Int. J. Plant Sci. 153(3):S171-S181. 1992.
C 1992 by The University of Chicago.All rightsreserved.
1058-5893/92/5303-0044$02.00
IN PLANTFORMANDFUNCTION
BACTERIAL-INDUCED
CHANGES
ANN M. HIRSCH,HEATHERI. McKHANN, AND MARIAN LOBLER
Departmentof Biology, 405 HilgardAvenue, University of California,Los Angeles,California90024
Numerous steps occur between the mutual recognitionof host and Rhizobiumand the establishment
of a nitrogen-fixingindeterminatenodule:root hair curlingand infection threadformation,corticalcell
divisions and initiation of a nodule primordium,nodule meristemformation,differentiationof specific
cell types and tissues, and induction of nitrogenfixation. Rhizobia with defined mutationsor AgrobacteriumtumefacienstransconjugantscarryingR. melilotisymbioticgeneshave been used to interruptthese
steps. Using this approach,we have detectedtranscriptsof the earlynodulinMsENOD2in alfalfanodules
induced by A. tumefacienstransconjugantscarryingone or both symbiotic plasmids of R. meliloti. We
have also specificallylocalizedMsENOD2transcriptsby in situ hybridizationto noduleparenchymacells
in nodules that form spontaneouslyon alfalfaroots. These bacteria-freenodules differentiatea discrete
nodule meristem and other tissues that are typical of normal nodules. Transcriptsfor MsENOD12, a
proline-richprotein normallyfound in the invasion zone of wildtypeR. meliloti-inducednodules, were
not detected in nodules induced by the Agrobacteriumtransconjugantsor in the spontaneouslyformed
nodules. However, we have detectedMsENOD12 mRNAs in nodules formedin responseto R. meliloti
exoH mutants. The exoH mutants, which induce nodules without a persistentnodule meristem, infect
host cells at a reduced frequencycomparedwith wildtype or with fix or nif mutant R. meliloti. These
resultsindicatethat infectionratherthan the formationof a persistentnodule meristemis correlatedwith
MsENOD12 gene expression.
Introduction
10-7 M, inducescorticalcell
and
nodule
primordium formation in
divisions
Plants grow indeterminatelybecause of meriet
al. 1991). It is not known
roots
(Truchet
alfalfa
stems, which are located at the tips of each stem
and transducesthis sigplant
perceives
how
the
and root. The continued mitotic activity of these
most likely the first
hairs
are
the
root
nal,
but
meristems produces all plant tissues, including
factor.
to
the
Nod
respond
cells
to
the reproductivestructures.In addition,the shoot
of a comsymptom
first
morphological
The
apical meristem controls the shape of the aerial
is
root
hair
deformation
(fig. 1).
infection
patible
part of the plant by maintainingdominance over
very
low
concentrations
at
factor
alone,
Nod
axillarymeristems.Growthand differentiationof
cells derived from the meristemoccurin a highly (10-"1 M), causes root hairs to deform (Truchet
et al. 1991). However, rhizobia are required to
organizedmanner in both root and shoot. However, pathogenicbacteriasuch as Agrobacterium initiatethe typical3600 curlingor shepherd'scrook
formation that is diagnostic of a compatible intumefacienscan disturbthe organizedpatternof
plant developmentby initiatinghyperplasiasthat fection (fig. 1). Rhizobia are also necessary for
lack apical meristems. The activity of hormone the development of infection threads through
which the bacteria invade the nodule primordibiosyntheticgenes on the T-DNA, integratedinto
um.
the host genome, results in the production of a
Depending on the legume host, either a detercrown gall, a disorganized, undifferentiatedtisminate
or an indeterminate nodule develops.
sue.
mungbean, common bean, or Lotus
Soybean,
In contrast, the interaction between host lenodules that have a globular
form
determinate
gumes and Rhizobiumresultsin the development
shape at maturity. In soybean, Bradyrhizobium
of a highly specialized organ, the nodule, which
is organized into distinct tissues. Before nodule japonicum induces cell divisions in the outer root
cortex resultingin the formation of a nodule pridevelopmentcommences,however,the two symmordium. Meristematic activity of the nodule
biotic partnersmust recognize each other. Rhiprimordium cells ceases 12-18 d after inoculazobium species are chemotactically attracted to
the roots of their respectivehost; their nod genes tion (Newcomb et al. 1979), and, thereafter,the
are induced by host-secretedflavonoids (Denarie derivatives of the nodule primordium give rise
to a centralbody of cells surroundedby peripheral
and Roche 1992; Long 1992). Induction of the
tissues that include nodule parenchyma,nodule
nod genes results in the formation of Nod facendodermis, and the nodule cortex (Rolfe and
tor(s), molecules that triggerdedifferentiationof
Gresshoff 1988). The central tissue consists of
mitotically quiescent root cortical cells and their
uninfected
cells and cells that contain bacteroids,
subsequentredifferentiationinto nodule initials.
rhizobia that convert atmothe
differentiated
The Nod factor producedby R. meliloti is a sulammonia.
into
nitrogen
spheric
fated glycolipid (Lerouge et al. 1990) that, at
Indeterminatenodules, like those of pea and
alfalfathat are induced by R. leguminosarumbv.
viciae or by R. meliloti, respectively, originate
ManuscriptreceivedApril 1992.
concentrationsof
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INTERNATIONAL JOURNAL OF PLANT SCIENCES
2
n.p.
4
Fig.1 Diagramof alfalfaroot noduledevelopment.Afterplant-bacterialrecognition,the root haircurls,forminga shepherd's
crook, and the infection thread(i.t.) invades the root hair cell (1). Anticlinalcell divisions take place in the inner root cortex
opposite a protoxylempole as the infection thread continues its growth(2). A meristem is initiated at the distal end of the
nodule primordium(n.p.) and the infection threadelongatestowardthe meristem(3). A nodule develops with its distinctive
histologicalzonation:meristem(m.), invasion zone (i.z.), and centralzone (c.z.) consistingof infectedand uninfectedcells and
surroundedby peripheraltissues (4).
from cell divisions in the inner root cortex (fig.
1) (Libbenga and Harkes 1973; Dudley et al.
1987). Following the initiation of the nodule primordium and penetration of infection threads
into host cells, a persistent nodule meristem becomes organizedat one end (fig. 1). Continuous
activity of the meristem at the distal end gives
rise to a cylindricalnodule with an indeterminate
growthpattern.Therefore,an indeterminatenodule exhibits a gradientof differentiationfrom the
distal end to the proximalpoint of attachmentto
the root. Like determinate nodules, indeterminate nodules have a centralbacteroid-containing
tissue surroundedby peripheraltissues consisting
of nodule parenchyma,nodule endodermis, and
nodule cortex. An invasion zone, whereinfection
threads extend and branch into host cells, is adjacent to the nodule meristem (fig. 1).
We are interested in elucidatingthe details of
the earlystagesof nodule development,especially
the host response to specific signals from Rhizobium. As has been found for other legumes,
alfalfanodulationcan be arrestedat variousstages
along the developmental pathway. The different
R. meliloti nif and fix mutants that have been
studied induce both cortical cell division and
nodule primordium formation. However, the
pattern of development beyond nodule primordium initiation may vary. For example, R. meliloti nif orfix mutantsinduce Fix- alfalfanodules
that have a discrete, persistent meristem and a
histologicalzonation like a Fix+nodule;i.e., they
have a centraltissue consisting of uninfectedand
infected cells with elongated bacteroids (Hirsch
et al. 1983; Hirschand Smith 1987). The R. meliloti mutant Rm5610 NS6, a spontaneous neomycin-resistantmutant, however, elicits nodules
with a similar histological organization,but the
central tissue does not contain elongated bacteroids (Reddy et al. 1992). Last, exopolysaccharide (exo) mutants of R. meliloti induce Fixnodules that are free of bacteria,lack a discrete,
persistent nodule meristem, and exhibit an altered arrangementof nodule tissues, especially
nodule parenchyma(Van de Wiel et al. 1990a;
Yang et al. 1992).
In addition to the bacteria-inducednodules, we
have found that chemicalsthat act as auxin transport inhibitors (ATIs), such as 2,3,5-triiodobenzoic acid (TIBA) and N-i -(naphthyl)phthalamic
acid (NPA) induce nodule-like structureson alfalfa and pea roots (Hirsch et al. 1989; Scheres
HIRSCH ET AL.-NODULE
et al., submitted). These nodule-like structures
lack a nodule meristem,but contain othertissues,
includingnodule parenchymaand nodule cortex.
Furthermore,certain alfalfa genotypes (Nodulation in the Absence of Rhizobium [NAR]) have
the potential to form nodules spontaneously in
the absence of bacteria (Truchet et al. 1989b).
These nodules are judged to be genuine nodules
based on their histologicalorganization.Like the
nodules induced by R. meliloti or by A. tumefaciens transconjugantscarryingR. meliloti symbiotic genes, spontaneousnodules originatefrom
inner cortical cells (Joshi et al. 1991). Moreover,
like the bacteria-inducednodules, spontaneous
nodule development is inhibited by the presence
of combined nitrogen (Truchetet al. 1989b).
We wanted to know whether the formation of
a persistentnodule meristem is a prerequisitefor
nodule morphogenesis in plants with indeterminate nodules and also whether the development of a nodule meristem is correlatedwith the
expressionof certainnodulingenes.We have used
cDNA clones of the early nodulin genes MsENOD2 and MsENOD12 as tissue-specific
markersfor examiningnodules with and without
meristems. ENOD2, a proline-rich protein, is
found in both determinate and indeterminate
nodules. It is most likely a cell wall component,
and its location in the nodule parenchymais consistent with the hypothesis that ENOD2 is part
of an oxygen barrierused to maintain a low concentrationof oxygen in the nodule (Van de Wiel
et al. 1990b). MsENOD12 transcriptsare found
in the invasion zone of alfalfa nodules; the derived amino acid sequence is also proline-rich,
indicatingthat this protein too is a cell wall protein (Lobler and Hirsch, submitted). The MsENOD 12 cDNA clone shows limited sequence
similarityto the PsENOD12 cDNA clone. Scheres
et al. (1990) have shown that PsENOD12 transcripts are localized to the invasion zone of pea
nodules and have suggestedthat PsENOD12 is
involved in the infection process.
Material
andmethods
BACTERIAL STRAINS
The Rhizobium meliloti strains used in this
study were Rm 1021, which is a streptomycinresistantNod+Fix+derivative of SU47 (Meadeet
al. 1982) and Rm7154, an exopolysaccharide
mutant of RmlO21 (exoH::Tn5) (Leigh et al.
1987). The Agrobacteriumtumefacienstransconjugants were Atl28 (GM19023, pRmeSU47a
Q30::Tn5-11) and Atl35 (GM19023, pRmeSU47a Q30::Tn5-11, pRmeSu47b, Q5007::Tn5oriT) (Finan et al. 1986). The rhizobia and
agrobacteriaweregrowneitheron platesof LuriaBertani(LB) medium (Maniatiset al. 1982) or of
Rhizobium-defined minimal medium (RDM
MERISTEMS
S173
[Vincent 1970]). Antibiotics were used at the following final concentrations (,g/ml): streptomycin, 250; neomycin, 50; spectinomycin, 100; and
gentamycin, 50.
PLANTS
Seeds of Medicago sativa L. cv Iroquois were
surfacesterilizedas describedin Yanget al. (1992)
and germinatedon water agarin the dark for 72
h before transferto modified Fahraeusslide assemblies(Bhuvaneswariand Solheim 1985).Root
hair curling was examined periodically as describedin Yanget al. (1992). Sterilizedseeds were
also germinated on nitrogen-free agar slants
(Meade et al. 1982) and inoculated with bacteria
after 72 h, or sown in autoclaved plastic pans
containing vermiculite/perlite watered with nitrogen-freemedium, and immediately inoculated. The seedlings were grown under 16h/8h, 21
C/i 9 C, day/night conditions for 3-4 wk.
MICROSCOPY
For electronmicroscopy,nodules were excised
from roots and fixed as describedin Hirsch et al.
(1983). The nodule tissue was embeddedin Spurr
low viscosity resin after dehydration in an acetone series. Ultrathin sections were stained with
uranyl acetate and Reynold's lead citrate. Semithin (0.5- 1,m) sections weremade of the plasticembedded material and stained in 0.05% toluidine blue 0 dissolved in 1%sodium borate.Nine
At128-induced nodules and 12 AtI 35-induced
nodules were examined by light microscopy.
Some tissue was fixed in FAA (Sass 1953), embedded in Paraplastafter dehydrationthrougha
tert-butyl alcohol series, and sectioned at 8 ,im.
These sections were subsequentlypreparedfor in
situ hybridization using 35S-UTP-labeledsense
and antisenseprobesas describedby Van de Wiel
at al. (1990a).
Spontaneously formed nodules were fixed in
FAA and clearedin lactic acid accordingto Dudley et al. (1987). The clearednoduleswere stained
in 0.01% methylene blue (Truchetet al. 1989a).
DETECTION
OFNODULINS
Alfalfa nodules were harvested 3-4 wk after
inoculation with bacteria,frozen immediately in
liquid nitrogen,and then stored at -75 C. Total
RNA was isolated from nodules following the
protocol of Goldberg et al. (1981), subjected to
electrophoresis,and blotted onto either Nytran
(Schleicher& Schuell) or GeneScreen(New England Nuclear) membranes following the manufacturers'directions.The RNA transferblots were
probed with a 32P-dCTP-labeledfragmentof either a 292 base pair (bp) insert of pA2ENOD2
(Dickstein et al. 1988) or a 509 bp insert of
MsENOD12-1 (L6blerand Hirsch, in press).Hybridizations were performed according to the
t
rn
I,'.
a_
's~~~~~~~~~~~~4
itSI
HIRSCH ET AL.-NODULE
MERISTEMS
S175
manufacturers'specifications,and the blots were
washed under high stringencyconditions (0.2 x
SSC, 50 C).
rowheads, fig. 2A). Mature nodules were small,
white, and Fix- and, in overall morphologyand
internal structure,closely resembledthe nodules
elicited by R. meliloti exo mutants (Finan et al.
1985; Dickstein et al. 1988; Reed and Walker
Results
1991; Yang et al. 1992). The nodules were genEARLY NODULIN GENE EXPRESSION IN NODULES
erally devoid of bacteria and lacked a discrete
WITHOUT A DEFINED MERISTEM
apical meristem. A broad zone of meristematic
Agrobacterium activity extendedover the distal end of the nodule
Agrobacteriumtransconjugants.
tumefacienstransconjugantAtI28, carryingthe
(fig. 2D).
Infection threads were detected in the periphRhizobium meliloti megaplasmid with the nod
and nif/fix genes (pSyma) or At 135, containing eral cells of Agrobacteriumtransconjugant-inboth pSyma and pSymb, the megaplasmid that duced nodulesand occasionallyin the nodule corcarriesthe exoABDFgenes, deformedalfalfaroot tex (fig. 2B, C). It is not known whether these
hairs but were delayed in eliciting shepherd's threadsare relatedto root hair infection or to an
crooks compared with wildtype R. meliloti. We atypical infection as described by Truchet et al.
detected slight distortions of root hairs 26 h after (1984). The infection threads contained an elecinoculation when strain Atl28 was used, and tron-dense matrix that enclosed the bacteria.
moderate root hair deformation when strain Some of the infection threadsappearedmultilayAti 35 was employed. In contrast, shepherd's ered and to consist of several components (arrows, fig. 2E, F).
crook formation was observed as early as 9.5 h
One Ati35-induced nodule contained agroafter inoculation with Rm 1021, the wildtype R.
bacteria that had been released into host cells
meliloti strain. By 26 h, extensive root hair dewithin the centraltissue of the nodule. However,
formation as well as a number of distinct shepherd's crooks were detected on roots that had few of the centralcells were infected, and in general, the cytoplasm of the infected cells appeared
been inoculated with Rm 1021. After 50 h, one
senescent(datanot shown). Uninfected cells conor two shepherd'scrooks could be found on each
alfalfaroot inoculatedwith Atl 35, but none were tained numerous amyloplasts. At the distal end
seen on roots inoculated with At128, although of this nodule, a discrete meristem was initiated.
root hair branching was observed. Shepherd's Proximalto this region,host cells were expanded
and densely cytoplasmic. However, no infection
crooks were observed 4 d after inoculation with
threadswere detected within these cells.
At128 in agreementwith Finan et al. (1986). By
MsENOD2mRNAs accumulatedin the At 1286 d after inoculation, both At128- and At135or Ati35-induced nodules (fig. 3). We had preincubatedroots exhibited extensive root hair deformation with conspicuous shepherd's crooks. viously localizedMsENOD2transcriptsby in situ
Like Truchet et al. (1984), who examined Agro- hybridizationto the proximal end of the nodule
bacteriumtransconjugantscarryingeither pSyma in cells surroundingthe vascularbundles, i.e., in
or large portions of pSyma, we could not detect a tissue homologous to the nodule parenchyma
infectionthreadsin root hairsinfectedwith At 128 of wildtype R. meliloti-induced nodules (Van de
or Atl 35. However, infection threadswere found Wiel et al. 1990a). We could not detect Msin nodules (see below).
ENOD 12 transcripts in nodules induced by
At128 or At135 (data not shown).
Both At 128 and At 135 formed nodules on alfalfa 3-4 wk after inoculation; this nodulation
RHIZOBIUMMELILOTIEXOH-INDUCEDNODwas delayed 4-7 d comparedwith Rm 1021. Like ULES. Root hair curling responses and the structhe exo mutant-induced nodules, the At 128 and tureof alfalfanodulesinducedby R. melilotiexoH
At 135 nodules were initiated from cell divisions mutants have been described previously (Leigh
in the inner root cortex next to the endodermis et al. 1987). Unlike the other exo mutants, which
(fig. 2A). Cell divisions were also evident in the completely lack exopolysaccharide (EPS), exoH
endodermis and in the pericycle (arrowsand ar- mutants fail to succinylate their EPS and have a
Fig.2 A, Cell divisions in the inner cortex (arrowhead)and pericycle (arrow)presage the development of the nodule
primordiumafterinoculationwith At 128. The root endodermis(e) is indicated.x = xylem. x 150. B, Abortedinfectionthread
(it) in an epidermalcell, adjacentto a root hair (rh). Bacteriaof strainAti35 (b) are externalto the epidermalcells. x 1,500.
C, An infection thread (it) extends into a peripheralcell. Bacteriaof strain Ati28 (b) are presenton the exteriorof the root.
x 600. D, Longitudinalsection of a root bearinga nodule inducedby At 128. Infectionthreads(arrows)are presentonly in the
peripheralcells of the nodule. There is no discrete nodule meristem, only a broad meristematiczone (m z). x 75. E, Cross
section of an infectionthreadcontainingAti 28. The infectionwall is multilaminate(doublearrowheads).x 9,200. F, Oblique
section of an infectionthreadfrom anotherAt 128-inducednodule.The doublearrowheadspoint to the multilaminateinfection
threadwall. x 19,000.
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Fig.3 RNA transferblot of RNA isolatedfrom roots, from
nodulesof two differentagesinducedby wildtype(wt)Rm 1021,
from nodules inducedby Atl28 and Atl35, and probedwith
A2ENOD2 (Dicksteinet al. 1988). A band of approximately
1,400 nucleotides is present in the nodule lanes. Less RNA
from the wt-inducednodules was included on the gel.
"haloless" phenotype when grown on agar medium containing Calcofluor.Rhizobium meliloti
exoH-induced nodules are usuallydevoid of bacteria because infection threads abort within peripheralcells of the nodule as is the case for the
other exo mutant-induced nodules and the nodules elicited by At 128 and At 135. However, approximately 10%-1 5%of the nodules examined
contained infection threads that had penetrated
into the central tissue of the nodule (Leigh et al.
1987). In these nodules, a broad zone of meristematic activity ratherthan a persistent nodule
meristem was evident at the distal end.
We previously reported that exoH-induced
nodules contain Nms-30, an in vitro translation
product found in other bacteria-freealfalfa nodules (Leigh et al. 1987). MsENOD2 transcripts
have also been detected in R. meliloti exoH-induced nodules (L6bler and Hirsch, unpublished
results).When total RNA was isolated from wildtype R. meliloti and exoH-induced nodules, prepared for northern analysis, and probed with
MsENOD 12-1, a transcript of 600 nucleotides
was detected in both RNA samples (fig. 4).
EARLY NODULIN GENE EXPRESSION IN
BACTERIA-FREE NODULES WITH
DEFINED MERISTEMS
We examined the structure of the spontaneously formed nodules by clearing them in lactic
acid. We found that the nodules possessed multiple apical meristems giving them a lobed appearance,a centraltissue devoid of bacteria,and
an endodermisjust inside the nodule cortex. The
vasculatureof the spontaneouslyformed nodules
is complex and may reflect differentiationpatterns that occurredrelative to the multiple mer-
OF PLANT SCIENCES
Fig.4 RNA transferblot of RNA isolatedfrom roots, from
nodules inducedby wildtype(wt) Rm 1021, from nodules induced by Rm7 154 (exoH), and from spontaneouslyformed
nodules. The blot was probed with MsENOD12-1 (L6bler
and Hirsch,in press).An RNA of 600 nucleotidesis detected
in RNA isolated from the wildtype and from the exoH mutant-induced nodules.
istems that typify these nodules (fig. 5A). The
vascular bundles frequently bifurcated at the
proximal end of a nodule lobe.
Because ENOD2 is expressed in bacteria-free
nodules, including nodules formed on NAR alfalfa (Truchet et al. 1989b), we localized MsENOD2 expressionby in situ hybridization.The
MsENOD2 gene was expressedin cells surrounding the vascular bundles within the confines of
the vascular endodermis, and also in nodule parenchymacells adjacentto the outer endodermis
(fig. 5B, C). However, when RNA isolated from
the spontaneous nodules was probed with MsENOD 12-1, we did not detect any hybridization
(fig. 4).
We have summarized the morphological responses of alfalfa plants, using the phenotypic
code established by Vincent (1980), and also the
patterns of nodulin gene expression after inoculation with various bacterialstrains(table 1). We
have also included the responses of the NAR alfalfa. All the Rhizobium strains as well as the
AgrobacteriumtransconjugantscarryingR. meliloti symbiotic sequences elicit root hair deformation and infection thread formation, albeit to
varying degrees.Other than the nodules induced
by the wildtype strain, none of the nodules are
capable of nitrogen fixation. In the case of the
NAR alfalfa,ineffectivenodules are also formed,
but the root hairs do not deform and infection
threadsdo not develop becausebacteriaare completely absent from these plants. However, the
NAR plants have a persistent nodule meristem,
unlike the nodules elicited by R. meliloti mutant
4;'t~~~~~~~
X
E9
vS.b
t~~~
pontt
tofok o bfrctdvsuabude(b.x7.BBrgtfedmcorpofoeleofapnaeulyomd
it
t
nodule.~~~~~
MsND.rncit,sona
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Fecig.5n,
aDistal
field of B.
ak
t.
ein
r
on
rudtevsuarbnl
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one-thirofah spntdlaneouslyfomaedwe
alflf
noduleclrted inlcti acid and
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INTERNATIONAL
JOURNAL
OF PLANT SCIENCES
Table1
SUMMARY
OFALFALFA
RESPONSES
TOINOCULATION
WITHVARIOUS
BACTERIAL
STRAINS
Host response
Bacterium
RmlO21 ..........
Rm5610NS6a
Rm7154 ..........
Atl28 .............
Atl35 ...
No bacteria
spontaneous
nodules
...........
.....
Had
Inf
Persistent
nodule
meristem
++
+
+
++
+b+
+c.d
+-b
Nif
Nms3O
ENOD2
ENOD 12
+
+
ND
+
+
+
+
+
+
_
d
_
+
.
+C
+e
-
-
_
+-
-
Note. Had = root hair deformation, Inf= bacterial infection, Nif=
Reddy et al. 1992.
b
Infection thread aborted in root cortex.
c Delayed.
dLeigh et al. 1987.
e Infection thread aborted in epidermal cell layer.
f van de Wiel et al. 1990a.
+f
+
ND
+
nitrogen fixation. ND = not determined.
a
in exoH and by the AgrobacteriumtransconjugantscarryingR. melilotisymbiotic plasmids.All
of the nodules tested contain mRNAs of Nms3O
and ENOD2, while only those nodules with infection threads penetratinginto the central cells
of the nodule express ENOD 12.
Discussion
Ourgoal in this investigationwas to determine
the necessity of nodule meristem formation for
the expression of the early nodulin genes MsENOD2 and MsENOD12. Nodules and nodulelike structures induced by Rhizobium meliloti
exoA, exoB, or exoF mutants or by treatment
with ATIs are bacteria-freeand lack a persistent,
focused nodule meristem but have been shown
to contain MsENOD2 transcripts(Dickstein et
al. 1988; Hirsch et al. 1989; Reddy et al. 1992;
L6bler and Hirsch, unpublished results). Similarly,nodulesinducedby Ati 28 and Ati 35, which
closely resemble the R. meliloti exo mutant-induced nodules, also contain MsENOD2 transcripts. The presence of MsENOD2 mRNAs in
these nodules suggeststhat the differentiationand
persistenceof a discrete nodule meristem is not
essential for the expressionof MsENOD2. Rather, MsENOD2 gene expressionappearsto be correlatedwith the differentiationof nodule tissues,
specificallynodule parenchyma(Van de Wiel et
al. 1990b).
The Agrobacteriumtransconjugant-induced
nodules, however, do not expressthe early nodulin MsENOD12. MsENOD12 transcriptsare also
not detected in RNA isolated from nodules induced by R. meliloti exoF or exoB mutants or by
treatment with NPA (Reddy et al. 1992; Lobler
and Hirsch, submitted). However, MsENOD12
transcriptshave been localized to the invasion
zone in wildtype R. meliloti-induced nodules
(Lobler and Hirsch, submitted) and have been
found in nodules elicited by the R. meliloti mutant Rm561ONS6 (Reddy et al. 1992). NS6 mutants induce nodules with a distinct, persistent
meristem at the apical end of the nodule. The
central tissue is devoid of differentiatedbacteroids but contains numerous infection threads.
Although these data indicate that nodule meristem development is correlated with MsENOD12 gene expression, our observations on
the spontaneously formed alfalfa nodules and
nodules elicited by exoH mutants do not support
this conclusion. We were unable to detect MsENOD 12 gene expression in spontaneously
formed alfalfanodules, many of which have multiple meristems. MsENOD12 transcripts,however, werefound in nodules elicited by R. meliloti
exoH mutants, indicatingthat ENOD 12 gene expression is correlatedwith invasion ratherthan
the development of a nodule meristem (Lobler
and Hirsch, in press), in agreementwith the results of Scheres et al. (1990) for pea.
Meristem formation in indeterminatenodules
may follow a "default"cascade,which startswith
cell divisions in the inner cortex, continues with
nodule primordiuminitiation and meristem development on one side of the primordium, and
ends with the outgrowthof a discrete, persistent
nodule meristem. Meristem initiation itself does
not seem to be dependent on the penetrationof
infection threads into the central tissue and the
subsequent release of rhizobia into host cells as
evidenced by the spontaneously formed alfalfa
nodules. The formationof multiple meristemsin
the spontaneously formed nodules may result
HIRSCH ET AL.-NODULE
MERISTEMS
S179
from the short life of each individual meristem. terized by discrete nodule meristems, meristem
Normal nitrogen-fixingalfalfanodules have one, persistence is not essential for nitrogen fixation
or at the most, two lobes, which persist through- as shown by the sphericalnodules of soybeanand
out the life of the nodule, indicating that meri- other legumes with determinatenodules. Nitrostem persistenceresults from continued interac- gen fixationin determinatenodules is temporally
tion between the host and the invading rhizobia. regulated:the rhizobia and the plant cells coorWe do not know why the exoH-induced nod- dinate their differentiationalong a temporalaxis.
ules lack a nodule meristem. There are at least Once the nodule becomes senescent,nitrogenfixtwo explanations. First, the exoH mutants, like ation ceases. An interesting observation is that
the other R. meliloti exopolysaccharidemutants, in determinate nodules, the nature of rhizobial
may inhibit nodule meristem activity by eliciting EPS does not seem as important as it is in ina host defense reaction. Piihleret al. (1991) have determinate nodules. EPS mutants of R. fredii,
shown that in exo mutant-induced nodules, the R. leguminosarumbv. phaseoli, and R. loti form
superficialcells, which contain abortedinfection normal Fix+ nodules on their respective hosts
(Borthakuret al. 1986; Kim et al. 1989; Hotter
threads,accumulatephenolics. Phenolicsmay inhibit persistent meristem formation. Second, and Scott 1991).Also, as mentionedearlier,Agrocontinuous rhizobialpenetrationmay be a signal bacterium transconjugantscarrying R. legumito the host to maintain the activity of the meri- nosarumbv. phaseoli plasmids induce nitrogenstem. This signal may be related to oligosac- fixingnodules on bean, althoughwhen compared
charides contained within rhizobial exopolysac- with inoculation with wildtype R. leguminosacharidethat are cleaved and then secretedwithin rum bv. phaseoli, fewer nodules, with lower nithe host, or to other unidentifiedmolecules. Bat- trogen-fixing ability, develop (Martinez et al.
tisti et al. (1992) have shown that R. meliloti 1987).
In contrast to determinate nodules, nitrogen
exoA, exoB, exoF, and exoH mutantsinduce normal nitrogen-fixingnodules with persistentmer- fixation is spatially regulated in indeterminate
istems if low molecular weight succinoglycanis
nodules. Nitrogenfixationby rhizobiatakesplace
suppliedto alfalfaroots. Only R. meliloti succin- in host cells at a defineddistance proximalto the
apical meristem.The continuedgrowthof an apioglycan restores nodule invasion ability to the
exo mutants, indicating that a specific chemical cal meristem means that nitrogen fixation constructureis required.Low molecularweightEPSs tinues within one nodule as long as the meristem
from other Rhizobium species or from A. tume- producesderivatives.Ongoingcell divisions may
provide cues for infection thread elongation and
faciens are inactive (Battisti et al. 1992).
The results with strain At135 are difficult to
bacterialinvasion as well as for the induction of
explain.One expectedresultfrom infectingalfalfa early nodulins that preparethe host cell for niroots with this Agrobacteriumtransconjugant, trogen fixation. In indeterminatenodules, if the
which harbors both symbiotic plasmids, would bacteriacannotinvade,a persistentmeristemdoes
be the development of infected nodules similar not develop, and the spatialbiochemicalgradient
to what has been reportedfor A. tumefacienscar- from the distal to proximal end of the nodule is
ryingthe R. trifoliisymbiotic plasmid (Hooykaas not established.Rhizobium meliloti exo mutants
et al. 1981). However, of the 1,000 or more plants and A. tumefaciens transconjugants do not invade
inoculated with Atl 35 and grown under the culalfalfanodule cells and do not establishthe spatial
ture conditions describedin this report,few nodgradient.Occasionally,we have found some evules were infected. Those that were infected conidence for release of these bacteria. However, it
tainedsenescenthost cells and agrobacteria.There may be that if they are released,they are released
are at least two possible explanationsfor the lack into a host cell that is not correctlylocated in the
of infection by Atl 35: (1) the indigenous exospatial gradient,and thus, they are unable to fix
polysaccharideof the Agrobacteriummasks any nitrogen.
We cannot easily test whether the nitrogenrhizobialEPS producedby the action of genes on
fixation ability of indeterminatenodules, such as
pSymb, and (2) no rhizobial EPS is producedby
At 135 because the genes on pSymb are not expea or alfalfa, is dependent on the initiation and
pressedin the A. tumefaciensgeneticbackground. persistenceof a meristem.Rhizobiummeliloti nif
An additional possibility is that infection by
orJix mutants, like the NS6 mutant, induce nodAgrobacteriumtransconjugantsrequiresdifferent ules with a distal meristem, but the nodules are
culture conditions than those described in this
ineffective because of defects in bacterial genes.
report. Martinez et al. (1987) observed that A.
The exopolysaccharide mutants or A. tumefatumefacienscarryingR. leguminosarumbv. phaciens transconjugants carrying nod/nif genes,
seoli plasmids elicited nitrogen-fixingnodules on
however, do not fix nitrogen in nodulo because
bean only when the plants were grown at 26-28
they either cannot invade the host cells or invade
C, and not at 21 C.
poorly. However, R. meliloti exo mutantsare caAlthough indeterminate nodules are charac- pable of fixing nitrogen if coinoculated with R.
S180
INTERNATIONAL
JOURNAL
meliloti nod or fix mutants (Klein et al. 1988).
Furthermore,Puhler and colleagues (A. Puhler,
personal communication) have found that nodules induced by R. meliloti exo mutants become
nitrogen-fixingwith prolonged incubation. The
bacteria recovered from these nitrogen-fixing
nodules are not revertants. We also have occasionally isolated Exo- rhizobia from nitrogenfixing,pink nodules. In these nodules, EPSII (also
called EPSb) may have substituted for succinoglycan and allowed nodule invasion (Glazebrook and Walker 1989; Zhan et al. 1989).
The triggersfor nodule meristem initiation in
indeterminatenodules are unknown,but it seems
that once a nodule meristem is established, infection threads elongate in the direction of the
meristem and release bacteriainto the host cells
just proximalto it. The relationshipbetweennodule meristem persistenceand bacterialreleaseremains to be elucidated. To analyze these rela-
OF PLANT SCIENCES
tionships, more molecular markers as well as
geneticallydefined plant mutants will be needed.
Acknowledgments
We acknowledgethe generosityof Agway,Inc.,
Syracuse,N.Y., for seeds of Medicago sativa Iroquois. The Agrobacteriumtransconjugantswere
generously provided by Turlough Finan. John
Leighand FredAusubel are thankedfor Rm7 154
and Rm 1021, respectively.The alfalfaA2ENOD2
clone was a gift from Rebecca Dickstein, Drexel
University. CarolA. Smith and Monica Eiserling
are acknowledgedfor their help with the sectioning. We thank MargaretKowalczyk for figure 1
and Jacob Seeler for the photography.We also
thank the members of our laboratory and Dr.
Stefan J. Kirchanskifor their helpful comments
on the manuscript.We acknowledgethe support
from National Science Foundation grants DCB8703297 and DCB-902 1587.
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