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World Journal of Microbiology and Biotechnology, 8 (SlJpple131ent 1), 120-123
Regulation of nodulation in Rhizobium
leguminosarum
B.J.J. Lugtenberg
Under conditions of nitrogen limitation, Rhizobium bacteria
infect leguminous plants, which then form root nodules. In
this symbiotic process the bacteria present in the nodules
(as bacteroids, differentiated forms of the bacteria) fix
atmospheric N 2 and convert this into ammonia which is used
as a nitrogen source by the plant. In return, the plant
provides the bacterium with a carbon source.
Nodulation by Rhizobium is a host-specific process: of the
various species and biovars of Rhizobium only one, or a
limited number, is able to nodulate a certain plant and fix
nitrogen efficiently. N2-fixation is an extremely important
process since, after water, nitrogen is the second most
important compound limiting for world crop production.
Since 1896, Rhizobium strains selected for their N2-fixing
ability have been commercially used as inoculants, to
increase the production of especially soybean and alfalfa.
Inoculation is often only moderately successful since
indigenous rhizobia are better adapted to the local
conditions. They therefore often outcompete the inoculant
strains.
The Nodulation Process
The following stages have been established or proposed.
(1) chemotaxis to root exudate.
(2) Attachment of bacteria to the root hair tip of the
plant.
(3) Induction of nod genes by flavonoids secreted by the
plant. The result is secretion of soluble, low molecular
weight components which are involved in hair
curling, release of additional nod-gene inducers,
meristem formation and elicitation of at least one
nodulin, ENOD12.
(4) Root hair curling leading to internalization of the
rhizobia.
B.J.J. Lugtenberg is at the Leiden University, Department of Plant Mok c u l a r Biology, Botanical Laboratory, Nonnensteeg 3, 2311 VJ Leiden,
The Netherlands.
9 1992 Rapid Communications of Oxford Ltd
120
World Journal of Microbiolo,ey and Biotechnolq,r
Vol 8 S~pplement 1 " 1992
(5)
(6)
Infection thread initiation.
Nodule initiation ahead of advancing infection thread
(meristemic growth).
(7) Growth and branching of infection thread followed
by invasion of (dividing) meristemic cells. Bacteria in
the infection thread have divided many times.
(8) Release of bacteria surrounded by a peribacteroid
membrane in plant cells.
(9) Differentiation of bacteria into bacteroids.
(10) Continued meristemic growth resulting in a nodule.
(11) Synthesis of nodulins by the plant (a.o. leghemoglobin).
(I2) Conversion of N 2 into ammonia.
Role of Sym Plasmid
Rhizobium strains usually contain one or more plasmids,
among which is the Sym(biosis) plasmid. Loss of this
plasmid, e.g. by growth at elevated temperatures, results in
strains which are no longer able to nodulate. Transfer of
another Sym plasmid into such a cured strain restores
nodulation ability. Even more interestingly, the hostspecificity of the resulting strain is the same as that of the
donor strain of the Sym plasmid. Also, if the Sym plasmid
is transferred to a cured Agrobacterium fumefaciens strain, the
resulting strain is able to form nodules, which usually are
ineffective.
Based on these data it must be concluded that (i) the Sym
plasmid carries importanf nodulation genes, and (ii) the Sym
plasmid determines the host range.
Sym-Plasmid-Localized Nodulation Genes
About 13 nodulation genes are localized on pRLIJI, the Sym
plasmid of the Rhizobium leguminosarumbv. viciae strain that
is mostly used in our studies. Only nodD, the positive
regulatory gene, is constu~ively transcribed, whereas the
Regulation of nodulation
operons nadABCIJ, nodFEL, n o d M N T and nodO are
transcribed only upon activation of NodD protein by certain
flavonoids.
Many of these nod genes are involved in the production
and/or secretion of soluble low molecular weight
extracellular compounds as described for R. meliloti by
D6nari6's group in Toulouse (Lerouge et al. 1990). With
respect to the function of nod genes one has to take into
account that some nod genes can functionally replace others,
e.g. node and nodO (Economou el al. 1990), although Node
protein is localized in the cytoplasmic membrane (Spaink et
al. 1989), whereas NodO protein is secreted (De Maagd et
al. 1989c).
Research in the Near Future
Attachment
Attachment is independent of the Sym plasmid. So far, three
factors have been implicated in attachment of R.
leguminosarum bv. viciae bacteria to the tips of pea root hairs:
a bacterial protein, bacterial cellulose fibrils and the plant's
lectin (Lugtenberg et al. I989). Once mutants lacking the
bacterial protein, rhicadhesin, have been generated, it can
be tested whether attachment is a prerequisite for
nodulation. Once antibodies are available it can be tested
whether rhicadhesin is just a cell surface protein or whether
it is organized in fimbriae-like structures. Finally, the
receptors of rhicadhesin and of lectin still have to be
identified.
Activation of NodO Protein
Evidence has been presented for a direct interaction between
NodD protein and inducing flavonoids (Spaink eta]. 1987),
presumably in the cytoplasmic membrane (Recourt et al.
1989; Schlaman el al. 1989). This interaction can play a role
in host-specificity. Components which inhibit activation of
NodD protein have also been identified (Firmin et al. 1986;
Djordjevic e~ al. 1987). It is not clear yet whether they play
a role in the nodulation process. It is hard to make a model
which includes all reported interactions of NodD protein,
namely those with the cytoplasmic membrane (Schlaman et
al. 1989), with a flavonoid inducer (Recourt et al. 1989) and
with nod box DNA (Fisher et al. 1988).
The Bacterial Answer upon nod Gene Expression
Many Sym plasmid-localized nod genes function in the
synthesis and/or secretion of soluble low molecular weight
molecules. In the case of R. meliloti, one such molecule has
been identified as a sulphated fi-l,4-tetrasaccharide of
D-glucosamine, in which three amino groups are acetylated
and one was acylated with a C-16 bis-unsaturated fatty acid
(Lerouge et aI. 1990). Future research will unfold the
identities of similar molecules secreted by other rhizobia.
Moreover, these studies will focus on (i) the function of
individual nod genes in the synthesis and secretion of these
molecules, (ii) the roles of these molecules in hair eurl!~g,
meristem formation and secretion of additional fla~onoid
inducers. Of particular interest will be the molecular
mechanism of signal perception and transduction, in which
ENOD12 elicitation (Scheres et al. 1990) could be used as
the response of the signal perception/transduction pathway.
Finally, it is tempting to speculate that the bacterial answer
molecules may interact with lectin (Dfaz et al. 1989) in order
to initiate infection thread formation.
Host-Specificity
R. leguminosarum by. viciae and R. leguminosarum bv. trifolii
are closely related rhizobia which differ in host-specificity
in that the former nodulates pea and Vicia, whereas the latter
nodulates clovers. Several molecules are involved in the
determination of this host-specificity.
(i)
NodD proteins of the two biovars become activated
by different sets of flavonoids which largely overlap
but which also contain activators of only one of the
two NodD proteins (Spaink et al. I987).
(ii) Of the inducible nod genes node has been shown to
be the only gene that determines the difference in host
specificity (Spaink et al. 1989).
(iii) When the gene encoding pea lectin is expressed in
white clover hairy roots, R. Ieguminosarum bv. viciae
can form effective nodules on the transgenic hairy
roots. The same bacterium is unable to form stable
infection threads on white clover plants or hairy roots
carrying only its own lectin gene (Diaz et al. 1987).
Future research will focus on unravelling the molecular
mechanism of host-specificity.
Functional Common Roles of Different nod Genes
Mutations in some nod genes have hardly any effect on
nodulation except when, in addition, other nod genes are
also missing (Van Brussel et al. 1988). The molecular basis
of this compensation effect is not understood. The
best-known example is the couple nodO/nodE (Economou et
al. 1990). NodO encodes a secreted flavonoid-inducibte
protein (De Maagd et aI. I989c,d) which binds to roots of
~'cia hirsuta and V. saliva (Lugtenberg et al. 1990). It binds
Ca 2+ ions (Economou et al. 1990). NodE encodes a
cytoplasmic membrane protein (Spaink et aI. 1989) which is
involved in synthesis or secretion of a low molecular weight
soluble molecule which is a major determinant of
host-specificity (Spaink et al. 1989) and which elicits nodulin
ENOD12 synthesis (Scheres et al. 1990). We have
interpreted these data as indicating that these are at least
two different nodulation pathways (Lugtenberg et al. 1990).
World Journal of Microbiology and Biotechnology, Vo] 8 Supplement 1 9 1992
1 )~ 1
B.].J. Lugtenberg
Role of Lipopolysaccharide in Nodulation
The exact stage of the nodulation process in which
lipopolysaccharide (LPS) mutants are defective seems to
differ between biovars. Biovar phaseoli mutants form
abortive infection threads and are not released into bean
plant cells (De Maagd & Lugtenberg 1989). Biovar viciae
mutants lacking O-antigen are released from the infection
thread (although possibly less frequently than wild type
cells), and form (some) bacteroid-filled V. hirsuta plant cells
(De Maagd et al. 1988, 1989b; Priefer I989). In all these
cases the nodules are small, white and devoid of nitrogenase
activity. Thus, it appears that complete LPS is not essential
for the early steps of symbiosis, but that, depending on the
bacterium-host combination, complete LPS is required for
normal infection thread development, bacterial release and
bacteroid differentiation (De Maagd & Lugtenberg 1989).
One can speculate on the role of LPS at the molecular
level (De Maagd & Lugtenberg 1989).
(I)
(2)
(3)
(4)
LPS may contain, either constitutively or induced, a
structure that acts as a signal towards the plant. This
signal may be released by a plant enzyme. The signal
may (i) be involved in infection thread initiation, (ii)
induce endocytosis of the bacteria, or (iii) inhibit plant
defence reactions. The enormous variation in Oantigen chain sugar composition make the involvement
of this LPS-moiety in the generation of a specific signal
not very likely.
LPS may contain a structure that acts as a receptor for
a plant signal. The interaction may trigger the next
step in symbiosis. A possible candidate for a plant cell
wall receptor is root lectin (Wolpert & Albersheim
1976). The disadvantage mentioned under (1) for such
a specific role also applies here.
Normal symbiosis may require a minimal bacterial cell
surface density of (lipo)polysaccharide molecules, e.g.
because of requirements for surface change or
hydrophobicity (De Maagd et aI. 1989b). Such a
relaxed requirement can be fulfilled by many
structurally different (lipo)polysaccharides.
The effect of LPS mutations on symbiosis may be
indirect, e.g. due to the lack of another component
which shares part of the same pathway, or due to a
role of LPS in outer membrane stability.
Differentiation from Bacterium to Bacteroid
During development of bacteria to bacteroids a number of
cell surface changes take place, particularly in LPS and outer
membrane protein composition (De Maagd et aI. 1989a).
The latter two changes are probably initiated in the stage
of release from the infection thread (Lugtenberg et al. 1990).
Another recent finding is that nod genes are apparently
switched off in bacteroids of R. mellioti (Scharma & Signer
1990) and R. legaminosarum bv. viciae (Lugtenberg et aI.
1,22
WorldJournalof Microbiologyand Biotechnology,VoI 8 Supplement1 91992
1990). Future research will discover whether changes in cell
surface composition and in nod gene expression are switched
off by the same (plant) signal and what the nature of this
signal is.
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World Journal of Microbiology and Biot~chnology, Vol 8 Supplement I 9 1992
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