Alfalfa Enod12 Genes Are Differentially Regulated during Nodule Development by Nod Factors and Rhizobium Invasion

Abstract

MsEnod12A and MsEnod12B are two early nodulin genes from alfalfa (Medicago sativa). Differential expression of these genes was demonstrated using a reverse transcription-polymerase chain reaction approach. MsEnod12A RNA was detected only in nodules and not in other plant tissues. In contrast, MsEnod12B transcripts were found in nodules and also at low levels in roots, flowers, stems, and leaves. MsEnod12B expression was enhanced in the root early after inoculation with the microsymbiont Rhizobium meliloti and after treatment with purified Nod factors, whereas MsEnod12A induction was detected only when developing nodules were visible. In situ hybridization showed that in nodules, MsEnod12 expression occurred in the infection zone. In empty Fix- nodules the MsEnod12A transcript level was much reduced, and in spontaneous nodules it was not detectable. These data indicate that MsEnod12B expression in roots is related to the action of Nod factors, whereas MsEnod12A expression is associated with the invasion process in nodules. Therefore, alfalfa possesses different mechanisms regulating MsEnod12A and MsEnod12B expression.

Full text

Plant
Physiol.
(1994)
105:
585-592
Alfalfa
Enodl2
Cenes Are Differentially Regulated during
Nodule Development
by
Nod
Factors and
Rhizobium
I
nvasion'
Petra Bauer, Martin
D.
Crespi, Judit Szécsi, Lori A. Allison*, Michael Schultze, Pascal Ratet,
Eva Kondorosi, and Adam Kondorosi*
lnstitut des Sciences Végétales, Centre National de Ia
Recherche
Scientifique,
F-91198
Gif sur Yvette Cedex,
France (P.B.,
M.D.C.,
J.S.,
L.A.A.,
M.S.,
P.R.,
E.K., A.K.);
and lnstitute
of
Genetics, Biological Research Center,
Hungarian Academy
of
Sciences, Szeged P.O.
Box
521,
H-6701 Hungary
(A.K.)
MsEnodlZA and MsEnodlZB are two early nodulin genes from
alfalfa (Medicago
sativa).
Differential expression of these genes
was demonstrated using a reverse
transcription-polymerase
chain
readion approach. MsEnodlZA RNA was detected only in nodules
and not in other plant tissues. In contrast, MsEnodlZB transcripts
were found in nodules and also at low levels
in
roots, flowers,
stems, and leaves. MsfnodlZB expression was enhanced in the
root early after inoculation with the microsymbiont
Rhizobium
meliloti
and after treatment with purified Nod factors, whereas
MsEnodlZA induction was detected only when developing nod-
ules were visible. In situ hybridization showed that in nodules,
MsfnodlZ expression occurred in the infection zone.
In
empty Fix-
nodules the MsEnodlZA transcript leve1 was much reduced, and
in
spontaneous nodules it was not detectable. These data indicate
that MsEnodlZB expression in roots
is
related to the adion of Nod
fadors, whereas MsFnodlZA expression
is
associated with the
invasion process in nodules. Therefore, alfalfa possesses different
mechanisms regulating MsEnodlZA and
MsfnodlZB
expression.
Early symbiotic interactions of leguminous plants and rhi-
zobia comprise signal exchanges between the
two
partners.
Nodulation of host plants requires bacterial factors and com-
pounds identified through the extensive study of
Rhizobium
mutants.
Rhizobium
nodulation factors (Nod factors), pro-
duced after induction of
nod
genes by plant flavonoids, direct
the initiation of nodule morphogenesis and infection through
curled root hairs via infection threads (for review, see Hirsch,
1992). Nod factors of various
Rhizobium
species are lipooli-
gosaccharides differing by modifications of a common oli-
'
Research support was provided
by
the Commission
of
the Eu-
ropean Countries (BRIDGE BIOT-900159-C) contract to A.K. P.B.
received a fellowship from the Gottlieb Daimler and Carl Benz
Foundation. M.C. was the recipient of an European Economic Com-
munity fellowship (ECLAIR program),
J.S.
received a fellowship from
the Ministerie des Enseignements Supéneurs et de Ia Recherche
(France). L.A.A. was supported through a postdoctoral fellowship
from the Natural Sciences and Engineering Research Council
of
Canada. M.S received a
F.
Lynen grant from the Alexander v.
Humboldt Foundation.
Present address: State University of New Jersey, Rutgers Waks-
man Institute, P.O. Box 759, Piscataway, NJ 08855-0759.
*
Corresvonding author; fax 33-1-69-82-36-95.
gomeric /3-1,4-linked N-acetyl-D-glucosamine backbone.
Rhi-
zobium meliloti
produces a sulfated tetramer containing a
CI6
acyl chain with two double bonds, NodRm-IV(C16:2,S), that
can be acetylated (Lerouge et al., 1990; Schultze et al., 1992).
Purified Nod factors induce root hair deformations, preinfec-
tion thread formation, and cortical cell division (reviewed by
Spaink, 1992). Furthermore, complex polysaccharides of the
rhizobial outer surface are necessary for successful invasion
of nodule cells, such as exopolysaccharides and lipopolysac-
charides synthesized by the products of the
exo
and
Ips
genes,
respectively (reviewed by Gray and Rolfe, 1990).
The sequential expression of plant nodulin genes is asso-
ciated with the onset of the early symbiotic events (Scheres
et al., 1990b). Early nodulin genes, e.g.
Enod5
and
Enodl2,
are induced during nodule development, whereas activation
of late nodulin genes, e.g. the leghemoglobin genes, is cor-
related with nodule function (Nap and Bisseling, 1990;
Scheres et al., 1990b). Analyzing early nodulin gene expres-
sion may contribute to the understanding of the regulatory
pathways involved in nodulation.
Enodl2,
one of the most characterized early nodulin genes,
was first isolated from a 21-d-old pea nodule cDNA library
(Scheres et al., 1990a). The Enodl2 protein sequence is
composed of a putative signal peptide followed by a stretch
of Pro-rich repeats. It presumably represents -a Hyp-rich
glycoprotein of the cell wall, Two
Enodl2
genes were identi-
fied
in
pea,
PsEnodl2A
and
PsEnodlZB,
that showed the same
expression pattem (Govers et al., 1991). In situ hybridization
of segments of inoculated pea roots and nodules
of
different
ages revealed that PsEnodl2 might be involved in the infec-
tion process (Scheres et al., 1990a). Expression was found in
cells containing infection threads and in cells preparing the
passage for these structures.
PsEnodl2A
and
PsEnodl2B
were
expressed in root hairs after inoculation with wild-type
Rhi-
zobium leguminosarum
bv
viciae,
but not after infection with
mutants defective in Nod factor synthesis (Scheres et al.,
1990a). Recently, Horvath et al. (1993) demonstrated that in
pea roots, purified rhizobial lipooligosaccharides induced the
expression of both
Enodl2
genes.
In
Medicago,
three
Enodl2
genes have been identified as
Abbreviation: RT. reverse transcrivtion.

586 Bauer et
al.
Plant
Physiol.
Vol.
105, 1994
early nodulin genes,
MtEnod12
from the diploid
Medi-
cago truncatula
(Pichon et al., 1992) and
MsEnod12A
and
MsEnod12B
from the tetraploid
Medicago sativa
(Allison et al.,
1993). Like the
Enod12
gene from pea,
MtEnod12
is expressed
in the infection zone of nodules. Induction as early as 3 to 6
h after infection with
R. meliloti
was shown for the
MtEnod12
promoter-0-glucuronidase
fusion in transgenic
M. sativa
ssp.
varia
plants (Pichon et al., 1992).
We studied the expression pattem of the endogenous
M. sativa EnodZ2
genes. We found that
MsEnod12A
and
MsEnod1 2B
were differentially expressed not only
in
various
plant tissues but also during the early symbiotic stages. We
demonstrated that
MsEnodl2B
was induced in roots treated
with the cognate Nod factor, NodRm-IV(C16:2,S), whereas
MsEnod12A
was expressed in nodules being invaded by rhi-
zobia. Our results indicate that in alfalfa, expression of the
two
EnodZ2
genes are under different controls during sym-
biotic nodule development.
MATERIALS AND METHODS
Plant Material and lnfection
For root harveshg, plants were grown and treated in the
following ways. Alfalfa seeds
(Medicago sativa
ssp.
sativa
cv
Sitel) were washed for
10
min in 95% ethanol and subse-
quently sterilized in 0.5% sodium dichloroisocyanurate (Bay-
rochlor, Bayrol, GMBH, Munich, Germany),
0.1%
SDS (w/
v) for 15 min. A row of
10
1-d germinated seedlings was
placed on Petri plates containing nitrogen-free Gibson plant
medium as described by Schultze et al. (1992). After
2
d in a
growth chamber at 24OC under a 16-h light period, the roots
were treated with
Rhizobium meliloti
or with purified Nod
factors. For each treatment, about
15
to
20
plant roots were
harvested.
R. meliloti
was grown ovemight in nitrogen-depleted basal
medium supplemented with Glc and sodium succinate (GTS
medium)
(Kiss
et al., 1979) in the presence of
1
p~
luteolin
and resuspended at an
A540
of 0.35 in a liquified 0.8% agarose
solution containing
10
mM MgSO,. This solution (15
pL)
was
deposited in a spot on the root zone with growing root hairs.
Wild-type
R.
meliloti
strain 41 and the nonnodulating mutant
derivative 28138 (Kondorosi et al., 1984) were used. In this
assay, small, white nodules were first visible
4
d postinocu-
lation with strain
41.
For harvesting, foots were dissected
below the inoculation site and approximately
2
cm above the
meristem of the main root.
For experiments with Nod factors, 15
pL
of liquid nitrogen-
depleted Gibson medium, with or without
10-9
M
purified
NodRm-IV(C16:2,S) (Schultze et al., 1992), were distributed
along the root. Action of Nod factor was verified
2
d later by
root-hair deformation as described by Schultze et al. (1992).
The action of modified Nod factor molecules was tested using
Gibson medium supplemented with
10-9
M
nonsulfated
NodRm-IV(Cl6:Z) (Truchet et al., 1991; Baev et al., 1992)
and
10-9
M
tetraacetyl chitotetraose (Sigma)
in
addition to
10-9
M
NodRm-IV(Cl62,S) and the control without Nod
factors.
For harvesting nodules and other plant tissues, alfalfa
plants were grown under aeroponic conditions and treated
as described by Allison et al. (1993). Similarly, mature spon-
taneous nodules were obtained in the aeroponic system on
rooted shoot cuttings of alfalfa genotype A2 (NAR+ pheno-
type) after 5 weeks in the presence of low nitrogen-containing
solution.
Empty nodules induced by Fix-
R. meliloti
were collected
as follows. One-day-old sterile alfalfa seedlings were trans-
ferred in pairs onto 15-mL nitrogen-depleted Ciibson plant
medium slants. Each plant was infected after 3
cl
with 75
pL
of liquid Gibson medium containing
R. meliloti
strain PP553
(Putnoky et al., 1990) at an
A540
of 0.4. White
I+-
nodules
of diffeirent ages were collected after
3
weeks.
RT-PCR.
RNA was extracted using the guanidinium thiocyanate
method and centrifugation through cesium ch:loride. RNA
quality was checked on a formaldehyde gel (Sambrook et al.,
1989).
‘To
avoid any genomic DNA contamination, 5 to
10
rg
of total RNA were treated for 30 min at 37OC with
10
units
of
RNase-free DNase
I
in a volume of i14
pL
in the
presenc:e of 40 mM Tris-HC1 (pH 7.5), 6 mM M;;C12, and 12
units
of
RNAguard (Pharmacia). After heat inactivation of
the DNase, the RNA was precipitated with ethanol and
resuspended in diethyl pyrocarbonate-treated water.
Multiple transcript analysis by RT-PCR was performed
according to modified protocols of Sambrook et al. (1989)
and ChLelly et al. (1988). For greatest reproduci’bility, cDNA
and PCR reaction samples were prepared from a single
“master mix“ of the appropriate reagents. Two to 5
pg
of
DNase-treated RNA were reverse transcribed
by
50 units of
Moloney murine leukemia
virus
H-
Superscript reverse tran-
scriptase (Gibco
BRL)
in a 30-pL reaction mixture containing
100
pmol of oligo(pdT)12-18, corresponding buffer (Gibco
BRL),
10
mM DTT,
0.8
mM dNTP, and 14.4 units of RNAguard
(Pharmlacia) during 1.5 h at 37OC.
Equal amounts of cDNAs (in general, one-tenth of the
reaction mix) or 0.3
pg
of genomic DNA purified according
to Delliiporta et al. (1983) were used for amplification in
100
pL
of corresponding buffer (Promega), 1.5
m~
MgC12, 0.12
m~
dNTP, 150 pmol of 5’ and 3’
MsEnod12
priiners (Allison
et al., ‘L993),
10
pmol of
Rhe2
or
Msc27
primers (Allison et
al., 1993), and 1.5 units of Taq polymerase (Promega). The
MsEnod12
primers described by Allison et al. (1993) were
derived from the
MsEnod12A
sequence. The 24-bp long
3’
MslCnodl2
primer differs in one nucleotide from the
MsEnod12B
sequence, namely at position four in front of the
3’ end of this primer. Amplification from genoinic
M. sativa
DNA showed that this single-nucleotide difference did not
affect the amplification efficiency of the
MsEnodZ2B
fragment
(see Fig.
lB,
lane Ms). The sequence for the
Rhe2
5’ primer
is
5’-CAGCCCATGATCAGCTCCC-3’
and the sequence for
the 3’ primer is
5’-GAACCTGCTAGGCCAAGC-3’.
Ampli-
fication was performed during
20
to 30 cycles of I-min
denaturation at 92OC, 1-min primer annealing at 55OC, and
I-min elongation at 72OC. RT-PCR was controlled by co-
amplification of the endogenously expressed
Msc27
or
Rhe2.
Msc27
(Gyorgyey et al., 1991; Pay et al., 1992; Allison et al.,
1993; Csanadi et al., 1994) was similarly expressed
in
differ-
ent tissues and nodules, as demonstrated by a northem blot
with equal amounts of RNA loaded and also probed as

Differential
Regulation
of
Alfalfa
Enoc/72
Genes
587
control
to
ribosomal
DNA
(our unpublished results). Rhe2,
isolated
from
an
alfalfa
root hair cDNA library (L.A. Allison,
unpublished results),
was
used
as an
internal control
in
root
samples.
By
northern blot,
no
induction
of
this gene
was
detected
after
treatment with
R.
meliloti
or Nod
factors
(L.A.
Allison,
unpublished results).
Rhe2
is
homologous
to
genes
from
tobacco
and
Arabidopsis
coding
for
channel proteins
(Yamamoto
et
al., 1990).
The
exponential range
of the PCR
was
tested
by
removing aliquots
after
various numbers
of
cycles
from
trial
PCR
reactions.
After
20
cycles
the
amplifi-
cation
rate
was in a
linear range
for all PCR
products (data
not
shown).
Gel
Electrophoresis
and
Southern Blot
According
to
Sambrook
et al.
(1989), one-tenth
of the PCR
products
was
separated
on a 2%
Tris-borate-EDTA agarose
gel
and
transferred
to a
Hybond-N nylon membrane using
a
capillary
blot system.
The
membrane
was
first
hybridized
to
pBluescript
containing
an
MsEnodl2B
412-bp
PCR
fragment
(see
'In
Situ Hybridization").
Due to the 96%
sequence iden-
tity
of the
MsEnodllA
and
MsEnodllB
PCR
products, this
probe revealed both bands. Following
its
removal,
the
blot
was
rehybridized
to
either
an
Msc27
probe (Allison
et
al.,
1993)
or to
Rhe2
234-bp
PCR
fragments. Probes were labeled
with [32P]dCTP.
In
Situ
Hybridization
A
detailed description
of the
preparation
of
sections
of 20-
d-old
nodules,
fixation,
and in
situ
hybridization
to RNA
probes
was
reported previously
by
Grosskopf
et al.
(1993).
For
generating
the RNA
probe,
an
MsEnodllB
fragment
was
amplified
with
the
MsEnodll
5'
primer
and a
primer homol-
ogous
to the 3'
untranslated region
of
MsEnodllB (5'-CA-
ACTTGCCTTGCCCAT-3')
from
a
XEMBL4
genomic clone
containing
the
MsEnodl2B
gene.
The
412-bp
PCR
product
was
cloned into
the
EcoRV
site
of a
pBluescript vector.
The
antisense
RNA
probe
was
obtained
by in
vitro
transcription
according
to
Grosskopf
et al.
(1993).
RESULTS
Differential
Expression
of
MsEnod12A
and
MsEnod12B
MsEnodl2A
and
MsEnodl2B
(Allison
et
al., 1993)
and
MtEnodl2
(Pichon
et
al., 1992) show high sequence homology
among
each
other. Figure
1A
shows
the
alignment
of the
encoded amino acid sequences.
The
major
difference
between
the
derived proteins
is the
length
of the
Pro-rich repeat
region. MsEnodl2A
is the
smallest protein with
11
Pro-rich
repeats, each consisting
of
five
amino
acids,
followed
by
MtEnodl2
and
MsEnodl2B with
13 and 15
repeats,
respectively.
Due
to the
high
95% DNA
sequence identity
of
MsEnodllA
and
MsEnodl2B
in the
coding
as
well
as in the 3'
noncoding
region (Allison
et
al., 1993),
we
could
not
follow
the
expres-
sion
of the two
genes separately
by any
conventional hybrid-
ization technique. Therefore,
the
expression pattern
was
investigated
by
applying
an
RT-PCR
method, using oligo-
nucleotides
flanking
the
deletion
in the
Pro-rich repeat region
PCR
primer
1 T
PCR
primer
2
Ms Mt
299
bp -
MsEnodlZB
239
bp -
MsEnodl2A
MtEnodlZ
- 269 bp
Figure
T.
Comparison
of the
Enod12 sequences
in
Med/cago.
A,
Alignment
of the
amino
acid
sequences
of
MsEnod12B,
MtEnod12,
and
MsEnod12A. Black
outlining
indicates
identical
amino
acids,
gray
outlining
shows conservative substitutions. Dots
within
the
sequence show
deletions
in the
Pro-rich repeat
region.
The
arrow-
head marks
the end of the
putative signal
peptide.
The
positions
of
Msfnod/2
primers
1 and 2 are
indicated
by
arrows.
B, PCR
analysis
of
Enoc/72
genes
in M.
saliva
and M.
truncatula.
Enod/2
sequences
were
amplified
during
30
cycles
from
0.3 /jg of
genomic
DNA,
separated
by
electrophoresis,
and
transferred
to a
membrane
that
was
hybridized
to an
MsEnodT2B
probe.
Lanes:
Ms, M.
saliva
ssp.
saliva
cv
Nagyszenasi;
Mt, M.
truncatula
PCR
products
with
indi-
cated sizes.
as
indicated
in
Figure
1A. The PCR DNA
fragments
of
MsEnodllA
and
MsEnodllB
differ
in
size
by 60 bp as
shown
in
Figure
IB.
After
amplification
of
genomic
M.
saliva
DNA,
two PCR
bands were visible
of the
expected
sizes
of Ms-
EnodllA
and
MsEnodllB, namely
239 and 299 bp
(Fig.
IB,
lane
Ms). Genomic
DNA
from
M.
truncatula
gave
a
single
intermediate
PCR
band corresponding
to the
expected 269-
bp
size (Fig.
IB,
lane
Mt;
Pichon
et
al., 1992).
We
established
an
approach
for
multiple transcript analysis
that
proved
to be the
most
reliable
for our
experiments.
DNase-treated total
RNAs
from
different
plant tissue samples
were reverse transcribed with
an
oligo(dT) primer. From these
cDNA
mixtures, Enodll
and an
endogenously expressed con-
trol
gene, Mscl7
or
Rhel,
were simultaneously co-amplified
with
the
appropriate oligonucleotide primer pairs
in the
same
reaction tube.
The
intensity
of the DNA
fragment
of the
control
gene
after
PCR
reflected sample-to-sample variations
in RT and
PCR,
and
monitored
the
eventual extent
of RNA
degradation during
the
manipulations.
We
chose controls
generating single products
after
amplification.
The
constitu-
tively
expressed Mscl7 (Gyorgyey
et
al., 1991;
Pay et
al.,
1992; Allison
et
al., 1993; Csanadi
et
al., 1994)
was
used
as
an
internal control
in
different
tissue
and
nodule samples.
Rhel
(L.A.
Allison, unpublished results),
a
gene constitutively
expressed
in
roots,
was
used
to
control
the
RT-PCRs
in
root
samples
(see
'Materials
and
Methods").
For
quantisation,
the
ratios between control
and
Enodll amplification fragments
had to be
comparable (for
a
recent review about quantitative
RT-PCR,
see
Foley
et
al., 1993).
In our
case,
the
control
transcripts were more abundant than
the
Enodll sequences.
To
overcome this problem
and a
possible
effect
of
out-

588
Bauer
et al.
Plant
Physiol.
Vol. 105, 1994
titration
of the
low-abundant Enodl2 sequences (described
by
Murphy
et
al., 1990),
the
primer concentrations were
reduced
for the
controls (down
to 10% of the
optimum
concentration)
and
increased
for
Enod.11
(150%
of the
opti-
mum
concentration). Analyzing trial
PCRs
after
various num-
bers
of
cycles demonstrated that control
and
Enodll
ampli-
fications
were
in the
linear range
after
20 to 25
cycles (data
not
shown).
The
expression
of
MsEnodl
2A
and
MsEnodl2B
was
studied
in
various plant tissues.
MsEnodl
2A
was
expressed
in
nodules
but it was not
detected
in any
other tissue
as
seen
in
Figure
2.
In
contrast,
MsEnodl2B
transcripts were found
in
nodules
and low
amounts were also detected
in
roots, flowers, stems,
and
leaves (Fig.
2). In
5-d-old seedling roots (Fig.
2,
lane
r),
MsEnodl2B
was
more expressed than
in the
roots
of
4-week-
old
nodulated
plants.
The
expression
was
analyzed
in the
middle
and
lower parts
of
these roots
after
excision
of
nodules
(Fig.
2,
lanes
rm and
rl).
We
excluded
the
possibility that
the
faint
MsEnodl2B
PCR
bands were
due to
contaminating
genomic
DNA
fragments
because
no
MsEnodl2A
signals were
found
(compare
to
Fig.
IB,
lane Ms). These results indicated
that
MsEnodl
2A and
MsEnodl
2B
were differentially expressed
in
various plant tissues.
In
a
further
experiment,
the
induction
of the two
genes
was
followed during nodule development. Three-day-old
alfalfa
seedlings were inoculated with
the
wild-type strain
R.
meliloti
Rm41,
and as a
control, with
the
nodulation-deficient
mutant
ZB138.
After
2, 4, and 6 d, the
regions
of the
roots
below
the
spot-inoculation site were dissected
for
transcript
analysis.
Figure
3
shows that
a low
amount
of
MsEnodl
2B
transcripts
was
found
in all
root samples.
After
inoculation
with
Rrn41,
MsEnodl
2B
expression increased until
d 6,
whereas
after
inoculation with
the
nodulation-deficient
mu-
tant,
it
stayed
at a
constant,
low
level
during this same time
period. Expression
of
MsEnodl
2A was
detectable only
4 and
6 d
after
inoculation with Rm41, when nodules
first
became
visible (Fig.
3). The
control
for
RT-PCR
was
Rhel
amplifica-
ZB
138
2
4
st 1 fl r n7 n20 rm rl
MsEnodl2B
MsEnodl
2
A.
MsEnodl2B
MsEnodl2A
Msc27
Figure
2.
RT-PCR
analysis
of
MsEnodl2A
and
MsEnodl2B expres-
sion
in
different plant tissues. Enod/2
and the
control
Msc27
se-
quences were
co-amplified
during
25
cycles
from
RNA of
various
plant
tissues
of M.
sat/va
ssp. saliva. After
electrophoresis
and
transfer
blot,
they were subsequently
hybridized
to an
MsEnodl2B
or
Msc27
probe.
The
different lanes
contain
RT-PCR
products
from
stems
(st)
and
leaves
(I)
both
from
9-d-old
plants; flowers (fl);
untreated
roots
of
5-d-old seedlings (r);
7-
(n7)and
20-d-old
(n20)
nodules;
middle
(rm)
and
lower (rl)
root
parts
of
4-week-old
nodu-
lated
plants after excision
of
nodules.
A
short
(a) and a
long
(b)
exposure
of the
MsEnodl26
hybridization
filter
are
shown.
MsEnodl2B
MsEnodl2A
Rhe2
Figures.
RT-PCR
analysis
of
MsEnodl2A
and
MsEnod/2B
induction
during
nodule
development.
RNA
from
root
samples harvested
at
different
time
points
after
R.
meliloti
inoculation
was
used
for RT
and
co-amplification
during
20
cycles
of
MsEnodl2
and
Rhe2
se-
quences.
The PCR
products were
hybridized
subsequently
to an
Ms£nod/26
and an
Rhe2
probe.
The
first three lanes
(ZB138)
represent
control
roots harvested
at
several
time
points
after ZB138
inoculation,
namely after
2, 4, and 6 d; the
next three lanes
(Rm41)
show roots
collected
2, 4, and 6 d
after Rm41
infection.
tion.
Since only Rm41
but not
ZB138
is
capable
of
producing
Nod
factors, these results
indicated
that
the
lipooligosaccha-
ride molecules might
be
signals inducing
MsEnodl
2
gene
expression.
Effect
of
Nodulation
Factors
on
MsEnodl2A
and
MsEnodl2B
Expression
The
effect
of
purified
Nod
factors
on
MsEnodl2A
and
MsEnodl2B
gene expression
was
tested. Three-day-old plant
seedlings were treated with
the
cognate
Nod
factor
NodRm-
IV(C16:2,S)
at a
concentration
of
10~9
M for
different
time
periods.
As a
control, plants were mock-inoculated with plant
medium. Roots were
cut
into three zones: zone
1,
next
to the
meristem
and
devoid
of
root
hairs;
zone
2,
containing
grow-
ing
root hairs;
and
zone
3,
with mature root hairs,
as
shown
in
Figure
4C.
Expression
of
MsEnodl
2B
was
enhanced
in the
root
hair zones
2 and 3
after
Nod
factor
treatment (Fig. 4A).
The
highest
MsEnodl
2B
transcript level
in
zone
2 was de-
tected
6 h
after
treatment,
and in
zone
3 1 d
after
treatment.
In the
meristematic
region,
MsEnodl
2B
expression
was not
enhanced. Root-hair deformations were observed
after
1 d in
zones
2 and 3. At 2 and 3 d
after
Nod
factor
application,
the
MsEnodl
2B
transcript
level
was not
significantly
different
from
the
control
in any
zone (data
not
shown). Minor
differ-
ences
in the
control Rhel amplification were
not
sufficient
to
account
for the
relatively large differences observed
for
MsEnodl
2B,
indicating comparable inputs
of
cDNA quantities
and PCR
efficiencies
in the
different
samples (Fig. 4A).
No
major
differences
in the
level
of the
MsEnodl
2B
transcripts
were found
if Nod
factor
concentrations
from
10~13
M to
10~7
M
were used.
Nod
factor
below
a
concentration
of
10~13
M
did not
enhance
MsEnodl
2B
expression (data
not
shown).
After
a
longer exposure
of the
filter
hybridization
of
Figure
4A,
MsEnodl
2B
signals were visible
in all
root samples (not
shown) except
MsEnodl
M. At the
different
concentra-
tion
tested,
the
cognate
Nod
factor
was not
able
to
elicit
MsEnodl
2A
expression
in
roots
in any of the
tested times
(up
to 4 d).
In
further experiments, unsulfated
Nod
factor NodRm-

Differential
Regulation
of
Alfalfa
Enod12
Genes
589
co
6h
IV,S
6h co 26h
IV,S
26h
1
2 31
3 " 1 2 3
MsEnodl
2B
co
IV.S
IV NAG
I
/ "
Zone3
Mature
root
hairs
Zone2
Growing
root
hairs
Zonel
No
visible root hairs1
Figure
4.
RT-PCR
analysis
of
MsEnodl
2B
induction
after treatment
with
Nod
factors.
A,
RT-PCR
was
performed
on
roots
treated
with
10~9
M
NodRm-IV(C16:2,S)
for 6 and 26 h and on
mock-inoculated
control roots
cut
into three zones
as
shown
in C. The
co-amplified
Msfnod/2
and
Rhe2
fragments
(20 PCR
cycles)
were subsequently
hybridized
to an
MsEnod12B
and an
Rhe2
probe.
The
first
six
lanes
contain
the
root samples
after
mock-inoculation
(co 6h) and
treat-
ment
with
Nod
factor
for 6 h
(IV,S
6h)
harvested
from
zones
1, 2,
and 3; the
next
six
lanes
represent control roots
(co
26h)
and Nod
factor-treated
roots
after
26 h
(IV,S 26h).
1, 2, and 3
indicate
the
root zones from which
RNA was
prepared.
B,
Roots
were treated
with
10~9
M
modified
Nod
factors
for 6 h.
Root pieces
of
zone
2
were
dissected
and
used
for
RT-PCR
analysis
as
described above.
The
different
lanes represent
the PCR
products
of
mock-inoculated
control roots (co); NodRm-IV,S
(IV.S);
NodRm-IV (IV);
and
chitote-
traose-treated
roots (NAG).
C,
Schematic representation
of the
root
of an
alfalfa
seedling. Zone
1
corresponds
to the
meristematic zone,
zone
2 is the
zone
with
growing root hairs,
and
zone
3 is the
zone
with
mature root
hairs.
IV(C16:2)
and
N-acetyl glucosamine terrasaccharide were
tested
for
their capacity
to
enhance
the
expression
of the
MsEnodl2B
gene, both
at the
concentration
of
10~9
M.
After
a
treatment
of 6 h,
root
pieces
of
zone
2
were
harvested.
Nonsulfated
Nod
factor
with reduced abilities
for
root-hair
deformation
(Roche
et
al., 1992)
significantly
enhanced
MsEnodl2B
expression (Fig.
4B,
lane IV).
After
treatment with
chitotetraose,
no
enhancement
of
MsEnodl2B
expression
was
found
(Fig.
4B,
lane NAG). Again,
MsEnodl2A
transcripts
were
not
detectable
in
roots. These results indicate that
expression
of
MsEnodl2B
but not
MsEnodl2A
can be
modu-
lated
by Nod
factors
in the
root.
Expression
of
MsEnod12A
and
MsEnodl2B
Is
Associated
with
Infection
The
site
of
MsEnodl2
gene expression
in
nodules
was
localized
by in
situ hybridization
of a
20-d-old
M.
sativa
ssp.
sativa
nodule using
a
MsEnodl2B
probe (Fig.
5).
Hybridization
signals indicating
MsEnodl2
expression occurred
in
cells
of
the
infection zone near
the
nodule meristem where
the
plant
cells
are
invaded through infection threads.
In
addition,
hybridization
signals were observed
in the
peripheral tissue
of
the
nodule. These additional signals might
be
explained
by
cross-hybridizing
RNA
coding
for a
Pro-rich protein.
A
cross-hybridizing transcript
has
been
seen
on a
northern
blot
containing
nodule
RNAs
probed against
MsEnodl
2
sequences
(Allison
et
al., 1993).
To
study whether Enodl2 induction
is
related
to the
process
of
infection,
we
examined
its
expression
by
RT-PCR
in
nodules blocked
in the
invasion step. Empty
nodules
were obtained
by
inoculation with
the
Fix"
R.
meliloti
strain PP553 (Putnoky
et
al., 1990) mutated
in the
exoB
and
fix-23
genes
and
consequently lacking outer
surface
exo-
polysaccharides
and
capsular polysaccharides required
for
invasion.
In
nodules induced
by
this strain, infection threads
are
aborted
and
only very
few
cells
are
invaded (Putnoky
et
al., 1990). Fix" nodules
of
different developmental stages
were
harvested
3
weeks
after
inoculation.
MsEnodllA
was
expressed
at a
much lower level
in
these empty nodules than
in
7-d-old white
or
19-d-old nitrogen-fixing wild-type nod-
Figure
5.
Localization
of
MsEnod12
expression
in
mature alfalfa
nodules.
In
situ hybridization
was
performed
on
sections
of a 20-
d-old
M.
sativa
nodule.
A
35S-labeled MsEnodl28 antisense
probe
was
used.
The
infection zone showed
a
high density
of
silver grains
in
this
section.
B
shows
a
higher magnification
of the
infection
zone
than
A.

590
Bauer
et al.
Plant
Physiol.
Vol. 105, 1994
Fix
~
Spont 7dwt 19dwt
MsEnodl2B
MsEnodl2A
MsEnodl2B
MsEnodl2A
Msc27
Figure
6.
RT-PCR
analysis
of
MsEnod12A
and
MsEnodl2B expres-
sion
in
Fix"
and
spontaneous nodules.
Ms£nodI2
and
Msc27
se-
quences were
co-amplified
from
various
nodule
samples. After
gel
electrophoresis they were transferred
to a
membrane
and
hybrid-
ized
subsequently
to an
MsEnod12B
and an
Msc27
probe.
The
lanes
show amplification products from Fix" nodules (Fix"); mature spon-
taneous
M.
sativa
ssp.
varia
nodules (Spont);
7- and
19-d-old
wild-
type
nodules
(7dwt
and
19dwt,
respectively),
a and b
show
the
Ms£nod!2A
and
Ms£nod!2B
amplification
after
20 and 25 PCR
cycles,
respectively.
The
Msc27 signals were
obtained
after
20
cycles.
ules (Fig.
6,
lanes
FbT,
7dwt,
and
19dwt).
The
MsEnodUB
transcript level
in
Fix"
nodules
was
comparable
to the one in
19-d-old wild-type
and
slightly lower than
in
7-d-old wild-
type nodules (Fig.
6).
Reduced expression
in
Fix" nod-
ules compared
to
wild-type
nodules
was
more
striking
for
MsEnodl2A
than
for
MsEnodUB.
We
also analyzed
the
expression
in
spontaneous nodules, obtained
after
nitrogen
starvation
on
certain
genotypes
of M.
sativa
in the
absence
of
bacteria (Truchet
et
al., 1989).
In
mature spontaneous
nodules
of M.
sativa
ssp.
varia
genotype
A2,
neither
of the
two
transcripts
was
detectable (Fig.
6,
lane Spont). Both
MsEnodl2
genes
can be
amplified readily
from
genomic
DNA
of
the
subspecies
varia
(not shown).
The
control
Msc27
bands
were amplified
in all
cases, which proved that
in the
samples
analyzed cDNA synthesis
and PCR had
taken place correctly
(Fig.
6).
These results indicate that
MsEnodllA
expression
correlates with
the
presence
of
rhizobia inside nodules.
DISCUSSION
We
demonstrated
here
that
the two
early nodulin
genes
MsEnodl2A
and
MsEnodUB
are
differentially
expressed dur-
ing
symbiosis, providing evidence
for
distinct mechanisms
regulating
these
two
genes
in
alfalfa.
Our
results
showed
that
R.
meliloti
Nod
factors
induce
the
expression
of
only
MsEnodUB.
Hence,
MsEnodl2A
exhibits
a
novel EnodU
expression
pattern,
different from
all
other EnodU
genes,
since
it is not
induced
by
Rhizobium
lipooligosaccharides.
MsEnodllA
expression
is
related
to the
invasion process
in
nodules.
After
infection with
R.
meliloti,
MsEnodl2B
was
rapidly
induced
in
roots, prior
to
induction
of
MsEnodl2A. This
MsEnodl2B
expression increased until nodules became visi-
ble. This early induction probably occurred
in the
epidermis
and in
root hairs
as
demonstrated
for the
Enodl2 genes
in
pea
(Scheres
et
al., 1990a)
and M.
truncatula
roots (Pichon
et
al.,
1992). Accordingly,
an
enhancement
of
MsEnodUB
tran-
scription
in
roots
was
detected
after
treatment with
the
cog-
nate
Nod
factor
at the
concentration
of
10"9
M.
This induction
took place
in the
root
zones
susceptible
for
root-hair defor-
mations. However,
after
application
of the Nod
factor,
the
effect
occurred transiently, reaching
a
maximum level
6 to 26
h
after
treatment.
The
transient
effect
indicates that
the
continuous presence
of Nod
factors might
be
necessary
for a
prolonged
MsEnodUB
expression,
as is the
case during con-
tact
with rhizobia. Degradation
of Nod
factors
due to
plant
chitinases
(Staehelin
et
al., 1994) could
be an
explanation
for
this
transient expression.
MsEnodl2B
was
induced
by the
sulfated
and the
nonsulfated
NodRm-IV(C16:2),
whereas
the
unsubstituted sugar backbone alone
was not
able
to
induce
the
gene. Recent studies
by
other groups also indicate that
Enodl2
genes
can be
induced
by
noncognate
Nod
factors.
Horvath
et al.
(1993) demonstrated
that
besides
R.
legumi-
nosarum
bv
viciae
NodRlv metabolites, lipooligosaccharides
from
R.
meliloti
were able
to
trigger
the
expression
of the
PsEnodl
2
genes
in
pea. Pichon
et al.
(1993) reported induction
of
MtEnodl2
by
nonsulfated
Nod
factor
at
concentrations
of
10"9
M and
higher.
In
this case, sulfated
NodRm-IV(C16:2,S)
was
active
at a
concentration range
4
orders
of
magnitude
lower,
indicating that
a
specific structure
was
required
for
MtEnodl2
induction
by low
Nod-factor concentrations.
MsEnodl2B
was
expressed
at a low
level
in
various
plant
organs,
and
therefore
is not a
true
early
nodulin gene,
ac-
cording
to the
definition
by van
Kammen (1984). Expression
in
nonsymbiotic
tissues
was
also
demonstrated
for
PsEnodl
2A
and
PsEnodl2B,
specifically
in
flowers
and
stems (Scheres
et
al.,
1990a; Covers
et
al., 1991).
In
contrast
to the pea
EnodU
genes
and to
MtEnodl2,
MsEnodUB
was
expressed
at a
basal
level
in
untreated roots.
The
expression
was
higher
in
3-d-
old
seedling roots than
in
roots
of
plants grown
for
more
than
a
month (Fig.
2). A
similar down-regulation
of Ms-
Enodl2B
expression
was
observed during
the
aging
of
wild-
type nodules (Allison
et
al., 1993).
The
very
low
level
of
expression
in
mature tissues could also explain
the
failure
to
detect
MsEnodUB
signals
in
mature spontaneous nodules.
During
formation
of
these spontaneous nodules,
a
higher
expression level might have occurred.
MsEnodl2A
was
induced
at a
later stage during nodule
development than
MsEnodl2B,
and
could
not be
induced
in
roots
after
application
of Nod
factors.
Therefore,
we
conclude
that
in
root cells,
MsEnodUA
cannot
be
regulated
by
Rhizo-
bium
lipooligosaccharides,
as
would
be the
case
for all
other
EnodU
genes characterized (Horvath
et
al., 1993; Pichon
et
al.,
1993).
MsEnodUA
expression
was
detectable only
4 d
after
infection with
R.
meliloti
when nodules
first
became
visible.
Presumably,
the
transcription started
in
nodule pri-
mordia, where
it
increased
rapidly
to a
high
level
during
nodule development.
In
situ hybridization
of a
nitrogen-
fixing
nodule with
a
MsEnodU
probe showed hybridization
signals
in the
infection zone
and in the
periphery
of the
nodule
but not in the
central symbiotic zone, providing
evidence
for the
role
of
MsEnodU
genes
in the
infection
process. However,
due to the
high homology
of the two
MsEnodU
genes, precise localization
of the
expression
of
each gene could
not be
addressed
by
this technique.
In
empty

Differential
Regulation
of
Alfalfa
Enodl2
Genes
591
nodules,
MsEnodl2A
expression
was
much reduced compared
to wild-type nodules, correlating with the low frequency of
invasion
observed
in nodules induced by
exoB
and
fix-23
double
mutants (Putnoky et al., 1988). The
low
leve1
of
MsEnodl2A
expression in empty nodules and the absence of
MsEnodl2A
transcripts
in
spontaneous nodules suggest that
MsEnodl2A
expression correlates with
R.
meliloti
invasion.
Moreover,
MsEnodl2A
is a
true
early
nodulin
gene
(van
Kammen,
1984),
with activation occumng
only
in
nodules
but not
in
roots or
any
other plant organs.
MsEnodl2A
and
MsEnodl2B
show
nove1 expression pat-
tems compared to the
Enodl2
genes
of
pea or
M
truncatula.
MsEnodl2B
expression
takes
place
in
a
way
similar
to
the
induction
of
MtEnodl2
during symbiosis. Most information
about the
M. truncatula Enodl2
gene
expression,
however,
is
derived
from
the
behavior
of
a
MtEnodl2
promoter-&glucu-
ronidase fusion in a
M. sativa
background.
Further
evidence
for
a close relationship
of
MsEnodl2B
with
MtEnodl2
was
recently provided
by
Csanadi et al. (1994),
who
traced the
evolution
of
Medicago
species based
on
Enodl2
sequences.
They
calculated that after divergence
of
Medicago
species,
MsEnodl2A
as
well
as
MtEnodl2
would
have evolved inde-
pendently through
deletions
from
an MsEnodl2B-type
gene
present
in
a
common
ancestor plant.
In
conclusion, early nodulin gene expression pattems
differ
among legume species. Alfalfa is interesting in the analysis
of
Enodl2
gene
transcriptional control, since
different
regu-
latory pathways
may
control the activation
of
MsEnodl2A
and
MsEnodl2B
during
symbiosis. Transgenic
M. sativa
ssp.
varia
plants containing
promoter-0-glucuronidase
fusions
for
both
MsEnodl2
genes
are
being constructed in
our
laboratory
to study the signals required
for
MsEnodl2A
and
MsEnodl2B
expression.
Tissue- and cell-specific expression pattems
were
also
found for members
of
other Hyp-rich glycoprotein
gene
families
whose
encoded proteins
differ
primarily in the com-
position
and
the
number
of
Pro-rich repeats (see,
for
example,
Hong
et
al.,
1989;
Wyatt
et
al., 1992).
The
importance
of
the
organization
of
the Pro-rich repeat region
for
the properties
of
Hyp-rich glycoproteins is still unknown. We speculate that
MsEnodl2A
and
MsEnodl2B
might
be
involved
in
distinct
cell-wall
structures
of
root
hairs,
roots,
and
nodules
arising
during
the infection
process
leading
to symbiosis.
ACKNOWLEDCMENT
The authors thank Dr. Heather
I.
McKhann for critical reading of
the manuscript.
Received December 7, 1993; accepted February 9, 1994.
Copyright Clearance Center:
0032-0889/94/l05/0585/08.
LITERATURE CITED
Allison LA, Kiss GB, Bauer P, Poiret M, Pierre M, Savoure A,
Kondorosi
E,
Kondorosi A
(1993) Identification of
two
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