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483
Plant responses to nodulation factors
J Allan Downie* and Simon A Walker†
The focus of research on signalling in Rhizobium–legume
interactions has moved from understanding the structure and
synthesis of rhizobially made Nod factors, towards an analysis
of how they function in plants. Nod-factor-induced changes in
ion fluxes across membranes, followed by establishment of an
oscillation of intracellular Ca2+ concentration, point to the
involvement of a receptor-mediated signal transduction
pathway. Progress towards the identification of components in
this pathway is being made by identifying Nod-factor binding
proteins, isolating plant mutants that are defective in signalling
and analysing plant responses to Nod factors.
characterised and it is evident that specificity (i.e. the
types of legume hosts nodulated by given rhizobia) is
determined, at least in part, by chemical modification of
the Nod factors. These modifications include the attachment of sulphate, acetate, carbamoyl groups; addition of
other sugars such as arabinose, mannose or fucose (and
substituted derivatives of fucose); changes to the acyl
chain; and variation of the chitin oligomer length
(Figure 1). (The role of rhizobial nod gene products in Nod
factor biosynthesis has been reviewed in detail [1,2,3•].)
In the appropriate legume, Nod factors can induce a variety of effects including deformation of root hairs, division
of root cortical cells, and nodule morphogenesis [1,4•,5••].
Some of these responses are induced at concentrations as
low as 10–12–10–13 M Nod factor; this finding alone points
to the probable existence of high-affinity receptors. Other
components may act to enhance nodulation; thus, for
example, a protein secreted by some rhizobia can extend
the range of legumes nodulated [6,7] possibly by forming
ion channels in the plant membranes [8]. Proteins secreted
by other rhizobia are also likely to play a role in nodulation
[9], although nothing is known about how such secreted
proteins act. There is indirect evidence for the uptake of
Nod factors into plant cells [10•,11] raising the possibility
that Nod factor recognition could occur within cells as well
as at the membrane surface. Analysis of infection by rhizobial mutants that do not make fully substituted Nod
factors has suggested that there may be different types of
Nod factor receptors, one controlling early responses and
another controlling invasion events [12,13]. Root chitinases, some of which are specifically induced during the
symbiosis, can degrade Nod factors and may play an important role in the regulation of their activity [14].
Addresses
John Innes Centre, Norwich Research Park, Colney, Norwich,
NR4 7UH, UK
*e-mail: downie@bbsrc.ac.uk
†e-mail: simon.walker@bbsrc.ac.uk
Current Opinion in Plant Biology 1999, 2:483–489
1369-5266/99/$ — see front matter © 1999 Elsevier Science Ltd.
All rights reserved.
Abbreviations
ENOD early nodulation
LCO
lipo-chitin oligomer
LNP
lectin nucleotide phosphohydrolase
NFBS Nod-factor-binding site
Introduction
Nod factors, which are signalling molecules made by rhizobia, initiate nodule development in legumes. They are
composed of lipo-chitin oligomers (LCOs) that usually
comprise four or five β, 1–4 linked N-acetyl glucosamine
residues, in which the N-acetyl group of the terminal (nonreducing) sugar is replaced by an acyl chain. Many Nod
factors, made by a diverse range of rhizobia, have been
Figure 1
Generic Nod factor structure with potential
sites for modification. Known modifications by
various rhizobial isolates include (1)
unsaturations in the fatty acyl chain and
variation in chain length. Substitution of the
following groups at the positions indicated:
(2) methyl; (3) carbamoyl; (4) carbamoyl; (5)
carbamoyl and acetyl; (6) acetyl; (7) fucosyl;
(8) sulphuryl, acetyl, fucosyl and other fucosyl
derivatives; (9) mannosyl and glycerol; (10)
arabinosyl. Details of the nod genes involved
and which strains of rhizobia make the various
modified Nod factors can be found in recent
reviews [1,3•,4•].
4
O
O
5
O
O
6
O
O
O
3
O
2
O
N
8
OH
O
7
O
N
O
HO
N
O
10
n=1-2
N
9
O
1
Current Opinion in Plant Biology
484
Cell biology
Figure 2
Nod factor
(v) Cytoskeletol rearrangements
Seconds
Hours
(i) Ion flux and plasma membrane depolarisation
(vi) Root-hair deformation
Ca2+
Depolarisation
R
Cl–
K+
(ii) Accumulation of Ca2+ at root hair tip
Minutes
(vii) Early nodulin gene expression
(iii) G-protein-mediated signal transduction?
(viii) Reactivation of cell cycle
Days
R
G
(ix) Root cortical cell divisions
Ca2+cyt
PLC
IP3
Ca2+
(Organelle)
(iv) Calcium spiking
[Ca2+]int
1 min
(x) Nodule formation
Current Opinion in Plant Biology
Temporal order of plant responses to Nod factors for a ‘typical’ legume.
This figure draws upon data from several different legume species and
the responses for any individual legume may therefore vary. No cause
and effect is necessarily implied in the order of events shown. The first
responses seen are (i) ion fluxes and associated depolarisation at the
plasma membrane [25••], and (ii) calcium accumulation at the root hair
tip [30]. Indirect evidence suggests that (iii) a G-protein-mediated
signal transduction pathway involving inositol phosphate (IP3) may be
operating [35••]; however, it is not known if this is related in any way to
the initial ion fluxes. (iv) Calcium spiking is induced [26], though it is
not known if this is a consequence of a putative G-protein pathway
and/or is associated with the ion fluxes. Calcium may play a role in the
(v) subsequent changes seen in the actin cytoskeleton [32•]. The actin
cytoskeleton does appear to influence (vi) root-hair morphogenesis
[33•]. (vii) Nodulation-specific gene induction is proposed to occur via
a G-protein pathway [35••] but whether calcium spiking and/or ion
fluxes play a role is not known. (viii) Reactivating the cell cycle initiates
(ix) cortical cell divisions [34] which lead to (x) subsequent nodule
formation but relating this back to the earliest recognition events has
yet to be achieved.
Our review primarily addresses recent work on Nod-factorinduced changes to ion movements in and around legume
root hairs and evaluates recent biochemical and genetic
experiments that may lead to an understanding of the signalling events several of which are summarised in
Figure 2. (Note that several reports on effects of Nod factors on cell division in non-legumes have recently been
withdrawn [15•].) We have not described work on the identification of the many genes induced by Nod factors or on
the role of plant hormones in nodule development [16,17];
recent reviews [4•,5••,18,19•,20• ] describe in detail various
aspects of the plant responses to Nod factors that are not
included here.
Roles for calcium
Nod factors induce a rapid and transient depolarisation of
the plasma membrane of root hairs [21–23] and an associated transient extracellular and sustained intracellular
alkalinisation [24,25••]. A few seconds after Nod factor
exposure, there is a transient influx of Ca2+ , which is rapidly followed by a Cl– efflux, and then efflux an of K+ to
balance the charge (Figure 2i). This K+/Cl– movement and
Plant responses to nodulation factors Downie and Walker
the associated movement of H+ ions seem to account for
the membrane depolarisation.
Calcium-sensitive fluorescent dyes have been used to
monitor intracellular Ca2+ in root hairs. Dyes linked to high
molecular weight dextran must be microinjected but are
often preferred as they remain within the cytoplasm.
Acetoxymethyl esters of dyes are permeant and so can be
loaded into cells by diffusion but imaging can be problematic because they are present at only small intracellular
concentrations, diffuse out of cells and can become
localised inside organelles (particularly in plant cells).
Microinjection of Calcium Green/dextran into alfalfa root
hairs revealed that Nod factors induced regular periodic
increases in intracellular calcium that were localised mostly around the nucleus [26]. These increases occurred about
once per minute and the rapid rise and defined cut-off to
the decrease in Ca 2+ defined these as Ca 2+ spikes
(Figure 2iv). The spiking initiated ~10 minutes after Nod
factor addition and continued for at least three hours. Since
Ca2+ spiking did not occur in a non-nodulating alfalfa
mutant and was not induced by Nod factors lacking a sulphate group that is essential for alfalfa nodulation, it is very
likely to form part of the normal plant response. Moreover,
similar Ca2+-spiking responses have been observed in different spp. of Vicia, Lotus and Medicago (S Long, personal
communication) and in pea root hairs (SA Walker, unpublished data). Ca2+ spiking plays an important role in
developmental gene regulation in mammalian cells [27•].
Analogous systems have yet to be defined in plants,
although Ca2+ oscillations are proposed to have a role in
regulating the opening of the stomatal guard cell aperture
and are also associated with pollen tube growth [28,29].
Other studies using Ca2+-responsive dyes suggest that the
dye type and/or its method of delivery may influence the
observable effects of Nod factors. Microinjection of dextranlinked dyes revealed Ca2+ spiking, whereas buffered
loading of acetoxymethyl derivatives in cowpea revealed a
rapid plateau-like increase in Ca2+ at the root hair tip, but
Ca2+ spiking was not observed [30]. In vetch, Nod factors
induced swelling of root hair tips and inhibited root hair
growth; after ~1-1½ hrs exposure to Nod factor, root hair
growth resumed and enhanced concentrations of Ca2+ were
seen (as detected by the Ca2+-sensitive dye Indo 1) in the
swollen root hair tip and in the newly formed growing root
hair tip [31•]. The relationships between, first, Nod-factorinduced Ca2+ influx measured using microelectrodes,
second, the onset of Ca2+ spiking and third, the accumulation of Ca2+ at the growing tip of the root hair have yet to be
resolved. The reasons for the different observations made
by different groups could relate to differences in methodology. Microinjection may disturb the ionic equilibrium and/or
the cytoarchitecture such that calcium accumulation at the
tip does not occur. The age and growth phase of the root hair
may be important and the temporal resolution of imaging
techniques may be insufficient to allow the observation of
the Ca2+ spiking (discussed in [20•,31•]).
485
Nod factors have been shown to induce disintegration of
the actin cytoskeleton and inhibit root hair tip growth
within 10 mins of Nod factor addition to Phaseolus vulgaris
[32•]. At the subsequent branch-like outgrowth of the root
hairs in vetch, actin filaments reappear [33•] in the region
where there is an increased level of Ca2+ (Figure 2vi) [31•]
and it was concluded that fine bundles of actin filaments
promote polar growth by releasing Golgi vesicles to a vesicle-rich region of the growing root hair tip [33 •].
Cytoskeletal changes may also be associated with the Nod
factor induced reactivation of the cell cycle; outer cortical
cells enter the cell cycle but do not divide, whereas inner
cortical cells divide (Figure 2ix), entering the cell cycle
from the G0/G1 phase [34]. At a later stage in the interaction, a transient disorganisation of microtubules was
correlated with the appearance of Nod factors within
infected cells, suggesting that Nod factors may also be
involved in cytoskeletal rearrangements that occur during
the differentiation of infected cells [10•].
The effects of pharmacological agents on the expression of
the reporter gene MtENOD12A-GUS, which is induced in
response to Nod factors, provided indirect evidence for a
role for Ca2+ in a G-protein-mediated signal transduction
pathway [35••]. Mastoparan, a G-protein agonist induced
MtENOD12A in a similar way to Nod factor, whereas the
G-protein antagonist, pertussis toxin, inhibited Nod factor
and mastoparan-induced gene expression. Inhibitors of
phospholipase C, such as neomycin, and of Ca 2+
influx/release such as ethylene glycol tetraacetic acid
(EGTA), La3+ and ruthenium red, also blocked both the
Nod factor and mastoparan-induced gene expression.
Although these results are consistent with a mammaliantype G-protein signalling pathway, coupled to the
activation of phosphoinositide and Ca2+ as a second messenger (Figure 2iii), great care has to be taken with the
interpretation of data generated by such pharmacological
techniques that may be having multiple effects. The combination of such studies with the use of plant mutants in
which signalling is blocked in the early stages should, however, give an insight into the nature of the pathways
involved. The identity of the Nod factor receptors are of
key interest.
Nod-factor-binding proteins
Radioactively labelled LCOs were used to identify Nod factor binding proteins from Medicago truncatula. A protein with
a relatively low affinity (Kd = 86 nM) for the Nod factor was
found but is unlikely to play a role in the symbiosis [36]. A
second component, NFBS2 (Nod-factor-binding site 2),
from a plasmalemma fraction [37••] has high affinity
(Kd = 4 nM) for Nod factors. Several synthetic LCOs with
structural similarity to Nod factors were used in competitive
binding assays to determine the effects of structural modifications on relative binding efficiency. Substitutions on the
non-reducing terminal sugar (i.e. the presence of an Oacetate group, or the type of fatty acid) and the oligomer
length both contributed to optimal binding but the presence
486
Cell biology
or absence of a sulphate group on the reducing sugar did not
affect Nod factor binding. This sulphate group is essential for
normal Nod factor activity on Medicago spp. and so the binding specificity of NFBS2 did not correspond with what
would be predicted (on the basis of in vivo studies) for a putative Nod factor receptor. Thus, if NFBS2 does correspond to
(part of?) a receptor, the perception of the sulphate group by
the Nod factor receptor would be complex, and could possibly involve another protein that confers additional specificity.
Isolation of proteins that bind chitin oligomers led to the
identification of a Nod factor binding lectin with apyrase
activity [38••]. As this lectin hydrolyses the phosphoanhydride bonds of nucleoside phosphates, it was called lectin
nucleotide phosphohydrolase (LNP). LNP, which was isolated from the legume Dolichos biflorus, has much greater
affinity for Nod factors than chitin oligomers and has greatest
affinity for those Nod factors from rhizobia that can nodulate
D. biflorus. Furthermore, the apyrase activity of LNP is
enhanced by Nod factors. These results, together with the
observations that LNP is located on root hairs and that antibody to LNP inhibited root-hair deformation, suggest a role
for LNP in the nodule symbiosis, but as with NFBS2 a direct
role for LNP in signalling has not yet been established.
Although LNP is a lectin (as defined by its sugar-binding
capability), its sequence shows that it does not belong to the
large family of typical legume lectins [39,40]. Several years
ago it was proposed that such ‘typical’ lectins might play a role
in recognition by acting as a kind of surface-binding receptor,
and several studies, including addition of lectins and demonstration of their presence on root hair tips, supported a role for
lectins in the early stages of infection [40]. Furthermore,
transgenic clover plants expressing a pea lectin gene can be
nodulated by R. leguminosarum bv. viciae — which normally
nodulates pea and not clover — and the sugar-binding
domain is necessary for this activity [41]. The sugar-binding
sites of such lectins, however, are not consistent with them
binding Nod factors. A more recent study with transgenic
plants that express an introduced lectin gene has led to a
reassessment of the original hypothesis and the proposal of a
different model for a role of such lectins in nodulation. Lotus
corniculatus expressing a soybean lectin gene induced nodulelike outgrowths, which were devoid of bacteria, in response to
the soybean symbiont Bradyrhizobium japonicum [42•]. In this
case, infection did not proceed normally and it was suggested
that the introduced lectin, which was located in root-hair tips,
may interact with a component of the bacterial surface polysaccharide. It was proposed that the lectin might increase the
attachment and aggregation of heterologous rhizobia
(B. japonicum) at the infection site. This, in turn, could result
in an increased level of Nod factors at the appropriate location, thereby potentiating a weak signal and stimulating the
early stages of nodule morphogenesis, even by heterologous
Nod factors. This model illustrates that there could be multiple inputs into recognition between rhizobia and legumes and
that dissecting out the signalling pathway from other recognition events may not be simple.
Legume nodulation mutants
Many nodulation-defective legume mutants have been
isolated but many are in species that do not lend themselves to gene isolation. Nevertheless, these mutants have
been used to gain insight into the events that occur during
nodulation. Nod– mutants of several species do not form a
mycorrhizal symbiosis [43]. The pea early nodulation transcripts ENOD5 and ENOD12A, which are induced in
response to Rhizobium, are also induced by the endomycorrhizal fungus Gigaspora margarita; no expression was
seen in a Nod– sym8 pea mutant when inoculated with
either symbiont [44•]. Other studies using transgenic peas
expressing an ENOD12A–GUS reporter gene demonstrated that the induction of the pea ENOD12A gene is also
blocked in the sym19 mutant [45]. Many gene transcripts
are induced early in nodulation [5••,19•,20•] and knowledge of which genes are switched off in various mutants
may help to determine the sequence of events that are initiated after Nod factor perception, and where divergence
between endomycorrhizal and rhizobial symbioses occurs.
One of the key transcripts that is induced during
rhizobium–legume symbiosis is ENOD40, which is expressed
prior to initiation of pericycle cell division opposite the protoxylem poles and is also expressed in the dividing cells
[46•,47]. Transgenic legumes expressing ENOD40 initiate cortical cell division at multiple sites indicating that this gene
influences the differentiation and division of root cells [48].
Sequence comparisons led to the realisation that ENOD40
encodes a short (10–13 residue) peptide and a conserved noncoding RNA domain that may play a regulatory role [46•,47].
Each region seems to contribute to nodule development. The
conservation of this gene in non-legumes [46•] suggests that
nodule signalling may have evolved from a more ancient pathway conserved in non-legumes. Nod factor was reported to
induce a M. truncatula ENOD12 gene in rice [49•], although
this observation has not yet been verified by other laboratories.
Recently, there has been a focussed effort on investigating
the genetics of ‘model’ legumes, an experimental counterpart to Arabidopsis for the study of symbioses. These legumes
are diploid, have relatively small genome sizes, are easy to
transform and have high seed yield. Such properties lend
themselves to gene-tagging strategies and positional cloning.
One candidate gene for positional cloning affects the ethylene sensitivity of nodulation [50]; the mutant of M. truncatula
which does not express this gene has enhanced nodulation
and altered position of nodule development. These effects
are due to its insensitivity to ethylene which normally downregulates nodule development [17]. A bacterial artificial
chromosome library of M. truncatula genomic DNA has been
created and used to identify ethylene responsive genes [51].
Other approaches to gene identification involve a root-hairspecific cDNA library from M. truncatula [52•]. In Lotus
japonicus, an essential nodulation gene Nin has been identified by gene tagging following mutagenesis using the maize
transposable element Ac [53••]. The mutant is not infected
by rhizobia, but root hair deformation and curling occur,
Plant responses to nodulation factors Downie and Walker
indicating that the Nin gene is unlikely to encode the primary Nod factor receptor. The Nin gene product is predicted to
be a membrane protein and four similar Arabidopsis proteins
(of unknown function) were identified in database searches.
Similarity was also found with a Chlamydomonas gene product
(the so-called minus-dominance protein) that regulates mating type during gametogenesis. All of these proteins share a
putative DNA-binding dimerisation domain and so a regulatory role for the Lotus nodulation gene was proposed,
although the mechanism of regulation and the regulated
genes are not yet known. This first characterisation of an
essential nodulation gene suggests that we are now on the
threshold of a phase identifying genes required to establish
symbiotic interactions.
Conclusions
There is strong evidence to suggest that the early events in
Nod factor recognition involve movements of ions across
the plant plasma membrane, followed by the establishment
of intracellular Ca2+ oscillations. Circumstantial evidence
suggests that a G-protein-type receptor that could act via an
inositol-phosphate and phospholipase based signalling
pathway may be involved. Much work remains to be done,
however, to integrate the various observations that have
been made on the legume symbiosis. The key objective in
the future will be to exploit various nodulation mutants to
clone and characterise genes that are essential for nodule
development. The ease of the isolation (and maintenance)
of legume nodulation mutants, the availability of the Nodfactor-signalling molecules, and the ability to analyse ion
movements in epidermal cells mean that nodule development is a particularly good system for the analysis of a
developmental pathway. Understanding how nodules are
formed may well give us an insight into how other morphogenetic events are activated in plants.
Acknowledgements
We thank NJ Brewin for his constructive comments on the manuscript and
various colleagues for sending copies of recent and ‘in press’ publications.
The authors are supported by the Biotechnology and Biological Sciences
Research Council.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
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•
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487
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•
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•
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•
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•
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••
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•
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29. Franklin-Tong VE, Drobak BK, Allan AC, Watkins PAC, Trewavas AJ:
Growth of pollen tubes of Papaver rhoeas is regulated by a slowmoving calcium wave propagated by inositol 1,4,5-trisphosphate.
Plant Cell 1996, 8:1305-1321.
30. Gehring CA, Irving HR, Kabbara AA, Parish RW, Boukli NM,
Broughton WJ: Rapid, plateau-like increases in intracellular free
calcium are associated with nod-factor-induced root-hair
deformation. Mol Plant-Microbe Interact 1997, 10:791-802.
31. de Ruijter NCA, Rook MB, Bisseling T, Emons AMC: Lipochito
•
oligosaccharides re-initiate root hair tip growth in Vicia sativa with
high calcium and spectrin-like antigen at the tip. Plant J 1998,
13:341-350.
Cytological changes induced in root hairs by Nod factors were examined by
microscopy. Root hair cells with terminating growth reacquired tip-growing
characteristics including a tip gradient of Ca2+ and accumulation of a spectrin-like antigen. The results indicate that the root hairs respond to Nod factors by recovering cytoplasmic polarity and exocytosis.
32. Cárdenas L, Vidali L, Domínguez J, Pérez H, Sánchez F, Hepler PK,
•
Quinto C: Rearrangement of actin microfilaments in plant root
hairs responding to Rhizobium etli nodulation signals. Plant
Physiol 1998, 116:871-877.
In this paper, researchers describe fragmentation of the Phaseolus vulgaris
root hair actin cytoskeleton upon exposure to Rhizobium etli Nod factors.
This was observed 5–10 minutes after Nod factor application which could
correlate the response to observed changes in intracellular calcium. This
work indicates that Nod factors alter the organisation of actin microfilaments
in root hairs.
33. Miller DD, de Ruijter NCA, Bisseling T, Emons AMC: The role of actin
•
in root hair morphogenesis: studies with lipochitooligosaccharide as a growth stimulator and cytochalasin as an
actin perturbing drug. Plant J 1999, 17:141-154.
The authors propose a model for root hair morphogenesis whereby actin filaments co-ordinate growth by releasing Golgi vesicles to a vesicle-rich region
of the growing root hair tip. The model relies on the influence of Nod factors
on root hair growth and the interference of cytochalasin D with actin function.
34. Yang WC, Deblank C, Meskiene I, Hirt H, Bakker J, van Kammen A,
Franssen H, Bisseling T: Rhizobium Nod factors reactivate the cellcycle during infection and nodule primordium formation, but the
cycle is only completed in primordium formation. Plant Cell 1994,
6:1415-1426.
35. Pingret JL, Journet EP, Barker DG: Rhizobium Nod factor signaling:
•• evidence for a G protein-mediated transduction mechanism. Plant
Cell 1998, 10:659-671.
Using a GUS reporter system to reflect early nodulin gene induction this
work describes the use of various agonists and antagonists to provide indirect evidence for a G-protein-mediated signalling pathway operating downstream of the Nod factor receptor.
Gressent F, Drouillard S, Mantegazza N, Samain E, Geremia RA,
Canut H, Niebel A, Driguez H, Ranjeva R, Cullimore J, Bono JJ:
Ligand specificity of a high-affinity binding site for lipochitooligosaccharidic Nod factors in Medicago cell suspension
cultures. Proc Natl Acad Sci USA 1999, 96:4704-4709.
This work demonstrates that a high affinity binding protein copurifies with the
plasma-membrane fraction from Medicago cell suspension cultures. Binding
of LCOs with different substitutions is analysed and the presence of a sulphate group, that is essential for nodulation, has no effect on binding affinity. Therefore there is some question as to whether the partially purified
protein is a Nod factor membrane receptor or some other protein that happens to bind LCOs.
38. Etzler ME, Kalsi G, Ewing NN, Roberts NJ, Day RB, Murphy JB: A
•• Nod factor binding lectin with apyrase activity from legume roots.
Proc Natl Acad Sci USA 1999, 96:5856-5861.
Isolation of a chitin-binding protein fortuitously led to the identification of this
novel type of lectin that has both a high affinity for Nod factors and an
apyrase activity. It is structurally distinct from the large family of legume
lectins previously described [39,40] and a potential signalling receptor.
39. Brewin NJ, Kardailsky IV: Legume lectins and nodulation by
Rhizobium. Trends Plant Sci 1997, 2:92-98.
40. Kijne JW, Bauchrowitz MA, Diaz CL: Root lectins and Rhizobia. Plant
Physiol 1997, 115:869-873.
41. van Eijsden RR, Díaz CL, de Pater BS, Kijne JW: Sugar-binding
activity of pea (Pisum sativum) lectin is essential for heterologous
infection of transgenic white dover hairy roots by Rhizobium
leguminosarum biovar viciae. Plant Mol Biol 1995, 29:431-439.
42. van Rhijn P, Goldberg RB, Hirsch AM: Lotus corniculatus nodulation
•
specificity is changed by the presence of a soybean lectin gene.
Plant Cell 1998, 10:1233-1249.
Transfer of a soybean lectin gene enables B. japonicum to induce uninfected nodules on Lotus corniculatus. A model is proposed suggesting that
lectins enhance bacterial attachment to root hairs thereby enhancing Nod
factor signalling by increasing localised concentration.
43. Albrecht C, Geurts R, Bisseling T: Legume nodulation and
mycorrhizae formation; two extremes in host specificity meet.
EMBO J 1999, 18:281-288.
44. Albrecht C, Geurts R, Lapeyrie F, Bisseling T: Endomycorrhizae and
•
rhizobial Nod factors both require SYM8 to induce the expression
of the early nodulin genes PsENOD5 and PsENOD12A. Plant J
1998, 15:605-614.
The authors find that the same plant early nodulation genes are induced by
rhizobia and mycorrhizal fungi. Gene induction by both endosymbionts is
blocked in a nodulation mutant of pea.
45. Schneider A, Walker SA, Poyser S, Sagan M, Ellis THN, Downie JA:
Genetic mapping and functional analysis of a nodulationdefective mutant (sym19) of pea (Pisum sativum L). Mol Gen
Genet 1999, 262:1-11.
46. Kouchi H, Takane K, So RB, Ladha JK, Reddy PM: Rice ENOD40:
•
isolation and expression analysis in rice and transgenic soybean
root nodules. Plant J 1999, 18:121-129.
The authors of this paper describe the cloning of a rice gene homologous to
the early nodulin ENOD40. A promoter–GUS fusion study indicated the rice
gene may play a similar role in vascular differentiation.
47.
Fang YW, Hirsch AM: Studying early nodulin gene ENOD40
expression and induction by nodulation factor and cytokinin in
transgenic alfalfa. Plant Physiol 1998, 116:53-68.
48. Charon C, Johansson C, Kondorosi E, Kondorosi A, Crespi M:
enod40 induces dedifferentiation and division of root cortical cells
in legumes. Proc Natl Acad Sci USA 1997, 94:8901-8906.
49. Reddy PM, Ladha JK, Ramos MC, Maillet F, Hernandez RJ, Torrizo LB,
•
Oliva NP, Datta SK, Datta K: Rhizobial lipochitooligosaccharide
nodulation factors activate expression of the legume early
nodulin gene ENOD12 in rice. Plant J 1998, 14:693-702.
One of the very few papers describing activation of a legume nodulation
gene by Nod factors in a transgenic non-legume.
50. Penmetsa RV, Cook DR: A legume ethylene-insensitive mutant
hyperinfected by its rhizobial symbiont. Science 1997,
275:527-530.
51. Nam YW, Penmetsa RV, Endre G, Uribe P, Kim D, Cook DR:
Construction of a bacterial artificial chromosome library of
Plant responses to nodulation factors Downie and Walker
Medicago truncatula and identification of clones containing
ethylene-response genes. Theor Appl Genet 1999, 98:638-646.
52. Covitz PA, Smith LS, Long SR: Expressed sequence tags from a
•
root-hair-enriched Medicago truncatula cDNA library. Plant Physiol
1998, 117:1325-1332.
An expressed sequence tag (EST) library of root hair genes has been established and is growing rapidly. This paper describes many genes that are
expressed in root hairs.
489
53. Schauser L, Roussis A, Stiller J, Stougaard J: A plant regulator
•• controlling development of symbiotic root nodules. Nature 1999,
in press.
The first paper to describe cloning of a gene essential for some of the earliest stages in the rhizobia-legume symbiosis. Transposon-tagged mutants of
Lotus japonicus led to the isolation of a gene required for both bacterial entry
into the host plant and the establishment of the nodule primordium. The
encoded protein contains transcription factor-like domains and could define
a new class of developmental regulators.
Plant responses to nodulation factors
J Allan Downie* and Simon A Walker†
The focus of research on signalling in Rhizobium–legume
interactions has moved from understanding the structure and
synthesis of rhizobially made Nod factors, towards an analysis
of how they function in plants. Nod-factor-induced changes in
ion fluxes across membranes, followed by establishment of an
oscillation of intracellular Ca2+ concentration, point to the
involvement of a receptor-mediated signal transduction
pathway. Progress towards the identification of components in
this pathway is being made by identifying Nod-factor binding
proteins, isolating plant mutants that are defective in signalling
and analysing plant responses to Nod factors.
characterised and it is evident that specificity (i.e. the
types of legume hosts nodulated by given rhizobia) is
determined, at least in part, by chemical modification of
the Nod factors. These modifications include the attachment of sulphate, acetate, carbamoyl groups; addition of
other sugars such as arabinose, mannose or fucose (and
substituted derivatives of fucose); changes to the acyl
chain; and variation of the chitin oligomer length
(Figure 1). (The role of rhizobial nod gene products in Nod
factor biosynthesis has been reviewed in detail [1,2,3•].)
In the appropriate legume, Nod factors can induce a variety of effects including deformation of root hairs, division
of root cortical cells, and nodule morphogenesis [1,4•,5••].
Some of these responses are induced at concentrations as
low as 10–12–10–13 M Nod factor; this finding alone points
to the probable existence of high-affinity receptors. Other
components may act to enhance nodulation; thus, for
example, a protein secreted by some rhizobia can extend
the range of legumes nodulated [6,7] possibly by forming
ion channels in the plant membranes [8]. Proteins secreted
by other rhizobia are also likely to play a role in nodulation
[9], although nothing is known about how such secreted
proteins act. There is indirect evidence for the uptake of
Nod factors into plant cells [10•,11] raising the possibility
that Nod factor recognition could occur within cells as well
as at the membrane surface. Analysis of infection by rhizobial mutants that do not make fully substituted Nod
factors has suggested that there may be different types of
Nod factor receptors, one controlling early responses and
another controlling invasion events [12,13]. Root chitinases, some of which are specifically induced during the
symbiosis, can degrade Nod factors and may play an important role in the regulation of their activity [14].
Addresses
John Innes Centre, Norwich Research Park, Colney, Norwich,
NR4 7UH, UK
*e-mail: downie@bbsrc.ac.uk
†e-mail: simon.walker@bbsrc.ac.uk
Current Opinion in Plant Biology 1999, 2:483–489
1369-5266/99/$ — see front matter © 1999 Elsevier Science Ltd.
All rights reserved.
Abbreviations
ENOD early nodulation
LCO
lipo-chitin oligomer
LNP
lectin nucleotide phosphohydrolase
NFBS Nod-factor-binding site
Introduction
Nod factors, which are signalling molecules made by rhizobia, initiate nodule development in legumes. They are
composed of lipo-chitin oligomers (LCOs) that usually
comprise four or five β, 1–4 linked N-acetyl glucosamine
residues, in which the N-acetyl group of the terminal (nonreducing) sugar is replaced by an acyl chain. Many Nod
factors, made by a diverse range of rhizobia, have been
Figure 1
Generic Nod factor structure with potential
sites for modification. Known modifications by
various rhizobial isolates include (1)
unsaturations in the fatty acyl chain and
variation in chain length. Substitution of the
following groups at the positions indicated:
(2) methyl; (3) carbamoyl; (4) carbamoyl; (5)
carbamoyl and acetyl; (6) acetyl; (7) fucosyl;
(8) sulphuryl, acetyl, fucosyl and other fucosyl
derivatives; (9) mannosyl and glycerol; (10)
arabinosyl. Details of the nod genes involved
and which strains of rhizobia make the various
modified Nod factors can be found in recent
reviews [1,3•,4•].
4
O
O
5
O
O
6
O
O
O
3
O
2
O
N
8
OH
O
7
O
N
O
HO
N
O
10
n=1-2
N
9
O
1
Current Opinion in Plant Biology
484
Cell biology
Figure 2
Nod factor
(v) Cytoskeletol rearrangements
Seconds
Hours
(i) Ion flux and plasma membrane depolarisation
(vi) Root-hair deformation
Ca2+
Depolarisation
R
Cl–
K+
(ii) Accumulation of Ca2+ at root hair tip
Minutes
(vii) Early nodulin gene expression
(iii) G-protein-mediated signal transduction?
(viii) Reactivation of cell cycle
Days
R
G
(ix) Root cortical cell divisions
Ca2+cyt
PLC
IP3
Ca2+
(Organelle)
(iv) Calcium spiking
[Ca2+]int
1 min
(x) Nodule formation
Current Opinion in Plant Biology
Temporal order of plant responses to Nod factors for a ‘typical’ legume.
This figure draws upon data from several different legume species and
the responses for any individual legume may therefore vary. No cause
and effect is necessarily implied in the order of events shown. The first
responses seen are (i) ion fluxes and associated depolarisation at the
plasma membrane [25••], and (ii) calcium accumulation at the root hair
tip [30]. Indirect evidence suggests that (iii) a G-protein-mediated
signal transduction pathway involving inositol phosphate (IP3) may be
operating [35••]; however, it is not known if this is related in any way to
the initial ion fluxes. (iv) Calcium spiking is induced [26], though it is
not known if this is a consequence of a putative G-protein pathway
and/or is associated with the ion fluxes. Calcium may play a role in the
(v) subsequent changes seen in the actin cytoskeleton [32•]. The actin
cytoskeleton does appear to influence (vi) root-hair morphogenesis
[33•]. (vii) Nodulation-specific gene induction is proposed to occur via
a G-protein pathway [35••] but whether calcium spiking and/or ion
fluxes play a role is not known. (viii) Reactivating the cell cycle initiates
(ix) cortical cell divisions [34] which lead to (x) subsequent nodule
formation but relating this back to the earliest recognition events has
yet to be achieved.
Our review primarily addresses recent work on Nod-factorinduced changes to ion movements in and around legume
root hairs and evaluates recent biochemical and genetic
experiments that may lead to an understanding of the signalling events several of which are summarised in
Figure 2. (Note that several reports on effects of Nod factors on cell division in non-legumes have recently been
withdrawn [15•].) We have not described work on the identification of the many genes induced by Nod factors or on
the role of plant hormones in nodule development [16,17];
recent reviews [4•,5••,18,19•,20• ] describe in detail various
aspects of the plant responses to Nod factors that are not
included here.
Roles for calcium
Nod factors induce a rapid and transient depolarisation of
the plasma membrane of root hairs [21–23] and an associated transient extracellular and sustained intracellular
alkalinisation [24,25••]. A few seconds after Nod factor
exposure, there is a transient influx of Ca2+ , which is rapidly followed by a Cl– efflux, and then efflux an of K+ to
balance the charge (Figure 2i). This K+/Cl– movement and
Plant responses to nodulation factors Downie and Walker
the associated movement of H+ ions seem to account for
the membrane depolarisation.
Calcium-sensitive fluorescent dyes have been used to
monitor intracellular Ca2+ in root hairs. Dyes linked to high
molecular weight dextran must be microinjected but are
often preferred as they remain within the cytoplasm.
Acetoxymethyl esters of dyes are permeant and so can be
loaded into cells by diffusion but imaging can be problematic because they are present at only small intracellular
concentrations, diffuse out of cells and can become
localised inside organelles (particularly in plant cells).
Microinjection of Calcium Green/dextran into alfalfa root
hairs revealed that Nod factors induced regular periodic
increases in intracellular calcium that were localised mostly around the nucleus [26]. These increases occurred about
once per minute and the rapid rise and defined cut-off to
the decrease in Ca 2+ defined these as Ca 2+ spikes
(Figure 2iv). The spiking initiated ~10 minutes after Nod
factor addition and continued for at least three hours. Since
Ca2+ spiking did not occur in a non-nodulating alfalfa
mutant and was not induced by Nod factors lacking a sulphate group that is essential for alfalfa nodulation, it is very
likely to form part of the normal plant response. Moreover,
similar Ca2+-spiking responses have been observed in different spp. of Vicia, Lotus and Medicago (S Long, personal
communication) and in pea root hairs (SA Walker, unpublished data). Ca2+ spiking plays an important role in
developmental gene regulation in mammalian cells [27•].
Analogous systems have yet to be defined in plants,
although Ca2+ oscillations are proposed to have a role in
regulating the opening of the stomatal guard cell aperture
and are also associated with pollen tube growth [28,29].
Other studies using Ca2+-responsive dyes suggest that the
dye type and/or its method of delivery may influence the
observable effects of Nod factors. Microinjection of dextranlinked dyes revealed Ca2+ spiking, whereas buffered
loading of acetoxymethyl derivatives in cowpea revealed a
rapid plateau-like increase in Ca2+ at the root hair tip, but
Ca2+ spiking was not observed [30]. In vetch, Nod factors
induced swelling of root hair tips and inhibited root hair
growth; after ~1-1½ hrs exposure to Nod factor, root hair
growth resumed and enhanced concentrations of Ca2+ were
seen (as detected by the Ca2+-sensitive dye Indo 1) in the
swollen root hair tip and in the newly formed growing root
hair tip [31•]. The relationships between, first, Nod-factorinduced Ca2+ influx measured using microelectrodes,
second, the onset of Ca2+ spiking and third, the accumulation of Ca2+ at the growing tip of the root hair have yet to be
resolved. The reasons for the different observations made
by different groups could relate to differences in methodology. Microinjection may disturb the ionic equilibrium and/or
the cytoarchitecture such that calcium accumulation at the
tip does not occur. The age and growth phase of the root hair
may be important and the temporal resolution of imaging
techniques may be insufficient to allow the observation of
the Ca2+ spiking (discussed in [20•,31•]).
485
Nod factors have been shown to induce disintegration of
the actin cytoskeleton and inhibit root hair tip growth
within 10 mins of Nod factor addition to Phaseolus vulgaris
[32•]. At the subsequent branch-like outgrowth of the root
hairs in vetch, actin filaments reappear [33•] in the region
where there is an increased level of Ca2+ (Figure 2vi) [31•]
and it was concluded that fine bundles of actin filaments
promote polar growth by releasing Golgi vesicles to a vesicle-rich region of the growing root hair tip [33 •].
Cytoskeletal changes may also be associated with the Nod
factor induced reactivation of the cell cycle; outer cortical
cells enter the cell cycle but do not divide, whereas inner
cortical cells divide (Figure 2ix), entering the cell cycle
from the G0/G1 phase [34]. At a later stage in the interaction, a transient disorganisation of microtubules was
correlated with the appearance of Nod factors within
infected cells, suggesting that Nod factors may also be
involved in cytoskeletal rearrangements that occur during
the differentiation of infected cells [10•].
The effects of pharmacological agents on the expression of
the reporter gene MtENOD12A-GUS, which is induced in
response to Nod factors, provided indirect evidence for a
role for Ca2+ in a G-protein-mediated signal transduction
pathway [35••]. Mastoparan, a G-protein agonist induced
MtENOD12A in a similar way to Nod factor, whereas the
G-protein antagonist, pertussis toxin, inhibited Nod factor
and mastoparan-induced gene expression. Inhibitors of
phospholipase C, such as neomycin, and of Ca 2+
influx/release such as ethylene glycol tetraacetic acid
(EGTA), La3+ and ruthenium red, also blocked both the
Nod factor and mastoparan-induced gene expression.
Although these results are consistent with a mammaliantype G-protein signalling pathway, coupled to the
activation of phosphoinositide and Ca2+ as a second messenger (Figure 2iii), great care has to be taken with the
interpretation of data generated by such pharmacological
techniques that may be having multiple effects. The combination of such studies with the use of plant mutants in
which signalling is blocked in the early stages should, however, give an insight into the nature of the pathways
involved. The identity of the Nod factor receptors are of
key interest.
Nod-factor-binding proteins
Radioactively labelled LCOs were used to identify Nod factor binding proteins from Medicago truncatula. A protein with
a relatively low affinity (Kd = 86 nM) for the Nod factor was
found but is unlikely to play a role in the symbiosis [36]. A
second component, NFBS2 (Nod-factor-binding site 2),
from a plasmalemma fraction [37••] has high affinity
(Kd = 4 nM) for Nod factors. Several synthetic LCOs with
structural similarity to Nod factors were used in competitive
binding assays to determine the effects of structural modifications on relative binding efficiency. Substitutions on the
non-reducing terminal sugar (i.e. the presence of an Oacetate group, or the type of fatty acid) and the oligomer
length both contributed to optimal binding but the presence
486
Cell biology
or absence of a sulphate group on the reducing sugar did not
affect Nod factor binding. This sulphate group is essential for
normal Nod factor activity on Medicago spp. and so the binding specificity of NFBS2 did not correspond with what
would be predicted (on the basis of in vivo studies) for a putative Nod factor receptor. Thus, if NFBS2 does correspond to
(part of?) a receptor, the perception of the sulphate group by
the Nod factor receptor would be complex, and could possibly involve another protein that confers additional specificity.
Isolation of proteins that bind chitin oligomers led to the
identification of a Nod factor binding lectin with apyrase
activity [38••]. As this lectin hydrolyses the phosphoanhydride bonds of nucleoside phosphates, it was called lectin
nucleotide phosphohydrolase (LNP). LNP, which was isolated from the legume Dolichos biflorus, has much greater
affinity for Nod factors than chitin oligomers and has greatest
affinity for those Nod factors from rhizobia that can nodulate
D. biflorus. Furthermore, the apyrase activity of LNP is
enhanced by Nod factors. These results, together with the
observations that LNP is located on root hairs and that antibody to LNP inhibited root-hair deformation, suggest a role
for LNP in the nodule symbiosis, but as with NFBS2 a direct
role for LNP in signalling has not yet been established.
Although LNP is a lectin (as defined by its sugar-binding
capability), its sequence shows that it does not belong to the
large family of typical legume lectins [39,40]. Several years
ago it was proposed that such ‘typical’ lectins might play a role
in recognition by acting as a kind of surface-binding receptor,
and several studies, including addition of lectins and demonstration of their presence on root hair tips, supported a role for
lectins in the early stages of infection [40]. Furthermore,
transgenic clover plants expressing a pea lectin gene can be
nodulated by R. leguminosarum bv. viciae — which normally
nodulates pea and not clover — and the sugar-binding
domain is necessary for this activity [41]. The sugar-binding
sites of such lectins, however, are not consistent with them
binding Nod factors. A more recent study with transgenic
plants that express an introduced lectin gene has led to a
reassessment of the original hypothesis and the proposal of a
different model for a role of such lectins in nodulation. Lotus
corniculatus expressing a soybean lectin gene induced nodulelike outgrowths, which were devoid of bacteria, in response to
the soybean symbiont Bradyrhizobium japonicum [42•]. In this
case, infection did not proceed normally and it was suggested
that the introduced lectin, which was located in root-hair tips,
may interact with a component of the bacterial surface polysaccharide. It was proposed that the lectin might increase the
attachment and aggregation of heterologous rhizobia
(B. japonicum) at the infection site. This, in turn, could result
in an increased level of Nod factors at the appropriate location, thereby potentiating a weak signal and stimulating the
early stages of nodule morphogenesis, even by heterologous
Nod factors. This model illustrates that there could be multiple inputs into recognition between rhizobia and legumes and
that dissecting out the signalling pathway from other recognition events may not be simple.
Legume nodulation mutants
Many nodulation-defective legume mutants have been
isolated but many are in species that do not lend themselves to gene isolation. Nevertheless, these mutants have
been used to gain insight into the events that occur during
nodulation. Nod– mutants of several species do not form a
mycorrhizal symbiosis [43]. The pea early nodulation transcripts ENOD5 and ENOD12A, which are induced in
response to Rhizobium, are also induced by the endomycorrhizal fungus Gigaspora margarita; no expression was
seen in a Nod– sym8 pea mutant when inoculated with
either symbiont [44•]. Other studies using transgenic peas
expressing an ENOD12A–GUS reporter gene demonstrated that the induction of the pea ENOD12A gene is also
blocked in the sym19 mutant [45]. Many gene transcripts
are induced early in nodulation [5••,19•,20•] and knowledge of which genes are switched off in various mutants
may help to determine the sequence of events that are initiated after Nod factor perception, and where divergence
between endomycorrhizal and rhizobial symbioses occurs.
One of the key transcripts that is induced during
rhizobium–legume symbiosis is ENOD40, which is expressed
prior to initiation of pericycle cell division opposite the protoxylem poles and is also expressed in the dividing cells
[46•,47]. Transgenic legumes expressing ENOD40 initiate cortical cell division at multiple sites indicating that this gene
influences the differentiation and division of root cells [48].
Sequence comparisons led to the realisation that ENOD40
encodes a short (10–13 residue) peptide and a conserved noncoding RNA domain that may play a regulatory role [46•,47].
Each region seems to contribute to nodule development. The
conservation of this gene in non-legumes [46•] suggests that
nodule signalling may have evolved from a more ancient pathway conserved in non-legumes. Nod factor was reported to
induce a M. truncatula ENOD12 gene in rice [49•], although
this observation has not yet been verified by other laboratories.
Recently, there has been a focussed effort on investigating
the genetics of ‘model’ legumes, an experimental counterpart to Arabidopsis for the study of symbioses. These legumes
are diploid, have relatively small genome sizes, are easy to
transform and have high seed yield. Such properties lend
themselves to gene-tagging strategies and positional cloning.
One candidate gene for positional cloning affects the ethylene sensitivity of nodulation [50]; the mutant of M. truncatula
which does not express this gene has enhanced nodulation
and altered position of nodule development. These effects
are due to its insensitivity to ethylene which normally downregulates nodule development [17]. A bacterial artificial
chromosome library of M. truncatula genomic DNA has been
created and used to identify ethylene responsive genes [51].
Other approaches to gene identification involve a root-hairspecific cDNA library from M. truncatula [52•]. In Lotus
japonicus, an essential nodulation gene Nin has been identified by gene tagging following mutagenesis using the maize
transposable element Ac [53••]. The mutant is not infected
by rhizobia, but root hair deformation and curling occur,
Plant responses to nodulation factors Downie and Walker
indicating that the Nin gene is unlikely to encode the primary Nod factor receptor. The Nin gene product is predicted to
be a membrane protein and four similar Arabidopsis proteins
(of unknown function) were identified in database searches.
Similarity was also found with a Chlamydomonas gene product
(the so-called minus-dominance protein) that regulates mating type during gametogenesis. All of these proteins share a
putative DNA-binding dimerisation domain and so a regulatory role for the Lotus nodulation gene was proposed,
although the mechanism of regulation and the regulated
genes are not yet known. This first characterisation of an
essential nodulation gene suggests that we are now on the
threshold of a phase identifying genes required to establish
symbiotic interactions.
Conclusions
There is strong evidence to suggest that the early events in
Nod factor recognition involve movements of ions across
the plant plasma membrane, followed by the establishment
of intracellular Ca2+ oscillations. Circumstantial evidence
suggests that a G-protein-type receptor that could act via an
inositol-phosphate and phospholipase based signalling
pathway may be involved. Much work remains to be done,
however, to integrate the various observations that have
been made on the legume symbiosis. The key objective in
the future will be to exploit various nodulation mutants to
clone and characterise genes that are essential for nodule
development. The ease of the isolation (and maintenance)
of legume nodulation mutants, the availability of the Nodfactor-signalling molecules, and the ability to analyse ion
movements in epidermal cells mean that nodule development is a particularly good system for the analysis of a
developmental pathway. Understanding how nodules are
formed may well give us an insight into how other morphogenetic events are activated in plants.
Acknowledgements
We thank NJ Brewin for his constructive comments on the manuscript and
various colleagues for sending copies of recent and ‘in press’ publications.
The authors are supported by the Biotechnology and Biological Sciences
Research Council.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
Dénarié J, Debellé F, Promé JC: Rhizobium lipo-chitooligosaccharide
nodulation factors: signaling molecules mediating recognition and
morphogenesis. Annu Rev Biochem 1996, 65:503-535.
2.
Kamst E, Spaink HP, Kafetzopoulos D: Biosynthesis and secretion
of rhizobial lipochitin-oligosaccharide signal molecules. Subcell
Biochem 1998, 29:29-71.
3.
•
Downie JA: Functions of rhizobial nodulation genes. In The
Rhizobiaceae, edn 1. Edited by Spaink HP, Kondorosi A, Hooykaas PJJ.
Dordrecht: Kluwer academic publishers; 1998:387-402.
The most comprehensive list of rhizobial Nod gene products currently available. This chapter is most useful to specialists in the area.
4.
•
Bladergroen MR, Spaink HP: Genes and signal molecules involved
in the rhizobia-Leguminoseae symbiosis. Curr Opin Plant Biol
1998, 1:353-359.
A good overview of hormone signalling and the possible effects of flavanoids
on auxins during infection.
487
5. Schultze M, Kondorosi A: Regulation of symbiotic root nodule
•• development. Annu Rev Gen 1998, 32:33-57.
An excellent and comprehensive review of signalling between plants and
bacteria focusing on plant responses.
6.
Economou A, Davies AE, Johnston AWB, Downie JA: The Rhizobium
leguminosarum biovar viciae nodO gene can enable a nodE
mutant of Rhizobium leguminosarum biovar trifolii to nodulate
vetch. Microbiology 1994, 140:2341-2347.
7.
Vlassak KM, Luyten E, Verreth C, van Rhijn P, Bisseling T,
Vanderleyden J: The Rhizobium sp. BR816 nodO gene can function
as a determinant for nodulation of Leucaena leucocephala,
Phaseolus vulgaris and Trifolium repens by a diversity of
Rhizobium spp. Mol Plant–Microbe Interact 1998, 11:383-392.
8.
Sutton JM, Lea EJA, Downie JA: The nodulation-signaling protein
NodO from Rhizobium leguminosarum biovar viciae forms ion
channels in membranes. Proc Natl Acad Sci USA 1994,
91:9990-9994.
9.
Viprey V, DelGreco A, Golinowski W, Broughton WJ, Perret X:
Symbiotic implications of type III protein secretion machinery in
Rhizobium. Mol Microbiol 1998, 28:1381-1389.
10. Timmers ACJ, Auriac MC, de Billy F, Truchet G: Nod factor
•
internalization and microtubular cytoskeleton changes occur
concomitantly during nodule differentiation in alfalfa. Development
1998, 125:339-349.
The authors of this work describe the immunolocalisation of Nod factors
and tubulin, revealing a transient disorganisation of the cytoskeleton in
association with Nod factor internalisation. This implies a direct association between Nod-factor-regulated symbiotic differentiation and the coordination of the cytoskeleton.
11. Philip-Hollingsworth S, Dazzo FB, Hollingsworth RI: Structural
requirements of Rhizobium chitolipooligosaccharides for uptake
and bioactivity in legume roots as revealed by synthetic analogs
and fluorescent probes. J Lipid Res 1997, 38:1229-1241.
12. Ardourel M, Demont N, Debellé FD, Maillet F, de Billy F, Promé JC,
Dénarié J, Truchet G: Rhizobium meliloti lipooligosaccharide
nodulation factors: different structural requirements for bacterial
entry into target root hair-cells and induction of plant symbiotic
developmental responses. Plant Cell 1994, 6:1357-1374.
13. Geurts R, Heidstra R, Hadri AE, Downie JA, Franssen H, van
Kammen A, Bisseling T: Sym2 of pea is involved in a nodulation
factor-perception mechanism that controls the infection process
in the epidermis. Plant Physiol 1997, 115:351-359.
14. Schultze M, Staehelin C, Brunner F, Genetet I, Legrand M, Fritig B,
Kondorosi E, Kondorosi A: Plant chitinase/lysozyme isoforms
show distinct substrate specificity and cleavage site preference
towards lipochitooligosaccharide Nod signals. Plant J 1998,
16:571-580.
15. Schell J, Bisseling T, Dülz M, Franssen H, Fritze K, John M, Kleinow T,
•
Lessnick A, Miklashevichs E, Pawlowski K et al.: Re-evaluation of
phytohormone-independent division of tobacco protoplastderived cells. Plant J 1999, 17:461-466.
Several previous reports suggested that Nod factors induce cell division in
tobacco protoplasts. This paper retracts these observations and cites all of
the work containing unreproducible data.
16. Hirsch AM, Fang Y, Asad S, Kapulnik Y: The role of phytohormones
in plant-microbe symbioses. Plant Soil 1997, 194:171-184.
17.
Spaink HP: Ethylene as a regulator of Rhizobium infection. Trends
Plant Sci 1997, 2:203-204.
18. Hirsch AM, Larue TA: Is the legume nodule a modified root or stem
or an Organ sui generis? Crit Rev Plant Sci 1997, 16:361-392.
19. Hadri AE, Bisseling T: Responses of the plant to Nod factors. In The
•
Rhizobiaceae, edn 1. Edited by Spaink HP, Kondorosi A, Hooykaas PJJ.
Dordrecht: Kluwer academic publishers; 1998:403-416.
An in-depth review of various aspects of plant responses to Nod factors
describing the many genes induced.
20. Irving HR, Boukli NM, Kelly MN, Broughton WJ: Nod factors in
•
symbiotic development of root hairs. In Cell and Molecular Biology of
Root Hairs, edn 1. Edited by Ridge RW, Emons AM. Berlin: SpringerVerlag; 1999: in press.
A comprehensive review of root hair responses to Nod factors with an interesting discussion on potential signalling models.
21. Ehrhardt DW, Atkinson EM, Long SR: Depolarisation of alfalfa root
hair membrane potential by Rhizobium meliloti Nod factors.
Science 1992, 256:998-1000.
488
Cell biology
22. Felle HH, Kondorosi E, Kondorosi A, Schultze M: Nod signal-induced
plasma membrane potential changes in alfalfa root hairs are
differentially sensitive to structural modifications of the
lipochitooligosaccharide. Plant J 1995, 7:939-947.
36. Bono JJ, Riond J, Nicolaou KC, Bockovich NJ, Estevez VA,
Cullimore JV, Ranjeva R: Characterization of a binding-site for
chemically synthesized lipo- oligosaccharidic NodRm factors in
particulate fractions prepared from roots. Plant J 1995, 7:253-260.
23. Kurkdjian AC: Role of the differentiation of root epidermal cells in
Nod factor (from Rhizobium meliloti)-induced root-hair
depolarization of Medicago sativa. Plant Physiol 1995, 107:783-790.
37.
••
24. Felle HH, Kondorosi E, Kondorosi A, Schultze M: Rapid alkalinization
in alfalfa root hairs in response to rhizobial
lipochitooligosaccharide signals. Plant J 1996, 10:295-301.
25. Felle HH, Kondorosi E, Kondorosi A, Schultze M: The role of ion
•• fluxes in Nod factor signalling in Medicago sativa. Plant J 1998,
13:455-463.
The very earliest plant responses to Nod factors are investigated. Nod factors induce a rapid influx of Ca2+ that is followed by Cl– and K+ efflux. These
latter ion movements can explain the Nod-factor induced depolarisation of
the root hair plasma membrane.
26. Ehrhardt DW, Wais R, Long SR: Calcium spiking in plant root hairs
responding to Rhizobium nodulation signals. Cell 1996, 85:673-681.
27. Meldolesi J: Calcium signalling — oscillation, activation,
•
expression. Nature 1998, 392:863-865.
It has long been suspected that oscillations in intracellular calcium may play
a role in regulating gene expression in mammalian cells. This commentary
highlights the importance of two papers published in the same issue
describing the first hard evidence to support this hypothesis.
28. Staxén I, Pical C, Montgomery LT, Gray JE, Hetherington AM,
McAinsh MR: Abscisic acid induces oscillations in guard-cell
cytosolic free calcium that involve phosphoinositide-specific
phospholipase C. Proc Natl Acad Sci USA 1999, 96:1779-1784.
29. Franklin-Tong VE, Drobak BK, Allan AC, Watkins PAC, Trewavas AJ:
Growth of pollen tubes of Papaver rhoeas is regulated by a slowmoving calcium wave propagated by inositol 1,4,5-trisphosphate.
Plant Cell 1996, 8:1305-1321.
30. Gehring CA, Irving HR, Kabbara AA, Parish RW, Boukli NM,
Broughton WJ: Rapid, plateau-like increases in intracellular free
calcium are associated with nod-factor-induced root-hair
deformation. Mol Plant-Microbe Interact 1997, 10:791-802.
31. de Ruijter NCA, Rook MB, Bisseling T, Emons AMC: Lipochito
•
oligosaccharides re-initiate root hair tip growth in Vicia sativa with
high calcium and spectrin-like antigen at the tip. Plant J 1998,
13:341-350.
Cytological changes induced in root hairs by Nod factors were examined by
microscopy. Root hair cells with terminating growth reacquired tip-growing
characteristics including a tip gradient of Ca2+ and accumulation of a spectrin-like antigen. The results indicate that the root hairs respond to Nod factors by recovering cytoplasmic polarity and exocytosis.
32. Cárdenas L, Vidali L, Domínguez J, Pérez H, Sánchez F, Hepler PK,
•
Quinto C: Rearrangement of actin microfilaments in plant root
hairs responding to Rhizobium etli nodulation signals. Plant
Physiol 1998, 116:871-877.
In this paper, researchers describe fragmentation of the Phaseolus vulgaris
root hair actin cytoskeleton upon exposure to Rhizobium etli Nod factors.
This was observed 5–10 minutes after Nod factor application which could
correlate the response to observed changes in intracellular calcium. This
work indicates that Nod factors alter the organisation of actin microfilaments
in root hairs.
33. Miller DD, de Ruijter NCA, Bisseling T, Emons AMC: The role of actin
•
in root hair morphogenesis: studies with lipochitooligosaccharide as a growth stimulator and cytochalasin as an
actin perturbing drug. Plant J 1999, 17:141-154.
The authors propose a model for root hair morphogenesis whereby actin filaments co-ordinate growth by releasing Golgi vesicles to a vesicle-rich region
of the growing root hair tip. The model relies on the influence of Nod factors
on root hair growth and the interference of cytochalasin D with actin function.
34. Yang WC, Deblank C, Meskiene I, Hirt H, Bakker J, van Kammen A,
Franssen H, Bisseling T: Rhizobium Nod factors reactivate the cellcycle during infection and nodule primordium formation, but the
cycle is only completed in primordium formation. Plant Cell 1994,
6:1415-1426.
35. Pingret JL, Journet EP, Barker DG: Rhizobium Nod factor signaling:
•• evidence for a G protein-mediated transduction mechanism. Plant
Cell 1998, 10:659-671.
Using a GUS reporter system to reflect early nodulin gene induction this
work describes the use of various agonists and antagonists to provide indirect evidence for a G-protein-mediated signalling pathway operating downstream of the Nod factor receptor.
Gressent F, Drouillard S, Mantegazza N, Samain E, Geremia RA,
Canut H, Niebel A, Driguez H, Ranjeva R, Cullimore J, Bono JJ:
Ligand specificity of a high-affinity binding site for lipochitooligosaccharidic Nod factors in Medicago cell suspension
cultures. Proc Natl Acad Sci USA 1999, 96:4704-4709.
This work demonstrates that a high affinity binding protein copurifies with the
plasma-membrane fraction from Medicago cell suspension cultures. Binding
of LCOs with different substitutions is analysed and the presence of a sulphate group, that is essential for nodulation, has no effect on binding affinity. Therefore there is some question as to whether the partially purified
protein is a Nod factor membrane receptor or some other protein that happens to bind LCOs.
38. Etzler ME, Kalsi G, Ewing NN, Roberts NJ, Day RB, Murphy JB: A
•• Nod factor binding lectin with apyrase activity from legume roots.
Proc Natl Acad Sci USA 1999, 96:5856-5861.
Isolation of a chitin-binding protein fortuitously led to the identification of this
novel type of lectin that has both a high affinity for Nod factors and an
apyrase activity. It is structurally distinct from the large family of legume
lectins previously described [39,40] and a potential signalling receptor.
39. Brewin NJ, Kardailsky IV: Legume lectins and nodulation by
Rhizobium. Trends Plant Sci 1997, 2:92-98.
40. Kijne JW, Bauchrowitz MA, Diaz CL: Root lectins and Rhizobia. Plant
Physiol 1997, 115:869-873.
41. van Eijsden RR, Díaz CL, de Pater BS, Kijne JW: Sugar-binding
activity of pea (Pisum sativum) lectin is essential for heterologous
infection of transgenic white dover hairy roots by Rhizobium
leguminosarum biovar viciae. Plant Mol Biol 1995, 29:431-439.
42. van Rhijn P, Goldberg RB, Hirsch AM: Lotus corniculatus nodulation
•
specificity is changed by the presence of a soybean lectin gene.
Plant Cell 1998, 10:1233-1249.
Transfer of a soybean lectin gene enables B. japonicum to induce uninfected nodules on Lotus corniculatus. A model is proposed suggesting that
lectins enhance bacterial attachment to root hairs thereby enhancing Nod
factor signalling by increasing localised concentration.
43. Albrecht C, Geurts R, Bisseling T: Legume nodulation and
mycorrhizae formation; two extremes in host specificity meet.
EMBO J 1999, 18:281-288.
44. Albrecht C, Geurts R, Lapeyrie F, Bisseling T: Endomycorrhizae and
•
rhizobial Nod factors both require SYM8 to induce the expression
of the early nodulin genes PsENOD5 and PsENOD12A. Plant J
1998, 15:605-614.
The authors find that the same plant early nodulation genes are induced by
rhizobia and mycorrhizal fungi. Gene induction by both endosymbionts is
blocked in a nodulation mutant of pea.
45. Schneider A, Walker SA, Poyser S, Sagan M, Ellis THN, Downie JA:
Genetic mapping and functional analysis of a nodulationdefective mutant (sym19) of pea (Pisum sativum L). Mol Gen
Genet 1999, 262:1-11.
46. Kouchi H, Takane K, So RB, Ladha JK, Reddy PM: Rice ENOD40:
•
isolation and expression analysis in rice and transgenic soybean
root nodules. Plant J 1999, 18:121-129.
The authors of this paper describe the cloning of a rice gene homologous to
the early nodulin ENOD40. A promoter–GUS fusion study indicated the rice
gene may play a similar role in vascular differentiation.
47.
Fang YW, Hirsch AM: Studying early nodulin gene ENOD40
expression and induction by nodulation factor and cytokinin in
transgenic alfalfa. Plant Physiol 1998, 116:53-68.
48. Charon C, Johansson C, Kondorosi E, Kondorosi A, Crespi M:
enod40 induces dedifferentiation and division of root cortical cells
in legumes. Proc Natl Acad Sci USA 1997, 94:8901-8906.
49. Reddy PM, Ladha JK, Ramos MC, Maillet F, Hernandez RJ, Torrizo LB,
•
Oliva NP, Datta SK, Datta K: Rhizobial lipochitooligosaccharide
nodulation factors activate expression of the legume early
nodulin gene ENOD12 in rice. Plant J 1998, 14:693-702.
One of the very few papers describing activation of a legume nodulation
gene by Nod factors in a transgenic non-legume.
50. Penmetsa RV, Cook DR: A legume ethylene-insensitive mutant
hyperinfected by its rhizobial symbiont. Science 1997,
275:527-530.
51. Nam YW, Penmetsa RV, Endre G, Uribe P, Kim D, Cook DR:
Construction of a bacterial artificial chromosome library of
Plant responses to nodulation factors Downie and Walker
Medicago truncatula and identification of clones containing
ethylene-response genes. Theor Appl Genet 1999, 98:638-646.
52. Covitz PA, Smith LS, Long SR: Expressed sequence tags from a
•
root-hair-enriched Medicago truncatula cDNA library. Plant Physiol
1998, 117:1325-1332.
An expressed sequence tag (EST) library of root hair genes has been established and is growing rapidly. This paper describes many genes that are
expressed in root hairs.
489
53. Schauser L, Roussis A, Stiller J, Stougaard J: A plant regulator
•• controlling development of symbiotic root nodules. Nature 1999,
in press.
The first paper to describe cloning of a gene essential for some of the earliest stages in the rhizobia-legume symbiosis. Transposon-tagged mutants of
Lotus japonicus led to the isolation of a gene required for both bacterial entry
into the host plant and the establishment of the nodule primordium. The
encoded protein contains transcription factor-like domains and could define
a new class of developmental regulators.