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Annu. Rev. Microbiol. 1992.46:497-531. Downloaded from www.annualreviews.org by University of Utah - Marriot Library on 05/05/13. For personal use only.
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© 1992
1992.46:497-531
by Annual Reviews Inc. All rights reserved
SIGN.ALING AND HOST RANGE VARIATION IN NODULATION
Jean Derzarie, Frederic Debelle, and Charles Rosenberg Laboratoire de Biologie Moleculaire des Relations Plantes-Microorganismes, CNRS INRA, BP27, 31326 Castanet-Tolosan Cedex, France KEY WORDS:
nitrogen fixation, symbiosis, legumes, nodulation factors, host specificity,
Rhizobium, Bradyrhizobium
CONTENTS Abstract. ........ .... ......... ........................................................ ... .......... ..
498
INTRODUCTION.....................................................................................
498
Diversity of Legumes and Rhizobia . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . ........ . . . . . . . . ... Infection and Nodulation Patterns . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . .............. . . . . . ... A Variety of Host Ranges ............................................................ ........... Rhizobial Genes Controlling Infection, Nodulation, and Host Range ....... . . . . . . . . .. .
498 499 500 501
REGULATORY nod GENES AS HOST-RANGE DETERMINANTS.....................
502
Basic Elements of the nodD Regulatory Circuit............................................ NodD Proteins Are Specifically Activated by Flavonoids.................. . . . . . . ...... . . . Plant Exudates Contain Inducers and Antiinducers . . . . . . . . . . . . . . . ........... . .. . . . ........ Additional Elements of the nodD Regulatory Circuit...................................... Other nod Regulatory Genes .......................................... ...... . . . . . . . . . .... . . . . . .
502 503 505 507 508 509
STRUCTURAL nod GENES AS HOS T-RANGE DETERMINANTS...................... Common nod Gen es ....................... ................... ........................... ......... Specific nod Genes . . . . . . . . . . . . . ....... . . . ................................ . . . . . . . . . . . . . ... . . . . . . .. Extracellular Specific Nod Factors . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. . . . ... Nod-Fact.or Chemical Characterization and Biological Activity . . . . . . . . . . . . . ... . . . . . .... The Common nod Genes and the Molecule Backbone ......................... ........... Synthesis of Specific N-Acyl Chains. . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . .. . ... . . . . ........... Sulfation of the R. meliloti No d Factors. . . . ... . . . . . . . . . . ..... ..... . . . .... ... . . . . . . . . .. . .. . . . a-Acetylation and Other Substitutions of Nod Factors. . . . . . . . ............ . . . . . . . . . . . . . . . . A Secreted Nod Protein . . . . . ........ . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . .. Signal E:x.change and Plant Determinants of Specificity .... ....... . . . . . . . . . ...... . . . . ....
509 513 514 517 518 519 520 521 522
CONCLUSIONS AND PERSPECTIVES.........................................................
523
0066-4227/92/1001-0497$02.00
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Annu. Rev. Microbiol. 1992.46:497-531. Downloaded from www.annualreviews.org by University of Utah - Marriot Library on 05/05/13. For personal use only.
Abstract Rhizobium, Bradyrhizobium, and Azorhizobium strains, collectively referred to as rhizobia, elicit on their leguminous hosts , in a specific manner, the formation of nodules in which they fix nitrogen. Rhizobial nod genes , which determine host specificity, infection, and nodulation , are involved in the exchange of low molecular weight signal molecules between the plant and the bacteria as follows. Transcription of the nod operons is under the control of NodD regulatory proteins , which are specifically activated by plant flavonoid signals. The common and species-specific structural nod genes are involved in tum in the synthesis of specific lipo-oligosaccharides that signal back to the plant to elicit root-hair deformations, cortical-cell divisions, and nodule meristem formation . INTRODUCTION
Legumes and their symbiotic bacteria (collectively referred to as rhizobia) make the largest contribution to global biological nitrogen fixation (62). Rhizobia elicit on their hosts, in a specific manner, the formation of special ized organs , the nodules, in which they reduce molecular nitrogen into ammonia (11). The Rhizobium-legume symbiosis, because of its agricultural importance, has ensured continuing research support world-wide (62) and is presently the best understood of all plant-microbe interactions (73, 154). The symbiosis is specific: every strain has a definable host range (164). Considerable progress has been made during the past few years in understand ing the mechanisms underlying this specificity thanks to interdisciplinary approaches bringing together bacterial molecular genetics, plant physiology and cytology, and biochemistry. These studies have revealed that in the course of infection and nodulation both partners exchange low-molecular weight signal molecules. The plant controls the expression of bacterial nodulation genes via phenolic compounds (93, 108) whereas the rhizobia signal back to the plant by secreting specific lipooligosaccharides (27, 1 1 8).
Diversity of Legumes and Rhizobia The ability to establish a nitrogen-fixing symbiosis with rhizobia is restricted to legumes with one exception , the genus Parasponia of the Ulmaceae family (164). The Leguminosae family comprises three subfamilies, Caesalpi nioideae, Mimosoideae, and Papilionoideae, each of which contains genera able to form root nodules (2, 124). Leguminous plants (approximately 15,000 species) exhibit very diverse morphology , habitat , and ecology ranging from arctic annuals to tropical trees. As the great majority of legumes are nodulated by rhizobia, the symbiosis with rhizobia is apparently not an adaptation to a specialized ecological niche but rather depends on some genetic particularity
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of legumes that is sufficiently complex that it has rarely evolved elsewhere in the plant kingdom ( 1 64) . Rhizobia were first classified, according to their host range, into cross inoculation groups. The use of modem methods of bacterial systematics such as numerical taxonomy and nucleic acid hybridization and sequencing led to the definition of three genera, Rhizobium (fast growing) , Bradyrhizobium (slow growing), and Azorhizobium (Nrdependent growth in free-living con ditions) (39, 84, 1 64) (Table 1 ) . These genera are quite distinct and are much closer to nonsymbiotic relatives than they are to each other. For example, Rhizobium is closely related to the plant-associated Agrobacterium, while Rhodopseudomonas palustris, a soil phototroph, and Xanthobacter spp. are the closest relatives of Bradyrhizobium and Azorhizobium, respectively (39, 72, 84). Thus, rhizobia do not form a monophyletic group. The reason why such diverse bacteria are grouped into a single family , the Rhizobiaceae, is their common ability to establish a nitrogen-fixing symbiosis with legumes . For the sake of brevity , in the following text, Rhizobium leguminosarum bv. phaseoli, bv. trifolii, and bv. viciae (see Table 1 ) are referred to as R . phaseoli, R . trifolii, and R . leguminosarum, respectively.
Infection and Nodulation Patterns Rhizobia multiply in the rhizosphere and on the root surface of legumes. The mechanisms of root infection vary and include simple entry through in tercellular spaces in the epidermis or in the middle lamella (crack entry) as in Arachis (peanut) and Stylosanthes, or entry through root hairs as in alfalfa and vetch (see 1 1 , 30). In the latter case, rhizobia elicit root-hair curling, the Table 1
Rhizobia-plant associations
Rhizobia
Host plants
Rhizobium meliloti
Alfalfa (Medicago)
Rhizobium leguminosarum biovar viciae
Pea (Pisum), vetch (Vida)
biovar trifolii
Clover (Trifolium)
biovar phaseoli
Rhizobium loti
Bean (Phaseolus)
Lotus
Rhizobium fredii
Soybean (Glycine)
Rhizobium sp. NGR234
Tropical legumes, Parasponia (nonlegume)
Rhizobium tropici
Bean (Phaseo/us), Leucaena
Bradyrhizobium japonicum
Soybean (Glycine)
Bradyrhizobium "cowpea"
Tropical legumes
Azorhizobium caulinodans
Sesbania (stem-nodulating)
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formation of an infection focus within the curl , and the development of a tubular structure, the infection thread, which grows through the root-hair cell and into the root cortex where it ramifies. Associated with the infection is the induction of cell division in the cortex and the formation of nodule primordia. The development of primordia gives rise to nodules that are genuine organs, not mere tumors or deformed roots. Bacteria multiply in infection threads and are then released into plant cells where they differentiate into nitrogen-fixing bacteroids. Nodules are always formed on the roots; however, some aquatic legumes belonging to the genera Aeschynomene and Sesbania also exhibit stem nodulation. Nitrogen-fixing nodules of a given plant species have a characteristic ontogeny , morphology, and anatomy. They also have a defined development that is either indeterminate (with a persistent meristem) as in alfalfa, pea, or vetch, or determinate (with a transient meristem) in soybeans or Phaseolus (see recent reviews 1 1 , 1 6). The route of infection is a characteristic of the host because the same bacteria can penetrate different host species by either crack entry or root-hair infection threads , and a given legume is infected by the same type of mechanism whatever the strain (see 27). Similarly, the structural and de velopmental characteristics of an efficient nodule are specified by the plant and not by the rhizobial strain, indicating that the host possesses the genetic information for symbiotic infection and nodulation and that the role of the bacteria is to tum on this plant symbiotic program (see 27) .
A Variety of Host Ranges The bacterial symbiont forms nodules only on a restricted number of hosts , and each host is only nodulated by a restricted number of microsymbionts . However, the degree of specificity varies greatly among rhizobia (for a review , see 1 64) . Some isolates can have a broad host range. For example, some tropical Bradyrhizobium strains nodulate legumes in different tribes and subfamilies (Papilionoideae and Mimosoideae) (164), and Rhizobium sp. strain NGR234 nodulates at least 35 different legume genera, belonging to l 3 tribes, as well as the nonlegume Parasponia (S. G. Pueppke & W . J. Broughton, unpublished data) . In contrast, some isolates have a narrow host range. For example, Rhizobium meliloti strains elicit nitrogen-fixing nodules only on species of the genera Medicago, Melilotus, and Trigonella, and R . trifolii only on species of Trifolium (clover). Some strains even discriminate between genotypes within a legume species. For example, most isolates of R . leguminosarum nodulate European pea varieties but not certain peas from Afghanistan that require special strains with an extended host range ( 106). Rhizobial strains have been reported to form effective nodules on one plant species (or genus) and ineffective ones on another, showing that specificity is not limited to nodulation but may also affect the late stages of nodule
SIGNAL EXCHANGE IN NODULATION
501
development and the establishment of a nitrogen-fixing symbiosis . In this chapter, however, we only consider the specificity of the early steps of symbiosis , rhizospheric life, infection, and nodulation.
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Rhizobial Genes Controlling Infection, Nodulation, and Host Range Molecular genetics , combined with the study of the symbiotic behavior of bacterial mutant strains , has led to the identification of two major classes of rhizobial genes involved in infection and nodulation (for reviews see 8 , 30, 108, 109, 1 12 , 153 , 1 54). One class includes genes in which mutations result in both: (a) alterations in the synthesis, transport, and assembly of surface components such as exopolysaccharides, lipopolysaccharides, and f3- 1 ,2glucans and (b) alterations in the infection process, such as the inability to elicit the formation of infection threads , resulting in the formation of nonfix ing empty nodules (Nod+Fix- phenotype) ( 1 1 9 , 1 27). These results show that these polysaccharides are involved in the infection process, and a possible role of exopolysaccharides and lipopolysaccharides in the determination of host specificity has been suggested (60, 6 1 , 1 1 9 , 1 27). However, no clear genetic evidence, such as gain-of-function experiments, has yet shown that rhizobial surface components are major determinants of host range. The role of surface components in infection (60, 6 1 , 1 00a, 1 1 9) and root adhesion (90-92) has been reviewed recently and is not discussed here. The second class consists of the nodulation (nod and not) genes (27 , 93, 1 08) . Because of the many different nodulation genes described in the various rhizobial species, all the letters of the alphabet have been used for the nomenclature of nod genes. Therefore, researchers have designated the re cently identified nodulation genes as nol genes. The nod and nol genes are involved in infection and nodulation, and their inactivation can result in various in planta phenotypes such as the absence of nodules (Nod-) or a delayed but effective (nitrogen-fixing) nodulation (Nodd , Fix + ) on homologous hosts , or changes in host range. Some nod genes are common to all rhizobia whereas others are specific. The nod genes arc regulated in a similar way in all rhizobia (27 , 46, 47 , 50, 93 , 1 08, 1 1 1 , 1 44) . Their regulation involves the following three elements . (a) Transcription is acti vated by trans-acting regulatory NodD proteins; (b) NodD proteins bind to promoter r'egions that contain extended conserved sequences, the nod boxes; (c) activation of nod gene transcription by the NodD proteins requires the presence of plant signals, flavonoids , or other phenolic compounds exuded by legume roots . The presence of highly-conserved nod boxes upstream of all known common and host-specific nod operons of R. leguminosarum and R. meliloti and the general presence of nod boxes among rhizobia has led to a definition of nod genes as those genes that are part of an operon preceded by a
Annu. Rev. Microbiol. 1992.46:497-531. Downloaded from www.annualreviews.org by University of Utah - Marriot Library on 05/05/13. For personal use only.
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ROSENBERG
nod box (5 1 , 1 35). This definition, however, may not be valid for all rhizobia because in Bradyrhizobium japonicum and Rhizobium sp . NGR234, some nodulation genes are indeed preceded by a nod box, whereas other genes specifying host range do not seem to be preceded by these conserved se quences (8, 1 2) . In those Rhizobium species studied to date, the nod genes reside on large symbiotic plasmids (pSym) that also carry the fix and nif nitrogen-fixation genes. In Bradyrhizobium and Azorhizobium, the symbiotic genes are likely to be chromosomal. Both regulatory and structural nod genes are major determinants of host range, as determined with gain-of-function experiments . For example, muta tions in the regulatory nodD gene of R. trifolii ( 1 1 5) and transfer of the nodDl gene of Rhizobium sp . NGR234 into other Rhizobium species (79) result in an extension of the host range . Similarly, mutations in the structural nodE gene of R. trifolii (33) and nodQ gene of R. meliloti ( 1 8 , 43 , 142) extend the host range. Moreover, the transfer of species-specific structural nod genes between rhizobia is associated with the transfer of specificity for particular hosts (24, 3 1 , 43 , 1 05, 1 56). In this chapter, we review the present knowledge of the mechanisms by which the regulatory and structural nod genes determine the host specificity of infection and nodulation. REGULATORY nod GENES AS HOST-RANGE DETERMINANTS
Several lines of genetic evidence have established that nodD genes are determinants of host specificity . (a) Some mutations in nodD cannot be complemented by nodD DNA from other Rhizobium species (79, 1 52) . (b) Some point mutations in the nodD gene of R. trifolii result in an extended host-range ( 1 1 5 ) . (c) A nodD hybrid gene, constructed in vitro from R . meliloti and R. trifolii nodD genes, causes constitutive expression of nod genes and extends the host range to include tropical legumes ( 1 48) . (d) The transfer of the nodDl gene from Rhizobium sp. NGR234 into R . meliloti results in the transfer of the ability to nodulate Siratro (79) . We now examine how these regulatory genes are involved in the control of host specificity.
Basic Elements of the DodD Regulatory Circuit The regulatory nodD genes are found in all of the strains of Rhizobium, Bradyrhizobium, and A zorhizobium and are required for nodulation ( l08) because mutants that do not contain a functional nodD gene cannot nodulate their hosts (78, 94) . Some species like R. leguminosarum and R . trifolii have only one nodD gene (94) , while R. meliloti (57 , 78) , R. phaseoli (21), R. fredii (3) , and B. japonicum (58) carry two or three copies of nodD . The different nodD genes are conserved at the nucleotide sequence level and share
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homology with the lysR family of prokaryotic regulatory genes (71). In common with the LysR-family proteins, the N-terminal end of NodD contains a helix-tum-helix motif characteristic of DNA-binding proteins (71, 1 68). The NodD proteins are transcriptional activators that bind to the nod box, a consensus sequence present in the promoter of the nod genes inducible by plant exudates (94) . This nod box was originally defined in R. meliloti as a 47-bp consensus sequence required for nod gene induction (135). In the genetically distant Azorhizobium caulinodans and B . japonicum, less con served nod boxes have been identified and new shorter consensus sequences have been proposed to account for these divergent nod boxes (54, 1 63). These short consensus sequences are also found in promoter sequences of genes regulated by proteins of the LysR family. The long nod boxes contain repetitions of these short consensus sequences and could be bound by multi meric forms of NodD. The activation of nod gene expression by NodD proteins requires the presence of compounds present in plant exudates (108). The inducing molecules have been purified from seed or root exudates. They are flavonoids or related compounds derived from phenylpropanoid metabol ism, which is also known to provide molecules involved in plant defense ( 1 32). The nod-inducing compounds can be active at concentrations as low as 10-9 M (1 1 3) .
NodD Proteins Are Specifically Activated by Flavonoids The R. meliloti nodDl gene does not restore the ability to nodulate Siratro in a nodDl mutant of the broad host range strain Rhizobium sp. MPIK3030 ( NGR234) (79). Similarly, the R . meliloti nodDl gene does not restore the ability to nodulate red clover in a nodD mutant of R. trifolii ( 1 52) . This lack of complementation results from the inability of R. meliloti nodD 1 to respond to the compounds exuded by Siratro (79) and red clover (152). In addition, when different nodD genes are introduced into the same Rhizobium strain, the structural nod genes are induced by different sets of flavonoids ( 1 52) . Such a phenomenon is likely responsible for the fact that R. meliloti or R. trifolii strains carrying the nodD 1 gene of MPIK3030 exhibit an extended host range including Siratro (79). These experiments indicate that different NodD pro teins have different inducer specificities. Generally, a NodD protein from a given Rhizobium species is most active in the presence of exudates from the plants nodulated by that Rhizobium species ( 1 52, 1 66). Using synthetic or natural compounds of known structure investigators have detennined the structural features of flavonoids necessary for nod gene induction by different NodD proteins (Table 2). NodD proteins are generally active in the presence of a family of related flavonoids (Table 2). NodDs from narrow-host-range rhizobia CR . meliloti, R. leguminosarum, R. trifolii) re spond to few flavonoids, while NodDs from the broad-host-range Rhizobium =
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DENARrE, DEBELLE
Table 2
Effects of flavonoids and chalcones, in conjunction with various NodD proteins, on nod
&
ROSENBERG
gene expression COMPOUNDS
SUBSTITUTION POSITION
3 Flavones
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luteolin apigenin
chrysoeriol
5
7
OH OH
OH OH OH OH
OH
OH OH OH OH OH OH
OH OH OH
OH OH
OH OH OH OH OH OH OH OH
hesperetin
I:iQflil�Df;l:i
OH
genistein
4'
5'
OH OH OH OH OH 0cH30H OH OH OH OH OH
OH
chrysin Flayonols myricetin quercetin kaempferol EISlysiDQD�� eriodictyol naringenin
3'
OH OH
daidzein
OH OH OH
At
NodD PROTEINa RI Am Rm NGR Bj 01 D2
++
++
++
++
++
+
++
+
n
++
++
n
n
n
++
n
n n
n n
++
++
n
+
+
n
n
n
++
+
++
OH
+
n
++ ++
OH OH
0CH3
+
n
+
++
++
++
+
++
++
++
OH OH
Chalcooe
n
++
++
++
++
n
n
4,4'dihydroxy-
OH
2'-methoxychalcone
OH
n
n
++
++
3'
3'
6 flavones and flayonols
O
7276 8
6
I
5
0
jsoflayoD9s
•
2
6'
flayaDoDes
3 2' 5'
3' 1 4'
4' 5 o
cbalcQnes
The nod inducing activities of various flavonoids and chalcone were recorded for rhizobial strains harboring
the nodD gen es of R. trifolii (Rt), R. leguminosarum (Rl), R. meliloti (Rm), NGR234 (NGR), and B.japonicum
(Bj). The different activities are related to the maximum inducing activity recorded 50-100%; + = 10-50%; - = 0-10% approximately of tbe maximum induction. n = 10,32,45,66,98,99,103,111,1l3,122, 165, 167.)
for these strains. + + = not determined. (After
7,
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SIGNAL EXCHANGE IN NODULATION
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NGR234 have a larger spectrum of inducing molecules including even monocyclic aromatic compounds (03) (Table 2). The activity of a given flavonoid compound varies widely according to the rhizobial species consid ered. For example, the isoflavone daidzein is a strong activator of the B. japonicum nod genes but does not activate the R . melitoli. R. leguminosarum. or R . lrifolii nod genes. Though the specific binding of flavonoids to NodD has not been biochemi cally demonstrated, genetic data suggest a direct interaction of these com pounds wilth NodD. Point mutations in nodD have been obtained that result in nod gene activation with a broader range of inducers ( 1 3 , 1 4 , 1 1 5). Further more, hybrid nodD genes have been constructed from nodD genes of different Rhizobium species and show modified flavonoid sensitivity (79 , 1 5 1). The hybrid NodD proteins exhibit the flavonoid specificity of the NodD product constituting their C-terminal end (79 , 1 5 1). These experiments indicate that the C-terminal part of NodD, which is less conserved than the N-terminal end, is involved in determining flavonoid specificity ( 1 68). In addition, this C terminal region shows homology to ligand-binding domains of animal steroid receptors that are known to bind flavonoids (65, 1 68). However, some of the mutations that modify the flavonoid specificity map in the N-terminal region ( 1 1 5 , 1 5 1 ) . Thus, the flavonoid specificity also depends on the overall tertiary structure of NodD . Naringenin, which is an inducer of R. leguminosarum nod genes , has been shown to accumulate in the cytoplasmic membrane ( 1 25) where the R . leguminosarum NodD protein has been localized ( 1 38). This observation has led to the suggestion that the site of interaction between NodD and the flavonoids is the bacterial inner membrane (138). Experiments with R . legu minosarum and R. meliloti NodD have shown that binding of NodD to the nod box does not require the presence of flavonoids (46, 76). However, the binding of A. caulinodans NodD to the nod box is enhanced by the flavonoid naringenin (54). Therefore, the mechanism of activation of NodD proteins by flavonoids is still unknown.
Plant Exudates Contain Inducers and Antiinducers A given legume releases several flavonoids with or without inducing proper ties for its specific Rhizobium . The nature and amounts of the compounds exuded depend on the plant and its stage of development. For alfalfa, bean, or soybean, the spectrum of flavonoids present in seed exudates is different from that present in root exudates (59, 68, 69 , 8 1 , 82, 1 2 1 ) . Flavonoids are released as aglycones or glycosidic conjugates of lower activity but higher solubility (69, 1 14). These glycoconjugates can be converted to active forms by bacterial glycosidases . The availability of nod inducing flavonoids can -
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sometimes limit nodulation: Nodulation of a eultivar of alfalfa was enhanced when the inducer luteolin was supplied to the plant (88). The different flavonoids present in exudates can interfere with each other's ability to induce nod gene expression. In several cases , flavonoids without inducing properties have been shown to inhibit nod gene activation by effec tive inducers (32, 45 , 66, 99, 1 22). The antiinducers generally have structures similar to those of the inducers , and the inhibition can be overcome by increasing the concentration of inducers ( 1 22). Thus, one can consider the antiinducers to be competitive inhibitors. Some of these inhibitors are strain specific , acting only on a few strains belonging to the same cross-inoculation group (99). Synergistic interactions between inducers have also been observed (68, 69) . This effect could be explained if one considers that NodD acts as a multimer: A multimeric complex interacting with different flavo noids could have a higher activity . The spatio-temporal distribution of flavonoids in the rhizosphere and at the root surface is likely to determine the levels of induction of the Rhizobium nod genes. This effect can be directly observed by placing a legume seedling in a Petri dish on a lawn of bacteria carrying a nod-laeZ fusion (32,5 3 , 1 22 , 1 26) . Zones of nod gene induction or inhibition can be distinguished. Nodules generally appear in the zone of maximum induction corresponding to the zone of emerging root hairs . Flavonoids present in root exudates might influence rhizobial infection by mechanisms other than nod gene regulation. Rhizobia respond by positive chemotaxis to plant-root exudates and move towards localized sites on legume roots (34 , 49, 63). Both Bradyrhizobium and Rhizobium spp. are attracted by phenolic compounds that are potential nutrients ( 1 20, 123), but also, at very low concentrations, by compounds that may not be of nutritional value, such as flavonoids ( 1 , 4, 1 5 , 87). In R. meliloti, the chemotactic effect of flavo noids and their nod gene-inducing ability are closely correlated, and this chemo-attraction requires functional nodD and nodABC genes ( 1 5) . In R. leguminosarum and R. phaseoli, this correlation is less pronounced as is the dependence of chemotaxis upon nod genes ( 1 , 4). Chemotaxis toward flavo noids may facilitate the contact between the bacterium and the root surface of its specific host. Surprisingly, growth of R. meliloti in a minimal medium is strongly stimulated by micromolar concentrations of the nod gene inducer luteolin and other 5 ,7-dihydroxyl substituted flavones naturally released in alfalfa exudates (67). This intriguing growth stimulation does not depend on the presence of the nod genes and is also observed for flavonoids , such as quer cetin, that are not inducers of the R. meliloti nod genes (67). Quercetin and luteolin also increase the growth rate of R. trifolii and Pseudomonas putida, but not of Agrobacterium tumefaciens, which is taxonomically close
507
SIGNAL EXCHANGE IN NODULAnON
to R. meliloti. The mechanism by which micromolar concentrations of specif ic flavonoids could reduce the bacterial doubling time so drastically (by a factor of three to five times) is not known. Like chemotaxis, this growth stimulating phenomenon may play a critical role in structuring the microbial community around the developing root (67).
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Additional Elements of the nodD Regulatory Circuit R. meliloti carries three functional nodD genes whose relative importance in the nodulation process depends on the host plant considered (57, 64, 78) . NodD proteins have different sensitivities to the root exudates of various hosts that contain different sets of flavonoids (64, 66, 77). Studies using purified compounds showed that nodD 1 but not nodD2 responds to luteolin (77 , 1 1 1 , 1 17) whereas nodD2 is active in the presence of 4,4' dihydroxy 2' methoxy chalcone (70). In addition, the three nodD genes interfere with each other in the activatiion of the common and host-specific nod genes (77, 1 1 1 , 1 17 ) . The significance, in terms of control of specificity, of these nodD reiterations is not clear, because there is no correlation between the number of nodD genes and the broadness of the host range: The narrow-host-range R. meliloti species has three copies , whereas the broad-host-range strain NGR234 possesses two copies . Note that the role of NodD proteins is not limited to regulating nod gene expression in response to flavonoid signal molecules. For example, in R . meliloti, nodD3 i s also involved in the regulation o f nod gene expression in the presence of an excess of combined nitrogen (40) (see Figure 1).
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Annu. Rev. Microbiol. 1992.46:497-531. Downloaded from www.annualreviews.org by University of Utah - Marriot Library on 05/05/13. For personal use only.
Copyright
© 1992
1992.46:497-531
by Annual Reviews Inc. All rights reserved
SIGN.ALING AND HOST RANGE VARIATION IN NODULATION
Jean Derzarie, Frederic Debelle, and Charles Rosenberg Laboratoire de Biologie Moleculaire des Relations Plantes-Microorganismes, CNRS INRA, BP27, 31326 Castanet-Tolosan Cedex, France KEY WORDS:
nitrogen fixation, symbiosis, legumes, nodulation factors, host specificity,
Rhizobium, Bradyrhizobium
CONTENTS Abstract. ........ .... ......... ........................................................ ... .......... ..
498
INTRODUCTION.....................................................................................
498
Diversity of Legumes and Rhizobia . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . ........ . . . . . . . . ... Infection and Nodulation Patterns . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . .............. . . . . . ... A Variety of Host Ranges ............................................................ ........... Rhizobial Genes Controlling Infection, Nodulation, and Host Range ....... . . . . . . . . .. .
498 499 500 501
REGULATORY nod GENES AS HOST-RANGE DETERMINANTS.....................
502
Basic Elements of the nodD Regulatory Circuit............................................ NodD Proteins Are Specifically Activated by Flavonoids.................. . . . . . . ...... . . . Plant Exudates Contain Inducers and Antiinducers . . . . . . . . . . . . . . . ........... . .. . . . ........ Additional Elements of the nodD Regulatory Circuit...................................... Other nod Regulatory Genes .......................................... ...... . . . . . . . . . .... . . . . . .
502 503 505 507 508 509
STRUCTURAL nod GENES AS HOS T-RANGE DETERMINANTS...................... Common nod Gen es ....................... ................... ........................... ......... Specific nod Genes . . . . . . . . . . . . . ....... . . . ................................ . . . . . . . . . . . . . ... . . . . . . .. Extracellular Specific Nod Factors . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. . . . ... Nod-Fact.or Chemical Characterization and Biological Activity . . . . . . . . . . . . . ... . . . . . .... The Common nod Genes and the Molecule Backbone ......................... ........... Synthesis of Specific N-Acyl Chains. . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . .. . ... . . . . ........... Sulfation of the R. meliloti No d Factors. . . . ... . . . . . . . . . . ..... ..... . . . .... ... . . . . . . . . .. . .. . . . a-Acetylation and Other Substitutions of Nod Factors. . . . . . . . ............ . . . . . . . . . . . . . . . . A Secreted Nod Protein . . . . . ........ . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . .. Signal E:x.change and Plant Determinants of Specificity .... ....... . . . . . . . . . ...... . . . . ....
509 513 514 517 518 519 520 521 522
CONCLUSIONS AND PERSPECTIVES.........................................................
523
0066-4227/92/1001-0497$02.00
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DENARIE, DEBELLE & ROSENBERG
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Abstract Rhizobium, Bradyrhizobium, and Azorhizobium strains, collectively referred to as rhizobia, elicit on their leguminous hosts , in a specific manner, the formation of nodules in which they fix nitrogen. Rhizobial nod genes , which determine host specificity, infection, and nodulation , are involved in the exchange of low molecular weight signal molecules between the plant and the bacteria as follows. Transcription of the nod operons is under the control of NodD regulatory proteins , which are specifically activated by plant flavonoid signals. The common and species-specific structural nod genes are involved in tum in the synthesis of specific lipo-oligosaccharides that signal back to the plant to elicit root-hair deformations, cortical-cell divisions, and nodule meristem formation . INTRODUCTION
Legumes and their symbiotic bacteria (collectively referred to as rhizobia) make the largest contribution to global biological nitrogen fixation (62). Rhizobia elicit on their hosts, in a specific manner, the formation of special ized organs , the nodules, in which they reduce molecular nitrogen into ammonia (11). The Rhizobium-legume symbiosis, because of its agricultural importance, has ensured continuing research support world-wide (62) and is presently the best understood of all plant-microbe interactions (73, 154). The symbiosis is specific: every strain has a definable host range (164). Considerable progress has been made during the past few years in understand ing the mechanisms underlying this specificity thanks to interdisciplinary approaches bringing together bacterial molecular genetics, plant physiology and cytology, and biochemistry. These studies have revealed that in the course of infection and nodulation both partners exchange low-molecular weight signal molecules. The plant controls the expression of bacterial nodulation genes via phenolic compounds (93, 108) whereas the rhizobia signal back to the plant by secreting specific lipooligosaccharides (27, 1 1 8).
Diversity of Legumes and Rhizobia The ability to establish a nitrogen-fixing symbiosis with rhizobia is restricted to legumes with one exception , the genus Parasponia of the Ulmaceae family (164). The Leguminosae family comprises three subfamilies, Caesalpi nioideae, Mimosoideae, and Papilionoideae, each of which contains genera able to form root nodules (2, 124). Leguminous plants (approximately 15,000 species) exhibit very diverse morphology , habitat , and ecology ranging from arctic annuals to tropical trees. As the great majority of legumes are nodulated by rhizobia, the symbiosis with rhizobia is apparently not an adaptation to a specialized ecological niche but rather depends on some genetic particularity
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SIGNAL EXCHANGE IN NODULATION
499
of legumes that is sufficiently complex that it has rarely evolved elsewhere in the plant kingdom ( 1 64) . Rhizobia were first classified, according to their host range, into cross inoculation groups. The use of modem methods of bacterial systematics such as numerical taxonomy and nucleic acid hybridization and sequencing led to the definition of three genera, Rhizobium (fast growing) , Bradyrhizobium (slow growing), and Azorhizobium (Nrdependent growth in free-living con ditions) (39, 84, 1 64) (Table 1 ) . These genera are quite distinct and are much closer to nonsymbiotic relatives than they are to each other. For example, Rhizobium is closely related to the plant-associated Agrobacterium, while Rhodopseudomonas palustris, a soil phototroph, and Xanthobacter spp. are the closest relatives of Bradyrhizobium and Azorhizobium, respectively (39, 72, 84). Thus, rhizobia do not form a monophyletic group. The reason why such diverse bacteria are grouped into a single family , the Rhizobiaceae, is their common ability to establish a nitrogen-fixing symbiosis with legumes . For the sake of brevity , in the following text, Rhizobium leguminosarum bv. phaseoli, bv. trifolii, and bv. viciae (see Table 1 ) are referred to as R . phaseoli, R . trifolii, and R . leguminosarum, respectively.
Infection and Nodulation Patterns Rhizobia multiply in the rhizosphere and on the root surface of legumes. The mechanisms of root infection vary and include simple entry through in tercellular spaces in the epidermis or in the middle lamella (crack entry) as in Arachis (peanut) and Stylosanthes, or entry through root hairs as in alfalfa and vetch (see 1 1 , 30). In the latter case, rhizobia elicit root-hair curling, the Table 1
Rhizobia-plant associations
Rhizobia
Host plants
Rhizobium meliloti
Alfalfa (Medicago)
Rhizobium leguminosarum biovar viciae
Pea (Pisum), vetch (Vida)
biovar trifolii
Clover (Trifolium)
biovar phaseoli
Rhizobium loti
Bean (Phaseolus)
Lotus
Rhizobium fredii
Soybean (Glycine)
Rhizobium sp. NGR234
Tropical legumes, Parasponia (nonlegume)
Rhizobium tropici
Bean (Phaseo/us), Leucaena
Bradyrhizobium japonicum
Soybean (Glycine)
Bradyrhizobium "cowpea"
Tropical legumes
Azorhizobium caulinodans
Sesbania (stem-nodulating)
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formation of an infection focus within the curl , and the development of a tubular structure, the infection thread, which grows through the root-hair cell and into the root cortex where it ramifies. Associated with the infection is the induction of cell division in the cortex and the formation of nodule primordia. The development of primordia gives rise to nodules that are genuine organs, not mere tumors or deformed roots. Bacteria multiply in infection threads and are then released into plant cells where they differentiate into nitrogen-fixing bacteroids. Nodules are always formed on the roots; however, some aquatic legumes belonging to the genera Aeschynomene and Sesbania also exhibit stem nodulation. Nitrogen-fixing nodules of a given plant species have a characteristic ontogeny , morphology, and anatomy. They also have a defined development that is either indeterminate (with a persistent meristem) as in alfalfa, pea, or vetch, or determinate (with a transient meristem) in soybeans or Phaseolus (see recent reviews 1 1 , 1 6). The route of infection is a characteristic of the host because the same bacteria can penetrate different host species by either crack entry or root-hair infection threads , and a given legume is infected by the same type of mechanism whatever the strain (see 27). Similarly, the structural and de velopmental characteristics of an efficient nodule are specified by the plant and not by the rhizobial strain, indicating that the host possesses the genetic information for symbiotic infection and nodulation and that the role of the bacteria is to tum on this plant symbiotic program (see 27) .
A Variety of Host Ranges The bacterial symbiont forms nodules only on a restricted number of hosts , and each host is only nodulated by a restricted number of microsymbionts . However, the degree of specificity varies greatly among rhizobia (for a review , see 1 64) . Some isolates can have a broad host range. For example, some tropical Bradyrhizobium strains nodulate legumes in different tribes and subfamilies (Papilionoideae and Mimosoideae) (164), and Rhizobium sp. strain NGR234 nodulates at least 35 different legume genera, belonging to l 3 tribes, as well as the nonlegume Parasponia (S. G. Pueppke & W . J. Broughton, unpublished data) . In contrast, some isolates have a narrow host range. For example, Rhizobium meliloti strains elicit nitrogen-fixing nodules only on species of the genera Medicago, Melilotus, and Trigonella, and R . trifolii only on species of Trifolium (clover). Some strains even discriminate between genotypes within a legume species. For example, most isolates of R . leguminosarum nodulate European pea varieties but not certain peas from Afghanistan that require special strains with an extended host range ( 106). Rhizobial strains have been reported to form effective nodules on one plant species (or genus) and ineffective ones on another, showing that specificity is not limited to nodulation but may also affect the late stages of nodule
SIGNAL EXCHANGE IN NODULATION
501
development and the establishment of a nitrogen-fixing symbiosis . In this chapter, however, we only consider the specificity of the early steps of symbiosis , rhizospheric life, infection, and nodulation.
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Rhizobial Genes Controlling Infection, Nodulation, and Host Range Molecular genetics , combined with the study of the symbiotic behavior of bacterial mutant strains , has led to the identification of two major classes of rhizobial genes involved in infection and nodulation (for reviews see 8 , 30, 108, 109, 1 12 , 153 , 1 54). One class includes genes in which mutations result in both: (a) alterations in the synthesis, transport, and assembly of surface components such as exopolysaccharides, lipopolysaccharides, and f3- 1 ,2glucans and (b) alterations in the infection process, such as the inability to elicit the formation of infection threads , resulting in the formation of nonfix ing empty nodules (Nod+Fix- phenotype) ( 1 1 9 , 1 27). These results show that these polysaccharides are involved in the infection process, and a possible role of exopolysaccharides and lipopolysaccharides in the determination of host specificity has been suggested (60, 6 1 , 1 1 9 , 1 27). However, no clear genetic evidence, such as gain-of-function experiments, has yet shown that rhizobial surface components are major determinants of host range. The role of surface components in infection (60, 6 1 , 1 00a, 1 1 9) and root adhesion (90-92) has been reviewed recently and is not discussed here. The second class consists of the nodulation (nod and not) genes (27 , 93, 1 08) . Because of the many different nodulation genes described in the various rhizobial species, all the letters of the alphabet have been used for the nomenclature of nod genes. Therefore, researchers have designated the re cently identified nodulation genes as nol genes. The nod and nol genes are involved in infection and nodulation, and their inactivation can result in various in planta phenotypes such as the absence of nodules (Nod-) or a delayed but effective (nitrogen-fixing) nodulation (Nodd , Fix + ) on homologous hosts , or changes in host range. Some nod genes are common to all rhizobia whereas others are specific. The nod genes arc regulated in a similar way in all rhizobia (27 , 46, 47 , 50, 93 , 1 08, 1 1 1 , 1 44) . Their regulation involves the following three elements . (a) Transcription is acti vated by trans-acting regulatory NodD proteins; (b) NodD proteins bind to promoter r'egions that contain extended conserved sequences, the nod boxes; (c) activation of nod gene transcription by the NodD proteins requires the presence of plant signals, flavonoids , or other phenolic compounds exuded by legume roots . The presence of highly-conserved nod boxes upstream of all known common and host-specific nod operons of R. leguminosarum and R. meliloti and the general presence of nod boxes among rhizobia has led to a definition of nod genes as those genes that are part of an operon preceded by a
Annu. Rev. Microbiol. 1992.46:497-531. Downloaded from www.annualreviews.org by University of Utah - Marriot Library on 05/05/13. For personal use only.
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&
ROSENBERG
nod box (5 1 , 1 35). This definition, however, may not be valid for all rhizobia because in Bradyrhizobium japonicum and Rhizobium sp . NGR234, some nodulation genes are indeed preceded by a nod box, whereas other genes specifying host range do not seem to be preceded by these conserved se quences (8, 1 2) . In those Rhizobium species studied to date, the nod genes reside on large symbiotic plasmids (pSym) that also carry the fix and nif nitrogen-fixation genes. In Bradyrhizobium and Azorhizobium, the symbiotic genes are likely to be chromosomal. Both regulatory and structural nod genes are major determinants of host range, as determined with gain-of-function experiments . For example, muta tions in the regulatory nodD gene of R. trifolii ( 1 1 5) and transfer of the nodDl gene of Rhizobium sp . NGR234 into other Rhizobium species (79) result in an extension of the host range . Similarly, mutations in the structural nodE gene of R. trifolii (33) and nodQ gene of R. meliloti ( 1 8 , 43 , 142) extend the host range. Moreover, the transfer of species-specific structural nod genes between rhizobia is associated with the transfer of specificity for particular hosts (24, 3 1 , 43 , 1 05, 1 56). In this chapter, we review the present knowledge of the mechanisms by which the regulatory and structural nod genes determine the host specificity of infection and nodulation. REGULATORY nod GENES AS HOST-RANGE DETERMINANTS
Several lines of genetic evidence have established that nodD genes are determinants of host specificity . (a) Some mutations in nodD cannot be complemented by nodD DNA from other Rhizobium species (79, 1 52) . (b) Some point mutations in the nodD gene of R. trifolii result in an extended host-range ( 1 1 5 ) . (c) A nodD hybrid gene, constructed in vitro from R . meliloti and R. trifolii nodD genes, causes constitutive expression of nod genes and extends the host range to include tropical legumes ( 1 48) . (d) The transfer of the nodDl gene from Rhizobium sp. NGR234 into R . meliloti results in the transfer of the ability to nodulate Siratro (79) . We now examine how these regulatory genes are involved in the control of host specificity.
Basic Elements of the DodD Regulatory Circuit The regulatory nodD genes are found in all of the strains of Rhizobium, Bradyrhizobium, and A zorhizobium and are required for nodulation ( l08) because mutants that do not contain a functional nodD gene cannot nodulate their hosts (78, 94) . Some species like R. leguminosarum and R . trifolii have only one nodD gene (94) , while R. meliloti (57 , 78) , R. phaseoli (21), R. fredii (3) , and B. japonicum (58) carry two or three copies of nodD . The different nodD genes are conserved at the nucleotide sequence level and share
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SIGNAL EXCHANGE IN NODULATION
503
homology with the lysR family of prokaryotic regulatory genes (71). In common with the LysR-family proteins, the N-terminal end of NodD contains a helix-tum-helix motif characteristic of DNA-binding proteins (71, 1 68). The NodD proteins are transcriptional activators that bind to the nod box, a consensus sequence present in the promoter of the nod genes inducible by plant exudates (94) . This nod box was originally defined in R. meliloti as a 47-bp consensus sequence required for nod gene induction (135). In the genetically distant Azorhizobium caulinodans and B . japonicum, less con served nod boxes have been identified and new shorter consensus sequences have been proposed to account for these divergent nod boxes (54, 1 63). These short consensus sequences are also found in promoter sequences of genes regulated by proteins of the LysR family. The long nod boxes contain repetitions of these short consensus sequences and could be bound by multi meric forms of NodD. The activation of nod gene expression by NodD proteins requires the presence of compounds present in plant exudates (108). The inducing molecules have been purified from seed or root exudates. They are flavonoids or related compounds derived from phenylpropanoid metabol ism, which is also known to provide molecules involved in plant defense ( 1 32). The nod-inducing compounds can be active at concentrations as low as 10-9 M (1 1 3) .
NodD Proteins Are Specifically Activated by Flavonoids The R. meliloti nodDl gene does not restore the ability to nodulate Siratro in a nodDl mutant of the broad host range strain Rhizobium sp. MPIK3030 ( NGR234) (79). Similarly, the R . meliloti nodDl gene does not restore the ability to nodulate red clover in a nodD mutant of R. trifolii ( 1 52) . This lack of complementation results from the inability of R. meliloti nodD 1 to respond to the compounds exuded by Siratro (79) and red clover (152). In addition, when different nodD genes are introduced into the same Rhizobium strain, the structural nod genes are induced by different sets of flavonoids ( 1 52) . Such a phenomenon is likely responsible for the fact that R. meliloti or R. trifolii strains carrying the nodD 1 gene of MPIK3030 exhibit an extended host range including Siratro (79). These experiments indicate that different NodD pro teins have different inducer specificities. Generally, a NodD protein from a given Rhizobium species is most active in the presence of exudates from the plants nodulated by that Rhizobium species ( 1 52, 1 66). Using synthetic or natural compounds of known structure investigators have detennined the structural features of flavonoids necessary for nod gene induction by different NodD proteins (Table 2). NodD proteins are generally active in the presence of a family of related flavonoids (Table 2). NodDs from narrow-host-range rhizobia CR . meliloti, R. leguminosarum, R. trifolii) re spond to few flavonoids, while NodDs from the broad-host-range Rhizobium =
504
DENARrE, DEBELLE
Table 2
Effects of flavonoids and chalcones, in conjunction with various NodD proteins, on nod
&
ROSENBERG
gene expression COMPOUNDS
SUBSTITUTION POSITION
3 Flavones
Annu. Rev. Microbiol. 1992.46:497-531. Downloaded from www.annualreviews.org by University of Utah - Marriot Library on 05/05/13. For personal use only.
luteolin apigenin
chrysoeriol
5
7
OH OH
OH OH OH OH
OH
OH OH OH OH OH OH
OH OH OH
OH OH
OH OH OH OH OH OH OH OH
hesperetin
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OH
genistein
4'
5'
OH OH OH OH OH 0cH30H OH OH OH OH OH
OH
chrysin Flayonols myricetin quercetin kaempferol EISlysiDQD�� eriodictyol naringenin
3'
OH OH
daidzein
OH OH OH
At
NodD PROTEINa RI Am Rm NGR Bj 01 D2
++
++
++
++
++
+
++
+
n
++
++
n
n
n
++
n
n n
n n
++
++
n
+
+
n
n
n
++
+
++
OH
+
n
++ ++
OH OH
0CH3
+
n
+
++
++
++
+
++
++
++
OH OH
Chalcooe
n
++
++
++
++
n
n
4,4'dihydroxy-
OH
2'-methoxychalcone
OH
n
n
++
++
3'
3'
6 flavones and flayonols
O
7276 8
6
I
5
0
jsoflayoD9s
•
2
6'
flayaDoDes
3 2' 5'
3' 1 4'
4' 5 o
cbalcQnes
The nod inducing activities of various flavonoids and chalcone were recorded for rhizobial strains harboring
the nodD gen es of R. trifolii (Rt), R. leguminosarum (Rl), R. meliloti (Rm), NGR234 (NGR), and B.japonicum
(Bj). The different activities are related to the maximum inducing activity recorded 50-100%; + = 10-50%; - = 0-10% approximately of tbe maximum induction. n = 10,32,45,66,98,99,103,111,1l3,122, 165, 167.)
for these strains. + + = not determined. (After
7,
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SIGNAL EXCHANGE IN NODULATION
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NGR234 have a larger spectrum of inducing molecules including even monocyclic aromatic compounds (03) (Table 2). The activity of a given flavonoid compound varies widely according to the rhizobial species consid ered. For example, the isoflavone daidzein is a strong activator of the B. japonicum nod genes but does not activate the R . melitoli. R. leguminosarum. or R . lrifolii nod genes. Though the specific binding of flavonoids to NodD has not been biochemi cally demonstrated, genetic data suggest a direct interaction of these com pounds wilth NodD. Point mutations in nodD have been obtained that result in nod gene activation with a broader range of inducers ( 1 3 , 1 4 , 1 1 5). Further more, hybrid nodD genes have been constructed from nodD genes of different Rhizobium species and show modified flavonoid sensitivity (79 , 1 5 1). The hybrid NodD proteins exhibit the flavonoid specificity of the NodD product constituting their C-terminal end (79 , 1 5 1). These experiments indicate that the C-terminal part of NodD, which is less conserved than the N-terminal end, is involved in determining flavonoid specificity ( 1 68). In addition, this C terminal region shows homology to ligand-binding domains of animal steroid receptors that are known to bind flavonoids (65, 1 68). However, some of the mutations that modify the flavonoid specificity map in the N-terminal region ( 1 1 5 , 1 5 1 ) . Thus, the flavonoid specificity also depends on the overall tertiary structure of NodD . Naringenin, which is an inducer of R. leguminosarum nod genes , has been shown to accumulate in the cytoplasmic membrane ( 1 25) where the R . leguminosarum NodD protein has been localized ( 1 38). This observation has led to the suggestion that the site of interaction between NodD and the flavonoids is the bacterial inner membrane (138). Experiments with R . legu minosarum and R. meliloti NodD have shown that binding of NodD to the nod box does not require the presence of flavonoids (46, 76). However, the binding of A. caulinodans NodD to the nod box is enhanced by the flavonoid naringenin (54). Therefore, the mechanism of activation of NodD proteins by flavonoids is still unknown.
Plant Exudates Contain Inducers and Antiinducers A given legume releases several flavonoids with or without inducing proper ties for its specific Rhizobium . The nature and amounts of the compounds exuded depend on the plant and its stage of development. For alfalfa, bean, or soybean, the spectrum of flavonoids present in seed exudates is different from that present in root exudates (59, 68, 69 , 8 1 , 82, 1 2 1 ) . Flavonoids are released as aglycones or glycosidic conjugates of lower activity but higher solubility (69, 1 14). These glycoconjugates can be converted to active forms by bacterial glycosidases . The availability of nod inducing flavonoids can -
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DENARIE, DEBELLE & ROSENBERG
sometimes limit nodulation: Nodulation of a eultivar of alfalfa was enhanced when the inducer luteolin was supplied to the plant (88). The different flavonoids present in exudates can interfere with each other's ability to induce nod gene expression. In several cases , flavonoids without inducing properties have been shown to inhibit nod gene activation by effec tive inducers (32, 45 , 66, 99, 1 22). The antiinducers generally have structures similar to those of the inducers , and the inhibition can be overcome by increasing the concentration of inducers ( 1 22). Thus, one can consider the antiinducers to be competitive inhibitors. Some of these inhibitors are strain specific , acting only on a few strains belonging to the same cross-inoculation group (99). Synergistic interactions between inducers have also been observed (68, 69) . This effect could be explained if one considers that NodD acts as a multimer: A multimeric complex interacting with different flavo noids could have a higher activity . The spatio-temporal distribution of flavonoids in the rhizosphere and at the root surface is likely to determine the levels of induction of the Rhizobium nod genes. This effect can be directly observed by placing a legume seedling in a Petri dish on a lawn of bacteria carrying a nod-laeZ fusion (32,5 3 , 1 22 , 1 26) . Zones of nod gene induction or inhibition can be distinguished. Nodules generally appear in the zone of maximum induction corresponding to the zone of emerging root hairs . Flavonoids present in root exudates might influence rhizobial infection by mechanisms other than nod gene regulation. Rhizobia respond by positive chemotaxis to plant-root exudates and move towards localized sites on legume roots (34 , 49, 63). Both Bradyrhizobium and Rhizobium spp. are attracted by phenolic compounds that are potential nutrients ( 1 20, 123), but also, at very low concentrations, by compounds that may not be of nutritional value, such as flavonoids ( 1 , 4, 1 5 , 87). In R. meliloti, the chemotactic effect of flavo noids and their nod gene-inducing ability are closely correlated, and this chemo-attraction requires functional nodD and nodABC genes ( 1 5) . In R. leguminosarum and R. phaseoli, this correlation is less pronounced as is the dependence of chemotaxis upon nod genes ( 1 , 4). Chemotaxis toward flavo noids may facilitate the contact between the bacterium and the root surface of its specific host. Surprisingly, growth of R. meliloti in a minimal medium is strongly stimulated by micromolar concentrations of the nod gene inducer luteolin and other 5 ,7-dihydroxyl substituted flavones naturally released in alfalfa exudates (67). This intriguing growth stimulation does not depend on the presence of the nod genes and is also observed for flavonoids , such as quer cetin, that are not inducers of the R. meliloti nod genes (67). Quercetin and luteolin also increase the growth rate of R. trifolii and Pseudomonas putida, but not of Agrobacterium tumefaciens, which is taxonomically close
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SIGNAL EXCHANGE IN NODULAnON
to R. meliloti. The mechanism by which micromolar concentrations of specif ic flavonoids could reduce the bacterial doubling time so drastically (by a factor of three to five times) is not known. Like chemotaxis, this growth stimulating phenomenon may play a critical role in structuring the microbial community around the developing root (67).
Annu. Rev. Microbiol. 1992.46:497-531. Downloaded from www.annualreviews.org by University of Utah - Marriot Library on 05/05/13. For personal use only.
Additional Elements of the nodD Regulatory Circuit R. meliloti carries three functional nodD genes whose relative importance in the nodulation process depends on the host plant considered (57, 64, 78) . NodD proteins have different sensitivities to the root exudates of various hosts that contain different sets of flavonoids (64, 66, 77). Studies using purified compounds showed that nodD 1 but not nodD2 responds to luteolin (77 , 1 1 1 , 1 17) whereas nodD2 is active in the presence of 4,4' dihydroxy 2' methoxy chalcone (70). In addition, the three nodD genes interfere with each other in the activatiion of the common and host-specific nod genes (77, 1 1 1 , 1 17 ) . The significance, in terms of control of specificity, of these nodD reiterations is not clear, because there is no correlation between the number of nodD genes and the broadness of the host range: The narrow-host-range R. meliloti species has three copies , whereas the broad-host-range strain NGR234 possesses two copies . Note that the role of NodD proteins is not limited to regulating nod gene expression in response to flavonoid signal molecules. For example, in R . meliloti, nodD3 i s also involved in the regulation o f nod gene expression in the presence of an excess of combined nitrogen (40) (see Figure 1).
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