. Note the exclusive nuclear localization for the ERN proteins in

the nucleoplasm ([C] and [E] to [H]) or in discrete nuclear bodies (D)

compared with the cytoplasm (arrows) and the nucleus-localized YFP

control ([A] and [B]). In all panels, chloroplast fluorescence appears red.

Similar results were obtained with green fluorescent protein (GFP) fusion

proteins (data not shown).

2876 The Plant Cell

Figure 8. Transactivation and Repressor Properties of ERN Factors in N. benthamiana Cells.

(A) Schematic representation of the 3xHA-tagged ERN effector proteins and the target GUS reporters used for trans-activation studies in A.

tumefaciens–infiltrated N. benthamiana cells. The P35Smin-GUS reporter construct alone or fused to the tetramer of the NF box (4xNF box) was used as

target GUS reporter. The effector constructs were under the control of the 35S promoter (P35S), and all contained a 3xHA 59 tag upstream of the ERN

sequences, deleted (ERN1D, ERN2D, and ERN3D) or not (ERN1, ERN2, and ERN3), for their respective AP2/ERF (AP2) DNA binding domains.

(B) to (E) Histochemical and fluorimetric GUS activities of N. benthamiana leaf discs at 24 h following infiltration with the reporters alone (�) or in

combination with the different effectors. ERN1 to -3 or ERN1D to -3D effectors were coinfiltrated with P35Smin (B) or 4xNF box ([C] and [D]) target GUS

reporters. Transcriptional activation of the 4xNF box reporter in (E) was measured following expression of individual ERN1 or ERN3 effectors or

combined expression of ERN1/ERN2, ERN1/ERN3, or ERN1/ERN3D effectors. GUS activity levels were measured using 1 mg of total protein extracts.

Error bars represent 6SD of mean GUS activity values derived from either three or four ([B] to [D]) or seven to nine (E) independent experiments. In (E),

data are compiled from measurements of 15 to 18 individual samples that were used for Student’s t test statistical analysis. Asterisks indicate

statistically significant differences (P < 0.05, Student’s t test) compared with the reference ERN1 effector construct (see Supplemental Table 2 online).


ERN factors bind specifically to the NF box sequence, which

contains the essential GCC-like sequence motif (GCAGGCC).

Although >107 yeast clones were screened for NF box binding, it

is significant that we were only able to identify the three ERN

factors, despite the fact that other AP2/ERF clones are repre-

sented in the root hair–specific cDNA libraries used for the one-

hybrid screen (F. de Carvalho-Niebel and A. Niebel, unpublished

data). Taken together, these results argue that ERN-type

transcription factors specifically recognize the atypical GCC-

like motif within the NF box.

Detailed structural information concerning the interaction be-

tween the transcription factor and its target DNA is only available

for a few members of the AP2/EREBP family. NMR analysis of the

solution structure of Arabidopsis ERF1 bound to the GCC box

has revealed that an antiparallel three-stranded b-sheet binds

within the DNA major groove (Allen et al., 1998). This interaction

involves direct base contacts with a total of seven amino acids

present in the b-sheet of the conserved AP2 domain of ERF1

(Arg-150, Arg-152, Trp-154, Glu-160, Arg-162, Arg-170, and Trp-

172). The AP2 domain of the three ERN factors shares a high level

of amino acid identity with that of RAP2.11 but significantly less

with ERF1 (Figure 5B). A comparison between the AP2 domains

of ERF1 and ERN factors reveals amino acid substitutions at two

key positions involved in DNA binding (Trp-154/Ser and Arg-

162/Lys). We propose that these amino acid substitutions

modify the binding specificity of the ERN factors and presumably

correlate with the atypical NF box GCC-like motif. For example,

Fukazawa et al. (2000) showed that the substitution of a highly

conserved Arg residue by Lys changes the optimal binding site

of the yeast bZIP protein GCN4 from the palindromic site

(GACGTC) to the pseudopalindromic site (TGACTCA).

We have demonstrated that the ERN genes are preferentially

expressed in root hairs (10-fold higher compared with the whole

root) and that in ERN1 and ERN2 are significantly upregulated

(7.3- and 3.5-fold, respectively) following the application of

purified NFs. Importantly, NF-elicited ERN gene induction is

dependent on the NFP genetic locus, an essential component of

the NF signaling pathway in the root epidermis, which likely

encodes a component of the NF receptor (Ben Amor et al., 2003).

Thus, in agreement with similar findings by Middleton et al.

(2007), our data support the proposition that ERN factors con-

stitute novel downstream components of the epidermal NF

signaling pathway. Interestingly, we note that ERN factors and

the Arabidopsis ortholog RAP2.11 appear to be preferentially

expressed in root hairs and the differentiation zone of the root

epidermis (this study and Genevestigator Gene Atlas; Zimmermann

et al., 2004). Furthermore, WIN1/SHN1, another member of the

group V Arabidopsis ERF factors, is involved in epidermal surface

cuticle biosynthesis and cell wall structural modification (Aharoni

et al., 2004; Broun et al., 2004). It is conceivable, therefore, that

ERNs may have been recruited from ERF regulatory proteins that

function in processes associated with root epidermal cell differ-

entiation.

ERN Factor Interplay May Coordinate NF-Elicited ENOD

Gene Activation

As a complement to the yeast one-hybrid–based in vivo system,

we studied the transactivation properties of individual ERN

factors using a heterologous plant transient expression assay.

We have shown that ERN-GFP/YFP fusion proteins transiently

expressed in N. benthamiana cells localize to the nucleus,

despite the fact that typical nuclear localization signal domains

are not present in the predicted protein sequences. It is possible

that basic amino acid clusters are responsible for nuclear tar-

geting of ERN factors, as reported for other AP2/ERF proteins

Figure 9. Model for ERN Factor–Mediated Regulation of Early Nodulin Gene Expression in M. truncatula Root Hairs.

In the absence of the rhizobial partner, we propose that constitutive binding of ERN3 to the NF box sequence represses Mt ENOD11 gene expression

in the root epidermis (A). During the early preinfection stages of the symbiotic association (B), NFs secreted by S. meliloti are perceived and transduced

by the NFP-dependent NF signaling pathway. Transcription activation and accumulation of the ERN1/2 activators, probably accompanied by

modifications of ERN3 protein levels/activities, lead to transactivation of the Mt ENOD11 gene in root hairs via the NF box regulatory unit (B). Upon

bacterial root infection, de novo accumulation of active ERN3 presumably leads to the progressive repression of ERN1/ERN2 transcription activities.

2878 The Plant Cell

(Chen et al., 2003; Krizek and Sulli, 2006; Saleh et al., 2006). We

observed that the ERN1 fusion protein is also associated with

discrete nuclear bodies (Figure 7). Previous studies have de-

scribed a similar subnuclear compartmentalization for certain

transcription factors and have associated this with functional

regulation, including phosphorylation and targeted degradation

via the proteasome pathway (Yang et al., 2005; Al-Sady et al.,

2006; Saleh et al., 2006). Additional experiments in Medicago

roots using native promoter and nuclear domain–associated

markers will now be required to further study distinct ERN1 sub-

nuclear localization.

We have found that the nucleus-targeted ERN1 and ERN2 act

as positive regulators of the NF box in N. benthamiana cells and

that this activity is dependent on the presence of the AP2/ERF

DNA binding domain. It should also be noted that ERN1 and

ERN2 possess a well-conserved C-terminal sequence that is

similar to the C-terminal CMV-4 domain of Arabidopsis and rice

(Oryza sativa) RAP2.11 clade members (Nakano et al., 2006),

whereas ERN3 lacks this C-terminal sequence and is unable to

activate reporter gene expression. Furthermore, when ERN3 is

expressed together with either ERN1 or ERN2, there is a clear

reduction in transcriptional activation of the 4xNF box reporter,

suggesting that ERN3 may function as a repressor-type ERF

factor. Coordinated induction of both activator- and repressor-

type AP2/ERFs has been proposed to fine-tune the regulation of

gene expression and to modulate antagonistic signaling path-

ways (Fujimoto et al., 2000; McGrath et al., 2005; Nakano et al.,

2006).

Our results suggest that activator and repressor ERN proteins

function to fine-tune NF-mediated root hair expression of plant

genes regulated via NF boxes. In the model presented in Figure

9, we propose that, in the absence of rhizobia, the highly

expressed ERN3 repressor limits the expression of target host

genes by binding to the NF box (Figure 9A). Following Rhizobium

inoculation, NF signal perception and transduction via the NFP-

dependent pathway results in enhanced transcription and protein

Table 1. Primers Used in This Study

Name Sequence 59 to 39

�549E9-for 59-GGAAGTCAGACAATAAGAATTCAATGAAATAGCAGG-39

�523E9-for 59-GGCCTTTAAGAATTCTTTAGACCAAGCTTATGG-39

�411E11EcoRI-for 59-AATAAAGAATTCGACATTAAATTTGAGGG-39

411E11XbaI-for 59-TAAAAATCTAGACACTTAAATTTGAGG-39

�411E11DGC-for 59-GAGGGTCATAATAACATAAAATAAT*AAAGCTATTATTTTACCAGCC-39

�411E11DGC-rev 59-GGCTGGTAAAATAATAGCTTT*ATTATTTTATGTTATTATGACCCTC-39

�411E11DGAT-for 59-GGCCAGTGAATTCGACACTTA*ATAATAACATAAAATAATTGCAG-39

�411E11DGAT-rev 59-CTGCAATTATTTTATGTTATTAT*TAAGTGTCGAATTCACTGGCC-39

�411E11dledel-for 59-GATTAATAAAAGAATTCGACACTTA*ATAATAACATAAAATAATAAAGC-39

�411E11dbledel-rev 59-GCTTTATTATTTTATGTTATTAT*TAAGTGTCGAATTCTTTATTAATC-39

�392E11EcoRI-for 59-TGGAATTCATAATAACATAAAATAATTG-39

�392E11XbaI-for 59-TGTCTAGAATAATAACATAAAATAATTG-39

�380E11NheI-rev 59-CTGCAAGCTAGCTATGTTATTATGACC-39

�380E11HindIII-rev 59-CTGCAAAAGCTTTATGTTATTATGACC-39

�367E11mutDOF-for 59-AGGCCTGCGACTATTATTTTACC-39

�367E11mutGC-for 59-CATATGTAAAGCTATTATTTTACC-39

�367E11mutGC-rev 59-CATATGCAATTATTTTATGTTATTATGAC-39

�367E11mutHDZ-rev 59-AGGCCTGCACTTAGTTTATGTTATTATGAC-39

�358E11HindIII-rev 59-GGCTGGTAAAATAAAAGCTTTAGGCC-39

�358E11NheI-rev 59-GGCTGGTAAAATGCTAGCTTTAGG-39

�350E11-for 59-GGTACCAGCCTTTTAATTTGACC-39

�257E11-rev 59-GGAATCTAGATTCTTGGC-39

�257E11-rev 59-GGAATCTAGATTCTTGGC-39

�0E11-rev 59-TGCCCACAGGCCGTCGAG-39

ERN1-for 59-GGAAGATGGTGCTGTTGCTT-39

ERN1-rev 59-TGTTGGATTGTGAACCTGACTC-39

ERN2-for 59-ATGATTCCCCTCTCGCTTCT-39

ERN2-rev 59-CACTGGCTGTGCCAATACAG-39

ERN3-for 59-TGGCCATTACCTCAACAAAGG-39

ERN3-rev 59-CCTTCCACATAGCACTCATTCA-39

ERN1delAP2-for 59-CACTCGAACCAGAATTCTCACTCATG-39

ERN1delAP2-rev 59-GCCTTTGCCGAATTCCAACAAAC-39

ERN2delAP2-for 59-CTCGTACCAGAATTCTCACACATG-39

ERN2delAP2-rev 59-CTTTGTCTCAGAATTCCAAACTTGTCTC-39

ERN3delAP2-for 59-TACTCGTAGAATTCTCTCTACTACTC-39

ERN3delAP2-rev 59-GCTCTTTGTCGAATTCCAACAAAC-39

Nucleotides in boldface correspond to substitutions in order to generate new restriction sites (underlined).


accumulation of the ERN1 and ERN2 transcriptional activators

(Figure 9B). Middleton et al. (2007) has reported that while the

ERN1 deletion mutant bit1-1 is defective in nodulation, infection,

and Mt ENOD11 gene activation, it is still capable of partial

transduction of the NF signal and of limited infection thread

initiation. This could be due to partial redundancy with respect to

ERN2, which may also be required to regulate NF-elicited ENOD

gene activation. The NF-dependent accumulation of ERN1/ERN2

activators may also be accompanied by a change in the level or

activity of the ERN3 repressor protein (Figure 9B), although any

model to explain this must be consistent with our demonstration

that ERN3 transcript levels remain constant following NF treat-

ment. In this respect, it is interesting that ubiquitin-conjugated

downregulation of a tobacco (Nicotiana tabacum) repressor-type

ERF3 factor has been proposed by Koyama et al. (2003). Addi-

tional regulatory steps must be postulated to account for the

expression of Mt ENOD11 and other genes in the subsequent

steps of infection. Once rhizobia are enclosed within the curled

root hair, activation of the entry receptor pathway, essential for

bacteria root infection (Ardourel et al., 1994), requires transcrip-

tional factor(s) that mediate Mt ENOD11 expression via the 257-

bp proximal promoter region (Boisson-Dernier et al., 2005). We

suggest that de novo accumulation of active ERN3 factors may

displace ERN1/ERN2 activators from the NF box by an active

repressor mechanism (Fujimoto et al., 2000; Ohta et al., 2001;

Song et al., 2005), as reported for other class II ERF factors, thus

turning off gene expression in adjacent noninfected epidermal

cells (Figure 9C). Our findings argue that ERN family members

contribute to both positive and negative regulatory fine-tuning of

host gene expression during preinfection stages of the legume/

Rhizobium symbiotic association.

METHODS

Plant Material, Growth Conditions, and NF Treatments

Medicago truncatula cv Jemalong A17 and the NF signaling mutant nfp-2

(Arrighi et al., 2006) were used in this study. Seeds were surface-sterilized

and germinated according to Boisson-Dernier et al. (2001) and either

used for Agrobacterium rhizogenes transformation or transferred to

nitrogen-starved medium for 3 d before subsequent NF treatment.

Aeroponically grown wild-type and mutant nfp-2 seedlings were trans-

ferred to Falcon tubes (Becton Dickinson Labware) containing either

water (control) or a 10�9 M aqueous solution of purified Sinorhizobium

meliloti NFs (Roche et al., 1991) and incubated for 6 h before tissue

harvest for subsequent RNA extraction. NF treatment of seedling roots

grown on pouch paper support (Mega International) was performed as

described by Sauviac et al. (2005) by immersing roots for 16 to 18 h in

either water (control) or an aqueous solution of purified NFs (10�8 M).

Seedling roots were then used for RNA extraction from intact roots or

from purified root hair fractions as described by Sauviac et al. (2005).

Treatments of M. truncatula Roots Generated by

A. rhizogenes Transformation

Germinated seedlings were inoculated with A. rhizogenes as described by

Boisson-Dernier et al. (2001). At ;3 weeks after inoculation, composite

kanamycin-resistant plants were transferred to pouch paper/Farhaeus

agar plates (nitrogen- and antibiotic-free) as described by Boisson-

Dernier et al. (2005). Transgenic roots growing on the paper support

were treated with either water (control) or an aqueous solution of purified

NFs (10�9 M) or inoculated with a liquid culture of S. meliloti strain

RCR2011 (GMI6526) (Ardourel et al., 1994) harboring the hemA-lacZ

transgene (5 3 10�5 bacteria/mL), as described by Boisson-Dernier et al.

(2005).

Chimeric Promoter–GUS Constructs

Promoter–GUS Fusion and Deletion Constructs

Mt ENOD12 promoter deletions �531E12 and �327E12 were generated

using ScaI and SphI internal restriction sites, respectively. Mt ENOD9

promoter deletions �558E9 and �532E9 were generated by PCR using

the 59 primers �549E9-for and �523E9-for (Table 1) in combination with

the T7 universal primer as 39 primer. Mt ENOD11 promoter deletions

�411E11, �367E11, and �257E11 were described previously (Boisson-

Dernier et al., 2005). The �350E11 deletion construct was generated by

PCR using 350E11-for and 0E11-rev primers (Table 1). The �411E11

construct lacking the �357 to �258 promoter region was obtained by

cloning the�411 to�358 promoter region (generated by PCR with primer

pairs �411E11EcoRI-for and �358E11HindIII-rev) (Table 1) between the

EcoRI and HindIII sites upstream from the �257E11 deletion construct.

Internal and Block Deletion Constructs

Block deletions of the two E11 motifs, named GCC-like (TGCAGGCCT) or

G(A/T)-rich (AAATTTGAGGGTC), were generated using the QuickChange

site-directed mutagenesis kit (Stratagene) according to the manufac-

turer’s instructions. Single block deletions were generated using a

�411E11-containing plasmid as a template and the corresponding

complementary primer pairs �411E11DGC-for/�411E11DGC-rev (GC-

rich motif deletion) and �411E11DGAT-for/�411E11DGAT-rev (G[A/T]-

rich motif deletion) (Table 1).

Site-Directed Mutagenesis

Motif substitutions within the NF-box sequence were introduced into the

�411E11 deletion construct. Point mutations in the HD-ZIP–like motif

(AAATAATTG to ACATAAGTG) were obtained by PCR using�411E11EcoRI-

for and �367mutHDZ-rev primers (Table 1). The resulting �411 to �367

mutated PCR fragment was cloned into the EcoRI and StuI sites in front of

a �367E11 deletion to reconstitute the �411E11mHDZ construct. Sub-

stitution in the GC-rich motif (TGCAGGCCT to TGCATATGT) was gener-

ated by PCR using primer pairs �411E11EcoRI-for/�367mutGC-rev and

�367mutGC-for/�257E11-rev (Table 1). PCR with these primers gener-

ated two DNA fragments (�411 to�367 and�367 to�257, respectively)

that after restriction digestion with EcoRI/NdeI and NdeI/XbaI enzymes,

respectively, were ligated together with the EcoRI/XbaI sites in front of the

�257E11 deletion to reconstitute the �411E11mGCcore construct. Sub-

stitution in the DOF motif (TAAAG to TGCAG) was introduced by PCR

using�367mutDOF-for and�257E11-rev primers (Table 1). The mutated

�367 to �257 PCR fragment was subsequently inserted between StuI/

XbaI sites of a �411E11 deletion construct to generate the �411E11m-

DOF construct.

Gain-of-Function Constructs

The tetramers of the NFE region (�411 to�358), of NFE59 (�411 to�380),

and of NFE39 (NF box) (�392 to �358) were inserted into EcoRI/HindIII

sites upstream from the minimal CaMV 35S promoter (�47) (Pontier et al.,

2001). Four tandem copies of each promoter region were obtained as

described by Rushton et al. (2002). Briefly, each promoter region (NFE

region, NFE59, or NF box) was amplified by PCR using the primer pairs

listed in Table 1 to generate for each sequence three PCR fragments

named A, B, and C bordered by EcoRI/NheI, XbaI/NheI, and XbaI/HindIII

2880 The Plant Cell

sites. After cloning into the pGEM-T vector (Promega), the resulting

plasmids were digested with ScaI (which cuts outside the Mt ENOD11

promoter sequences) together with either NheI (A and B) or XbaI (B and C).

Ligation of the digested plasmids 59-ScaI/NheI-39 and 59-XbaI/ScaI-39

recreated an intact plasmid with dimers AB and BC of the respective

promoter regions. Plasmids AB and BC, which have lost the internal NheI/

XbaI sites after ligation, were digested with ScaI/NheI and ScaI/XbaI as

before. Ligation of two such fragments then creates the tetrameric ABBC

constructs, which were then inserted into the EcoRI and HindIII sites

upstream from the minimal �47 CaMV 35S promoter.

All deletion, mutation, and gain-of-function promoter constructs were

fused to GUS in the binary vector pLP100 (Szabados et al., 1995) and

verified by sequencing using the GUS�1 primer (59-TGCCCACAGG-

CCGTCGAG-39). pLP100 binary vectors were introduced into A. rhizo-

genes strain ARqua1 by the freeze-thaw method as described by Hofgen

and Willmitzer (1998).

Histochemical and Fluorimetric GUS Assays

Histochemical GUS staining was performed as described previously

(Journet et al., 2001) by incubation of transgenic root samples in X-glcA

buffer solution (5-bromo-4-chloro-3-indolyl glucuronide, cyclohexylam-

monium salt; Biosynth). Stained samples were examined with a stereo-

microscope (Leica Microsystems) and a Zeiss Axiophot microscope (Carl

Zeiss). Double staining for both GUS and b-galactosidase activities after

inoculation with a S. meliloti strain carrying a constitutive hemA-lacZ

fusion was performed as described by Vernoud et al. (1999). For enzy-

matic GUS assays, pools of 7 to 10 independent transgenic roots were

ground in liquid nitrogen and homogenized in 13 GUS extraction buffer

(50 mM sodium phosphate, pH 7.5, 10 mM 2-mercaptoethanol, 10 mM

Na2EDTA, 0.1% Triton X-100, and 0.1% sodium lauryl-sarcosine) for total

protein extraction. GUS activities were measured fluorimetrically using

1 mg of total protein extract as described previously (Boisson-Dernier

et al., 2005).

A visual-based quantification method adapted from Sidorenko et al.

(2000) was used to evaluate GUS staining intensities for different pro-

moter deletion constructs. Briefly, roots were analyzed at 6 h after

staining and classified according to overall visual GUS staining intensities

into two distinct categories: moderate/strong staining (þ) or undetect-

able/weak staining (�). For each construct, ;40 to 150 independent

roots from three separate experiments were analyzed by histochemical

and fluorimetric methods. For all constructs tested, there is a linear

correlation between the results obtained from the two quantification

methods (0.95 < r < 0.98). Statistical data analyses were performed using

Student’s t test, in which a significant difference was defined for P < 0.05.

Electrophoretic Mobility Shift Assay

A pGEM-T vector containing the NF box sequence or its mutated

derivative was digested by EcoRI/SstI restriction enzymes, and the

respective DNA fragments were purified using the JETSORB gel extrac-

tion kit (Genomed). The NF box probe was radiolabeled with [a-33P]dCTP

(3000 Ci mmol�1) by filling in with the Klenow fragment of DNA poly-

merase I (Promega) and purified on MicroSpin columns (Amersham

Biosciences), according to the manufacturer’s instructions. Unlabeled

competitor DNA was filled in with nonradioactive nucleotides. Total

protein extract from isolated root hair was prepared according to Busk

and Pages (1997). One microgram of protein extract was incubated with

the radioactive probe (50,000 cpm) in 12 mL of 13 binding buffer (25 mM

HEPES, pH 7.8, 75 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 0.2 mM DTT,

and 10% glycerol) and 0.1 mg of poly(dA-dT) for 30 min at 48C before

loading on a 0.53 Tris-borate-EDTA, 6% polyacrylamide gel. Electro-

phoresis was run at 90 V for 1 h at 48C. In competition assays, the protein

was incubated first with unlabeled oligonucleotide for 10 min prior to

adding the radioactive probe and continuing with incubation for a further

30 min.

Quantitative RT-PCR Analysis

For NF-treated samples, total RNA was extracted from intact roots or

isolated root hairs of M. truncatula using the Macherey-Nagel total RNA

isolation kit according to the manufacturer’s instructions and the tech-

nique described by Sauviac et al. (2005). M. truncatula organs and

isolated nodule samples used for the quantitative RT-PCR analysis were

described previously (Godiard et al., 2007). Genomic DNA was removed

via on-column DNase treatment following basic kit instructions, and the

absence of DNA was confirmed by PCR amplification of the rip1 gene

(Cook et al., 1995) using primer pairs spanning an intronic region (Sauviac

et al., 2005). The DNA-free RNA samples were quantified, and RNA

integrity was checked on a 1% (w/v) agarose gel. First-strand cDNA

synthesis was performed using 0.7 to 1 mg of total RNA with an anchored

oligo(dT) (17TþV) and SuperScript II reverse transcriptase (Invitrogen)

following the manufacturer’s protocol. Thirty nanograms 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 (data not

shown). Quantitative RT-PCR was performed with the Light Cycler Fast

Start Reaction Mix MasterPLUS SYBR Green I (Roche) on a Roche light

cycler real-time PCR machine according to the manufacturer’s instruc-

tions. Each reaction was performed with 2 mL of a 1:20 (v/v) dilution of the

first cDNA strand, with 0.5 mM of each primer in a total reaction volume of

10 mL. The cycling conditions were as follows: 958C for 9 min followed by

45 cycles of denaturation at 958C for 5 s, annealing at 608C for 10 s, and

extension at 728C for 10 to 15 s. PCR amplification specificity was verified

by analysis of a dissociation curve at the end of the PCR cycles by heating

samples from 55 to 608C to 958C as well as by agarose gel resolution and

sequencing of the corresponding PCR products. Primer pairs to amplify

the elongation factor EF1-a were described previously (Sauviac et al.,

2005), and the primers used to amplify ERN1, ERN2, and ERN3 are listed

in Table 1 (ERN1-for, ERN1-rev, ERN2-for ERN2-rev, ERN3-for, and

ERN3-rev primers). Transcript levels for each of the target genes (ERN1,

ERN2, and ERN3) were normalized to the endogenous elongation factor

EF1-a transcript level. The data shown represent means of values ob-

tained from either two or three independent biological experiments and

quantitative RT-PCR.

Yeast One-Hybrid Screening

DNA fragments containing four tandem copies of NFE (�411 to �358) or

the NF box (�392 to �358) were inserted into the EcoRI/MluI sites of the

pHISi vector (Clontech) to create the tetramer NFE-HIS3 and tetramer NF-

box-HIS3 constructs. These and the control p53-HIS3 (Matchmaker one-

hybrid system; Clontech) plasmids were linearized by XhoI digestion and

used to transform the YM4271 yeast strain. Genome integration of the

corresponding constructs was verified by both the complementation of

the HIS� auxotrophic phenotype and the ability to PCR-amplify insert-

specific DNA fragments. Yeast colony PCR was performed using 2 mL of

yeast suspension obtained after 30 min of incubation at 378C within a

Lyticase/TE solution (catalog No. L4025; Sigma-Aldrich). A positive yeast

recombinant showing basal low HIS level, for which growth was blocked

at 2 to 5 mM 3-AT, was chosen for screening of the M. truncatula root hair

cDNA libraries. cDNA libraries were prepared using the Creator Smart

cDNA library construction kit (Clontech) from pooled samples of 10�8 M

NF-treated root hairs (2, 6, and 18 h) by directional cloning in SfiI sites of

a pGADT7-modified vector (Sauviac et al., 2005; F. de Carvalho-Niebel,

L. Sauviac, and L. Deslandes, unpublished data). Two types of libraries

were generated, using either the Powerscript reverse transcriptase

(RNaseH�), producing preferentially full-length cDNAs, or the wild-type

Moloney murine leukemia virus reverse transcriptase, with its RNaseH


activity, giving rise to shorter average size cDNAs. Primary libraries were

Seaprep-amplified following the manufacturer’s instructions (Cambrex

Bio Science). Powerscript and Moloney murine leukemia virus libraries

had 106 independent clones with an average insert size of 1.1 kb and 2.8

3 106 clones with an average insert size of 0.7 kb, respectively. cDNA

libraries were screened with selection set at 5 mM 3-AT, using the

YM4271 (tetramer NF box-HIS3) yeast strain. HISþ candidates able to

grow at 5 and 10 mM 3-AT were selected, amplified by PCR, and se-

quenced. Plasmids extracted from HISþ colonies were amplified in

Escherichia coli before retransformation into YM4271 (tetramer NF box-

HIS3), YM4271 (tetramer NFE-HIS3), and YM4271 (p53-HIS3) strains.

ERN Plasmid Constructs and Transient Expression in

N. benthamiana Leaves

In vitro BP and LR recombination reactions were performed according to

the manufacturer’s instructions (Invitrogen) between ERN1, ERN2, and

ERN3 pGADT7 clones and the respective donor plasmid pDONR 207

(Invitrogen Life Sciences) and destination vectors PAM-PAT35S-GFP/

YFP-GTW and PAM-PAT35S-3xHA-GTW (a kind gift of L. Deslandes,

Laboratory of Plant Microbe Interactions) to generate the respective 35S-

GFP-ERN, 35S-YFP-ERN, and 35S-3xHA-ERN fusion constructs. A PCR

strategy was used to create ERNs deleted for the respective AP2/ERF

DNA binding domains. For each ERN, PCR was performed using ERN-

PDONR 207 plasmids as templates in two independent PCRs with

PDONR 207 forward and reverse primers (Invitrogen Life Sciences)

combined, respectively, with the ERNdelAP2-rev or ERNdelAP2-for

primers listed in Table 1. These reactions generated two DNA fragments

lacking the region containing the AP2/ERF domain of each ERN, which

after restriction digestion with SfiI and EcoRI, respectively, were ligated

together within SfiI sites of PDONR 207 plasmids to reconstitute the

ERN1D, ERN2D, and ERN3D PDONR 207 plasmids. These ERN-AP2–

deleted donor plasmids were recombined with PAM-PAT35S-YFP-GTW

and PAM-PAT35S-3xHA-GTW destination vectors to generate 35S-YFP-

ERND and 35S-3xHA-ERND fusion constructs. These binary vectors

were introduced into Agrobacterium tumefaciens strains GV3101 or

GV3103 by electroporation following the Invitrogen protocol. A. tumefa-

ciens strains harboring the various constructs with or without p19 (Voinnet

et al., 2003) were grown in Luria-Bertani medium under antibiotic selec-

tion at 288C before harvesting and resuspension in 10 mM MgCl2, 10 mM

MES, pH 5.6, with 100 mM acetosyringone (catalog No. D13440-6;

Sigma-Aldrich). After incubation at room temperature for 2 h, A. tume-

faciens cultures were infiltrated into leaves of 3-week-old Nicotiana

benthamiana plants using a 1-mL needleless syringe. For coagroinfiltra-

tion, equal volumes of each A. tumefaciens culture adjusted to an OD600

of 0.5 were mixed prior to infiltration. The subcellular distribution patterns

of YFP- or YFP/GFP-fused proteins were analyzed by fluorescence

optical and confocal microscopy from 30 to 60 h after infiltration. Each

construct was analyzed in three distinct infiltrated leaves of six to eight

plants in at least three independent experiments. For transactivation

studies, leaf discs were collected at 24 h after inoculation and either used

directly for histochemical GUS assays or frozen in liquid nitrogen and

stored at �808C until processing. For the protein gel blot analysis, one-

tenth volume of proteins extracted from two leaf discs per sample was

loaded per lane. For enzymatic GUS assays, total proteins were extracted

and quantified as described above, and 1 mg of total protein was used in

replicates to measure enzymatic activities of individual samples.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers EU038802 (ERN1), EU038803 (ERN2),

and EU038804 (ERN3).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Histochemical Analysis of GUS Activity

Driven by the Gain-of-Function NF Box Construct at 4 d after

Inoculation with S. meliloti in Transgenic Roots and in Young Nodules.

Supplemental Figure 2. Expression Profiles of ERN1, ERN2, and

ERN3 Genes in Different Plant Organs.

Supplemental Figure 3. Expression of ERN Proteins in A. tumefa-

ciens–Infiltrated N. benthamiana Cells.

Supplemental Figure 4. Histochemical and Fluorimetric GUS Activ-

ities of N. benthamiana Leaf Discs following Infiltration with the 4xNF

Box Reporter Alone or in Combination with ERN2, ERN3, ERN2/

ERN3, or ERN2/ERN3D Effectors.

Supplemental Table 1. Statistical Analysis of GUS Activity Directed

by Different Promoter Constructs.

Supplemental Table 2. Statistical Analysis of GUS Activity Measured

in Transactivation Studies Described in Figure 8E and Supplemental

Figure 4.

ACKNOWLEDGMENTS

We thank Fabienne Maillet and Jean Denarie for providing S. meliloti

Nod factors and Etienne Journet for providing seeds of the M. truncatula

nfp-2 mutant line. We thank Laurent Deslandes for helpful advice

concerning yeast and N. benthamiana transformation and for providing

binary PAM-PAT destination vectors, and members of Pascal Gamas’s

group at the Laboratory of Plant Microbe Interactions for providing

M. truncatula organs and nodule samples for quantitative RT-PCR

analyses. We also thank Giles Oldroyd for providing the ERN paper

(Middleton et al., 2007) before publication.

Received May 21, 2007; revised July 26, 2007; accepted August 14, 2007;

published September 7, 2007.

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DOI 10.1105/tpc.107.052944; originally published online September 7, 2007; 2007;19;2866-2885Plant Cell

Barker and Fernanda de Carvalho-NiebelAndry Andriankaja, Aurélien Boisson-Dernier, Lisa Frances, Laurent Sauviac, Alain Jauneau, David G.

-Regulatory MotifcisHairs via a Novel Activation in RootENOD11Dependent Mt −AP2-ERF Transcription Factors Mediate Nod Factor

This information is current as of December 3, 2020

Supplemental Data /content/suppl/2007/09/07/tpc.107.052944.DC1.html

References /content/19/9/2866.full.html#ref-list-1

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