Directory UMM :Data Elmu:jurnal:P:PlantScience:Plant Science_BioMedNet:141-160:
305
Genealogy of legume-Rhizobium symbioses
William J Broughton* and Xavier Perret
Accumulating evidence suggests that lateral transfer of
nodulation capacity is an important driving force in symbiotic
evolution. As a consequence, many distantly related soil
bacteria have acquired the capacity to invade plants and fix
nitrogen within them. In addition to these proteins required
for bacteroid development and nitrogen fixation, core
symbiotic competence seems to require flavonoids, NodD
proteins, lipochitooligosaccharidic Nod-factors, extra-cellular
polysaccharides, as well as various exported proteins. Plants
respond to different levels and combinations of these
substances in species specific ways. After contact has been
initiated by flavonoids and NodD proteins, constant signal
exchange fine-tunes these symbiotic demands, especially to
overcome defence reactions.
Addresses
Laboratoire de Biologie Moléculaire des Plantes Supérieures
(LBMPS), Université de Genève, 1 ch. de l’Impératrice, 1292
Chambésy/Genève, Switzerland
*e-mail: [email protected]
Current Opinion in Plant Biology 1999, 2:305–311
http://biomednet.com/elecref/1369526600200305
© Elsevier Science Ltd ISSN 1369-5266
Abbreviations
Hac
root-hair curling
Had
root-hair deformation
Hai
root-hair formation
Introduction
In the latter part of the nineteenth century, inquiry into
the nature of plant–microbe interactions turned from the
observational to the experimental. Beyerinck proved
that bacteria elicit nodules on the roots of legumes by
preparing pure cultures from nodules of Vicia faba and
using them to infect Faba beans growing in sterile soil
[1]. Prazmowski [2] inoculated Pisum sativum with axenic
cultures and observed that micro-organisms entered into
the plant via infection threads in root-hairs. Hiltner [3]
prepared aqueous, bacteria-free filtrates from mature
P. sativum nodules and demonstrated that they contain a
substance that induces root-hair formation (Hai) and
deformation of the root-hairs (Had). Ninety years later
Lerouge et al. [4] showed that the root-hair deforming
substances of R. meliloti are N-acylated oligomers of
N-acetyl-D-glucosamine (known as Nod-factors) and
implied that variations in Nod-factor structure are derminants of host specificity. In turn, Spaink et al. [5]
demonstrated that the principal differences between the
Nod-factors of R. leguminosarum biovar viciae and those of
R. meliloti lie in the degree of unsaturation of the acyl
chains, as well as the presence or absence of a sulphate
group on the reducing-terminal glucosamine.
Common threads link these milestones in symbiotic nitrogen fixation research — all were performed by European
scientists on local legumes and their associated rhizobia.
Unfortunately, the plants they used (Medicago, Pisum, and
Vicia) are very closely related and belong to only two
(Vicieae and Trifolieae) of the thirty-nine recognised tribes
(three of the 666 genera) of the Leguminosae (see [6] and
Figure 1). Furthermore, their respective symbionts,
R. meliloti, R. leguminosarum bv. viciae and R. leguminosarum
bv. trifolii, associate with only few plants (i.e. have a narrow
host-range). It is thus possible that nitrogen-fixing symbioses in temperate, herbaceous legumes are not
representative of Rhizobium–legume associations as a
whole. To seek a more general understanding of the molecular bases of symbioses, we have examined the
relationship between promiscuous (i.e. able to nodulate
many different legumes) rhizobia and their hosts. This
raises the question of rhizobial taxonomy. Below, we will
argue that rhizobia are heterogenous because, like other
Gram-negative bacteria, they have acquired large blocks of
genetic information by lateral-transfer. In some cases these
modules represent pathogenicity islands, whereas in others
they carry symbiotic genes (see note added in proof). Once
acquired these ‘symbiotic islands’ have been subject to
constant re-arrangements, amplifications and deletions.
Rhizobia: phylogenetically distant bacteria
containing related symbiotic genes
Hiltner and Störmer [7] were probably the first to attempt
classification of rhizobia on the basis of their plant origins.
Host-based classifications would work if rhizobial hostranges mirrored legume taxonomy, but some rhizobia form
symbioses across taxonomic divisions. Wilson [8], for example, tested the host-ranges of rhizobia isolated from 31
different genera of legumes on 160 different legume
species. The average number of plant species nodulated by
a particular isolate was 33%, and four of the isolates formed
nodules on more than half the legumes tested. Likewise,
Rhizobium sp. NGR234 nodulates at least 112 genera of
legumes, whereas the closely related R. fredii USDA257
nodulates an exact subset (77 genera) of the NGR234 hosts
[9•]. Widespread existence of such broad host-range rhizobia, as well as promiscuous hosts such as Vigna [10] renders
any host-based classification scheme inadequate.
Recent taxonomic proposals based on DNA sequences of
highly conserved genes suggest that symbiotic rhizobia are
much more diverse than was previously thought [11], and the
reasons for this diversity are becoming clearer. Probably,
what permits many different Gram-negative bacteria to be
symbiotic is a group of less than 400 genes, and some basic
‘compatibility’ of their chromosomal backgrounds. This is
best seen in the ‘symbiosis islands’ of bacteria associated
with Mesorhizobium loti. Genetically diverse ‘mesorhizobia’
306
Biotic interactions
Figure 1
Loteae
P.13
Trifolieae
P.21
Psoraleeae
P.12
Desmodieae
P.11
Indigofereae
P.9
Phaseoleae
P.10
Amorpheae
P.6
o
Cicereae
P.20
o
o
o
o
O=C
Sesbanieae
P.8
O=C
Galegeae
P.16
Vicieae
P.19
Robinieae
P.8
Hedysareae
P.18
Aeschynomeneae
P.14
Millettieae
P.7
Adesmieae
P.15
Abreae
P.5
Crotalarieae
P.27
Brongniartieae
P.22
Bossiaeeae
P.23
Mirbelieae
P.24
Podalyrieae
P.25
Genisteae
P.30
Dalbergieae
P.4
Thermopsideae
P.29
Sophoreae
P.2
Acacieae M.4
& Ingeae M.5
Cassiinae
C2.D
Mimoseae
M.3
H3COO
HO
H2NCOO
CH2-OH
O
H3C N
CH3
O
HO3S-O
O-CONH2
CH2
Swartzieae
P.1
O
HO
CH3-O
O
CH 2
O
O
HO
CH3CONH
3
O=C
(C16:0 or C16:1or C14:1
)
CH3
O
OH
CH3CONH
Caesalpinioideae
Current Opinion in Plant Biology
Relationships amongst tribes of the Leguminosae, and the ability of
Rhizobium sp. NGR234 and R. fredii USDA257 to nodulate them. All
substituents found in the complete family of NodNGR factors are shown
near the bottom of the figure, and those structural requirements which
NGR234/USDA257 lack, but which seem to be necessary for nodulation
of the Cicereae, Trifolieae, and Vicieae (α-β-unsaturated fatty acids) are
shown in the top, left-hand portion. Tribes marked in green are nodulated
by both NGR234 and USDA257, whereas those marked in red are only
nodulated by NGR234. Tribes that are Nod– with both bacteria, are boxed
in blue. This figure was re-drawn from Pueppke and Broughton [9•].
Genealogy of legume–Rhizobium symbioses Broughton and Perret
were isolated from nodules of Lotus corniculatus in an area
devoid of indigenous rhizobia, but which had been inoculated with a single M. loti isolate [12]. All contained an identical
500 kb ‘symbiotic island’ integrated into the chromosomal
phenyl-alanine transfer RNA gene (phe-tRNA gene) [13•].
At the present moment, the exact nature of the soil bacteria
(‘the mezorhizobial recipients’) which have aquired these
‘symbiosis islands’ is not clear. Nevertheless, their aquisition
converts formerly saprophytic bacteria into symbionts. At
536 kb, pNGR234a, the symbiotic plasmid of the broad
host-range Rhizobium sp. NGR234, is about the same size as
the ‘symbiosis island’, and it confers the ability to nodulate
on Agrobacterium tumefaciens. Sequencing of this plasmid
highlighted that pNGR234a is particularly interesting, not
only because it is a repository of symbiotic promiscuity, but
also because of its mosaic genetic structure with origins of
replication and transfer very similar to those of Agrobacterium
species [14]. These features not only suggest a common origin for symbiotic replicons and tumour-inducing-plasmids,
but also exemplify the combinatorial possibilities offered by
lateral transfer of genetic information.
Genetic exchange also occurs between symbionts in the
soil [15,16]. Once acquired, amplification of the newly
inherited genetic information may generate pools of related
strains, some of which are better adapted to particular conditions than others [17]. Abundant and highly conserved
repeats found throughout rhizobial genomes are the sites of
amplification. When formed these ‘amplicons’ can be
translocated to other plasmids or the chromosome, where
they are more stably maintained. Thus, in populations of
soil bacteria, natural genetic mechanisms exist which are
capable of transforming isolates with widely different chromosomal backgrounds into pools of nodulating bacteria.
Nod-factors and host-specificity
What are the bacterial determinants that specify hostrange? It has been clear for almost 100 years that rhizobial
secretions provoke Had. Some purified Nod-factors provoke the extreme curling of the root-hairs (shepherd’s
crook formation or Hac), which is the most specific of the
morphological responses induced by rhizobia (see [18]).
They also induce cortical cell division [19], and permit rhizobia to penetrate host-tissues [20,21•]. Since rhizobia that
are unable to synthesise Nod-factors cannot nodulate
(i.e. Nod–), these signal molecules are obviously essential
to the symbiosis (see also A Hirsch, this issue pp 320–326).
It is thus possible that structural variations in Nod-factors
are the determinants of host-specificity. Indeed, mutation
of nodH, which encodes a sulphotransferase, leads to the
absence of the sulphate group from R. meliloti LCOs, and
loss of nodulation of its homologous host M. sativa. Yet,
these NodH– mutants are now able to nodulate the nonhost V. sativa [22]. Interestingly, almost the only legumes
that NGR234 is unable to nodulate are the temperate,
herbaceous genera Cicereae, Trifolieae, and Vicieae
(Figure 1). This suggests that in evolutionary terms, nodulation of this group seems to require a shift away from
307
Nod-factors with highly modified termini and simple fatty
acids to α-β-unsaturated fatty acids and unmodified termini
[23] (Figure 1). It should be noted, however, that although
nodFE mutants of R. meliloti secrete Nod-factors in which
the C16 unsaturated fatty acids are replaced by vaccenic
acid, the mutants still form nodules on various Medicago
cultivars [24]. Even though a few other correlations
between Nod-factor substituents and host-range have
been reported (Figure 2), other examples contradict the
dogma that Nod-factors determine host-specificity. For
instance, Nod-factors of heterologous rhizobia (i.e. isolated
from a different legume and unable to nodulate the plant
tested) also provoke Hac on root-hairs of Macroptilium
atropurpureum [25]. Despite the fact that the Nod-factors
produced by R. etli and M. loti are identical, the two species
have distinct host ranges (Phaseolus sp. and Lotus sp.
respectively) [26]. Similarly, the major Nod-factors secreted by R. leguminosarum bv. trifolii are identical to two of the
predominant Nod-factors produced by R. leguminosarum
bv. viciae [27,28], yet these two bacteria also have distinct
host ranges. In contrast, two rhizobia that nodulate the
same plant may secrete different Nod-factors: R. tropici
and R. etli produce sulphated and acetylfucosylated Nodfactors respectively, but both effectively nodulate Phaseolus
vulgaris [29,30]. All symbionts of Glycine max produce Nodfactors fucosylated by NodZ, yet null mutations in nodZ
have no discernible effect on nodulation of this and many
other hosts [31,32]. Thus, although Nod-factors are essential to nodulation and their modification may contribute to
host-specificity, this family of signal molecules is probably
just one of several elements specifying host range.
NodD proteins and plant exudates
Undoubtedly nodD and its alleles, which regulate the
transcription of nodulation genes (nod, nol, noe), modulate the first level of host specificity. NodD acts both as
a sensor of the plant signal and a transcriptional regulator
of the nod-genes. NodD proteins belong to the LysR
family of transcriptional activators, and bind to specific
sequences (nod-boxes) present in the promoter regions
of inducible nodulation genes. Mutations in nodD cannot
always be complemented by nodD from other species,
while point mutations can cause extension of the host
range. Several Lotus species are only nodulated when R.
etli is provided with an additional flavonoid-independent
nodD-gene [26]. Transfer of nodD1 of NGR234 into
R. meliloti results in host range extension to M. atropurpureum and Vigna unguiculata, whereas R. meliloti nodD1 is
incapable of restoring the ability of an NGR234 nodD1
mutant to nodulate M. atropurpureum [33]. Conjugation
of NGR234 nodD1 into R. leguminosarum bv. trifolii
extends its host-range to the non-legume Parasponia
andersonii (an NGR234 host), whereas a nodD1 mutant of
R. trifolii did not regain the ability to nodulate Trifolium
repens when it was complemented with nodD1 of
R. meliloti [34]. The capacities of different NodD proteins to interact with various phenolic components of
plant root exudates (particularly flavonoids) are thought
308
Biotic interactions
Figure 2
O-R3
NolO CH2
H2NCO NodU
CH3CO NodL
NodC
O
CH2-OH
CH3CONH
?
NodE , NodF and NodA
Acyl
CH3CONH
n
R1
R2
NodX
NodH
NodZ
NoeI
NoeE
OH 3- or 4-O-acetyl NolL
2-O-methyl
3-O-sulfate
R5-O
CH3CONH
Strains / Plasmids
O
O
HO
NodS CH3 R1- N
CH2
O
O
R6
Ac yl
CH2-OH
O
O
R2
CH3CO
SO3H
Fucose
O-R4
NoeC
R3
B. japonicum USDA110
R4
R5
R6
n
MeFuc
Macroptilium
M. loti E1R
C18:1, C18:0
Me
Cb
H
AcFuc
H
OH
2
R. etli CE3
C18:1, C18:0
Me
Cb, OH
H
AcFuc
H
OH
2
R. leguminosarum
bv. viciae 8401 (pRL1JI)
Ac
Lathyrus, Lens, Pisum
bv. trifolii LPR5045
(pRtRF101)
C18:3, C20:2
C20:3, C20:4
Trifolium
bv. viciae RBL5560
C18:4
Vicia
Fuc∗
Ac∗
A
Pisum-sym2
bv. viciae 248
R. meliloti RCR2011
Rhizobium sp. NGR234
C16:1, C16:2
C16:3
Ac
Medicago
Me
Leucaena
S
Medicago
SMeFuc
Pachyrhizus
Current Opinion in Plant Biology
Structures of Nod-factors and substituents which alter host-range.
Generalised Nod-factor structures are presented in the upper part of
the figure, and the table below shows those substituents (shaded)
which are necessary for nodulation of the legumes marked in colour.
Only those substituents whose presence is essential for nodulation of
the genera immediately below are listed. With the exception of the
Nod-factors of Mesorhizobium loti and R. etli, those substituents that
have not been shown to play a role in host-specificity are not listed.
Although M. loti and R. etli secrete identical Nod-factors (boxed), their
host-ranges are different. For example, M. loti strain E1R nodulates
Lotus corniculatus but not Phaseolus vulgaris, whereas R. etli strain
CE3 is Nod+ on P. vulgaris and Nod– on L. corniculatus (Cárdenas et
al. [26]). If either strain harbours a flavonoid independent nodD gene,
it can nodulate the heterologous host (see text). *The absence of the
acetyl group in Nod-factors produced by nodX– mutants can be
phenotypically complemented by the addition of a fucose group (nodZ
encodes a fucosyltransferase)(Ovtsyna et al. [48•]). Ac, acetyl;
Cb, carbamoyl; Fuc, fucosyl; H, hydrogen; Me, methyl; S, sulphate;
MeFuc, methylfucose; AcFuc, acetylated fucose;
SmeFuc, sulphated methylfucose.
to be responsible for the observed phenotypes. Thus
NodD of narrow host-range-rhizobia such as R. meliloti,
R. leguminosarum bv. viciae, and R. leguminosarum bv. trifolii responds to few flavonoids, whereas NodD1 of the
broad host-range NGR234 reacts with a large spectrum
of inducing compounds. These include simple phenolic
substances such as vanillin and iso-vanillin but also compounds that have been identified as inhibitors in other
rhizobia. Yet again, these correlations alone are insufficient to explain host-specificity.
Genealogy of legume–Rhizobium symbioses Broughton and Perret
Levels and mixtures of Nod-factors
Perhaps the fact that flavonoid–NodD complexes activate
nod-gene expression and trigger a chain of events which lead
to the secretion of Nod-factors is not enough to account for
nodulation of some legumes. Possibly the composition of
the Nod-factor mixtures and the levels of Nod-factors in the
rhizosphere are also important. USDA257 not only produces
a less complicated subset of the NGR234 factors, but it
secretes only one-fortieth as many [33]. Amongst the structural differences between the two sets of factors is the
absence of an N-methyl group on the non-reducing terminus of NodRf factors [9•]. Mutation of nodS, the gene that
encodes the N-methyltranferase deprives NGR234 of the
ability to nodulate tropical trees of the genus Leucaena.
Conjugation of the nod-box::nodS of NGR234 into
USDA257 leads to a twenty-two fold increase in the amount
of NodRf factors secreted by USDA257. Due to the Nmethyltransferase encoded by nodS [35], these factors are
also N-methylated, and the transconjugant gains the ability
to nodulate Leuceana spp. It is not possible to say, however,
whether this host-range extension is due to the presence of
the N-methyl group or to the elevated Nod-factor levels,
although the latter seems more likely given the fact that
some micro-symbionts of L. leucocephala do not possess nodS
homologues [36].
Co-operation amongst Nod-factors and related signal molecules has also been demonstrated. Minami et al. [37,38]
examined the effects of LCOs on changes in the expression
of early nodulin genes. Many plant genes are up-regulated
during nodule development (nodulins). Amongst them are
early nodulin genes, which are activated soon after rhizobia
enter the plant. One such gene, SrEnod2, encodes a
hydroxyproline-rich cell-wall protein, that is expressed in
the parenchyma cells of Sesbania rostrata nodules [39•].
Using expression of GmEnod2 in G. soya roots as a marker of
nodule initiation, Minami et al. showed that any combination of one active Nod-factor with a non-specific LCO was
sufficient to induce GmEnod2 mRNA expression. In contrast, both synthetic Nod-factors and the chitin pentamer
PACT, which is unable to provoke Had on G. soja, induced
the transient accumulation of GmEnod40 mRNA. It would
thus seem that not only are absolute levels of Nod-factors
important for the induction of different components of the
nodulation pathway, but also the composition and relative
proportions of the mixtures excreted by rhizobia.
Other keys to the legume doors
It seems fairly clear from the above that combinations of
flavonoids/NodD proteins/Nod-factors are insufficient to
explain host-specificity. Additional factors must, therefore,
help control symbiotic development. As Nod-factors permit
rhizobia to enter their hosts [20,21•], these additional factors
probably act within the plant (e.g. inside infection threads).
Early work showed that the relationship between the numbers of infection-threads initiated and nodules which
develop from them varies from 1.5% to almost 100%, suggesting that abortion of inappropriate infections helps control
309
nodulation [18]. But, what triggers abortion? Mounting evidence suggests that there is a fine line between symbiosis
and pathogenicity, as successful micro-symbionts must also
either evade or neutralise the plant defence systems. Specific
constituents of the rhizobial cell wall are required for infection-thread initiation and elongation [40•]. These
constituents in R. meliloti include polymers of succinoglycan
(EPS I) or galactoglucan (EPS II), and capsular polysaccharides containing 3-deoxy-D-manno-2-octulosonic acid, (see
[18]). Infection threads are composed of plant material,
although rhizobia may contribute to the matrix material
found inside the threads [41]. Nevertheless null mutations in
several genes involved in synthesis of EPS have no effect on
nodulation and nitrogen fixation in certain plant/bacteria
combinations, suggesting that other elements may substitute
for these deficiencies [18]. One possibility is that proteins
exported by the type three secretion system (TTSS) may be
synergistic with or complementary to extra-cellular polysaccharides in certain symbioses [42•]. In any event, the
principal remains the same — cell wall components and possibly secreted proteins of rhizobia are required for infection
thread formation and nodule development. Whether these
carbohydrates/proteins play regulatory or structural roles
remains to be determined, but it is interesting to note that
the rhizobia which produce Nod-factors containing α-βpolyunsaturated fatty acids apparently required for
nodulation of temperate herbaceous legumes, also seem to
lack the TTSS pathway [42•]. Type I dependent secreted
proteins may also contribute to host-specificity. NodO of
R. leguminasarum bv. viciae allows a nodE mutant of R. leguminosarum bv. trifolii to nodulate the non-host V. hirsuta [43],
whereas nodO of Rhizobium sp. BR816 functions as a nodulation determinant for L. leucocephala, P. vulgaris and T. repens in
diverse rhizobia [44•]. Thus, the existence of two types of
secretion systems, as well as the growing list of known symbiotically active substances secreted by rhizobia, suggests
that the molecular dialogue continues throughout nodule
development. Furthermore, the very heterogeneity of
secreted substances (acylated and non-acylated oligo- and
poly-saccharides, proteins, hormones etc) seems to preclude
unique roles for the individual compounds in host-specificity. Rather, we propose that some of these substances
complement an important symbiotic function in certain
plants, that in other hosts they could trigger defence responses, whereas in a third group they are unnecessary because
they are either not sensed by the plant or because functional equivalents already exist in the host. In such a scenario, the
secreted substances could play both structural (e.g. in infection-thread development) and signaling roles.
Conclusions
Symbiotic nitrogen fixing organisms seem to evolve from
non-symbiotic ancestors by acquiring nodulation (nod) and
nitrogen-fixation (nif, fix) genes. As a result, the chromosomal
backgrounds of rhizobia vary considerably, although some
basic ‘commonalities’ which permit co-ordinate expression
of nod, fix and nif loci as well as differentiation into bacteroids
exist. Recurring formation of ‘symbiosis islands’ in M. loti
310
Biotic interactions
confirms that horizontal transfer of symbiotic information frequently occurs in the soil. In this respect, interesting parallels
can be drawn to pathogenicity islands (Pais) of virulent bacteria (see [45]). Both carry either many virulence or
nod–fix–nif genes, are relatively large (up to several 100 kb),
present indications of lateral transfer (G and C content and
codon-usage differ significantly from the rest of the genome),
and are prone to genomic rearrangements. Thus, lateral
transfer of symbiotic (or pathogenic) functions followed by
adaptation to the new ecological niche probably drives evolution of many plant-interacting bacteria. If this is true, strict
correlations between rhizobial phylogeny, Nod-factors and
host-specificity will remain illusory.
Although it is possible that some major modulator of the
interaction between rhizobia and its hosts has been overlooked, it seems more likely that host-specificity is
controlled at various levels. The importance of each individual step would depend on the specific legume–Rhizobium
combination and would include levels of Nod-factors in the
rhizosphere (and, therefore, of their stability), composition
of the families of Nod-factors excreted, as well as post-entry
abortion of infection threads triggered by inappropriate
extra-cellular polysaccharides or proteins. In any event, a
sequence of reactions of variable specificity begins with different spectra of phenolic compounds in the rhizosphere of
the various hosts. Some of these may or may not interact
with NodD protein(s), that in turn bind to quite variable
nod-boxes [46,47•], so activating the transcription of nodgenes which produce varying amounts of different
Nod-factor families that may or may not be stable, but
which help control rhizobial entry into root-hairs. There,
they may provoke abortion, depending on the nature of the
extra-cellular carbohydrates or proteins. This sequence of
events provides ample flexibility to explain all the nodulation patterns observed.
Note added in proof
Two relevant papers on lateral transfer have recently been
published [49•,50•].
Acknowledgements
We would like to thank J Dénarié, JA Downie, M Holsters, E MartínezRomero, R Palacious, CW Ronson, HP Spaink, and G Stacey for helpful
comments and ideas during preparation of the manuscript. Dora Gerber
gave general support and financial assistance was provided by the Swiss
National Fond for Scientific Research (Grant # 31-45921.95).
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been annotated.
Forstwirthschaft am Kaiserlichen Gesundheitsamte, Berlin 1900,
1:177-222. [Title translation: The causes which determine the size,
number, position and effect of root nodules on legumes.]
4.
Lerouge P, Roche P, Faucher C, Maillet F, Truchet G, Promé J-C,
Dénarié J: Symbiotic host-specificity of Rhizobium meliloti is
determined by a sulphated and acylated glucosamine
oligosaccharide signal. Nature 1990, 344:781-784.
5.
Spaink HP, Sheeley DM, van Brussel AAN, Glushka J, York WS, Tak T,
Geiger O, Kennedy EP, Reinhold VN, Lugtenberg BJJ: A novel highly
unsaturated fatty acid moiety of lipo-oligosaccharide signals
determines host-specificity of Rhizobium. Nature 1991, 354:125-130.
6.
Polhill RM: Classification of the Leguminosae. In Phytochemical
Dictionary of the Leguminosae, vol. 1, Plants and their Constituents.
Edited by Bisby FA, Buckingham J, and Harborne JB. London:
Chapman and Hall; 1994: xvi-xxxvii.
7.
Hiltner L, Störmer K: Neue Untersuchungen über die
Wurzelknöllchen der Leguminosen und deren Erreger. Arbeiten
aus der Biologischen Abteilung für Land- und Forstwirthschaft,
am Kaiserlichen Gesundheitsamte (Berlin) 1903, 3:151-307. [Title
translation: New investigations into the root nodules of the
Leguminosae and their causitive agents.]
8.
Wilson JK: Leguminous plants and their associated organisms.
Cornell Univ Agric Exp Station, Memoir 221. Cornell University
Press; 1939.
Pueppke SG, Broughton WJ: Rhizobium sp. strain NGR234 and
R. fredii USDA257 share exceptionally broad, nested host-ranges.
Mol Plant–Microbe Interact 1999, 12:293-318.
Nodulation capacities of two promiscuous rhizobia were tested on 452
species of legumes. Correlations between Nod-factor structure and the ability to nodulate, the geographical origins of the legumes, their growth habit,
etc were not found.
9.
•
10. Lewin A, Rosenberg C, Meyer zA H, Wong CH, Nelson L, Manen J-F,
Stanley J, Dowling DN, Dénarié J, Broughton WJ: Multiple hostspecificity loci of the broad host range Rhizobium sp. NGR234
selected using the widely compatible legume Vigna unguiculata.
Plant Mol Biol 1987, 8:447-459.
11. Martínéz-Romero E, Caballero-Mellado J: Rhizobium phylogenies and
bacterial genetic diversity. Critical Rev Plant Sci 1996, 15:113-140.
12. Sullivan JT, Patrick HN, Lowther WL, Scott DB, Ronson CW:
Nodulating strains of Rhizobium loti arise through chromosomal
symbiotic gene transfer in the environment. Proc Natl Acad Sci
USA 1995, 92:8985-8989.
13. Sullivan JT, Ronson CW: Evolution of rhizobia by acquisition of a
•
500-kb symbiosis island that integrates into a phe-tRNA gene.
Proc Natl Acad Sci USA 1998, 95:5145-5149.
Convincing evidence that a mobile 500 kb ‘symbiosis island’ is transferred
to associated rhizospheric saprophytes rendering them symbiotic.
14. Freiberg C, Fellay R, Bairoch A, Broughton WJ, Rosenthal A, Perret X:
Molecular basis of symbiosis between Rhizobium and legumes.
Nature 1997, 387:394-401.
15. Broughton WJ, Samrey U, Stanley J: Ecological genetics of
Rhizobium meliloti: symbiotic plasmid transfer in the Medicago
sativa rhizosphere. FEMS Microbiol Lett 1987, 40:251-255.
16. Schofield PR, Gibson, AH, Dudman WF, Watson JM: Evidence for
genetic exchange and recombination of Rhizobium symbiotic
plasmids in a soil population. Appl Environ Microbiol. 1987,
53:2942-2947.
17.
Mavingui P, Flores M, Romero D, Martínez-Romero E, Palacios R:
Generation of Rhizobium strains with improved symbiotic
properties by random DNA amplification (RDA). Nat Biotech 1997,
15:564-569.
18. Perret X, Staehelin C, Broughton WJ: Molecular basis of symbiotic
promiscuity. Microbiol Mol Biol Rev 1999, in press.
1.
Beyerinck MW: Künstliche Infection von Vicia Faba mit Bacillus
radicicola. Ernährungsbedingungen dieser Bacterie. Botanische
Zeitung 1890, 52:837-843. [Title translation: Artificial infection of Vicia
Faba with Bacillus radicicola. Culture conditions of these bacteria.]
19. Truchet G, Roche P, Lerouge P, Vasse J, Camut S, de Billy F,
Promé J-C, Dénarié J: Sulphated lipo-oligosaccharide signals of
Rhizobium meliloti elicit root nodule organogenesis in alfalfa.
Nature 1991, 351:670-673.
2.
Prazmowski A: Die Wurzelknöllchen der Erbse. Landwirtschaftlichen
Versuchs-Stationen, Berlin 1890, 37:161-238. [Title translation: Root
nodules of peas.]
20. Relić B, Perret X, Estrada-Garcia MT, Kopcinska J, Golinowski W,
Krishnan HB, Pueppke SG, Broughton WJ: Nod-factors of Rhizobium
are a key to the legume door. Mol Microbiol 1994, 13:171-178.
3.
Hiltner L: Über die Ursachen, welche die Grösse, Zahl, Stellung
und Wirkung der Wurzelknöllchen der Leguminosen bedingen.
Arbeiten aus der Biologischen Abteilung für Land- und
21. D’Haeze W, Gao M-S, De Rycke R, Van Montagu M, Engler G,
•
Holsters M: Roles for azorhizobial Nod factors and surface
polysaccharides in intercellular invasion and nodule penetration,
Genealogy of legume–Rhizobium symbioses Broughton and Perret
respectively. Mol Plant–Microbe Interact 1998, 11:999-1008.
Functional complementation of an Azorhizobium nodA-mutant by NodAc-factors
confirmed that LCOs are necessary for intercellular infection in stem-nodulation.
22. Roche P, Debellé F, Maillet F, Lerouge P, Faucher C, Truchet G,
Dénarié J, Promé J-C: Molecular basis of symbiotic host specificity in
Rhizobium meliloti: nodH and nodPQ genes encode the sulfation of
lipochito-oligosaccharide signals. Cell 1991, 67:1131-1143.
23. Yang GP, Debellé F, Ferro M, Maillet F, Schiltz O, Vialas C, Savagnac A,
Promé J-C, Dénarié J: Rhizobium Nod factor structure and the
phylogeny of temperate legumes. In Biological Nitrogen Fixation for
the 21st Century. Edited by Elmerich C, Kondorosi A and Newton WE.
Kluwer Academic Publishers, Dordrecht; 1998:185-188.
24. Ardourel M, Demont N, Debellé F, Maillet F, de Billy F, Promé J-C,
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.
25. Relić B, Talmont F, Kopcinska J, Golinowski W, Promé J-C,
Broughton WJ: Biological activity of Rhizobium sp. NGR234 Nodfactors on Macroptilium atropurpureum. Mol Plant–Microbe Interact
1993, 6:764-774.
26. Cárdenas L, Dominguez J, Quinto C, Lopez-lara IM, Lugtenberg BJJ,
Spánk HP, Rademaker GJ, Haverkamp J, Thomas-Oates JE: Isolation,
chemical structure and biological activity of the lipo-chitin
oligosaccharide nodulation signals from Rhizobium etli. Plant Mol
Biol 1995, 29:453-464.
27.
Spaink HP, Bloemberg GV, van Brussel AAN, Lugtenberg BJJ, van der
Drift KMGM, Haverkamp J, Thomas-Oates JE: Host specificity of
Rhizobium leguminosarum is determined by the hydrophobicity of
highly unsaturated fatty acyl moieties of the nodulation factors.
Mol Plant–Microbe Interact 1995, 8:155-164.
28. Orgambide GG, Lee JI, Hollingsworth RI, Dazzo FB: Structurally
diverse chitolipooligosaccharide Nod factors accumulate
primarily in membranes of wild-type Rhizobium leguminosarum
biovar trifolii. Biochemistry 1995, 34:3832-3840.
29. Poupot R, Martínez-Romero E, Promé J-C: Nodulation factors from
Rhizobium tropici are sulfated or non-sulfated
chitopentasaccharides containing an N-methyl-N-acylglucosamine
terminus. Biochemistry 1993, 32:10430-10435.
30. Poupot R, Martínez-Romero E, Gautier N, Promé J-C: Wild-type
Rhizobium etli, a bean symbiont, produces acetyl-fucosylated, Nmethylated, and carbamoylated nodulation factors. J Biol Chem
1995, 270:6050-6055.
31. Stacey G, Luka S, Sanjuan J, Banfalvi Z, Nieuwkoop AJ, Chun JY,
Forsherg LS, Carlson R: nodZ a unique host-specific nodulation
gene, is involved in the fucosylation of the lipooligosaccharide
nodulation signal from Bradyrhizobium japonicum. J Bacteriol
1994, 176:620-633.
32. Quesada-Vincens D, Fellay R, Nasim T, Viprey V, Burger U, Promé J-C,
Broughton WJ, Jabbouri S: Rhizobium sp. NGR234 NodZ protein is
a fucosyltransferase. J Bacteriol 1997, 179:5087-5093.
33. Relić B, Staehelin C, Fellay R, Jabbouri S, Boller T, Broughton WJ: Do
Nod-factor levels play a role in host-specificity? In Proceedings of
the First European Nitrogen Fixation Conference. Edited by Kiss GB
and Endre G. Officina Press: Szeged, Hungary; 1994:69-75.
34. Spaink HP, Wijffelman CA, Pees E, Okker RJH, Lugtenberg BJJ:
Rhizobium nodulation gene nodD as a determinant of host
specificity. Nature 1987, 328:337-340.
35. Jabbouri S, Fellay R, Talmont F, Kamalaprija P, Burger U, Relić B,
Promé J-C, Broughton WJ: Involvement of nodS in N-methylation
and nodU in 6-O-carbamoylation of Rhizobium sp. NGR234 Nod
factors. J Biol Chem 1995, 270:22968-22973.
36. Krishnan HB, Lewin A, Fellay R, Broughton WJ, Pueppke SG:
Differential expression of nodS accounts for the varied abilities of
Rhizobium fredii USDA 257 and Rhizobium sp. NGR234 to
nodulate Leucaena spp. Mol Microbiol 1992, 6:3321-3330.
37.
Minami E, Kouchi H, Carlson RW, Cohn JR, Kolli VK, Day RB,
Ogawa T, Stacey G: Cooperative action of lipo-chitin nodulation
signals on the induction of the early nodulin, ENOD2 in soybean
roots. Mol Plant–Microbe Interact 1996, 9:574-583.
38. Minami E, Kouchi H, Cohn JR, Ogawa T, Stacey G: Expression of the
early nodulin, ENOD40, in soybean roots in response to various
lipo-chitin signal molecules. Plant J 1996, 10:23-32.
311
39. Chen R, Silver DL, de Brujn F: Nodule parenchyma-specific
•
expression of the Sesbamia rostrata early nodulin gene SrEnod2 is
mediated by the 3¢ untranslated region. Plant cell 1998, 10:1585-1602.
The 3′ untranslated region of SrEnod is required for its expression in the
parenchyma cell layer of the nodules. Expression of SrEnod2 is inducible by
cytokinins, adding plant hormones to the list of substances known to modulate expression of this gene (see [38]). Thus Nod-factors and cytokinins jointly modulate the activity of some of the same components of the signal
response pathway.
40. Cheng H-P, Walker GC: Succinoglycan is required for initiation and
•
elongation of infection threads during nodulation of alfalfa by
Rhizobium meliloti. J Bacteriol 1998, 180:5183-5191.
Shows clearly that individual exo-genes, which are involved in the synthesis
of succinoglycan, affect infection-thread development.
41. Kijne JW: The Rhizobium infection process. In Biological Nitrogen
Fixation. Edited by Stacey G, Burris RH, and Evans HJ: Chapman and
Hall; New York; 1992:349-398.
42. Viprey V, Del Greco A, Golinowski W, Broughton WJ, Perret X:
•
Symbiotic implications of type III protein secretion machinery in
Rhizobium. Mol Microbiol 1998, 28:1381-1389.
Sequencing of pNGR234a revealed a gene cluster which encodes a
flavonoid inducible type III protein secretion system. Builds on data which
showed that protein secretion plays a role in host-specificity.
43. Economau A, Davies AE, Johnston AWB, Downie JA: The Rhizobium
leguminosarum biovar viciae nod0 gene can enable a nodE
mutant of Rhizobium leguminosarum biovar trifolií to nodulate
vetch. Microbiology 1994, 140:2341-2347.
44. 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 leucocephaia,
Phaseolus vulgaris and Trifolium repens by a diversity of
Rhizobium spp. Mol Plant–Microbe Interact 1998, 11:383-392.
Shows that nodO is not confined to strains that nodulate Cicereae,
Trifolieae, and Vicerea, but is also present in rhizobia that associate with
tropical legumes. Functionally complements mutants in nodS of NGR234
for nodulation of L. leucoceplaia.
45. Hacker J, Blum Oehier G, Mühldorfer I, Tschäpe H: Pathogenicity
islands of virulent bacteria: structure, function and impact on
microbial evolution. Mol Microb 1997, 23:1089-1097.
46. van Rhijn P, Vanderleyden J: The Rhizobium-plant symbiosis.
Microbiol Rev 1995, 59:124-142.
Perret X, Freiberg C, Rosenthal A, Broughton WJ, Fellay R: Highresolution transcriptional analysis of the symbiotic replicon of
Rhizobium sp. NGR234. Mol Microbiol 1999, 32:415-425.
pNGR234a was divided into 441 segments representing all coding and intergenic regions. Each segment was amplified by PCR, transferred to filters and
probed with radioactively labelled-RNA. Genes involved in the synthesis of
Nod-factors were induced rapidly after the addition of flavonoids, while others
thought to act within the plant (e.g. those encoding the type three secretion
machine) responded more slowly. Many more transcripts were found in bacteroids of determinate- as opposed to indeterminate-nodules.
47.
•
48. Ovtsyna AO, Geurts R, Bisseling T, Lugtenberg BJJ, Tikhonovich IA,
•
Spaink H: Restriction of host range by the sym2 allele of Afghan
pea is nonspecific for the type of modification at the reducing
terminus of nodulation signals. Mol Plant–Microbe Interact 1998,
11:418-422.
A clear demonstration that the exact nature of Nod-factor substitutents is less
important than their position. Nodulation of the primitive P. sativum cultivar
Afghanistan was thought to require a 6-O-acetate group on the reducing terminus (added by NodX), but it can be functionally replaced by a 6-O-fucose
group (nodZ encodes a fucosyltransferase).
49. Karaolis DKR, Somara S, Maneval DR, Johnson JA, Kaper JB: A
•
bacteriophage encoding a pathogenicity island, a type-IV pilus and
a phage receptor in cholera bacteria. Nature 1999, 399:375-379.
Many bacteria carry pathogenicity islands — DNA sequences that encode virulence related proteins. These include specialised molecular machines to injectbacterial products into their animal or plant hosts, molecules involved in signal
transduction and toxins. Here the authors demonstrate an interplay between
two pathogenicity islands of the bacterium Vibrio cholerae and its bacteriophage CTXψ, which carries the cholera toxin genes.
50. Nelson KE, Clayton RA, Gill SR, Gwinn ML, Dodson RJ, Haft DH,
•
Hickley EK, Peterson JD, Nelson WC, Ketchum KA et al.: Evidence for
lateral gene transfer between archea and bacteria from genome
sequence of Thermotoga maritima. Nature 1999, 399:323-329.
The thermophilic eubacterium T. maritima has a single chromosome (G+C content 46%). Of the 1,877 predicted coding regions, 24% are most similar to
those of archea. Evidence is presented suggesting that much of the similarity
between T. maritima and archea is the result of extensive lateral gene transfer.
Genealogy of legume-Rhizobium symbioses
William J Broughton* and Xavier Perret
Accumulating evidence suggests that lateral transfer of
nodulation capacity is an important driving force in symbiotic
evolution. As a consequence, many distantly related soil
bacteria have acquired the capacity to invade plants and fix
nitrogen within them. In addition to these proteins required
for bacteroid development and nitrogen fixation, core
symbiotic competence seems to require flavonoids, NodD
proteins, lipochitooligosaccharidic Nod-factors, extra-cellular
polysaccharides, as well as various exported proteins. Plants
respond to different levels and combinations of these
substances in species specific ways. After contact has been
initiated by flavonoids and NodD proteins, constant signal
exchange fine-tunes these symbiotic demands, especially to
overcome defence reactions.
Addresses
Laboratoire de Biologie Moléculaire des Plantes Supérieures
(LBMPS), Université de Genève, 1 ch. de l’Impératrice, 1292
Chambésy/Genève, Switzerland
*e-mail: [email protected]
Current Opinion in Plant Biology 1999, 2:305–311
http://biomednet.com/elecref/1369526600200305
© Elsevier Science Ltd ISSN 1369-5266
Abbreviations
Hac
root-hair curling
Had
root-hair deformation
Hai
root-hair formation
Introduction
In the latter part of the nineteenth century, inquiry into
the nature of plant–microbe interactions turned from the
observational to the experimental. Beyerinck proved
that bacteria elicit nodules on the roots of legumes by
preparing pure cultures from nodules of Vicia faba and
using them to infect Faba beans growing in sterile soil
[1]. Prazmowski [2] inoculated Pisum sativum with axenic
cultures and observed that micro-organisms entered into
the plant via infection threads in root-hairs. Hiltner [3]
prepared aqueous, bacteria-free filtrates from mature
P. sativum nodules and demonstrated that they contain a
substance that induces root-hair formation (Hai) and
deformation of the root-hairs (Had). Ninety years later
Lerouge et al. [4] showed that the root-hair deforming
substances of R. meliloti are N-acylated oligomers of
N-acetyl-D-glucosamine (known as Nod-factors) and
implied that variations in Nod-factor structure are derminants of host specificity. In turn, Spaink et al. [5]
demonstrated that the principal differences between the
Nod-factors of R. leguminosarum biovar viciae and those of
R. meliloti lie in the degree of unsaturation of the acyl
chains, as well as the presence or absence of a sulphate
group on the reducing-terminal glucosamine.
Common threads link these milestones in symbiotic nitrogen fixation research — all were performed by European
scientists on local legumes and their associated rhizobia.
Unfortunately, the plants they used (Medicago, Pisum, and
Vicia) are very closely related and belong to only two
(Vicieae and Trifolieae) of the thirty-nine recognised tribes
(three of the 666 genera) of the Leguminosae (see [6] and
Figure 1). Furthermore, their respective symbionts,
R. meliloti, R. leguminosarum bv. viciae and R. leguminosarum
bv. trifolii, associate with only few plants (i.e. have a narrow
host-range). It is thus possible that nitrogen-fixing symbioses in temperate, herbaceous legumes are not
representative of Rhizobium–legume associations as a
whole. To seek a more general understanding of the molecular bases of symbioses, we have examined the
relationship between promiscuous (i.e. able to nodulate
many different legumes) rhizobia and their hosts. This
raises the question of rhizobial taxonomy. Below, we will
argue that rhizobia are heterogenous because, like other
Gram-negative bacteria, they have acquired large blocks of
genetic information by lateral-transfer. In some cases these
modules represent pathogenicity islands, whereas in others
they carry symbiotic genes (see note added in proof). Once
acquired these ‘symbiotic islands’ have been subject to
constant re-arrangements, amplifications and deletions.
Rhizobia: phylogenetically distant bacteria
containing related symbiotic genes
Hiltner and Störmer [7] were probably the first to attempt
classification of rhizobia on the basis of their plant origins.
Host-based classifications would work if rhizobial hostranges mirrored legume taxonomy, but some rhizobia form
symbioses across taxonomic divisions. Wilson [8], for example, tested the host-ranges of rhizobia isolated from 31
different genera of legumes on 160 different legume
species. The average number of plant species nodulated by
a particular isolate was 33%, and four of the isolates formed
nodules on more than half the legumes tested. Likewise,
Rhizobium sp. NGR234 nodulates at least 112 genera of
legumes, whereas the closely related R. fredii USDA257
nodulates an exact subset (77 genera) of the NGR234 hosts
[9•]. Widespread existence of such broad host-range rhizobia, as well as promiscuous hosts such as Vigna [10] renders
any host-based classification scheme inadequate.
Recent taxonomic proposals based on DNA sequences of
highly conserved genes suggest that symbiotic rhizobia are
much more diverse than was previously thought [11], and the
reasons for this diversity are becoming clearer. Probably,
what permits many different Gram-negative bacteria to be
symbiotic is a group of less than 400 genes, and some basic
‘compatibility’ of their chromosomal backgrounds. This is
best seen in the ‘symbiosis islands’ of bacteria associated
with Mesorhizobium loti. Genetically diverse ‘mesorhizobia’
306
Biotic interactions
Figure 1
Loteae
P.13
Trifolieae
P.21
Psoraleeae
P.12
Desmodieae
P.11
Indigofereae
P.9
Phaseoleae
P.10
Amorpheae
P.6
o
Cicereae
P.20
o
o
o
o
O=C
Sesbanieae
P.8
O=C
Galegeae
P.16
Vicieae
P.19
Robinieae
P.8
Hedysareae
P.18
Aeschynomeneae
P.14
Millettieae
P.7
Adesmieae
P.15
Abreae
P.5
Crotalarieae
P.27
Brongniartieae
P.22
Bossiaeeae
P.23
Mirbelieae
P.24
Podalyrieae
P.25
Genisteae
P.30
Dalbergieae
P.4
Thermopsideae
P.29
Sophoreae
P.2
Acacieae M.4
& Ingeae M.5
Cassiinae
C2.D
Mimoseae
M.3
H3COO
HO
H2NCOO
CH2-OH
O
H3C N
CH3
O
HO3S-O
O-CONH2
CH2
Swartzieae
P.1
O
HO
CH3-O
O
CH 2
O
O
HO
CH3CONH
3
O=C
(C16:0 or C16:1or C14:1
)
CH3
O
OH
CH3CONH
Caesalpinioideae
Current Opinion in Plant Biology
Relationships amongst tribes of the Leguminosae, and the ability of
Rhizobium sp. NGR234 and R. fredii USDA257 to nodulate them. All
substituents found in the complete family of NodNGR factors are shown
near the bottom of the figure, and those structural requirements which
NGR234/USDA257 lack, but which seem to be necessary for nodulation
of the Cicereae, Trifolieae, and Vicieae (α-β-unsaturated fatty acids) are
shown in the top, left-hand portion. Tribes marked in green are nodulated
by both NGR234 and USDA257, whereas those marked in red are only
nodulated by NGR234. Tribes that are Nod– with both bacteria, are boxed
in blue. This figure was re-drawn from Pueppke and Broughton [9•].
Genealogy of legume–Rhizobium symbioses Broughton and Perret
were isolated from nodules of Lotus corniculatus in an area
devoid of indigenous rhizobia, but which had been inoculated with a single M. loti isolate [12]. All contained an identical
500 kb ‘symbiotic island’ integrated into the chromosomal
phenyl-alanine transfer RNA gene (phe-tRNA gene) [13•].
At the present moment, the exact nature of the soil bacteria
(‘the mezorhizobial recipients’) which have aquired these
‘symbiosis islands’ is not clear. Nevertheless, their aquisition
converts formerly saprophytic bacteria into symbionts. At
536 kb, pNGR234a, the symbiotic plasmid of the broad
host-range Rhizobium sp. NGR234, is about the same size as
the ‘symbiosis island’, and it confers the ability to nodulate
on Agrobacterium tumefaciens. Sequencing of this plasmid
highlighted that pNGR234a is particularly interesting, not
only because it is a repository of symbiotic promiscuity, but
also because of its mosaic genetic structure with origins of
replication and transfer very similar to those of Agrobacterium
species [14]. These features not only suggest a common origin for symbiotic replicons and tumour-inducing-plasmids,
but also exemplify the combinatorial possibilities offered by
lateral transfer of genetic information.
Genetic exchange also occurs between symbionts in the
soil [15,16]. Once acquired, amplification of the newly
inherited genetic information may generate pools of related
strains, some of which are better adapted to particular conditions than others [17]. Abundant and highly conserved
repeats found throughout rhizobial genomes are the sites of
amplification. When formed these ‘amplicons’ can be
translocated to other plasmids or the chromosome, where
they are more stably maintained. Thus, in populations of
soil bacteria, natural genetic mechanisms exist which are
capable of transforming isolates with widely different chromosomal backgrounds into pools of nodulating bacteria.
Nod-factors and host-specificity
What are the bacterial determinants that specify hostrange? It has been clear for almost 100 years that rhizobial
secretions provoke Had. Some purified Nod-factors provoke the extreme curling of the root-hairs (shepherd’s
crook formation or Hac), which is the most specific of the
morphological responses induced by rhizobia (see [18]).
They also induce cortical cell division [19], and permit rhizobia to penetrate host-tissues [20,21•]. Since rhizobia that
are unable to synthesise Nod-factors cannot nodulate
(i.e. Nod–), these signal molecules are obviously essential
to the symbiosis (see also A Hirsch, this issue pp 320–326).
It is thus possible that structural variations in Nod-factors
are the determinants of host-specificity. Indeed, mutation
of nodH, which encodes a sulphotransferase, leads to the
absence of the sulphate group from R. meliloti LCOs, and
loss of nodulation of its homologous host M. sativa. Yet,
these NodH– mutants are now able to nodulate the nonhost V. sativa [22]. Interestingly, almost the only legumes
that NGR234 is unable to nodulate are the temperate,
herbaceous genera Cicereae, Trifolieae, and Vicieae
(Figure 1). This suggests that in evolutionary terms, nodulation of this group seems to require a shift away from
307
Nod-factors with highly modified termini and simple fatty
acids to α-β-unsaturated fatty acids and unmodified termini
[23] (Figure 1). It should be noted, however, that although
nodFE mutants of R. meliloti secrete Nod-factors in which
the C16 unsaturated fatty acids are replaced by vaccenic
acid, the mutants still form nodules on various Medicago
cultivars [24]. Even though a few other correlations
between Nod-factor substituents and host-range have
been reported (Figure 2), other examples contradict the
dogma that Nod-factors determine host-specificity. For
instance, Nod-factors of heterologous rhizobia (i.e. isolated
from a different legume and unable to nodulate the plant
tested) also provoke Hac on root-hairs of Macroptilium
atropurpureum [25]. Despite the fact that the Nod-factors
produced by R. etli and M. loti are identical, the two species
have distinct host ranges (Phaseolus sp. and Lotus sp.
respectively) [26]. Similarly, the major Nod-factors secreted by R. leguminosarum bv. trifolii are identical to two of the
predominant Nod-factors produced by R. leguminosarum
bv. viciae [27,28], yet these two bacteria also have distinct
host ranges. In contrast, two rhizobia that nodulate the
same plant may secrete different Nod-factors: R. tropici
and R. etli produce sulphated and acetylfucosylated Nodfactors respectively, but both effectively nodulate Phaseolus
vulgaris [29,30]. All symbionts of Glycine max produce Nodfactors fucosylated by NodZ, yet null mutations in nodZ
have no discernible effect on nodulation of this and many
other hosts [31,32]. Thus, although Nod-factors are essential to nodulation and their modification may contribute to
host-specificity, this family of signal molecules is probably
just one of several elements specifying host range.
NodD proteins and plant exudates
Undoubtedly nodD and its alleles, which regulate the
transcription of nodulation genes (nod, nol, noe), modulate the first level of host specificity. NodD acts both as
a sensor of the plant signal and a transcriptional regulator
of the nod-genes. NodD proteins belong to the LysR
family of transcriptional activators, and bind to specific
sequences (nod-boxes) present in the promoter regions
of inducible nodulation genes. Mutations in nodD cannot
always be complemented by nodD from other species,
while point mutations can cause extension of the host
range. Several Lotus species are only nodulated when R.
etli is provided with an additional flavonoid-independent
nodD-gene [26]. Transfer of nodD1 of NGR234 into
R. meliloti results in host range extension to M. atropurpureum and Vigna unguiculata, whereas R. meliloti nodD1 is
incapable of restoring the ability of an NGR234 nodD1
mutant to nodulate M. atropurpureum [33]. Conjugation
of NGR234 nodD1 into R. leguminosarum bv. trifolii
extends its host-range to the non-legume Parasponia
andersonii (an NGR234 host), whereas a nodD1 mutant of
R. trifolii did not regain the ability to nodulate Trifolium
repens when it was complemented with nodD1 of
R. meliloti [34]. The capacities of different NodD proteins to interact with various phenolic components of
plant root exudates (particularly flavonoids) are thought
308
Biotic interactions
Figure 2
O-R3
NolO CH2
H2NCO NodU
CH3CO NodL
NodC
O
CH2-OH
CH3CONH
?
NodE , NodF and NodA
Acyl
CH3CONH
n
R1
R2
NodX
NodH
NodZ
NoeI
NoeE
OH 3- or 4-O-acetyl NolL
2-O-methyl
3-O-sulfate
R5-O
CH3CONH
Strains / Plasmids
O
O
HO
NodS CH3 R1- N
CH2
O
O
R6
Ac yl
CH2-OH
O
O
R2
CH3CO
SO3H
Fucose
O-R4
NoeC
R3
B. japonicum USDA110
R4
R5
R6
n
MeFuc
Macroptilium
M. loti E1R
C18:1, C18:0
Me
Cb
H
AcFuc
H
OH
2
R. etli CE3
C18:1, C18:0
Me
Cb, OH
H
AcFuc
H
OH
2
R. leguminosarum
bv. viciae 8401 (pRL1JI)
Ac
Lathyrus, Lens, Pisum
bv. trifolii LPR5045
(pRtRF101)
C18:3, C20:2
C20:3, C20:4
Trifolium
bv. viciae RBL5560
C18:4
Vicia
Fuc∗
Ac∗
A
Pisum-sym2
bv. viciae 248
R. meliloti RCR2011
Rhizobium sp. NGR234
C16:1, C16:2
C16:3
Ac
Medicago
Me
Leucaena
S
Medicago
SMeFuc
Pachyrhizus
Current Opinion in Plant Biology
Structures of Nod-factors and substituents which alter host-range.
Generalised Nod-factor structures are presented in the upper part of
the figure, and the table below shows those substituents (shaded)
which are necessary for nodulation of the legumes marked in colour.
Only those substituents whose presence is essential for nodulation of
the genera immediately below are listed. With the exception of the
Nod-factors of Mesorhizobium loti and R. etli, those substituents that
have not been shown to play a role in host-specificity are not listed.
Although M. loti and R. etli secrete identical Nod-factors (boxed), their
host-ranges are different. For example, M. loti strain E1R nodulates
Lotus corniculatus but not Phaseolus vulgaris, whereas R. etli strain
CE3 is Nod+ on P. vulgaris and Nod– on L. corniculatus (Cárdenas et
al. [26]). If either strain harbours a flavonoid independent nodD gene,
it can nodulate the heterologous host (see text). *The absence of the
acetyl group in Nod-factors produced by nodX– mutants can be
phenotypically complemented by the addition of a fucose group (nodZ
encodes a fucosyltransferase)(Ovtsyna et al. [48•]). Ac, acetyl;
Cb, carbamoyl; Fuc, fucosyl; H, hydrogen; Me, methyl; S, sulphate;
MeFuc, methylfucose; AcFuc, acetylated fucose;
SmeFuc, sulphated methylfucose.
to be responsible for the observed phenotypes. Thus
NodD of narrow host-range-rhizobia such as R. meliloti,
R. leguminosarum bv. viciae, and R. leguminosarum bv. trifolii responds to few flavonoids, whereas NodD1 of the
broad host-range NGR234 reacts with a large spectrum
of inducing compounds. These include simple phenolic
substances such as vanillin and iso-vanillin but also compounds that have been identified as inhibitors in other
rhizobia. Yet again, these correlations alone are insufficient to explain host-specificity.
Genealogy of legume–Rhizobium symbioses Broughton and Perret
Levels and mixtures of Nod-factors
Perhaps the fact that flavonoid–NodD complexes activate
nod-gene expression and trigger a chain of events which lead
to the secretion of Nod-factors is not enough to account for
nodulation of some legumes. Possibly the composition of
the Nod-factor mixtures and the levels of Nod-factors in the
rhizosphere are also important. USDA257 not only produces
a less complicated subset of the NGR234 factors, but it
secretes only one-fortieth as many [33]. Amongst the structural differences between the two sets of factors is the
absence of an N-methyl group on the non-reducing terminus of NodRf factors [9•]. Mutation of nodS, the gene that
encodes the N-methyltranferase deprives NGR234 of the
ability to nodulate tropical trees of the genus Leucaena.
Conjugation of the nod-box::nodS of NGR234 into
USDA257 leads to a twenty-two fold increase in the amount
of NodRf factors secreted by USDA257. Due to the Nmethyltransferase encoded by nodS [35], these factors are
also N-methylated, and the transconjugant gains the ability
to nodulate Leuceana spp. It is not possible to say, however,
whether this host-range extension is due to the presence of
the N-methyl group or to the elevated Nod-factor levels,
although the latter seems more likely given the fact that
some micro-symbionts of L. leucocephala do not possess nodS
homologues [36].
Co-operation amongst Nod-factors and related signal molecules has also been demonstrated. Minami et al. [37,38]
examined the effects of LCOs on changes in the expression
of early nodulin genes. Many plant genes are up-regulated
during nodule development (nodulins). Amongst them are
early nodulin genes, which are activated soon after rhizobia
enter the plant. One such gene, SrEnod2, encodes a
hydroxyproline-rich cell-wall protein, that is expressed in
the parenchyma cells of Sesbania rostrata nodules [39•].
Using expression of GmEnod2 in G. soya roots as a marker of
nodule initiation, Minami et al. showed that any combination of one active Nod-factor with a non-specific LCO was
sufficient to induce GmEnod2 mRNA expression. In contrast, both synthetic Nod-factors and the chitin pentamer
PACT, which is unable to provoke Had on G. soja, induced
the transient accumulation of GmEnod40 mRNA. It would
thus seem that not only are absolute levels of Nod-factors
important for the induction of different components of the
nodulation pathway, but also the composition and relative
proportions of the mixtures excreted by rhizobia.
Other keys to the legume doors
It seems fairly clear from the above that combinations of
flavonoids/NodD proteins/Nod-factors are insufficient to
explain host-specificity. Additional factors must, therefore,
help control symbiotic development. As Nod-factors permit
rhizobia to enter their hosts [20,21•], these additional factors
probably act within the plant (e.g. inside infection threads).
Early work showed that the relationship between the numbers of infection-threads initiated and nodules which
develop from them varies from 1.5% to almost 100%, suggesting that abortion of inappropriate infections helps control
309
nodulation [18]. But, what triggers abortion? Mounting evidence suggests that there is a fine line between symbiosis
and pathogenicity, as successful micro-symbionts must also
either evade or neutralise the plant defence systems. Specific
constituents of the rhizobial cell wall are required for infection-thread initiation and elongation [40•]. These
constituents in R. meliloti include polymers of succinoglycan
(EPS I) or galactoglucan (EPS II), and capsular polysaccharides containing 3-deoxy-D-manno-2-octulosonic acid, (see
[18]). Infection threads are composed of plant material,
although rhizobia may contribute to the matrix material
found inside the threads [41]. Nevertheless null mutations in
several genes involved in synthesis of EPS have no effect on
nodulation and nitrogen fixation in certain plant/bacteria
combinations, suggesting that other elements may substitute
for these deficiencies [18]. One possibility is that proteins
exported by the type three secretion system (TTSS) may be
synergistic with or complementary to extra-cellular polysaccharides in certain symbioses [42•]. In any event, the
principal remains the same — cell wall components and possibly secreted proteins of rhizobia are required for infection
thread formation and nodule development. Whether these
carbohydrates/proteins play regulatory or structural roles
remains to be determined, but it is interesting to note that
the rhizobia which produce Nod-factors containing α-βpolyunsaturated fatty acids apparently required for
nodulation of temperate herbaceous legumes, also seem to
lack the TTSS pathway [42•]. Type I dependent secreted
proteins may also contribute to host-specificity. NodO of
R. leguminasarum bv. viciae allows a nodE mutant of R. leguminosarum bv. trifolii to nodulate the non-host V. hirsuta [43],
whereas nodO of Rhizobium sp. BR816 functions as a nodulation determinant for L. leucocephala, P. vulgaris and T. repens in
diverse rhizobia [44•]. Thus, the existence of two types of
secretion systems, as well as the growing list of known symbiotically active substances secreted by rhizobia, suggests
that the molecular dialogue continues throughout nodule
development. Furthermore, the very heterogeneity of
secreted substances (acylated and non-acylated oligo- and
poly-saccharides, proteins, hormones etc) seems to preclude
unique roles for the individual compounds in host-specificity. Rather, we propose that some of these substances
complement an important symbiotic function in certain
plants, that in other hosts they could trigger defence responses, whereas in a third group they are unnecessary because
they are either not sensed by the plant or because functional equivalents already exist in the host. In such a scenario, the
secreted substances could play both structural (e.g. in infection-thread development) and signaling roles.
Conclusions
Symbiotic nitrogen fixing organisms seem to evolve from
non-symbiotic ancestors by acquiring nodulation (nod) and
nitrogen-fixation (nif, fix) genes. As a result, the chromosomal
backgrounds of rhizobia vary considerably, although some
basic ‘commonalities’ which permit co-ordinate expression
of nod, fix and nif loci as well as differentiation into bacteroids
exist. Recurring formation of ‘symbiosis islands’ in M. loti
310
Biotic interactions
confirms that horizontal transfer of symbiotic information frequently occurs in the soil. In this respect, interesting parallels
can be drawn to pathogenicity islands (Pais) of virulent bacteria (see [45]). Both carry either many virulence or
nod–fix–nif genes, are relatively large (up to several 100 kb),
present indications of lateral transfer (G and C content and
codon-usage differ significantly from the rest of the genome),
and are prone to genomic rearrangements. Thus, lateral
transfer of symbiotic (or pathogenic) functions followed by
adaptation to the new ecological niche probably drives evolution of many plant-interacting bacteria. If this is true, strict
correlations between rhizobial phylogeny, Nod-factors and
host-specificity will remain illusory.
Although it is possible that some major modulator of the
interaction between rhizobia and its hosts has been overlooked, it seems more likely that host-specificity is
controlled at various levels. The importance of each individual step would depend on the specific legume–Rhizobium
combination and would include levels of Nod-factors in the
rhizosphere (and, therefore, of their stability), composition
of the families of Nod-factors excreted, as well as post-entry
abortion of infection threads triggered by inappropriate
extra-cellular polysaccharides or proteins. In any event, a
sequence of reactions of variable specificity begins with different spectra of phenolic compounds in the rhizosphere of
the various hosts. Some of these may or may not interact
with NodD protein(s), that in turn bind to quite variable
nod-boxes [46,47•], so activating the transcription of nodgenes which produce varying amounts of different
Nod-factor families that may or may not be stable, but
which help control rhizobial entry into root-hairs. There,
they may provoke abortion, depending on the nature of the
extra-cellular carbohydrates or proteins. This sequence of
events provides ample flexibility to explain all the nodulation patterns observed.
Note added in proof
Two relevant papers on lateral transfer have recently been
published [49•,50•].
Acknowledgements
We would like to thank J Dénarié, JA Downie, M Holsters, E MartínezRomero, R Palacious, CW Ronson, HP Spaink, and G Stacey for helpful
comments and ideas during preparation of the manuscript. Dora Gerber
gave general support and financial assistance was provided by the Swiss
National Fond for Scientific Research (Grant # 31-45921.95).
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been annotated.
Forstwirthschaft am Kaiserlichen Gesundheitsamte, Berlin 1900,
1:177-222. [Title translation: The causes which determine the size,
number, position and effect of root nodules on legumes.]
4.
Lerouge P, Roche P, Faucher C, Maillet F, Truchet G, Promé J-C,
Dénarié J: Symbiotic host-specificity of Rhizobium meliloti is
determined by a sulphated and acylated glucosamine
oligosaccharide signal. Nature 1990, 344:781-784.
5.
Spaink HP, Sheeley DM, van Brussel AAN, Glushka J, York WS, Tak T,
Geiger O, Kennedy EP, Reinhold VN, Lugtenberg BJJ: A novel highly
unsaturated fatty acid moiety of lipo-oligosaccharide signals
determines host-specificity of Rhizobium. Nature 1991, 354:125-130.
6.
Polhill RM: Classification of the Leguminosae. In Phytochemical
Dictionary of the Leguminosae, vol. 1, Plants and their Constituents.
Edited by Bisby FA, Buckingham J, and Harborne JB. London:
Chapman and Hall; 1994: xvi-xxxvii.
7.
Hiltner L, Störmer K: Neue Untersuchungen über die
Wurzelknöllchen der Leguminosen und deren Erreger. Arbeiten
aus der Biologischen Abteilung für Land- und Forstwirthschaft,
am Kaiserlichen Gesundheitsamte (Berlin) 1903, 3:151-307. [Title
translation: New investigations into the root nodules of the
Leguminosae and their causitive agents.]
8.
Wilson JK: Leguminous plants and their associated organisms.
Cornell Univ Agric Exp Station, Memoir 221. Cornell University
Press; 1939.
Pueppke SG, Broughton WJ: Rhizobium sp. strain NGR234 and
R. fredii USDA257 share exceptionally broad, nested host-ranges.
Mol Plant–Microbe Interact 1999, 12:293-318.
Nodulation capacities of two promiscuous rhizobia were tested on 452
species of legumes. Correlations between Nod-factor structure and the ability to nodulate, the geographical origins of the legumes, their growth habit,
etc were not found.
9.
•
10. Lewin A, Rosenberg C, Meyer zA H, Wong CH, Nelson L, Manen J-F,
Stanley J, Dowling DN, Dénarié J, Broughton WJ: Multiple hostspecificity loci of the broad host range Rhizobium sp. NGR234
selected using the widely compatible legume Vigna unguiculata.
Plant Mol Biol 1987, 8:447-459.
11. Martínéz-Romero E, Caballero-Mellado J: Rhizobium phylogenies and
bacterial genetic diversity. Critical Rev Plant Sci 1996, 15:113-140.
12. Sullivan JT, Patrick HN, Lowther WL, Scott DB, Ronson CW:
Nodulating strains of Rhizobium loti arise through chromosomal
symbiotic gene transfer in the environment. Proc Natl Acad Sci
USA 1995, 92:8985-8989.
13. Sullivan JT, Ronson CW: Evolution of rhizobia by acquisition of a
•
500-kb symbiosis island that integrates into a phe-tRNA gene.
Proc Natl Acad Sci USA 1998, 95:5145-5149.
Convincing evidence that a mobile 500 kb ‘symbiosis island’ is transferred
to associated rhizospheric saprophytes rendering them symbiotic.
14. Freiberg C, Fellay R, Bairoch A, Broughton WJ, Rosenthal A, Perret X:
Molecular basis of symbiosis between Rhizobium and legumes.
Nature 1997, 387:394-401.
15. Broughton WJ, Samrey U, Stanley J: Ecological genetics of
Rhizobium meliloti: symbiotic plasmid transfer in the Medicago
sativa rhizosphere. FEMS Microbiol Lett 1987, 40:251-255.
16. Schofield PR, Gibson, AH, Dudman WF, Watson JM: Evidence for
genetic exchange and recombination of Rhizobium symbiotic
plasmids in a soil population. Appl Environ Microbiol. 1987,
53:2942-2947.
17.
Mavingui P, Flores M, Romero D, Martínez-Romero E, Palacios R:
Generation of Rhizobium strains with improved symbiotic
properties by random DNA amplification (RDA). Nat Biotech 1997,
15:564-569.
18. Perret X, Staehelin C, Broughton WJ: Molecular basis of symbiotic
promiscuity. Microbiol Mol Biol Rev 1999, in press.
1.
Beyerinck MW: Künstliche Infection von Vicia Faba mit Bacillus
radicicola. Ernährungsbedingungen dieser Bacterie. Botanische
Zeitung 1890, 52:837-843. [Title translation: Artificial infection of Vicia
Faba with Bacillus radicicola. Culture conditions of these bacteria.]
19. Truchet G, Roche P, Lerouge P, Vasse J, Camut S, de Billy F,
Promé J-C, Dénarié J: Sulphated lipo-oligosaccharide signals of
Rhizobium meliloti elicit root nodule organogenesis in alfalfa.
Nature 1991, 351:670-673.
2.
Prazmowski A: Die Wurzelknöllchen der Erbse. Landwirtschaftlichen
Versuchs-Stationen, Berlin 1890, 37:161-238. [Title translation: Root
nodules of peas.]
20. Relić B, Perret X, Estrada-Garcia MT, Kopcinska J, Golinowski W,
Krishnan HB, Pueppke SG, Broughton WJ: Nod-factors of Rhizobium
are a key to the legume door. Mol Microbiol 1994, 13:171-178.
3.
Hiltner L: Über die Ursachen, welche die Grösse, Zahl, Stellung
und Wirkung der Wurzelknöllchen der Leguminosen bedingen.
Arbeiten aus der Biologischen Abteilung für Land- und
21. D’Haeze W, Gao M-S, De Rycke R, Van Montagu M, Engler G,
•
Holsters M: Roles for azorhizobial Nod factors and surface
polysaccharides in intercellular invasion and nodule penetration,
Genealogy of legume–Rhizobium symbioses Broughton and Perret
respectively. Mol Plant–Microbe Interact 1998, 11:999-1008.
Functional complementation of an Azorhizobium nodA-mutant by NodAc-factors
confirmed that LCOs are necessary for intercellular infection in stem-nodulation.
22. Roche P, Debellé F, Maillet F, Lerouge P, Faucher C, Truchet G,
Dénarié J, Promé J-C: Molecular basis of symbiotic host specificity in
Rhizobium meliloti: nodH and nodPQ genes encode the sulfation of
lipochito-oligosaccharide signals. Cell 1991, 67:1131-1143.
23. Yang GP, Debellé F, Ferro M, Maillet F, Schiltz O, Vialas C, Savagnac A,
Promé J-C, Dénarié J: Rhizobium Nod factor structure and the
phylogeny of temperate legumes. In Biological Nitrogen Fixation for
the 21st Century. Edited by Elmerich C, Kondorosi A and Newton WE.
Kluwer Academic Publishers, Dordrecht; 1998:185-188.
24. Ardourel M, Demont N, Debellé F, Maillet F, de Billy F, Promé J-C,
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.
25. Relić B, Talmont F, Kopcinska J, Golinowski W, Promé J-C,
Broughton WJ: Biological activity of Rhizobium sp. NGR234 Nodfactors on Macroptilium atropurpureum. Mol Plant–Microbe Interact
1993, 6:764-774.
26. Cárdenas L, Dominguez J, Quinto C, Lopez-lara IM, Lugtenberg BJJ,
Spánk HP, Rademaker GJ, Haverkamp J, Thomas-Oates JE: Isolation,
chemical structure and biological activity of the lipo-chitin
oligosaccharide nodulation signals from Rhizobium etli. Plant Mol
Biol 1995, 29:453-464.
27.
Spaink HP, Bloemberg GV, van Brussel AAN, Lugtenberg BJJ, van der
Drift KMGM, Haverkamp J, Thomas-Oates JE: Host specificity of
Rhizobium leguminosarum is determined by the hydrophobicity of
highly unsaturated fatty acyl moieties of the nodulation factors.
Mol Plant–Microbe Interact 1995, 8:155-164.
28. Orgambide GG, Lee JI, Hollingsworth RI, Dazzo FB: Structurally
diverse chitolipooligosaccharide Nod factors accumulate
primarily in membranes of wild-type Rhizobium leguminosarum
biovar trifolii. Biochemistry 1995, 34:3832-3840.
29. Poupot R, Martínez-Romero E, Promé J-C: Nodulation factors from
Rhizobium tropici are sulfated or non-sulfated
chitopentasaccharides containing an N-methyl-N-acylglucosamine
terminus. Biochemistry 1993, 32:10430-10435.
30. Poupot R, Martínez-Romero E, Gautier N, Promé J-C: Wild-type
Rhizobium etli, a bean symbiont, produces acetyl-fucosylated, Nmethylated, and carbamoylated nodulation factors. J Biol Chem
1995, 270:6050-6055.
31. Stacey G, Luka S, Sanjuan J, Banfalvi Z, Nieuwkoop AJ, Chun JY,
Forsherg LS, Carlson R: nodZ a unique host-specific nodulation
gene, is involved in the fucosylation of the lipooligosaccharide
nodulation signal from Bradyrhizobium japonicum. J Bacteriol
1994, 176:620-633.
32. Quesada-Vincens D, Fellay R, Nasim T, Viprey V, Burger U, Promé J-C,
Broughton WJ, Jabbouri S: Rhizobium sp. NGR234 NodZ protein is
a fucosyltransferase. J Bacteriol 1997, 179:5087-5093.
33. Relić B, Staehelin C, Fellay R, Jabbouri S, Boller T, Broughton WJ: Do
Nod-factor levels play a role in host-specificity? In Proceedings of
the First European Nitrogen Fixation Conference. Edited by Kiss GB
and Endre G. Officina Press: Szeged, Hungary; 1994:69-75.
34. Spaink HP, Wijffelman CA, Pees E, Okker RJH, Lugtenberg BJJ:
Rhizobium nodulation gene nodD as a determinant of host
specificity. Nature 1987, 328:337-340.
35. Jabbouri S, Fellay R, Talmont F, Kamalaprija P, Burger U, Relić B,
Promé J-C, Broughton WJ: Involvement of nodS in N-methylation
and nodU in 6-O-carbamoylation of Rhizobium sp. NGR234 Nod
factors. J Biol Chem 1995, 270:22968-22973.
36. Krishnan HB, Lewin A, Fellay R, Broughton WJ, Pueppke SG:
Differential expression of nodS accounts for the varied abilities of
Rhizobium fredii USDA 257 and Rhizobium sp. NGR234 to
nodulate Leucaena spp. Mol Microbiol 1992, 6:3321-3330.
37.
Minami E, Kouchi H, Carlson RW, Cohn JR, Kolli VK, Day RB,
Ogawa T, Stacey G: Cooperative action of lipo-chitin nodulation
signals on the induction of the early nodulin, ENOD2 in soybean
roots. Mol Plant–Microbe Interact 1996, 9:574-583.
38. Minami E, Kouchi H, Cohn JR, Ogawa T, Stacey G: Expression of the
early nodulin, ENOD40, in soybean roots in response to various
lipo-chitin signal molecules. Plant J 1996, 10:23-32.
311
39. Chen R, Silver DL, de Brujn F: Nodule parenchyma-specific
•
expression of the Sesbamia rostrata early nodulin gene SrEnod2 is
mediated by the 3¢ untranslated region. Plant cell 1998, 10:1585-1602.
The 3′ untranslated region of SrEnod is required for its expression in the
parenchyma cell layer of the nodules. Expression of SrEnod2 is inducible by
cytokinins, adding plant hormones to the list of substances known to modulate expression of this gene (see [38]). Thus Nod-factors and cytokinins jointly modulate the activity of some of the same components of the signal
response pathway.
40. Cheng H-P, Walker GC: Succinoglycan is required for initiation and
•
elongation of infection threads during nodulation of alfalfa by
Rhizobium meliloti. J Bacteriol 1998, 180:5183-5191.
Shows clearly that individual exo-genes, which are involved in the synthesis
of succinoglycan, affect infection-thread development.
41. Kijne JW: The Rhizobium infection process. In Biological Nitrogen
Fixation. Edited by Stacey G, Burris RH, and Evans HJ: Chapman and
Hall; New York; 1992:349-398.
42. Viprey V, Del Greco A, Golinowski W, Broughton WJ, Perret X:
•
Symbiotic implications of type III protein secretion machinery in
Rhizobium. Mol Microbiol 1998, 28:1381-1389.
Sequencing of pNGR234a revealed a gene cluster which encodes a
flavonoid inducible type III protein secretion system. Builds on data which
showed that protein secretion plays a role in host-specificity.
43. Economau A, Davies AE, Johnston AWB, Downie JA: The Rhizobium
leguminosarum biovar viciae nod0 gene can enable a nodE
mutant of Rhizobium leguminosarum biovar trifolií to nodulate
vetch. Microbiology 1994, 140:2341-2347.
44. 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 leucocephaia,
Phaseolus vulgaris and Trifolium repens by a diversity of
Rhizobium spp. Mol Plant–Microbe Interact 1998, 11:383-392.
Shows that nodO is not confined to strains that nodulate Cicereae,
Trifolieae, and Vicerea, but is also present in rhizobia that associate with
tropical legumes. Functionally complements mutants in nodS of NGR234
for nodulation of L. leucoceplaia.
45. Hacker J, Blum Oehier G, Mühldorfer I, Tschäpe H: Pathogenicity
islands of virulent bacteria: structure, function and impact on
microbial evolution. Mol Microb 1997, 23:1089-1097.
46. van Rhijn P, Vanderleyden J: The Rhizobium-plant symbiosis.
Microbiol Rev 1995, 59:124-142.
Perret X, Freiberg C, Rosenthal A, Broughton WJ, Fellay R: Highresolution transcriptional analysis of the symbiotic replicon of
Rhizobium sp. NGR234. Mol Microbiol 1999, 32:415-425.
pNGR234a was divided into 441 segments representing all coding and intergenic regions. Each segment was amplified by PCR, transferred to filters and
probed with radioactively labelled-RNA. Genes involved in the synthesis of
Nod-factors were induced rapidly after the addition of flavonoids, while others
thought to act within the plant (e.g. those encoding the type three secretion
machine) responded more slowly. Many more transcripts were found in bacteroids of determinate- as opposed to indeterminate-nodules.
47.
•
48. Ovtsyna AO, Geurts R, Bisseling T, Lugtenberg BJJ, Tikhonovich IA,
•
Spaink H: Restriction of host range by the sym2 allele of Afghan
pea is nonspecific for the type of modification at the reducing
terminus of nodulation signals. Mol Plant–Microbe Interact 1998,
11:418-422.
A clear demonstration that the exact nature of Nod-factor substitutents is less
important than their position. Nodulation of the primitive P. sativum cultivar
Afghanistan was thought to require a 6-O-acetate group on the reducing terminus (added by NodX), but it can be functionally replaced by a 6-O-fucose
group (nodZ encodes a fucosyltransferase).
49. Karaolis DKR, Somara S, Maneval DR, Johnson JA, Kaper JB: A
•
bacteriophage encoding a pathogenicity island, a type-IV pilus and
a phage receptor in cholera bacteria. Nature 1999, 399:375-379.
Many bacteria carry pathogenicity islands — DNA sequences that encode virulence related proteins. These include specialised molecular machines to injectbacterial products into their animal or plant hosts, molecules involved in signal
transduction and toxins. Here the authors demonstrate an interplay between
two pathogenicity islands of the bacterium Vibrio cholerae and its bacteriophage CTXψ, which carries the cholera toxin genes.
50. Nelson KE, Clayton RA, Gill SR, Gwinn ML, Dodson RJ, Haft DH,
•
Hickley EK, Peterson JD, Nelson WC, Ketchum KA et al.: Evidence for
lateral gene transfer between archea and bacteria from genome
sequence of Thermotoga maritima. Nature 1999, 399:323-329.
The thermophilic eubacterium T. maritima has a single chromosome (G+C content 46%). Of the 1,877 predicted coding regions, 24% are most similar to
those of archea. Evidence is presented suggesting that much of the similarity
between T. maritima and archea is the result of extensive lateral gene transfer.