Download Plant Physiology

Plant Physiol. (1992) 98, 143-151
0032-0889/92/98/01 43/09/$01 .00/0
Received for publication February 8, 1991
Accepted August 20, 1991
Nodules Initiated by Rhizobium meliloti Exopolysaccharide
Mutants Lack a Discrete, Persistent Nodule Meristem'
Cheng Yang, Ethan R. Signer, and Ann M. Hirsch*
Department of Biology, Peking University, Beijing, China (C. Y.), Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139 (E.R.S., C.Y.), and Department of Biology, University of California,
Los Angeles, California 90024 (A.M.H.)
ABSTRACT
pericycle after the initiation of mitoses in the inner cortex
(18).
R. meliloti mutants have been utilized extensively to characterize the earliest events in the establishment of alfalfa root
nodules. Although nodulation (nod) mutants attach to root
hairs (see references in Long [20]), they do not elicit shepherd's crook formation nor do they stimulate cell divisions
within the root cortex (7). On the other hand, exo mutants,
which are deficient in EPS,2 trigger nodule formation, but the
nodules that develop are atypical. The nodules are free of
bacteria ("empty") and clustered on secondary roots like
"beads on a string" (11).
We originally reported that EJ355, an exoB mutant in the
multiply marked genetic background of EJ312, deformed root
hairs but did not induce shepherd's crook formation (11). We
also noted that many of the nodules elicited by EJ355 developed on the primary root and had a defined apical meristem.
However, we have recently found that the EJ312 genetic
background affects the expression of R. meliloti symbiotic
genes (6). Accordingly, we have expanded our study of bacterial invasion and nodule initiation in response to exo mutations in the wild-type Rm 1021 background so that the point
of nodule arrest may be delineated more clearly.
We investigated infection thread development as well as
the earliest steps in nodule formation-cortical cell division,
nodule primordium initiation, emergence of the nodule-and
compared them with the published reports of wild-type R.
meliloti-elicited nodule development (7). We found that many
of the initial stages ofbacterial invasion and nodule formation
are similar for wild-type R. meliloti (exo+) and exo mutants.
However, unlike the EJ355-induced nodules we described
earlier (11), the empty nodules elicited by exo mutants in the
Rm 1021 background have a diffuse region of cell division
that extends over the distal end of the nodule, rather than a
discrete, persistent meristem. Furthermore, although infection
threads were not detected in the deformed root hairs of plants
inoculated with Rm 1021-derived exo mutants, they were
observed in sectioned material, where, as in EJ355-inoculated
plants, they were present in the outermost cells of the nodule.
We suggest that the lack of sustained meristematic activity in
exo mutant-induced nodules is directly related to the abortion
of infection threads in the superficial cells of the nodule.
Infection of alfalfa with Rhizobium meliloti exo mutants deficient
in exopolysaccharide results in abnormal root nodules that are
devoid of bacteria and fail to fix nitrogen. Here we report further
characterization of these abnormal nodules. Tightly curied root
hairs or shepherd's crooks were found after inoculation with
Rm1021-derived exo mutants, but curing was delayed compared
with wild-type Rm1O21. Infection threads were initiated in curled
root hairs by mutants as well as by wild-type R. meli/oti, but the
exo mutant-induced threads aborted within the peripheral cells of
the developing nodule. Also, nodules elicited by Rm1O21-derived
exo mutants were more likely to develop on secondary roots than
on the primary root. In contrast with wild-type R. meliloti-induced
nodules, the exo mutant-induced nodules lacked a well defined
apical meristem, presumably due to the abortion of the infection
threads. The relationship of these findings to the physiology of
nodule development is discussed.
Rhizobial invasion and infection thread penetration into
root hair cells are crucial steps leading to the establishment
and development of the Rhizobium-legume symbiosis. Although the early stages of nodule development have been
studied in a large number of legumes, many questions relating
to the entry of the bacteria into the root hair cell and the
initiation of the nodule remain.
In the alfalfa-R. meliloti symbiosis, one of the earliest
responses to Rhizobium inoculation is root hair curling. Infection threads are formed within the tightly curled root hairs
known as "shepherd's crooks" and then penetrate the cells of
the root (33). Several cells away from the growing infection
thread (5), in the inner root cortex in alfalfa, anticlinal cell
divisions take place, giving rise to the nodule primodium. The
infection thread penetrates deep into the root cortex and
invades the nodule primordium (18, 23). Bacteria are released
from infection thread branches into the cortical cell derivatives, which then stop dividing and expand. A persistent
nodule meristem is initiated at the distal (apical) end of the
nodule. Cell divisions also occur in the endodermis and
' Supported by National Institutes of Health grant ROI-GMI3 1030
(E.R.S.) and National Science Foundation grant DCB 87-03297
(A.M.H.)
2
Abbreviations: EPS, exo, exopolysaccharide; LPS, lipopolysaccharide; str, streptomycin resistance.
143
144
YANG ET AL.
MATERIALS AND METHODS
Growth of Seedlings
Seeds of alfalfa (Medicago sativa L. cv Iroquois) were either
surface sterilized in 70% (v/v) ethanol for 10 min, followed
by 0.1% (w/v) HgCl2 for 3 min and 2.6 % (v/v) sodium
hypochlorite for 20 min, or sterilized for 60 min in fullstrength commercial bleach (5.25% sodium hypochlorite)
after a 60-min pretreatment in 95% (v/v) ethanol. The seeds
were then washed 5 to 6 times with sterile distilled water and
germinated either on agar slants (22) under 16 h/8 h 21°C/
19°C day/night conditions or on water agar plates for 72 h in
the dark at room temperature.
Bacterial Strains
Rhizobium meliloti 1021 is a symbiotically effective, streptomycin-resistant (str-7) derivative of R. meliloti 2011 (22);
the str-7 mutation, also called str-21, does not affect the
symbiotic behavior of R. meliloti. Strains Rm7094 (exoB),
Rm7061 (exoA), and Rm7055 (exoF), all derived from R.
meliloti 1021, are TnS mutants that are Fix- on plants and
deficient in EPS I, as is strain Rm7029, which carries a
deletion of the exo genes (10) (exoE). EJ355 is an exoB
mutant of EJ312 (16), which is streptomycin-resistant (str-3).
The str-3 mutation, in contrast to str-7, affects symbiotic
function (1, 6). Rm5078 carries the exoB355 mutation transduced from EJ355 into the RmlO21 background (17).
Five milliliter cultures of rhizobia were grown overnight in
Lucia Bertani Broth with appropriate antibiotics (19), harvested by centrifugation, washed once with sterile PBS (per
liter: 8.0 g NaCl, 0.2 g KCI, 1.44 g Na2HPO4-2H20, 0.2 g
KH2PO4, pH 6.8), and centrifuged. The bacterial pellet was
resuspended in one-tenth strength PBS. The suspension was
further diluted with Fahraeus medium (9) to give an inoculum
density of approximately 1 x 107 cells/mL.
Plant Assays
Alfalfa seedlings were grown in agar slants (22) or in Fahraeus slide assemblies in liquid medium as described by
Bhuvaneswari and Solheim (3). The roots of plants grown in
the Fahraeus slide assemblies were examined under the light
microscope at daily intervals for 4 d. Roots were examined
every 4 to 6 h during the first day of culture in the Fahraeus
slide assemblies. The total root system of the seedling was
examined during the experiments. Photographs were taken of
almost fully elongated root hairs in the middle region of the
growing root hair zone as described by Wood and Newcomb
(33).
When plants were grown in agar slants, they were removed
from test tubes every other day, mounted on glass slides under
cover glasses, and examined under the light microscope for
the presence of infection threads. They were discarded after
Plant Physiol. Vol. 98, 1992
Petri dishes (Labtek) containing Jensen's (32) agar, and the
roots were inoculated as described by Dudley et al. (7). Root
sections were harvested and fixed for microscopy at varying
times after inoculation.
Microscopy
For electron microscopy, root and nodule tissue was fixed
in 3% (v/v) glutaraldehyde or 4% (v/v) glutaraldehyde, 1.5%
(w/v) paraformaldehyde in 0.1 M phosphate buffer, pH 6.8,
for 2 h at 4°C, then rinsed several times in fresh buffer. The
tissue was postfixed 1 h in 1% (w/v) OS04 at 4°C, rinsed
twice, dehydrated through acetone, and embedded in Spurr's
resin or Epon 812. Ultrathin sections for transmission electron
microscopy were stained in uranyl acetate and Reynold's lead
citrate.
For light microscopy, tissue was prepared as described
above. The plastic sections (0.5-1 ,um) were stained in 0.05%
(w/v) toluidine blue 0 dissolved in 1 % (w/v) sodium borate.
Serial sections were made of more than 10 different nodules.
Some tissue was embedded in paraffin as described by Van
de Wiel et al. (30) and sectioned serially at 7 to 8 ,m.
RESULTS
Root Hair Deformation
Root hairs of alfalfa grown in Fahraeus slide assemblies
deformed within 4 h after inoculation with wild-type strains
Rm 1021 or Rm2O 11. By 8 h, deformation was extensive, and
an occasional 3600 curl (shepherd's crook) was observed.
However, shepherd's crooks generally were not conspicuous
until 12 to 24 h after inoculation. Moreover, they were
difficult to detect among the population of deformed root
hairs (Fig. IA, open arrow).
Extensive root hair deformation occurred as early as 4 h
after inoculation with the exoB::Tn5, exoA::Tn5, or
exoF::Tn5 derivatives of Rm 1021. However, shepherd's
crooks were not observed on alfalfa roots inoculated with exo
mutants until 24 h after inoculation. By 48 h after inoculation,
3600 curls were infrequently observed in the growing root hair
zone (33) (Fig. 1B). By 7 d after inoculation, root hairs in the
growing root hair zone were extensively deformed (Fig. IC).
Like wild-type R. meliloti, exo mutant bacteria attached polarly to root hairs.
Previously, we reported that strain EJ355, a spontaneous
exoB mutant in an EJ312 background, deformed root hairs
but did not induce shepherd's crook formation or infection
thread formation (11). We reexamined the effect of EJ355 on
root hair deformation and found one or two shepherd's
crooks/root 4 to 5 d after inoculation. However, this was an
uncommon response compared with roots infected with
Rm 1021 TnS exo mutants, in which shepherd's crooks were
observed 1 to 2 d after inoculation (Fig. IB).
examination.
Invasion of Root Hairs
Spot Inoculation
Infection threads were found after wild-type R. meliloti
infection only in those hairs curled as shepherd's crooks.
However, infection threads were difficult to find in root hairs
of plants growing in the Fahraeus slide assemblies. Although
Alfalfa seeds were sterilized, planted on water agar, and
kept in the dark for 72 h. Seedlings were transferred to square
R. MELILOTI EXO MUTANT-INDUCED NODULES
145
we did not detect any infection threads in root hairs of living
plants after infection with exo mutants, infection threads were
commonly observed in the epidermal cells of exo mutantinduced nodules when nodule sections were examined under
the light microscope.
Invasion of the Root
Our observations suggested that there are two major modes
of invasion of the root by exo mutants: penetration via the
middle lamella (intercellular infection) and entry via infection
threads (intracellular infection).
Intercellular Infection
The exo mutant rhizobia were frequently sandwiched in
between epidermal cells, most likely a result of penetrating
the middle lamella (Fig. 2A, B). The bacteria within intercellular spaces were surrounded by a thin fibrillar material (Fig.
2C, arrow), which might be considered a modified infection
thread. Within this fibrillar boundary, numerous vesicular or
tubular-like structures were observed (Fig. 2C, D, open arrows). In some electron micrographs, the vesicles were closely
associated with the bacterial cell surface, appearing as extensions of the outer membrane (Fig. 2D, arrow).
Intracellular Infection
Infection threads were frequently found in the root hair
cells of 2-week-old sectioned nodules (Fig. 3A-C). Infection
threads rarely elongated beyond the peripheral cells, and many
seemed to end blindly in the outermost cells of the developing
nodule, appearing distended (Fig. 3B, C). Some exo mutant
rhizobia were also observed free within root hair cells. The
root hair cells, however, appeared to have senesced; no host
cytoplasm was visible (Fig. 3D). Mutant rhizobia were also
observed trapped between the outer and inner cell wall layers
(Fig. 3B, D, open arrows, and boxed region in Fig. 3D).
The external wall of the root hair cell appeared to consist
of an outer layer (a) and an inner layer (fl); both layers looked
fibrillar in electron micrographs (Fig. 3E). Some micrographs
illustrated what appeared to be rhizobial degradation of the
outer cell wall layer (Fig. 3E). Rhizobia were also observed
enclosed within the cell wall (Fig. 3E, asterisk). Bacteria
entrapped between the two wall layers were encapsulated by
a nonfibrillar material, presumably new wall material, from
which the thread appears to be derived (Fig. 3F). Figure 3F is
an enlargement of the boxed region in Figure 3D. The distended infection threads in Figure 3B and C (double arrowheads) appeared to have arisen in the same way-from new
wall material sandwiched between the original cell wall layers.
Figure
1.
Root hair deformation. A, The root hairs
deformed in response to R.
of inoculation. The
open
arrow
points
meflioti
photograph
to a
was
Rml 021
pictured
(wild type)
were
within 12 h
taken 24 h after inoculation. The
shepherd's crook and the
numerous
rhizobia
associated with the curled root hair. B, Root hairs 48 h after inoculation with exoA mutant Rm7061. Almost all of the root hairs
deformed, and several curled root hairs
are
are
visible. C, Root hairs
deformed in response to exoF mutant Rm7055 7 d after inoculation.
Almost all the root hairs
were
are
deformed.
taken of root hairs in the
Magnification
growing
x480. All
root hair zone
(33).
photos
Nodule Development
We spot-inoculated alfalfa roots or inoculated plants growing in agar slants with exoF, exoE, or exoB mutants of
RmlO21 to study the early stages of nodule development.
After inoculation, very few protrusions other than lateral root
primordia were detected by 5 to 7 d after inoculation under
the growth conditions described in "Materials and Methods."
However, in some regions where the root hairs were exten-
146
Plant Physiol. Vol. 98, 1992
YANG ET AL.
..
49'
.4..
*S*
0
F
I
.
1,0'.,, 4
"I
..:
,f'
'f
V
4:
rh
.
.s.
:,.
:.
1%,
4
A
ir
:;I.'
0
.:
W"".
:40,:,
'i
ml
A
'|-
iiii!
.
is
B1-If--
11r ''
4$A
9fE~~~~~~~'
..4.
.
Figure 2. Intercellular invasion. A, exoB R. melfloti (Rm7094) cells are found between the root hair (rh) cell and an adjacent epidermal cell. Some
rhizobia (r) appear to penetrate the middle lamella (ml) and are found in an intercellular space (is). x5600. B, Rhizobia (r) embedded in the middle
lamella between two adjacent cells (ml). This area is comparable to the region in A designated by the open arrow. x1 8,000. C, Rhizobia within
an intercellular space (is) comparable to that in A. A fine fibrillar (f) matrix encircles the bacteria, which are also surrounded by numerous vesicles
(open arrows). x40,000. D, Enlargement of one bacterium illustrating the vesicles (open arrow) associated with the bacterial surface. x1 02,000.
Bars = 1 gm except in panel D.
->3-or or--
.4
..
As
.#
r
''. \.
A- sLX
/r
l',,,
1k r h
:%
13
4
.4
f;
*;¢
o".
.-i
voor
.dE
0.5 urm
E
t
wS_
)
.... ..Hi;;
'.,.
,,,.
1 PM
Yn
a
I?
'*4.
D
t
15.21,I if,;z.1
,
-11;
t-.,
0.5
.... ...
ur
..
.-
ift.
4,
Figure 3. Intracellular invasion. A, Light micrograph of the periphery of a 2-week-old nodule induced by Rm7094 (exoB). An infection thread (it)
is visible within the root hair (rh). Some areas where intercellular invasions have occurred are indicated by the short arrows. x225. B, Root hairs
(rh) with aborted infection threads (it) containing R. mefiloti exoA mutants. The intercellular space (is) is packed with rhizobia. The open arrows
indicate rhizobia embedded between the outer and inner layers of the root hair cell wall, and the double arrowheads point to an expanding
infection thread. x1 500. C, A root hair (rh) with an aborted infection thread (it) containing R. mellloti exoA mutant bacteria. The double arrowheads
point to an expanding infection thread. x1500. D, Electron micrograph of the base of a root hair cell. Rhizobia are suspended within the cell and
are also embedded between the outer and inner cell wall layers (open arrow and boxed region). Rhizobia are also present within intercellular
spaces. x7000. Bar = 1 am. E, A polarly attached R. meliloti exo mutant bacterium appears to have degraded the outer layer (a) of the cell wall
(arrowhead). Another bacterial cell (*) is embedded between the outer and inner (fi) wall layers. x46,200. F, New cell wall material (nwm) is
deposited around the rhizobial cell (r) between the cell wall layers. x56,000.
YANG ET AL.
148
sively deformed, cell divisions were evident in both the pericycle (arrow) and the inner cortex (arrowhead) (Fig. 4A).
Serial sectioning of swollen roots 7 to 9 d after inoculation
with exo mutants confirmed that both inner cortical cells as
well as cells of the pericycle divided (Fig. 4B, arrow and
arrowheads). The root endodermis (open arrow) was interrupted at the site of these cell divisions (Fig. 4A-C). Derivatives of the pericycle gave rise to the proximal tissue of the
developing nodule, whereas the inner cortical cell derivatives
formed the nodule primordium. At the distal end of the
nodule primordium, several centers of mitotic activity were
frequently evident, but no localized, spatially restricted nodule
meristem differentiated (Fig. 4D). The epidermal cells appeared to keep pace with the developing nodule by dividing
and expanding in size, and infection threads aborted within
them.
DISCUSSION
The first response of the alfalfa plant to wild-type R. meliloti
is root hair deformation. Wood and Newcomb (33) have
made a detailed light microscopic study of the early infection
ofalfalfa root hairs by R. meliloti. They found that shepherd's
crooks were visible 6 to 8 h after inoculation and that only a
small number of deformed root hairs developed into tightly
curled shepherd's crooks, within which the rhizobial cells
became encapsulated. Infection threads were initiated within
a few of the tightly curled root hairs and infrequently in
branched or intertwined hairs. Wood and Newcomb (33)
estimated that, of the 80,000 root hairs present on 10 different
seedlings, 52 infection threads were initiated in two branched
hairs, 17 intertwined hairs, and 33 shepherd's crooks.
The details of rhizobial entry into the root hair cells have
been examined at the electron microscope level for only a few
legume species (4, 28). Electron micrographs of clover root
hairs show that the bacteria invade the root hairs by degrading
the outermost cell wall layer. The rhizobia then become
trapped between the inner and outer cell wall layers, and new
cell wall material, which forms the nascent infection thread,
is deposited over them. Although electron micrographs show
that the cell wall is degraded at invasion sites, the exact
mechanism of breakdown is unknown. Rhizobium species
have been reported to produce pectolytic enzymes (8, 14, 24,
27), but there is no firm evidence indicating that pectindegrading enzyme production is essential for nodule
development.
For R. meliloti exo mutants, although the initial stages of
thread formation appear to be similar to those for wild-type
infections, there are major differences. Infection threads do
not penetrate deep into the cortex but abort within the enlarged root hair cells. In the exo mutant-induced nodules,
numerous vesicle-like structures are associated with the bacteria. These structures are not observed in wild-type R. meliloti-induced nodules. However, Ridge and Rolfe (27) did
observe vesicle-like structures outside the infection threads in
Macroptilium nodules. There are at least two possible explanations for the lack of vesicles in sections of normal nitrogenfixing alfalfa nodules. The vesicles might be produced during
normal infection thread development but become visible only
in exo mutant-infected tissue, because they are normally
Plant Physiol. Vol. 98, 1992
covered by rhizobial EPS. Alternatively, the vesicle-like structures might accumulate only in the aborted infection threads,
either because the vesicles do not discharge their contents or
because they are deficient in contents that are secreted into
the normally growing infection thread.
Infection thread abortion is directly related to the production of EPS. R. meliloti that are mutant in exoA, exoB, exoC,
exoF, exoL, exoM, exoP, exoQ, or exoT produce no acidic
exopolysaccharides (see references in Reed and Walker [26]).
However, by mutation, R. meliloti can produce a second
exopolysaccharide (EPSII) even in exo strains; such bacteria
can elicit effective, nitrogen-fixing nodules on alfalfa roots
(12). Thus, EPS appears to be required for the establishment
of effective indeterminate nodules like those of alfalfa and
pea (2). In contrast, determinate nodules, like those of Phaseolus (2) and Lotus (13), are effective even when induced by
exo mutants. However, the situation is more complicated
because, even in R. meliloti exo mutants, other mutations
can restore effectiveness without restoring EPS-for example,
by modification of LPS (34). Whether the difference in the
effectiveness of nodules elicited by exo mutants is related to
the mode of invasion of Rhizobium in determinate versus
indeterminate nodules or to other differences related to nodule development remains to be established.
Although a number of roles for EPS in nodulation have
been suggested (e.g. 6, 12, 26, 34), its function remains
obscure. EPS might be part of the complex signaling process
that occurs between plant and bacteria. It might be involved
in positive recognition, enabling directional growth and penetration of the infection thread, or it might mask determinants
on the rhizobial surface, thereby protecting Rhizobium from
host attack. Recently, Puhler et al. (25) observed an increase
in phenolic materials in cell walls of peripheral cells containing aborted infection threads initiated by exoY R. meliloti.
This reaction is reminiscent of the hypersensitive response
whereby a host recognizes an incompatible pathogen. Alternatively, EPS might play a mechanical role, i.e. by filling the
interior of the developing infection thread. Whatever the role
of EPS, however, it can also be fulfilled by appropriately
modified mutant LPS (34).
Inoculation with exo bacteria initiates anticlinal cell divisions in the inner cortex, producing a region of cells equivalent
to the nodule primodium. The cells of the endodermis and
pericycle also divide, and vascular tissue from the root
branches and extends into the developing nodule. However,
no discrete, persistent nodule meristem is initiated, even
though small, densely cytoplasmic, apparently meristematic
cells are observed at the distal end of the nodule. Consequently, little peripheral and central tissue differentiates perpendicularly to the long axis of the nodule. The result is a
nodule that is compressed in length in contrast to the elongate,
cylindrical nodules elicited by wild-type R. meliloti.
The relationship of the differences we have observed between wild-type rhizobia and exo mutant-induced alfalfa nodules to EPS deficiency is not clear. There are several possible
explanations for the lack of initiation of a defined meristem.
Rhizobium EPS might include molecular domains that induce
directed infection thread growth and also meristem formation;
such domains could be present in mutant LPS as well. Adding
low mol wt EPS to R. meliloti exo mutants restores the
R. MELILOTI EXO MUTANT-INDUCED NODULES
-
*rh
r,
,
149
5O pm
-c
,
I
9
V
.I
KO
\Ct.
-
...
I
kl
X~
e
'A.
A
Figure 4. Early stages in nodule development elicited by exo mutants of R. melioti Rm7094. A, Transverse section of an alfalfa root 5 to 7 d
after inoculation with exoF mutant R. meliloti Rm7055. Cell divisions have taken place in both the pericycle (arrow) and the inner cortex
(arrowhead). The root endodermis (e) is interrupted. px, protoxylem; mx, metaxylem; rh, root hair. x310. B, Longitudinal section of an alfalfa
root at a stage comparable to that in A. Arrow points to a periclinal cell division in the pericycle, and the arrowhead indicates mitoses in the inner
cortex. The root endodermis (e) is indicated. x85. C, Transverse section of an alfalfa root 7 to 9 d after inoculation with Rm7029 (exoE). Cell
divisions in both the cortex and the pericycle have produced the nodule primordium (p). e, endodermis; px, protoxylem; rh, root hair. x1 20. D,
Longitudinal section of a root infected with an exoB mutant. Two sites of meristematic activity are observed within the confines of the root
cortex, but no defined, distal meristem is evident. x, xylem of the root. x85. Bar = 100 gm except in A.
YANG ET AL.
150
effective, elongate nodule phenotype, but at low efficiency.
Moreover, bacteroid development is not as extensive, and
nitrogen fixation levels are lower in the nodules elicited by
the exo mutants rescued by low mol wt EPS addition (J.
Leigh, personal communication; G. Walker, personal communication). Another possible explanation for the lack of
meristem initiation is that, by the time shepherd's crooks
form in response to exo mutant inoculation, the internal
cortical cells are no longer susceptible to Rhizobium signals
that trigger further nodule development. The cortical cells are
able to divide and establish a nodule primordium, but no
meristem forms and nodule formation is arrested.
A persistent nodule meristem in normal nitrogen-fixing
nodules develops after infection threads elongate. Thus, it is
likely that such a meristem does not develop in exo mutantinduced nodules because the infection threads abort within
the root hair cells and do not penetrate into internal tissues.
However, the exact mechanism of thread abortion is unknown; either chemical or mechanical cues could be critical
for thread elongation. Furthermore, as described by Puhler et
al. (25), the superficial cells of the developing nodule may
accumulate phenolic substances, which are considered to be
potent inhibitors of IAA oxidases (31). Increased levels of
phenolics could result in a hormonal imbalance that inhibits
the formation of a persistent, focused nodule meristem.
The observations reported here are consistent with other
evidence indicating limited meristematic activity of exo mutant-induced nodules. In wild-type R. meliloti-induced nodules, transcripts of MsENOD2, an early nodulin gene of
alfalfa, are detected in parenchyma cells at the base and along
the periphery of the nodule (30). In the exo mutant-induced
nodules, however, MsENOD2 transcripts are detected only at
the nodule base, around the vascular bundles.
Although nodules induced by wild-type R. meliloti are filled
with bacteria and exo mutant-induced nodules are "empty,"
that cannot be the reason for the difference in meristematic
activity. The "spontaneous" nodules described by Truchet et
al. (29) are also devoid of bacteria, but they have a distal,
persistent nodule meristem; in many cases, they have multiple
meristems. Furthermore, spontaneous nodule development,
like that of exo mutant-induced nodules, is delayed compared
with normal nitrogen-fixing nodule formation. Yet, spontaneous nodules have a focused meristem, thus arguing against
a delay in infection as the reason for the compressed nodule
phenotype of nodules elicited by exo mutant R. meliloti. In
addition, incubation of alfalfa roots with Io-' M quercetin or
luteolin, compounds that function as auxin transport inhibitors (15), increases the frequency of spontaneous nodules
with a discrete, terminal meristem (21). Thus, meristem formation is independent of bacterial invasion. The lack of a
persistent nodule meristem in exo mutant-induced nodules
appears to be directly related to infection thread abortion and
the physiological changes that occur in host cells in response
to invasion-deficient R. meliloti.
Plant Physiol. Vol. 98, 1992
Drs. Amala Reddy and Stefan J. Kirchanski for their helpful comments on the manuscript. We also thank Drs. J. Leigh and G.C.
Walker for communicating results prior to publication.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
ACKNOWLEDGMENTS
20.
We acknowledge the generosity of Agway, Inc., Syracuse, NY, for
providing us with alfalfa seeds. We thank Carol A. Smith and Cassie
Giedt for their help with analyzing root hairs, and we are grateful to
21.
LITERATURE CITED
Bent A (1989) Rhizobium meliloti suhR, RNA polymerase and
the heat shock response. PhD Thesis. Massachusetts Institute
of Technology, Cambridge
Borthakur D, Barber CE, Lamb JW, Daniels MJ, Downie JA,
Johnston AWB (1986) A mutation that blocks exopolysaccharide synthesis prevents nodulation of peas by Rhizobium
leguminosarum but not of beans by R. phaseoli and is corrected
by cloned DNA from Rhizobium or the phytopathogen Xanthomonas. Mol Gen Genet 203: 320-323
Bhuvaneswari TV, Solheim B (1985) Root hair deformation in
the white clover/Rhizobium trifolii symbiosis. Physiol Plant
63: 25-34
Callaham DA, Torrey JG (1981) The structural basis for infection
of root hairs of Trifolium repens by Rhizobium. Can J Bot 59:
1647-1664
Calvert HE, Pence MK, Pierce M, Malik NSA, Bauer WD
(1984) Anatomical analysis of the development and distribution of Rhizobium infections in soybean roots. Can J Bot 62:
2375-2383
Clover RH, Kieber J, Signer ER (1989) Lipopolysaccharide
mutants of Rhizobium meliloti are not defective in symbiosis.
J Bacteriol 171: 3961-3967
Dudley ME, Jacobs TW, Long SR (1987) Microscopic studies
of cell divisions induced in alfalfa roots by Rhizobium meliloti.
Planta 171: 289-301
Ervin SE, Hubbell DH (1985) Root hair deformations associated
with fractionated extracts from Rhizobium trifolii. Appl Environ Microbiol 49: 61-68
Fahraeus G (1957) The infection of white clover root hairs by
nodule bacteria studied by a simple glass slide technique. J
Gen Microbiol 16: 374-381
Finan TM (1988) Genetic and physical analyses of group E Exomutants of Rhizobium meliloti. J Bacteriol 170: 474-477
Finan TM, Hirsch AM, Leigh JA, Johansen E, Kuldau GA,
Deegan S, Walker GC, Signer ER (1985) Symbiotic mutants
of Rhizobium meliloti that uncouple plant from bacteria differentiation. Cell 40: 869-877
Glazebrook J, Walker GC (1989) A novel exopolysaccharide can
function in place of the calcofluor-binding exopolysaccharide
in nodulation of alfalfa by Rhizobium meliloti. Cell 55:
661-672
Hotter GS, Scott DB (1991) Exopolysaccharide mutants of Rhizobium loti are fully effective on a determinate nodulating host
but are ineffective on an indeterminate nodulating host. J
Bacteriol 173: 851-859
Hubbell DH, Morales VM, Umali-Garcia M (1978) Pectolytic
enzymes in Rhizobium. Appl Environ Microbiol 35: 210-213
Jacobs M, Rubery PH (1988) Naturally-occurring auxin transport regulators. Science 241: 346-349
Johansen E, Finan TM, Gefter ML, Signer ER (1984) Monoclonal antibodies to Rhizobium meliloti and surface mutants
insensitive to them. J Bacteriol 160: 454-457
Klein S, Walker GC, Signer ER (1988) All nod genes of Rhizobium meliloti are involved in alfalfa nodulation by exo mutants. J Bacteriol 170: 1003-1006
Libbenga KR, Bogers RJ (1974) Root-nodule morphogenesis. In
A Quispel, ed, The Biology of Nitrogen Fixation. NorthHolland Publishing Co, Amsterdam, pp 430-472
Leigh JW, Signer ER, Walker GC (1985) Exopolysaccharidedeficient mutants of Rhizobium meliloti that form ineffective
nodules. Proc Natl Acad Sci USA 82: 6231-6235
Long SR (1984) Genetics of Rhizobium nodulation. In T Kosuge,
E Nester, eds, Plant-Microbe Interactions, Vol 1. Macmillan,
New York, pp 265-306
McKhann HI, Jacobs M, Stine S, Hirsch AM (1990) Naturally
occurring NPA-like compounds in Rhizobium meliloti culture
R. MELILOTI EXO MUTANT-INDUCED NODULES
22.
23.
24.
25.
26.
27.
filtrate and alfalfa seed exudate. In 5th International Symposium on the Molecular Genetics of Plant-Microbe Interactions.
Interlaken, Switzerland, p 103
Meade HM, Long SR, Ruvkun GB, Brown SE, Ausubel FM
(1982) Physical and genetic characterization of symbiotic and
auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol 149: 114-122
Newcomb WE (1981) Nodule morphogenesis and differentiation.
Int Rev Cytol 13 (Suppl.): 247-248
Plazinski J, Rolfe BG (1985) Analysis of the pectolytic activity
of Rhizobium and Azospirillum strains isolated from Trifolium
repens. J Plant Physiol 120: 181-187
Puihler A, Arnold W, Buendia-Claveria A, Kapp D, Keller M,
Niehaus K, Quandt J, Roxlau A, Weng WM (1991) The role
of theRhizob,um meliloti exopolysacchandes EPSI and EPSII
in the infection process of alfalfa nodules. In H Hennecker,
DPS Verma, eds, Advances in Molecular Genetics of PlantMicrobe Interactions, Vol 1. Kluwer Academic Publishers,
Dordrecht, pp 189-194
Reed JW, Walker GC (1991) The exoD gene of Rhizobium
meliloti encodes a novel function needed for alfalfa invasion.
J Bacteriol 173: 664-677
Ridge RW, Rolfe BG (1985) Rhizobium sp. degradation of legume root hair cell wall at the site of infection thread origin.
151
Appl Environ Microbiol 50: 717-720
28. Turgeon G, Bauer WD (1985) Ultrastructure of infection-thread
development during the infection of soybean by Rhizobium
japonicum. Planta 163: 328-349
29. Truchet G, Barker DG, Camut S, de Billy F, Vasse J, Huguet T
(1989) Alfalfa nodulation in the absence of Rhizobium. Mol
Gen Genet 219: 65-68
30. Van de Wiel C, Norris JH, Bochenek B, Dickstein R, Bisseling
T, Hirsch AM (1990) Nodulin gene expression and ENOD2
localization in effective, nitrogen-fixing and ineffective, bacteria-free nodules of alfalfa. Plant Cell 2: 1009-1012
31. Vance CP (1978) Comparative aspects of root and root nodule
secondary metabolism in alfalfa. Phytochemistry 17:
1889-1891
32. Vincent JM (1970) A Manual for the Practical Study of RootNodule Bacteria: IBP Handbook No. 15. Blackwell Scientific
Publications, Oxford and Edinburgh
33. Wood SM, Newcomb W (1989) Nodule morphogenesis: the early
infection of alfalfa (Medicago sativa) root hairs by Rhizobium
meliloti. Can J Bot 67: 3108-3122
34. Williams MNV, Hollingsworth RI, Klein S, Signer ER (1990)
The symbiosis defect of Rhizobium meliloti exopolysaccharide
mutants is suppressed by IpsZ+, a gene involved in lipopolysaccharide biosynthesis. J Bacteriol 172: 2622-2632