Plant Physiol. (1998) 116: 53-68
Studying Early Nodulin Gene ENOD40 Expression and
Induction by Nodulation Factor and Cytokinin in Transgenic
Alfalfa1
Yiwen Fang and
Ann M. Hirsch*
Department of Molecular, Cell, and Developmental Biology (Y.F.,
A.M.H.), and Molecular Biology Institute (A.M.H.), University of
California, 405 Hilgard Avenue, Los Angeles, California 90095-1606
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ABSTRACT |
ENOD40, an early
nodulin gene, is expressed following inoculation with Rhizobium
meliloti or by adding R. meliloti-produced nodulation (Nod) factors or the plant hormone cytokinin to uninoculated roots. We isolated two MsENOD40 clones, designated
MsENOD40-1 and MsENOD40-2, with
distinct promoters from an alfalfa (Medicago sativa cv
Chief) genomic library. The promoters were fused to the reporter gene
uidA (gus), and the constructs were
introduced into alfalfa. We observed that the
MsENOD40-1 construct was expressed almost exclusively
under symbiotic conditions. The MsENOD40-2 construct
was transcribed under both symbiotic and nonsymbiotic conditions and in
nonnodular and nodular tissues. Both MsENOD40 promoter-gus constructs were similarly expressed as
nodules developed, and both were expressed in roots treated with
6-benzylaminopurine or purified Nod factor. However, no blue color was
detected in nodule-like structures induced by the auxin transport
inhibitor N-1-(naphthyl)phthalamic acid on roots of
plants containing the MsENOD40-1 promoter construct,
whereas pseudonodules from plants containing the
MsENOD40-2 promoter construct stained blue. A 616-bp region at the distal 5
end of the promoter is important for proper spatial expression of MsENOD40 in nodules and also
for Nod-factor and cytokinin-induced expression.
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INTRODUCTION |
The symbiotic interaction between leguminous plants and
rhizobia results in the development on plant roots of a novel organ, the nodule, in which rhizobia provide fixed nitrogen to the host in
exchange for carbon and protection. To establish this mutualistic relationship, the host plant and rhizobia must continually exchange molecular signals. Rhizobial nodulation (nod) gene
expression is induced in response to different flavonoids that are
excreted by plant roots or seeds (for reviews, see McKhann and Hirsch, 1994
; Long, 1996). In turn, nod gene products synthesize and
secrete nodulation signal molecules called nodulation factors (Nod
factors), which have been identified as modified
lipo-chitooligosaccharide molecules (Lerouge et al., 1990; Truchet et
al., 1991; for reviews, see van Rhijn and Vanderleyden, 1995;
Dénarié et al., 1996; Long, 1996). Nod factor is considered
a primary morphogenetic signal for nodulation because it triggers on a
compatible host the earliest stages of nodule development, including
root hair deformation and curling, as well as cortical cell divisions
(Lerouge et al., 1990; Spaink et al., 1991; Truchet et al., 1991).
Furthermore, Nod factor elicits the expression of several early nodulin
(ENOD) genes, such as ENOD12 and
ENOD40 (Horvath et al., 1993; Vijn et al., 1993, 1995a;
Bauer et al., 1994; Crespi et al., 1994; Journet et al., 1994; Hirsch
et al., 1997). Nodulins are plant-encoded proteins that are expressed
during nodule development. ENOD40 is one of the earliest
nodulins to be expressed upon Rhizobium inoculation, and has
been cloned from a number of legumes (Kouchi and Hata, 1993; Yang et
al., 1993; Asad et al., 1994; Crespi et al., 1994; Matvienko et al.,
1994; Vijn et al., 1995b; Papadopoulou et al., 1996) as well as one
nonlegume, tobacco (van de Sande et al., 1996). In alfalfa
(Medicago sativa) ENOD40 transcripts have been
detected in dividing cells or cells that are competent to divide (Asad
et al., 1994; Crespi et al., 1994).
The effects of Nod factor on legume plants can be phenocopied
by modulating the balance of endogenous plant hormones. For example,
applying 2,3,5-triiodobenzoic acid or NPA, both of which presumably
block auxin polar transport in the root, elicits the formation of
nodule-like structures (pseudonodules) on alfalfa, Afghanistan pea
roots, or sweetclover sym mutants (Hirsch et al., 1989;
Scheres et al., 1992; Wu et al., 1996). Transcripts for the early
nodulin genes, ENOD2, ENOD8, and
ENOD12, were detected in these pseudonodules. Similarly,
when alfalfa roots were inoculated with R. meliloti
nodulation mutants carrying the Agrobacterium tumefaciens
tzs gene (encoding DMA transferase, an enzyme which is associated
with trans-zeatin biosynthesis; [Beaty et al., 1986]), small, uninfected nodules developed. MsENOD2 (Cooper and
Long, 1994) and MsENOD40 (Hirsch et al., 1997) transcripts
were detected in these nodules. Likewise, cytokinin application
triggered ENOD2 expression in Sesbania rostrata
roots (Dehio and deBruijn, 1992) and ENOD2 and
ENOD12A expression in alfalfa roots (Bauer et al., 1996).
Furthermore, exogenous cytokinin can bypass the block of a
nonnodulating alfalfa mutant, MN1008, such that both MsENOD2 and MsENOD40 were expressed in the mutant roots, although at
a reduced level when compared with the wild-type alfalfa level (Hirsch et al., 1997). These results suggest that a change in endogenous hormone level and/or sensitivities may be the consequence of Nod factor
perception in a responsive host plant (Hirsch and Fang, 1994).
How the change of hormone (presumably auxin/cytokinin) balance is
brought about remains unknown. However, the fact that some early
nodulin genes are hormone responsive may lead to the identification of
specific transcription factors or cis-acting elements that are involved in regulating these genes upon Nod factor application. Thus, studying the promoter elements of MsENOD40 should help
us elucidate the cis-acting region(s) responsible for its
expression, as well as those necessary for induction by Nod factors
and/or cytokinin. In this way we can achieve a better understanding of the signal transduction pathway in the nitrogen-fixing symbiosis, as
well as plant hormone (especially cytokinin) regulation of plant genes.
In this report we cloned the promoter regions from two independent
genomic clones (1a and 6c) of MsENOD40 isolated from an alfalfa genomic library. The two discrete promoters were
individually fused to a reporter gene uidA
(gus), and introduced into alfalfa via
Agrobacterium tumefaciens-mediated transformation. We
followed the spatial and temporal expression patterns of the two
ENOD40 genes under both nonsymbiotic and symbiotic
conditions. The expression patterns of both promoter constructs were
also examined in NPA- or R. meliloti exopolysaccharide
mutant-induced nodules, both of which are bacteria free. To define the
cis-acting region of the MsENOD40 promoter
required for its induction by Nod factor or cytokinin, a series of
nested truncated promoters of the MsENOD40-1 promoter was
constructed and introduced into alfalfa plants. The activities of these
truncated promoters were studied in the transgenic plants.
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MATERIALS AND METHODS |
Isolation of MsENOD40 Genomic Clones and Sequencing
Analysis
One million plaques from an alfalfa (Medicago
sativa cv Chief) genomic library (Clontech, Palo Alto, CA) were
screened with a 32P-radiolabeled
MsENOD40 cDNA (Asad et al., 1994). Five individual plaques,
1a, 2b, 4B, 6c, and 9B, that hybridized to the probe were isolated and
the phage DNAs were purified. Two clones, 1a and 6c, were selected, and
the putative promoter regions located within the HindIII
fragments were subcloned into the pBluescript II KS vector (Stratagene)
to generate 1a1 (approximately 2.8 kb) and 6c1 (approximately 2.6 kb),
respectively. Fragment 1a1 was then designated as the
MsENOD40-1 promoter and 6c1 was designated as the
MsENOD40-2 promoter. A set of nested deletions from either the 5 or 3 end of both clones was generated using the method described by Henikoff (1984).
Both DNA strands were sequenced using the double-stranded dideoxy
chain-termination method according to the manufacturer's protocol
(Sequenase kit version 2, United States Biochemical). Sequence analyses
were performed on a VAX/VMS computer using the University of Wisconsin
Genetics Computer Group software package (Madison).
Southern Analysis
Phage DNAs were isolated according to the method described by
Kernodle et al. (1993). DNA was digested with different enzymes and
blotted for Southern analysis (Asad et al., 1994).
Construction of 5-Truncated MsENOD40-1 Promoters and Other
Plasmids
The HindIII-BclI fragments of 1a1 and
6c1 were individually ligated into the
HindIII-BamHI site of the binary vector pBI101.3
(Clontech) to create pBI1a1-1 and pBI6c1-1, respectively. The
BclI site is about 35 bp downstream of the putative TATA box of the MsENOD40 gene (Asad et al., 1994). A
SpeI-BclI (approximately 2.3 kb) and a
ClaI-BclI (approximately 1.6 kb) fragment from
1a1 were cloned into pBI101.3 to produce pBI1a1-2 and pBI1a1-4,
respectively. The EcoRV-BclI (231 bp) fragment
was used to create pBI1a1-5, which is considered to be the minimal
promoter in this paper. The SpeI-ClaI fragment of
1a1 was fused at the 5 end of pBI1a1-5 to produce pBI1a1-7, a
composite promoter for MsENOD40-1, consisting of the
minimal promoter and the 616-bp SpeI-ClaI fragment (see Fig.
2). All pBI plasmids were electroporated into Agrobacterium tumefaciens strain LBA4404 and then transformed into cv Regen using the alfalfa transformation and regeneration procedure described in Hirsch et al. (1995).
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| Figure 2.
The diagram shows the restriction map of the
constructs used for alfalfa transformation. The vector pBI101.3 is a
promoterless construct and is not shown here. The black boxes represent
the reporter gene uidA (gus). The unshaded boxes are the
regions shared by both promoters. The hatched box is the upstream
region in the MsENOD40-1 promoter that is only 40%
similar to the lined region in the MsENOD40-2 promoter.
Hind, HindIII; Spe, SpeI; Cla,
ClaI; Eco, EcoRV; Bcl,
BclI; Acc, AccI; and Bam,
BamHI.
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Transgenic Plant Propagation and Treatments
All of the transgenic plants used for the experiments were
primary transformants that were propagated vegetatively. Healthy stems
were cut and rooted in a vermiculite/perlite mixture soaked with
one-quarter-strength complete Hoagland plant medium. After 12 to
14 d the rooted plants were transferred into sterile 50-mL Falcon
tubes containing nitrogen-free Jensen's medium and placed in a growth
chamber (Conviron, Permbina, ND). The tubes were wrapped with aluminum
foil to exclude light from the roots. After 6 to 7 d the plants
were transferred to fresh Jensen's medium without any additives
(control) or inoculated with Rhizobium meliloti strain 1021, or treated with 10
8 m PNF
(NodRmIV[C16:1, S]) or 106
m BAP. These concentrations were determined
previously to give an optimal response (Hirsch et al., 1997). Roots
were harvested 4 d after treatment for GUS histochemical staining
or for total protein extraction. To obtain nodules at different stages,
the rooted plants were either spot inoculated (see Asad et al., 1994) or flood inoculated with R. meliloti strain 1021 (wild
type) or strain 7094 (exoB::Tn5), or were treated
with 105 m NPA. Nodules or other tissues
(e.g. roots) were harvested approximately 2 to 5 weeks postinoculation.
GUS Histochemical and Colorimetric Assay
We used the method developed by Jefferson (1987) for assaying GUS
activity histochemically. Plant tissues were placed in the fixing
buffer (Jefferson, 1987) for 45 min, rinsed three times with 50 mm phosphate buffer, and incubated with 1 mm 5-bromo-4-chloro-3-indolyl-
-glucuronic acid substrate
at 37°C from 1 h to overnight. After staining, the tissues were
rinsed in phosphate buffer and kept in 50% ethanol. Some tissues were
further cleared using a 20% bleach solution under a vacuum for 15 to
30 min. The blue-stained tissues were either used directly for
observation after washing in distilled water or embedded in paraffin
via the TBA series (McKhann and Hirsch, 1993). If paraffin embedded,
the tissues were sectioned at 15 to 25 µm and mounted onto slides for
observation and photography using an Axiophot microscope (Zeiss).
To assay the enzymatic activity of GUS protein, total protein was
extracted from roots 4 d following the various treatments (see
above). Twenty nanograms of protein was used for the GUS assays in
microtiter plates using N-nitrophenyl glucuronide as a
substrate. The assay was adapted from the method described by Breyne et
al. (1993). The plates were read in a Titertek plate reader (Eflab for
Flow Laboratories, Finland) at a wavelength of 405 nm after a 6-h
incubation at 37°C. Ten independent transgenic plants generated from
each construct were tested, and each plant was assayed three times with
three individual sets of plant cuttings per treatment. The colorimetric
GUS assay was repeated, and the readings were averaged and plotted
using Microsoft Excel and Cricket Graph programs.
| |
RESULTS |
There Are at Least Two Different MsENOD40 Genes
in Alfalfa
After three rounds of screening 1,000,000 plaques, five
independent phage clones were isolated from an alfalfa genomic library using the full-length MsENOD40 cDNA as a probe. Two
phage clones were found to be identical to each other; this was
confirmed later by sequence analysis (data not shown). The putative
promoter region, which was located within the HindIII
fragment in each phage clone, varied in size (data not shown). We knew
that the HindIII fragment contained the potential promoter
region based on our previous finding that there is a HindIII
site at the very 5 end of the MsENOD40 cDNA sequence (Asad
et al., 1994). This was verified by sequencing analysis (Fig. 1).
Genomic Southern analysis had suggested that one or two
MsENOD40 genes were present in the alfalfa genome (Asad et
al., 1994). Preliminary analysis showed that the putative promoters in
phages 1a and 6c were the largest fragments, and differed in their
restriction patterns (data not shown). On this basis, phages 1a and 6c
were selected for further characterization.
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| Figure 1.
Comparison of the promoter sequences of MsENOD40-1
(top sequence) and MsENOD40-2 (bottom sequence). The
lines between the two sequences represent the identical nucleotides
between them. Sequence analyses were performed on a VAX/VMS computer
using program GAP in the University of Wisconsin Genetics Computer
Group software package. Note that the first 1431 bp (1-1431) of the 3
end were identical between the two promoters, and they are at the
proximal end of the promoters (the unshaded regions in Fig. 2). The
remaining 5 distal sequences are only 40% similar to each other.
Approximately 200 bp of sequence of the 5 end of
MsENOD40-1 is not shown here, but the promoter sequence
of MsENOD40-2 is complete. The bold letters indicate
the HindIII restriction site. The single-underlined letters are the BclI site; and the double-underlined
letters indicate the putative TATA box in the promoter. The boxed
regions are the putative "nodule-specific motifs."
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Sequencing analysis indicated that the 2.8-kb (1a1)
HindIII fragment in clone 1a and the 2.6-kb (6c1)
HindIII fragment in clone 6c most likely contain the
promoters for the MsENOD40 genes. These HindIII
fragments also included the first 75-bp sequence found at the 5 end of
the MsENOD40 cDNA, including the TATA box. Sequence
comparison revealed that the proximal sequences of these two promoters,
which span approximately 1.4 kb, were identical. However, the upstream
sequences shared only 40% similarity (Fig. 1). The different
restriction sites in these regions also supported this conclusion
(Figs. 1 and 2). This finding suggested
that these two clones (1a and 6c) were likely to represent promoters of
two different MsENOD40 genes in alfalfa (Asad et al., 1994).
Thus, the designation MsENOD40-1 was assigned to clone 1a1,
and MsENOD40-2 was used for clone 6c1.
The Nonsymbiotic Expression of the MsENOD40
Promoters in Transgenic Alfalfa
To investigate the expression patterns of the two different
MsENOD40 genes, each promoter was fused to the
reporter gene uidA (gus) to produce plasmids
pBI1a1-1 and pBI6c1-1, respectively (Fig. 2). The putative
transcriptional start site in the promoter was determined by
primer-extension experiments (data not shown); thus, these constructs
were transcriptional fusions. Both constructs were subsequently
introduced into cv Regen via A. tumefaciens-mediated transformation to generate transgenic plants (Hirsch et al., 1995). More than 30 individual transgenic plants generated for each construct were examined by the histochemical assay, and approximately 70% of
them expressed the gus gene in nodules at locations
determined previously by in situ hybridization analysis (Asad et al.,
1994). On this basis, it seemed unlikely that sequences other than
those located within the MsENOD40 promoter were affecting
the spatial patterns of gene expression. Twenty of the 30 independent
transgenic lines from each construct were selected for more detailed
studies. Using Southern analysis we found that most lines contained
multiple inserts of the T-DNA. However, the level of gus
gene expression did not correlate with the copy number of the inserts
(data not shown).
By performing histochemical staining for GUS protein (Jefferson, 1987)
in various plant organs, the expression patterns of the two
MsENOD40 genes were shown to differ under nonsymbiotic conditions. During the process of somatic embryogenesis, we could not
detect any gus expression in callus cells or somatic embryos for either construct. In mature transgenic plants the
MsENOD40-1 promoter was usually not active in uninoculated
roots. For example, 16 of 20 plants showed no blue staining in roots,
whereas the remaining 4 plants expressed the gus gene in the
vascular tissues of the root and the stem (data not shown). Of the 20 plants, 9 of them (45%) showed the blue staining indicative of GUS
protein in emerging lateral root primordia. When the lateral root
emerged from the parent root, however, the blue color was no longer
apparent (data not shown).
In contrast, 17 of 21 (81%) of the transgenic plants containing the
MsENOD40-2 promoter-gus construct expressed
gus in the root stele and in the stem procambium/phloem
region even in the absence of R. meliloti (Fig.
3, A and B).
Furthermore, gus expression was detected at the root tip
(sometimes in the root cap) in 40% of the plants examined (Fig. 3C).
About 80% of the plants studied also expressed gus in
developing lateral roots (Fig. 3, D-F). The blue stain indicating GUS
protein was seen in the central cells of the lateral root primordium
(Fig. 3D). Upon lateral root elongation and emergence from the parent
root, gus expression became restricted to the central
vasculature at the proximal end of the lateral root, at the point where
it was attached to the parent root; however, it remained high in the
root tip (Fig. 3, E and F).
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| Figure 3.
The localization of GUS protein in the transgenic alfalfa plants
in the absence of R. meliloti. A, Transverse section of
a root of plant line A27 (MsENOD40-2). Blue color was
detected in the stele, in the pericycle, endodermis, and inner cortex;
bar = 55 µm. B, Cross-section of a stem of plant line A17
(MsENOD40-2). Blue color is localized to the stem
procambium and the protophloem; same magnification as in C. C,
Longitudinal section of a root tip of plant line A17
(MsENOD40-2). Note that the blue staining is present
even in the root cap; bar = 110 µm. D, Lateral root primordium on
transgenic plant line A27 (MsENOD40-2) same
magnification as in C. E and F, Elongated lateral roots on the same
plant shown in D; bar = 110 µm. G, Root of plant line a18
(MsENOD40-1) without any treatment; bar = 110 µm. H, Root of plant line a18 4 d after treatment with
106 m BAP. I, Root of plant line a18 4 d
after treatment with 108 m PNF. J, Root
of plant line A27 (MsENOD40-2) without any treatment. H
through J are the same magnification as in G. K, A 106
m BAP-treated root of line A27 after 4 d; bar = 110 µm. L, Root of line A27 treated with 108
m PNF for 4 d. M, Root of line A23 treated with
106 m 2,4-D for 4 d; no blue color is
present. L and M are the same magnification as in K. N, Nodule
primordium formed on plant line a18 (MsENOD40-1) after
BAP treatment for 4 d; bar = 55 µm. O, Cross-section of a
primordium in N that was induced by BAP. P, Primordium formed on plant
line A27 (MsENOD40-2) after BAP treatment for 4 d.
Same magnification as N. Q, Nodule primordium formed on plant line A27
(MsENOD40-2) following 4 d of PNF treatment; bar = 55 µm. Arrows indicate starch grains and the arrowhead
points to a root hair that is out of the plane of section.
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Despite the differences between the two promoters, their combined
expression patterns in transgenic alfalfa correlated with our previous
findings of MsENOD40 transcript localization using in situ
hybridization (Asad et al., 1994). Two new locations were detected: the
root tip and the node where the leaf is attached (data not shown). Only
the MsENOD40-2 promoter was active in the root tip and
occasionally in the root cap of some transgenic plants (Fig. 3C). The
MsENOD40-1 promoter construct was occasionally expressed in
the node but not in other parts of the stem (data not shown).
The Expression of the MsENOD40 Promoters during
Wild-Type Nodule Development
We then followed gus gene expression driven by
the MsENOD40 promoter constructs in the plants inoculated
with wild-type R. meliloti strain 1021. The two promoters
were found to respond to R. meliloti inoculation to
different extents. For the transgenic plants bearing the
MsENOD40-1 promoter-gus fusion construct, less than 50% of them expressed gus in the stele upon
inoculation (Fig. 4,
A-E), whereas nearly 90% of the transgenic alfalfa plants containing the MsENOD40-2 promoter responded in this way (Fig. 4,
F-I). However, both constructs demonstrated similar expression
patterns as the nodule developed. Before root cortical cell divisions
were apparent, gus expression was detected in the root outer
cortex, as well as in the epidermal cells (small arrowhead in Fig. 4C).
When the inner cortical cells were activated to divide, the blue color indicating GUS protein was found in the dividing inner cortical cells,
as well as in the pericycle (large arrowhead in Fig. 4, A and C; Fig.
4F). Blue color was subsequently detected in all cells of the nodule
primordium (Fig. 4, D, E, G, and H).
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| Figure 4.
The localization of GUS protein during wild-type
nodule development on roots of plants containing
MsENOD40-1 and MsENOD40-2 promoter-gus constructs. The roots illustrated here were
flood inoculated, but each developmental stage is comparable to that found by spot inoculation. Plants shown in A through E and J through M
contained the MsENOD40-1 promoter construct. Plants
shown in F through I contained the MsENOD40-2 promoter
construct. A, Two areas showing the blue color on a root of plant line
a18. The area indicated by the small arrowhead shows the blue color
mainly in the outer cortical cells and the epidermis. The area
indicated by the large arrowhead shows the blue color in the dividing
inner cortical cells, including the pericycle; bar = 114 µm. B,
An enlargement of the area indicated by the large arrowhead in A;
bar = 57 µm. C, Two adjacent areas show the blue color on a root
of plant line a33 at two stages similar to A; bar = 55 µm. D, A
small nodule primordium on a root of plant line a18 (cell divisions in
the inner cortical cells). The blue staining of GUS protein is
displayed mainly in the inner cortical cells and its derivatives as
well as in the root pericycle; bar = 110 µm. E, A well-developed
nodule primordium on a root of plant line a33. Same magnification as in
D. F through I, Nodule primordia/nodules at different developmental stages
for MsENOD40-2 transgenic plants. The blue staining for GUS protein is present in the cortex, as well as in the root stele. F
was from plant line A4, whereas G through I were from plant line A27.
F, Bar = 55 µm. G is the same magnification as B. H is the same
magnification as F. I, Bar = 110 µm. J through L, Nodules at
different stages (postprimordium) on plant line a18 or a33. J is the
same magnification as I. K, Bar = 110 µm. M, Longitudinal
section of a mature nodule on plant line b5-2 (containing construct
pBI1a1-2). L and M are the same magnification as K. Note that the blue
color is present in the nodule meristem, in peripheral infected cells,
and in cells associated with the nodule vascular bundles.
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Following initiation of a nodule meristem and the further development
of the nodule, gus expression became localized to the nodule
meristem (including the prefixing zone), to cells on the periphery of
the central region, and to cells associated with the nodule vascular
bundles (Fig. 4, I-L). This pattern persisted in the mature nodule
(Fig. 4M). For plants bearing the MsENOD40-2 promoter
construct, gus expression was much stronger. The time required to detect the appearance of the blue color was 1 to 6 h,
depending on the plant line, compared with 6 to 24 h for the MsENOD40-1 construct. In addition, blue staining indicating
GUS protein was detected in the root cortex following R. meliloti inoculation for the MsENOD40-2 construct
(Fig. 4, F-I). The localization of the reporter gene driven by the
MsENOD40 promoters in nodules formed on the transgenic
plants is consistent with the in situ hybridization results found
earlier (Asad et al., 1994).
For the transgenic plants containing the vector control pBI101.3, no
gus expression was detected in any tissue or organ, even after incubation in the solution with the
5-bromo-4-chloro-3-indolyl--glucuronic acid substrate for 2 to
3 d (more than 30 plants examined). Thus, gus
expression directed by the MsENOD40 promoters in the
transgenic alfalfa plants was specific.
The Expression Pattern of the MsENOD40 Promoters in Bacteria-Free
Nodules
Earlier we found using northern-blot analysis that
MsENOD40 is expressed in ineffective nodules elicited
by NPA and R. meliloti exo mutants (Asad et al., 1994). To
examine whether both promoter constructs are expressed in these
bacteria-free nodules, 16 individual plants that contained either the
MsENOD40-1- or the MsENOD40-2-gus
construct were tested. The NPA-induced nodule-like structures gave the
largest difference with respect to the expression patterns of the two
promoter constructs. No gus expression was detected in the
NPA-induced pseudonodules that were formed on the plants carrying the
MsENOD40-1 promoter construct, unless these plants
expressed the gus gene in the root stele constitutively (20% of the plants). In comparison, the majority (80%) of the plants
containing the MsENOD40-2 promoter expressed gus
in NPA-induced nodules. Following treatment with 105
m (Continued on p. 61.) NPA,
gus expression was detected in the dividing cortical
cells and pericycle (Fig.
5A). In an older
NPA-induced pseudonodule, GUS activity was detected in the area around
the central vascular bundle, as well as in the peripheral area
consisting of pericycle and cortical cell derivatives (Fig. 5, B and
C).
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| Figure 5.
GUS localization during the development of ineffective nodules (A
through F) and during nodule formation on plants containing the
composite promoter in pBI1a1-7 (see Fig. 2; plants with the composite
promoter are represented by the letter H). A through C represent
different stages of nodules induced by 105 m
NPA on plants containing the MsENOD40-2 promoter
construct (seven different lines were examined). The blue color is not
only found in the nodule primordium, but also in the root cortex. A, Very young nodule primordium on plant line A27. Arrowheads indicate starch grains. B, NPA-induced nodule on plant line A32. C, Dark-field picture of B. D through F show the different stages of R. meliloti exo (strain 7094) mutant-induced nodule development
(eight different lines were examined). D, Nodule primordium on plant
line a18 (MsENOD40-1); bar = 55 µm. E,
Well-formed nodule primordium on plant line a18 (MsENOD40-1). Arrowheads indicate starch grains. F,
Mature exo nodule on plant line A27. The blue color is
mainly located in the apical and peripheral area of the nodule. A
through C and E through F are the same magnification; bar = 110 µm. G, An infection thread formed in a curled root hair on plant line
H2. The picture was taken with Nomarski optics; bar = 92 µm. H,
Bright-field picture of G, showing the blue color in the cortical cells
where the infection thread has penetrated. The arrow indicates the
curled root hair. I, Very young nodule primordium on plant line H35.
Same magnification as A. J, Nodule primordium on plant line H2;
bar = 55 µm. K, Young nodule on plant line H15; bar = 460 µm. L, Section of a mature nodule formed on plant line H25; bar = 110 µm. M, Dark-field picture of L.
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|
In contrast, when roots of the transgenic plants containing either of
the promoter-gus constructs were inoculated with R. meliloti strain 7094 (exoB::Tn5), the blue
staining for gus protein was detected in the uninfected
nodules that developed on the roots. Both promoters, including the
composite MsENOD40-1 promoter (pBI1a1-7, Fig. 2),
expressed the gus gene in a pattern similar to that of wild-type nodules, i.e. in dividing cortical cells (Fig. 5D), in the
nodule primordium (Fig. 5E), and in the peripheral region that is
comparable to the meristem of the wild-type nodule (Yang et al., 1992)
in mature, bacteria-free nodules (Fig. 5F). In addition, we observed
amyloplast deposition in roots (arrowheads in Fig. 5, A and E)
following treatment with NPA or inoculation with R. meliloti
strain 7094.
The Expression Patterns of the Two Different
MsENOD40 Promoter Constructs in Cytokinin- or Nod
Factor-Treated Roots
Earlier we found using northern-blot analysis that
MsENOD40 was induced by BAP application (van Rhijn et al.,
1997). Treatment with 2,4-D or other plant hormones did not elicit
MsENOD40 expression (Hirsch et al., 1997; Fig. 3M). The
expression patterns of the two MsENOD40 promoter constructs
were observed after treating the transgenic plant roots with BAP or Nod
factor (six independent plant lines for each construct were examined).
For both constructs, Nod factor or cytokinin triggered similar
responses. In roots without any treatment (Jensen's medium alone), GUS
protein was not detected in roots of the MsENOD40-1
transgenic plants (Fig. 3G) or was seen in the root stele and root tip
of the MsENOD40-2 transgenic plants (Fig. 3J). In roots
treated with BAP or PNF for 4 d, blue staining indicating GUS
protein was present in the root cortex and epidermal cells for both
promoter constructs (Fig. 3, H, I, K, and L). In some cases,
gus expression was also found in root hairs (arrowheads in
Fig. 3, H and K). gus expression in the root cortex and
epidermis was seen mainly in the root-elongation zone, where the root
expanded laterally following BAP or PNF treatment (Fig. 3, H, I, K, and
L). This change in morphology and induction of gus
expression took place as early as 2 d after treatment (data not
shown). In the root treated with either BAP or PNF, small nodule
primordia resulting from inner cortical cell divisions developed; blue
staining indicated that GUS protein was present in all the cells of the
nodule primordium (Fig. 3, N-Q). Starch grain accumulation in BAP- or
PNF-treated roots was also observed (arrows in Fig. 3Q).
A 616-bp Region in the MsENOD40-1 Promoter Is
Essential and Sufficient for MsENOD40-1 Expression
in Nodules
To define the cis-acting region in the
MsENOD40 promoter that is important for its activity, a
series of 5-truncated promoters of MsENOD40-1 was
constructed, fused to uidA (Fig. 2), and subsequently transformed into alfalfa. The MsENOD40-1 promoter was
chosen for this study because its expression levels in roots and
nodules correlated well with our previous study (Asad et al., 1994),
and also because it was expressed almost exclusively under symbiotic conditions in contrast to the MsENOD40-2 promoter construct
(this report). By performing the GUS histochemical assay on 30 independent lines, the promoter in pBI1a1-4 (see Fig. 2) was found to
be completely inactive in any tissues or organs of the transgenic
plants, including nodules (data not shown). This promoter contained the
region common to both promoters. If the promoter contained the upstream
region up to SpeI site (pBI1a1-2; Fig. 2), the same
gus expression patterns as the full-length
MsENOD40-1 promoter were detected in 30 independent lines
(data not shown). The smallest construct from the
MsENOD40-1 promoter (231 bp, pBI1a1-5 in Fig. 2) behaved
exactly the same as the promoterless vector pBI101.3 (30 independent
lines examined for each construct). These results suggest that the
region between SpeI and ClaI site was required
for the MsENOD40-1 promoter activity.
To test this hypothesis, a composite promoter was created by directly
linking the SpeI-ClaI fragment (616 bp) to the
minimal promoter in pBI1a1-5 (pBI1a1-7, see Fig. 2). This construct
was introduced into alfalfa plants to produce transgenic lines. In 40%
of these transgenic plants gus expression was detected in the inner and outer root cortical cells at early stages of R. meliloti infection, but was absent or below detection levels in the root stele (including the pericycle) (see Fig. 5, G and H). When
the nodule primordium was formed, gus expression was found within the cells of the primordium (Fig. 5, I and K). In the nodule gus expression was detected at locations similar to those of
the full-length MsENOD40-1 promoter, e.g. in the nodule
meristem and in the cells associated with the nodule vascular bundle
(Fig. 5, J, L, and M).
Compared with the full-length promoter, only about 40% (versus >70%)
of the transgenic plants examined (11 out of 25 plants) demonstrated
the expected expression patterns in nodules. Like the full-length
promoter construct, the composite promoter construct was active in
nodules induced by R. meliloti exo mutants, but not by NPA
(data not shown). In addition, the composite promoter in pBI1a1-7 was
exclusively expressed in nodules. It completely lost its activity under
nonsymbiotic conditions, even in young lateral root primordia (data not
shown).
The Region Responsible for Inducing the MsENOD40-1
Expression by Nod Factors and Cytokinin Is Overlapping
Having the truncated promoters of the MsENOD40-1, we
were able to study the cis-acting region required for BAP
and/or PNF induction. Ten independent transgenic alfalfa plants from
each transformation with each construct, including that of the vector control, were tested. Rooted plants generated from the stem cuttings were treated with wild-type R. meliloti strain 1021, 106 m BAP, or
108 m PNF. Total protein was
extracted from roots 4 d following each treatment, and GUS
activity was assayed with a microtiter plate assay using
N-nitrophenyl glucuronide as a colorimetric
substrate (see ``Materials and Methods''). Both promoters
(MsENOD40-1 and MsENOD40-2) were
enhanced 2- to 3-fold on average in roots by exogenous BAP, as well
as by R. meliloti inoculation or addition of PNF (Fig.
6). Furthermore, the
average basal level of the MsENOD40-2 promoter activity was measured at approximately 3-fold higher than that of the
MsENOD40-1 promoter (compare the different scales in Fig.
6, A and B). When the MsENOD40-1 promoter was deleted from
the 5 end to the SpeI site (pBI1a1-2 in Fig. 2), it acted
like the full-length promoter (Fig. 6C). However, for the transgenic
plants containing the construct pBI1a1-4, gus expression
could no longer be induced by BAP, by PNF, or by R. meliloti
inoculation (Fig. 6D). The same result was obtained for the smallest
promoter (pBI1a1-5) and the vector control (pBI101.3) (Fig. 6, E and
F). These results suggest the 616-bp region in the
MsENOD40-1 promoter is involved in regulating expression
induced by either Nod factors or cytokinin.
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| Figure 6.
The results of GUS colorimetric assays in the transgenic alfalfa
roots. A, Plants with construct pBI6c1-1. B, Plants with construct
pBI1a1-1. C, Plants with construct pBI1a1-2. D, Plants with construct
pBI1a1-4. E, Plants with construct pBI1a1-5. F, Plants with vector
pBI101.3. GUS activity is expressed as per milligram per hour per
milliliter. Plants containing the same construct are represented with
the same letter, e.g. letter "A" for construct pBI6c1-1, "a"
for pBI1a1-1, "b" for pBI1a1-2, "c" for pBI101.3, "e" for
pBI1a1-4, and "f" for pBI1a1-5. The number following the letter
represents an individual plant line; a different number represents a
different transgenic plant. Plant roots were harvested after a 4-d
treatment with the following: C, Jensen's medium only;
106 m BAP; 108 m
PNF; Rm, wild-type R. meliloti strain 1021. The line
above and below the average values represent the sd. If
absent, the sds were too small to be displayed.
|
|
| |
DISCUSSION |
Here we have extended our investigations on
ENOD40 gene expression in alfalfa to studying
promoter-gus fusions in transgenic plants. Five independent
genomic clones were isolated from an alfalfa genomic library using the
MsENOD40 cDNA as a probe, and two of them were found to be
identical. The promoter sequences from two distinct clones, 1a and
6c, were cloned for detailed characterizations. Sequence analyses
revealed that the two promoters differed at their 5 distal end (40%
similarity), but were identical at the proximal region (1431 bp long)
(Fig. 1). Based on these results and our previous Southern analysis
(Asad et al., 1994), these two clones (1a and 6c) are proposed to
represent two distinct ENOD40 genes in the alfalfa genome.
We have designated them MsENOD40-1 and
MsENOD40-2, respectively.
More than one ENOD40 gene has also been isolated from other
legumes, such as soybean and French bean (Kouchi and Hata, 1993; Papadopoulou et al., 1996). A soybean GmENOD40-2
promoter-gus fusion has been introduced into a heterologous
plant, vetch, and found to be expressed only in the pericycle of the
nodule vascular bundle and at the region of the root where the nodule
was attached (Roussis et al., 1995). However, the spatial and
temporal expression patterns of the second soybean ENOD40
gene in a transgenic plant have not yet been investigated. We fused the
promoters of the two MsENOD40 genes individually to
gus, and subsequently transformed these constructs into
alfalfa plants. By doing so, we were able to investigate the
expression patterns of these two genes in a homologous transgenic plant
system with or without R. meliloti inoculation.
During the process of alfalfa transformation and regeneration, no
gus expression was detected in the callus cells or somatic embryos using the gus histochemical assay. When using
northern analysis, we found that MsENOD40 was not expressed
in alfalfa cell cultures, even after cytokinin treatment (data not
shown). Therefore, we conclude that the MsENOD40 gene is
probably not expressed during somatic embryogenesis or in cell
cultures.
Under nonsymbiotic conditions MsENOD40-1 was
usually not expressed in the uninoculated root of the
transgenic alfalfa plants, but was activated when lateral roots or
nodule primordia are initiated. In contrast, the expression of
MsENOD40-2 is likely to be constitutive, because the
promoter construct was expressed in the vascular tissues of the root
and the stem even in the absence of R. meliloti (see Fig.
3). In addition, the expression of the MsENOD40-2 promoter construct was also detected at the root tip and throughout lateral root
development (Fig. 3). The expression pattern of MsENOD40-2 in lateral roots is very similar to that found for PvENOD40
in bean (Papadopoulou et al., 1996), suggesting that one of the
PvENOD40 genes may be a MsENOD40-2 homolog.
Using in situ-hybridization analysis (Asad et al., 1994), we
detected the combined expression patterns of MsENOD40-1 and
MsENOD40-2. Transcripts were localized in the stem
procambium, in the root stele, in the margins of the young leaf
primordia, and in emerging lateral roots (see Table
I). We found two new sites of expression by using the GUS histochemical assay. Both promoter constructs were
expressed in the stem node at the point of petiole attachment in some
transgenic plants, a site which had not been examined previously using
in situ hybridizations. Moreover, MsENOD40-2 was expressed
in the root tip, including the root cap in some transgenic plants.
Comparing our previous findings (Asad et al., 1994) and the results
from studying the transgenic plants, the expression of the two
MsENOD40 genes in alfalfa appears to be differentially
regulated under nonsymbiotic conditions. Transcripts detected in the
stem and in the lateral roots by the in situ method are likely to be
derived from the expression of MsENOD40-2.
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|
Table I.
Summary of the expression patterns of the two
MsENOD40 genes in transgenic plants
Plasmids represent the different promoter constructs shown in Figure 3.
The percentage is the number of plants that demonstrated the indicated
GUS localization over the total number of the plants examined.  , Not
expressed; +, expressed.
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|
When transgenic plants were inoculated with wild-type R. meliloti strain 1021, both MsENOD40-1 and
MsENOD40-2 constructs were expressed in a similar
fashion throughout all stages of nodule development. Both promoter
constructs were activated in the cortical cells/pericycle in the
preinfection and infection stages. They were subsequently expressed in
the nodule primordium, and later in the nodule meristem and peripheral
cells around the central tissues, as well as in the nodule vascular
bundles of the mature nodule (see Fig. 4). Although
MsENOD40-2 expression appears to be stronger than
MsENOD40-1 (3-fold higher), we cannot distinguish between
the two promoters in terms of GUS localization as the nodule develops.
Also, because GUS localization correlates with our previous findings
obtained by in situ hybridization (Asad et al., 1994), we could not
determine whether only one or both genes are expressed during the
alfalfa-R. meliloti interaction.
On the other hand, even though the two promoter constructs
behaved similarly during nodule development, they are regulated differently in the NPA-induced pseudonodules. Only the
MsENOD40-2 promoter construct was expressed in the
nodule-like structures elicited by NPA treatment (Fig. 5, A-C). In
contrast, both promoter constructs were expressed in nodules induced by
R. meliloti exo mutants (Fig. 5, D-F). Part of the
explanation for the differential expression may be the fact that
NPA-elicited nodules morphologically and physiologically resemble
lateral roots more closely than nodules, whereas R. meliloti
exo mutant-induced nodules follow the wild-type nodule-developmental pathway (Yang et al., 1992; Asad et al., 1994).
Thus, the differential expression patterns of the two
MsENOD40s may reflect two distinct pathways for these
different types of ineffective nodules. MsENOD40-1 is not
usually active in the root stele or in the lateral root, and
consequently it is not expressed in NPA-induced nodules. In contrast,
the MsENOD40-2 gene is constitutively active in the main
root or lateral roots; thus, it is expressed in NPA-elicited nodules
(Fig. 5, A-C). Both genes show similar expression patterns during the
formation of wild-type nodules, and consequently both genes are
expressed in R. meliloti exo mutant-induced nodules (Fig. 5,
D-F). These results suggest that these two MsENOD40 genes
have different but complementary roles in the development of different
organs.
We have found that MsENOD40 gene expression was enhanced in
roots treated with either cytokinin or PNFs (Hirsch et al., 1997; van
Rhijn et al., 1997). Here we confirm this observation by testing gus gene expression in roots of transgenic plants carrying
either of the MsENOD40 promoters. The expression of
MsENOD40-1 as well as MsENOD40-2 was induced by
BAP or Nod factor treatment, and the extent of induction is similar for
both genes (2- to 3-fold), although the basal level of
MsENOD40-2 expression is generally higher (Fig. 6). In
addition, both genes were also expressed in a similar pattern in roots
following treatment with cytokinin or Nod factor for 4 d (see Fig.
3, G-Q). If both promoter constructs were induced by cytokinin or Nod
factors in alfalfa, we would expect that the expression levels of
MsENOD40 expression would increase at least 4- to 6-fold.
However, our previous northern analysis showed that MsENOD40
was enhanced only approximately 2-fold in roots after cytokinin or Nod
factor treatment. Two possible explanations are suggested. One is that
only one MsENOD40 gene is induced by cytokinin or Nod
factors in alfalfa. The second is that both genes are involved in
cytokinin or Nod factor induction, but they are coordinately regulated
so the overall level stays the same.
The differential expression patterns of MsENOD40-1 and
MsENOD40-2 may reflect their different sensitivities to
hormone changes or other signals. This is based on several
observations. Under nonsymbiotic conditions 80% of plants (compared
with 20% of the MsENOD40-1 transgenic plants) containing
the MsENOD40-2-gus construct expressed gus in
the root stele as well as in lateral roots. Following inoculation with
R. meliloti strain 1021, MsENOD40-2 was
expressed not only in nodules, but also in the root cortex; this latter location was not observed for the MsENOD40-1 construct.
MsENOD40-2 expression was also detected in the root cortex
after addition of either NPA or R. meliloti exo mutants.
This difference between the two genes may be dependent on the 5
upstream regions, which are only 40% similar between the two
promoters.
To define the cis-acting region involved in cytokinin or Nod
factor induction, a series of 5 deletions of the
MsENOD40-1 promoter was constructed and introduced into
plants to study their activities. By assaying GUS enzymatic activities
in roots after various treatments, a 616-bp
SpeI-ClaI region in the MsENOD40-1 promoter was found to be absolutely required for its expression. This
region plus the minimal promoter (the composite promoter) is
sufficient to drive reporter gene expression at similar locations in the nodule as the full-length promoter (see Fig. 5). Furthermore, this region is also involved in the induction of MsENOD40-1
gene by cytokinin or by Nod factors. However, this region does not contain any known consensus sequences needed for hormonal regulation, such as auxin-responsive elements, ethylene-responsive elements, G-boxes, etc., confirming our finding that MsENOD40 is not
induced by other plant hormones (Hirsch et al., 1997).
Similar to the GmENOD40-2 promoter, the two
MsENOD40 promoters also contain two conserved sequence
motifs, AAAGAT and CTCTT (see boxed regions in Fig. 1), that have been
identified in several nodulin gene promoters (Sandal et al., 1987; Bak
Ramlov et al., 1993; Miao and Verma, 1993; Roussis et al., 1995). Using
deletion analysis and site-specific mutagenesis, these sequences have
been found to be required for nodule-specific expression for certain genes, such as lbc3 and nodulin-26 (Stougaard et al., 1990;
Bak Ramlov et al., 1993; Miao and Verma, 1993; Szcyzglowski et al., 1994). Whether these motifs determine or are responsible for
nodule-specific expression of MsENOD40 will be investigated
in future work.
Our studies do not clarify whether the ENOD40 gene product
functions as a small peptide (van de Sande et al., 1996) or as a
riboregulator (Crespi et al., 1994). However, it is very likely that
the induction of expression of ENOD40 by cytokinin or other endogenous molecules upon R. meliloti inoculation could
serve as an amplification mechanism thereby triggering a localized
hormone imbalance, a state that initiates cell divisions in the root
cortex. We should caution that we have analyzed the expression patterns of transgenes in transgenic plants in this study. Most of our results
corresponded very well with our previous in situ results with few
exceptions. These may reflect the difference between detecting the
transcripts from the endogenous genes versus detecting the reporter
gene, i.e. gus, from the transgenes introduced.
| |
FOOTNOTES |
1
This research was supported by the National
Science Foundation (grant no. 90-23888 to A.M.H).
*
Corresponding author; e-mail ahirsch{at}ucla.edu; fax
1-310-206-5413.
Received June 27, 1997;
accepted September 17, 1997.
The GenBank accession numbers for the ENOD40-1 and ENOD40-2 promoters
reported in this article are AFO35556 and AFO35557, respectively.
| |
ABBREVIATIONS |
Abbreviations:
BAP, 6-benzylaminopurine.
NPA, N-1-(naphthyl)phthalamic acid.
PNF, purified Nod
factor.
| |
ACKNOWLEDGMENTS |
This paper was written in partial fulfillment of the Ph.D.
thesis of Y.F. to the Department of Molecular, Cell and Developmental Biology, University of California-Los Angeles (UCLA). We would like to
acknowledge the following UCLA undergraduate students for their help in
the experiments: Yousun Kim, Chrystene Ngyuen, Richard Na, John Chen,
and James Shih. We are grateful to Weigang Yang for taking care of the
transgenic plants in the greenhouse. We thank S.R. Long's lab
(Stanford University, CA) for their generous gift of PNF and B. Bonavida's lab (UCLA) for letting us use the plate reader for the GUS
colorimetric assay. We thank Margaret Kowalczyk at UCLA for preparing
the color plates.
| |
LITERATURE CITED |
Asad S,
Fang Y,
Wycoff KL,
Hirsch AM
(1994)
Isolation and characterization of cDNA and genomic clones of MsENOD40: transcripts are detected in meristematic cells of alfalfa.
Protoplasma
183:
10-23
[CrossRef]
Bak Ramlov K,
Bech Laursen N,
Stougaard J,
Marcker KA
(1993)
Site-directed mutagenesis of the organ-specific element in the soybean leghaemobloin lbc3 gene promoter.
Plant J
4:
577-580
[Medline]
Bauer P,
Crespi MD,
Szecsi J,
Allison LA,
Schultze M,
Ratet P,
Kondorosi E
(1994)
Alfalfa Enod12 genes are differentially regulated during nodule development by Nod factors and Rhizobium invasion.
Plant Physiol
105:
585-592
[Abstract]
Bauer P,
Ratet P,
Crespi MD,
Schultze M,
Kondorosi A
(1996)
Nod factors and cytokinins induce similar cortical cell division, amyloplast deposition and Msenod12A expression in alfalfa roots.
Plant J
10:
91-105
Beaty JS,
Powell GK,
Licia L,
Rigier DA,
MacDonald EMS,
Hommes NG,
Morris RO
(1986)
Tzs, a nopaline Ti plasmid gene from Agrobacterium tumefaciens associated with trans-zeatin biosynthesis.
Mol Gen Genet
203:
274-280
Breyne P,
De Loose M,
Dedonder A,
van Montagu M,
Depicker A
(1993)
Quantitative kinetic analysis of -glucuronidase activities using a computer-directed microtiter plate reader.
Plant Mol Biol
11:
21-31
Cooper JB,
Long SR
(1994)
Morphogenetic rescue of Rhizobium meliloti nodulation mutants by trans-zeatin secretion.
Plant Cell
6:
215-225
[Abstract]
Crespi MD,
Hurkevitch E,
Poiret M,
d'Aubenton-Carafa Y,
Petrovics G,
Kondorosi E,
Kondorosi A
(1994)
ENOD40, a gene expressed during nodule organogenesis, codes for a non-translatable RNA involved in plant growth.
EMBO J
13:
5099-5112
[ISI][Medline]
Dehio C,
deBruijn FJ
(1992)
The early nodulin gene SrEnod2 from Sesbania rostrata is inducible by cytokinin.
Plant J
2:
117-128
[ISI][Medline]
Dénarié J,
Debellé F,
Promé JC
(1996)
Rhizobium lipo-chitooligosaccharide nodulation factors: signaling molecules mediating recognition and morphogenesis.
Annu Rev Biochem
65:
503-535
[CrossRef][ISI][Medline]
Henikoff S
(1984)
Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing.
Gene
28:
351-359
[CrossRef][ISI][Medline]
Hirsch AM,
Bhuvaneswari TV,
Torrey JG,
Bisseling T
(1989)
Early nodulin genes are induced in alfalfa root outgrowths elicited by auxin transport inhibitors.
Proc Natl Acad Sci USA
86:
1244-1248
[Abstract/Free Full Text]
Hirsch AM,
Brill LM,
Lim PO,
Scambray J,
van Rhijn P
(1995)
Steps toward defining the role of lectins in nodule development in legumes.
Symbiosis
19:
155-173
Hirsch AM,
Fang Y
(1994)
Plant hormones and nodulation: what's the connection?
Plant Mol Biol
26:
5-9
[CrossRef][ISI][Medline]
Hirsch AM,
Fang Y,
Asad S,
Kapulnik Y
(1997)
The role of phytohormones in plant-microbe symbioses.
Plant Soil
194:
171-184
[CrossRef]
Horvath B,
Heidstra R,
Lados M,
Moerman M,
Spaink HP,
Promé J-C,
van Kammen A,
Bisseling T
(1993)
Lipooligosaccharides of Rhizobium induce infection-related early nodulin gene expression in pea root hairs.
Plant J
4:
727-733
[CrossRef][ISI][Medline]
Jefferson RA
(1987)
Assaying chimeric genes in plants: the gus gene-fusion system.
Plant Mol Biol Rep
5:
387-405
Journet EP,
Pichon M,
Dedieu A,
de Billy F,
Truchet G,
Barker DG
(1994)
Rhizobium meliloti Nod factors elicit cell-specific transcription of the ENOD12 gene in transgenic alfalfa.
Plant J
6:
241-249
[CrossRef][ISI][Medline]
Kernodle SP,
Cannon RE,
Scandalios JG
(1993)
Rapid and simple phage DNA isolation.
Biotechniques
14:
360-361
[Medline]
Kouchi H,
Hata S
(1993)
Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stages of soybean nodule development.
Mol Gen Genet
238:
106-119
[ISI][Medline]
Lerouge P,
Roché P,
Faucher C,
Maillet F,
Truchet G,
Promé J-C,
Dénarié J
(1990)
Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal.
Nature
344:
781-784
[CrossRef][Medline]
Long SR
(1996)
Rhizobium symbiosis: Nod factors in perspective.
Plant Cell
8:
1885-1898
[CrossRef][ISI][Medline]
Matvienko M,
van de Sande K,
Yang WC,
van Kammen A,
Bisseling T,
Franssen H
(1994)
Comparison of soybean and pea ENOD40 cDNA clones representing genes expressed during both early and late stages of nodule development.
Plant Mol Biol
26:
487-493
[Medline]
McKhann HI,
Hirsch AM
(1993)
In situ localization of specific mRNAs in plant tissues.
In
BR Glick,
JE Thompson,
eds, Methods in Plant Molecular Biology and Biotechnology.
CRC Press, Boca Raton, FL, pp 179-203
McKhann HI,
Hirsch AM
(1994)
Does Rhizobium avoid the host response?
In
J Dangl,
eds, Bacterial Pathogenesis in Plants and Animals. Current Topics in Microbiology and Immunology.
Springer-Verlag, Berlin, pp 132-162
Miao GH,
Verma DPS
(1993)
Soybean nodulin-26 gene encoding a channel protein is expressed only in the infected cells of nodules and is regulated differently in roots of homologous and heterologous plants.
Plant Cell
5:
781-784
[Abstract/Free Full Text]
Papadopoulou K,
Roussis A,
Katinakis P
(1996)
Phaseolus ENOD40 is involved in symbiotic and non-symbiotic organogenetic processes: expression during nodule and lateral root development.
Plant Mol Biol
30:
403-417
[CrossRef][ISI][Medline]
Roussis A,
van de Sande K,
Papadopoulos K,
Drenth J,
Bisseling T,
Franssen H,
Katinakis P
(1995)
Characterization of the soybean gene GmENOD40-2.
J Exp Bot
46:
719-724
[Abstract/Free Full Text]
Sandal NN,
Bojsen K,
Marcker KA
(1987)
A small family of nodule specific genes from soybean.
Nucleic Acids Res
15:
1507-1519
[Abstract/Free Full Text]
Scheres B,
McKhann HI,
Zalensky A,
Löbler M,
Bisseling T,
Hirsch AM
(1992)
The PsENOD12 gene is expressed at two different sites in Afghanistan pea pseudonodules induced by auxin transport inhibitors.
Plant Physiol
100:
1649-1655
[Abstract/Free Full Text]
Spaink HP,
Sheeley DM,
van Brussel AAN,
Glushka J,
York WS,
Tak T,
Geiger O,
Kennedy EP,
Reinhold VN,
Lugtenberg BJJ
(1991)
A novel highly saturated fatty acid moiety of lipo-oligosaccharide signals determines host specificity of Rhizobium.
Nature
354:
125-130
[CrossRef][Medline]
Stougaard J,
Jorgensen JE,
Christensen T,
Kuhle A,
Marcker KA
(1990)
Interdependence and nodule specificity of cis-acting regulatory elements in the soybean leghaemoglobin lbc3 and N23 gene promoters.
Mol Gen Genet
220:
353-360
[Medline]
Szczyglowski K,
Szabados L,
Fujimoto SY,
Silver D,
de Bruijn FJ
(1994)
Site-specific mutagenesis of the nodule-infected cell expression (NICE) element and the AT-rich element ATRE-BS2* of the Sesbania rostrata leghaemoglobin glb3 promoter.
Plant Cell
6:
317-332
[Abstract]
Truchet G,
Roche P,
Lerouge P,
Vasse J,
Camut S,
de Billy F,
Promé J-C,
Dénarié J
(1991)
Sulphated lipooligosaccharide signals from Rhizobium meliloti elicit root nodule organogenesis in alfalfa.
Nature
351:
670-673
[CrossRef][ISI]
van de Sande K, Pawlowski K, Czaja I, Wieneke U, Schell J, Schmidt J,
Walden R, Matvienko M, Wellink J, van Kammen A, and others (1996)
Modification of phytohormone response by a peptide encoded by ENOD40 of
legumes and a nonlegume. Science 273: 370-373
van Rhijn P,
Fang Y,
Galili S,
Shaul O,
Atzmon N,
Wininger S,
Eshed Y,
Lum M,
Li Y,
To V,
and others
(1997)
Expression of early nodulin genes in alfalfa mycorrhizae indicates that signal transduction pathways used in forming arbuscular mycorrhizae and Rhizobium-induced nodules may be conserved.
Proc Natl Acad Sci USA
94:
5467-5472
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Abstract
Introduction