Lipochitooligosaccharides and legume Rhizobium symbiosis--a new concept.

Indian Journal of Experimental Biology Vol. 39, May 2001, pp. 401-409

Review Article

Lipochitooligosaccharides and legume Rhizobium symbiosis-A new concept

Vivek Chi mote & LR Kashyap* National Research Center on Plant Biotechnology, Indian Agricultural Research Institute,

New Delhi 110 012, India

Legume-Rhizobium symbiosis is a multistep process characterized by the formation of root nodules on the host plant. A number of genes from both symbiotic partners share information during the interaction process. Nodulation genes (nod. not and noe) have been classified as common nodulation genes and host specific (hsn) nodulation genes. Though common nodulation genes are enough to form root nodules, host specific nodulation genes are needed for specific interaction leading to formation of functional nodules. Core lipochitooligosaccharides (LCOs), the products of common nodulation genes are modified by the action of host specific nodulation genes. LCOs seem to be present in legumes as well as nonlegume and are known to act as a morphogen by acting as auxin-transport inhibitor. The understanding of Nod factor may contribute to reveal complex biological functions such as developmental regulation, signal transduction and plant morphogenesis.

Legumes have root nodules, where atmospheric dinitrogen fixation is done by bacteria belonging to genera Rhizobium, Bradyrhizobium, Sino rhizobium, Azorhizobium and Mesorhizobium collectively termed as rhizobia. Nodulation involves symbiotic interaction between leguminous host plants and rhizobia. Attachment of rhizobia to root hairs is a two-step process I. In the first loose attachment step, bacterial rhicadhesin and ndv genes involved in production of exopolysaccharide, interact probably with a plant glycoprotein 2.3. In the second step called cap formation involves accumulation of bacterial aggregates and their firm attachment to plant lectins, to initiate the multi-step bi-directional signaling pathways between bacterium and host plants, that results in root hair curling, cortical cell division and nodule formation. Infection of root nodule by invading bacteria and its differentiation into bacteriods ultimately results in effective nodule formation. A gene associated with nodule development (ndv) in S. meliloti has been identified using Agrobaeterium virulence gene (ehv) probe. Mutants of S. meliloti in this gene lack infection thread formation and do not fix nitrogen3

. Infection of roots by invading bacteria and its differentiation into bacteriods ultimately results in effective root nodule formation.

Rhizobia are divided into cross-inoculation groups based. on their host-range specificity. Such host range is decided by specific flavonoids/chalcones in root exudates, which in turn induce nodulation genes

*Corresponding Author

whose products are involved in biosynthesis of specific signal molecules-Nod factors (NFs) that trigger the first step in nodule formation 4.8. Nod factors are involved in induction of host's nodule specific nodulin genes resulting in nodule formation. Nod factors are mono-N acylated chitinooligo­saccharides (LCOs) signal molecules comprising ~ 1-4 linked N acetyl D-glucosamine (GlcNAc) residues with terminal non-reducing sugar residue having N-acetyl group substituted with N-fatty-acyl (CI6-18) chain. Other substitutions are strain specific modification of the reducing and non-reducing GlcNAc molecules.

Nodulation genes and their organization-: Nodulation genes are designated as nod, nol and noe genes which are located along with nif and fix genes on large sym plasmids in Rhlzobium and Sino rhizobium, while in Mesorhizobium loti, Bradyrhizobium and Azorhizobium these genes are located on the chromosomes9

• These genes have been classified as common/structural and host specific genes (hsn). The inactivation of common nodulation (nod ABC) genes leads to lack of primary symbiotic responses and thereby lack of nodulation. They show inter-species complementation, since they are functionally conserved between different rhizobia. In R. etU nod A is separated by 20 kb from nod BC operon 10, in Rhizobium sp. (Oxytropis aretobia), an arctic rhizobia has nod A operon 4.1 kb upstream of nod BCn operon II, while M. loti has nod B operon separate from nod ACn operon 12. In our laboratory PCR based amplification of common nod genes of M. cieeri has indicated organization similar to M. loti (unpublished).

402 INDIAN J EXP BIOL, MAY 2001

The common nodulation genes are involved in biosynthesis of core lipochitino-oligosaccharide (LCO) molecules from uridyl-diphosphate-N-acetyl glucosamine (UDP-GlcNAc) precursor (Fig.l). UDP­GlcNAc may be used from peptidoglycan layer of cell may be synthesized either by nod M encoded glucosamine synthase or by housekeeping glucosamine synthase (Glm S). Nod C is a membrane bound UDP-GlcNAc transferase similar to chitin synthase 13

•'4 , cellulose synthase lS and Xenopus developmental protein DG_42(refl6). It forms tri, tetra

and pentameric chitin oligosaccharides with 131-4 linkage. Nod B protein is chitin deacetylase of terminal nonreducing GlcNAc residue I7

• Nod A is a N-acyl transferase adding acyl chain to core Nod factor, which may vary in length, degree of unsaturation and both termini of oligosaccharide chain carrying various substituents 18,19.

Nod enzymes are distributed between cytosol and inner membrane, and NF synthesis is likely to occur in enzyme-complexes located at the interface between cytoplasmic membrane and cytosol. Diagrammatic representation of the biosynthesis of a typical Nod factor biosynthesis has been shown in Fig. 1 and role of various nodulation genes in NF biosynthesis is mentioned in Table 1 and modifications occurring thereby are given in Table 2. Host specific nod (hsn) genes encode proteins that modify core Nod factor in host-specific recognizable manner. In addition there are genotype specific nodulation genes (gsn) allowing nodulation only to specific genotype within a given legume species e.g. nod X (R. leguminosarum bvs viceae) and nol A (B. japonicum serocluster 123) 20.

In R. leguminosarum, bv viciae and trifolii, R. etli, B. japonicum, and A. caulinodans nod IJ genes are present and may be present in S. melilotP'. Nod I is a inner membrane A TP binding transport protein and Nod J is a transmembrane protein involved in secretion of Nod factor (NF) at surface22

. In most cases Nod I and Nod J are involved in LCO secretion/excretion as suggested by inability of nod IJ mutant to accumulate NFs externally. However, in some cases nod IJ mutant have moderate effect suggesting existence of alternative transport system 23.

Regulation of nod gene expression--Inducible expression of common nod genes require Nod D (Lys-R family), positive regulator, which binds to a conserved promoter sequence called nod box in presence of host specific flavonoids or chalcones 24.

In most of fast growing rhizobia, nod D genes are expressed constitutively 25, while in some instances

they are autoregulatory 26, inducible27 or subject to repression by Nol R or NH4 28. Nod D homologue Syr M (a symbiotic regulator) is involved both in activation of nod genes and induction of exopolysaccharide biosynthesis 29. Additional nod gene activators include Nol A, a positive regulator in different Bradyrhizobium species, Nol R regulator acting negatively in different Rhizobium species, Nod D2 negative regulator in Rhizobium species NGR-234 and Nod V IW two component flavonoid sensors present in B. japonicum. Possible role of various nod genes products is mentioned in Table 1 and host­specific modifications present in different rhizobia is given in Table 2.

Conventional role of Nod factors in symbiosis Morphological and physiological roles-Cell free

extracts from rhizobia are known to have morphogenetic potential like root hair induction (hai) and hair deformation (had) since beginning of this

Fructose·6-P

INod ~/GlmS Glucosamine-6-P

+ OH

H~CZ OUDP

~o~

1 NHCOCH3

Nod C (UDP-GlcNAc transterase)

OH ~. o OH

HO H3COCHN n NHCOCH3

J Nod B (CMfn deacetylase)

Ho~H 0 OH 0 ~CZOH HO HO ~O~

NH2 H3COCHN NHCOCH3

Acyldonorl

+~"\ 0 o:~:~~=·~~ 0--.

HO HO ~~ ./' NH H3COCHN NHCOCH3

R1 I co

Fig. 1 Biosynthesis of nod factors

CHIMOTE & KASHY AP: LlPOCHITOOLlGOSACCHARIDES & LEGUME RHIZOBIUM 403

century31.32. Agrobacterium transconjugants for sym plasmids were shown to induce thick and short root phenotype33; later on it was shown that the genetic and physiological requirements for had and tsr are same i.e., core unmodified Nod factors34

• Similar reports from our laboratory have shown that Azotobacter chroococcum transconjugants express S. meliloti nod genes to form infection-thread and effective nodules35. Chemically synthesized S. meliloti Nod factors having necessary and a related non-modified LCO molecules have shown that modification is essential for its morphogenetic activity in a specific hose6

•37. Purified

Nod enzymes from cell extracts have been used for in

vitro LCO synthesis/modification. Nod factors have been purified from cell extracts using either butanol extraction38 or by C-18 reverse phase/styrene divinyl benzene cross-polymer columns39. Analytical techniques used to examine the presence of Nod factors include detection by autoradiography of TLC (reverse/direct phase) of C I4 or S35 radiolabelled Nod factors.

Nod factors have been previously believed to control cell division and nodule or~anogenesis by specifying phytohormone synthesis4. It was later proposed that in addition to classical plant hormones, plants may use LCOs to regulate physiological

Table I-Role of nodulation genes 4.30

nod gene Role of gene product nod gene Role of gene product

nod A nodB nodC

nodD

nodE

nodF

nodG

nodH

nodI

nodJ

nodK nodL

nodM nodN nod 0

nodP nodQ

nodR nodS nodT

nodU

nod V

nodW

nod X

nodY nodZ

Acyl transferase Chitin deacetylase UDP-GlcNAc transferase

Lys R type regulator

B-Ketoacyl type regulator

Acyl carrier protein

Homologous to ribito dehydrogenase. proposed to modify acyl chain (hsn) Sulfotransferase (O-substitution at reducing end)

ATP binding protein involved in secretion of Nod factors Membrane protein involved in secretion of Nod factors

6-0-Acetyl transferase at non reducing end

nol A nol B nolC

nol E

nol F

nolG

nol H

noll

nolK

nol L

nolO nol R

D-glucosamine synthase nol T Probably GlcNAc-Phosphate- Uridyl transferase [hsn] nol U Homologous to haemolysin. exported Ca 2+ binding nol V protein ATP sulfurylase nol W .TP sulfurylase lAPS kinase-Two domains with nol X different functions hsn in R. tropici S- adenosyl methionine methyl transferase ecretion of Nod factor (cultivar specific nodulation gene) 6-0-Carbamoyl transferase [O-substitution of reducing end] 2 Component flavonoid sensor

2 Component flavonoid dependent transcriptional activator protein Acetyl transferase at reducing end

hsn gene Fucosyl transferase

nolY nolZ noeC

noeD

noe E

noeK

noeL

syrM

Mer-A type positive transcriptional regulator Negatively acting gm gene in S. fredii Negatively acting genotype specific nodulation gene with homology to dna J Membrane proteins involved in secretion of Nod factor at surface Membrane proteins involved in secretion of Nod factor at surface Membrane proteins involved in secretion of Nod factor at surface Membrane proteins involved in secretion of Nod factor at surface Membrane proteins involved in secretion of Nod factor at surface Sugar epimerase causing O-substitution

Acetyl transferase to fucosyl residue in R. etli and M. loti

Methylation of fucose in B. japonicum Lys R type negative regulator of nod D gene in different Rhizobium species Negatively acting gsn gene in S. fredii Negati vely acting gsn gene in S. fredii Negatively acting gsn gene in S. fredii

Negati vely acting gsn gene in S. fredii Acetyl transferase. negatively acting gsn gene

gsn gene in B. japonicum gsn gene in B. japonicum Arabinosylation in A. caulinodans

Negatively acting gsn gene for soybean

Sulfotransferase to fucosyl residue in Rhizobium sp. NGR-234 Nucleotide sugar epimerasel dehydrogenase

Mannose dehydratase in S. fredii and Rhizobium sp. NGR-234 Inducer dependent transcriptional activator of nod genes

404 INDIAN J EXP mOL, MAY 2001

processes41• Since Ca2

+ is well known secondary messenger in plants, its role in nodulation is implicated based on Ca2

+ spiking in root hair cells and rapid increase in its cytoplasmic concentration in response to Nod factors42

.43.

Purified Nod factors on combined inoculation with nod ABC mutants stimulate depolarizations, root hair formation, and induction of several plant nodulin genes were complemented suggesting that Nod factors permitted entry of nod mutants in roots44

• Nod factors induce cortical cell division as well as nodulin gene expression in spatially controlled manner e.g. ENOD-12 and ENOD-40 are expressed in root primordium and inner cortex, respectively 45. Nod factors seem to alter cytokinin-auxin ratio to influence developmental changes. Induction of isoflavonoid biosynthesis by active Nod factors in alfalfa suspension culture indicates a possible feedback mechanism46

• Some flavonoids capable of inducing nod genes are also auxin transport inhibitors. Auxin transport inhibitors can induce nodule like structures expressing early nodulin in alfalfa47

.48, indicating the role of Nod factors as secondary signal molecules altering endogenous hormonal activities by counteracting auxin transport.

Earlier hypothesis based on alkanization of alfalfa suspension cultures has proposed that cortical cells get enriched in 0-2 phase in response to Nod factors46

.49. However rhizobial infection induced division has not been found correlated with 0-2 phase enrichment5o

• Rapid rearrangement of microtubular cytoskeleton in root hairs takes place in response to Nod factors in Rhizobium-Medicago interaction leading to infection thread formation51

•

RoLes in host pLant recognition--Nod signal recognition has been suggested to occur at two levels;

a low affinity binding (even to unmodified LCOs) for bacterial entry into root hair cells and transient expression of ENOD-40, while the high affinity binding recognizes only modified LCOs to give consistent expression of ENOD-40 triggering plants symbiotic responses of plants 52.53. Role of lectin as a possible Nod signal receptor has been in doubt for lack of direct evidences of Nod factor binding and further signaling inside the cells. Three distinct roles of lectin involved in nodulation are role in adhesion of root hair surface to aggregate rhizobia by associating with cellulose fibrils; to stimulate mitotic activity in nodule primodia by reducing threshold response to rhizobia that interact in nature; and in central mature nodule as a part of transient nitrogen reserve54

.

Transgenic lotus expressing soybean seed lectin gene results in nodule like swellings with B. japonicum 55.

Pea seed lectin gene when introduced into white clover creates a transgenic that responds to non-host LCOs by inducing cortical cell division 56. These studies indicate that expression of exogenous lectin gene somehow lowers the affinity of a Nod signal for host legume.

In addition to lectins-role of host chitinases in directing Nod signal specificity during symbiotic development has been suggested. Purified Nod signals with sulfate at reducing end shows no hydrolysis, while unmodified LCOs are hydrolyzed in alfalfa57

.

Similarly expression of Serratia chitinase gene in S. fredii has been shown to impede nodulation in soybean and alfalfa58

. LCOs induce production of LCO hydrolase, which does not degrade simple chitin. However the biological role of this specific LCO hydrolysis is still unclear59.Differential induction of chitinases (transient expression of class I and II chitinases during early stages, class IV chitinase

Table 2-Host- Specific modifications4,30

Species n Rl R2 R3 R4

S. meliloti 1,2,3 H C 16:2, C 16:3 H, Ac (0-6) Sulfate R. leguminosarum 2,3 H C 18:1, C 18:4 Ac (0-6) H B. japollicum 3 H C 16:0, C 16:1, C H, Ac (0-6) MeFuc

18:1 B. elkanii 2,3 HJMe C 18:1 H, Ac (0-6), Car Fuc, MeFuc R. sp. NGR-234 3 Me C 16:0, C 18: 1 Ac (0-6) MeFuc, AcMeFuc, MeFucS A. caulinodans 2,3 Me C 18:1 H,Car Ara,H R. tropici 3 Me C 18:1 H Sulfate S./reedii 1,2,3 H C 18:1 H Fuc, MeFuc M. loti 3 Me C 18:1 Car (0-4) Ac, Fuc R. tropici 1,2,3 H CI8:1, CI8:2, C 20:3, Ac (0-6) H

C20:4 Rl, R2, R3 and R4 represent modifications in groups as indicated in Fig 1. Similarly n represents number of non terminal glucosamine residues

CHIMOTE & KASHY AP: LIPOCHITOOLIGOSACCHARIDES & LEGUME RHIZOBIUM 405

expression even in matured nodules) was observed during Medicago nodulation60

• Similarly presence of fucosyl group affected the degradation rates and the accessibility of specific cleavage sites on the chitinoligosaccharide backbone to plant chitinase61

• It is possible that a hydrolase produced in response to a specific LCO may provide competitive edge to specific bacteria over another with different LCOs.

Novel role of Nod factors As an endogenous plant hormone-Ongoing

research work may provide evidence for LCO presence in both legumes and non-legumes. Rhizobia have developed into a molecular key to unlock the system of cell-cycle content, which may be common to all plants8

•62

. Earlier Parasponia genus is known to respond to Rhizobium thereby getting symbiotically fixed nitrogen, now LCOs are known to show morphogenetic responses in other non-legumes as well. Oligosaccharides having the potential of great structural diversity are ideally suited for encoding biological information like growth, development and defense even at extremely low concentration63

. Cell wall derived oligosaccharides like XXFG and oligogalacturonides are known to act In a counteracting manner against auxins.

To be a true plant hormone a compound must be endogenously existing in plants and showing plant responses that correlate with its abundance. A number of findings support the view that LCOs are true plant hormone. Presence of plant chitinases in absence of pathogen can be correlated with plant development indicating substrate (LCO) being present in healthy plant64

• Acidic chitinase65 and basic chitinases66 in flowers mainly in pedicel, sepals, anthers and ovaries in tobacco, indicate possible role of chitinases in sexual development of plants67

• Nodulin vf ENOD-32 from broad bean detectable in nodule zone and in much smaller amounts in flowers show homology to (alphalbeta) barrel-type seed protein narbonin and two conserved motifs characteristics of Class-III chitinases68

. Carrot mutants cell line ts-ll with arrested embryo development at 32°C can be rescued to some extent either by supplying Nod factors (NF)69 or purified endochitinases 70. All these findings suggest the presence of endogenous LCO in different part of plants.

Transgenics expressing nod A and nod B genes have been developed in tobacco, arabidopsis and alfalfa, and in all these cases plant morphology is grossly altered pointing towards presence of

endogenous LCOs (Ref. 71-73). Synthetic LCOs eliminate the requirement of auxins and cytokinins to sustain growth of tobacco protoplast culture74

. They have found that LCOs activate auxin responsive promoter and expression ofaxi-l gene indicating shared signal transduction pathway with auxins. LCOs with trans configuration show more activity than their cis counterpart indicating possible role of lipid domain. Nod factors (NF) have been reported to induce cortical cell-division, amyloplast deposition and ENOD-12 expression similar to cytokinin action in alfalfa roots 75. Lathyrus Elants were incubated with C I4 labelled acetate or S 5 labelled sulphate. TLC analysis of n-butanol extracts from flowers on TLC analysis showed that LCOs are present in treated plants 76. Spontaneous nodulation in absence of rhizobia (nar +) under nitrogen starvation in absence of exogenous Nod signals, suggests of an endogenous signal in alfalfa 77.

ENOD-40 nodulin, a peptide (10-13 ala) expressed by action of Nod factor, may be an endogenous plant hormone. It has a significant 3' untranslated region with a possible riboregulator role in initiating plant cell division78

•79

• Tobacco ENOD-40 homolog when applied exogenously to protoplast culture controls resistance to high level of auxins 79.

Rice suspension culture in presence of N-acetyl chitino-oligosaccharide shows many physiological and molecular changes8o

-84

• Chitin-oligosaccharide binding receptors have been identified in tomato, which show competitive inhibition of binding, by derivatives of chitin fragment and Nod factors in suspension culture of tomato cells85

. Similarly ionic changes in legumes root hairs responding to Nod factors were detected86

•

Universal role oj Nod like Jactors-Emergence of similarities between rhizobia-legume symbiosis and the arbuscular mycorrhizal (AM) symbiosis has stimulated investigations regarding expression of nodulin genes during AM symbiosis87

• In Medicago two nodulins, MsENOD-2 and MsENOD-40 have been induced in mycorrhizal root in the same tissue­specific pattern as in case of nodules88

• Both genes can be induced in roots in absence of a symbiosis via cytokinin application, and since cytokinin levels are elevated during both cases of symbiosis, it is speculated that cytokinin is a component during signal transduction leading to symbiosis88

• Studies from our laboratory have shown the presence of S. meliloti nod homologous genes in A. chroococcum and that these

406 INDIAN J EXP BIOL, MAY 2001

genes are expressed at level of root hair deformation on maize and wheat roots89

.90

.

Nod factor appears to be a part of an evolutionary conserved group of developmental regulators. Xenopus laevis DG-42 protein is a highly homologous to Nod C protein91

. In the predicted intracellular domain Nod C shows sequence similarity to other ~ 1---7 4 glycosyl transferases (Chs3, HasA, CeIA, Cps3S, Alg8)92. DG-42 expresses only between late midblastula and neuration stages of embryonic development in Xenopus and catalyzes Nod like chitin oligomer synthesis 17. Recent studies suggest that chitin oligosaccharide may be playing a direct role in zebrafish development. Homologs of DG-42 and Nod C have been found in zebrafish and mice. Microinjection of Nod Z (fucosyl transferase) from B. japonicum or an antibody raised against DG-42 into fertile egg of zebrafish lead to several embryo defects in trunk and tail development93

.

Conclusion Rhizobia interact in host specific manner with

legume host and this specificity is in modification of core Nod factors through the action of different host specific nod genes. Legume-rhizobial symbiosis has largely been viewed for the purpose of strain improvement for harvesting more nitrogen from the atmosphere. A number of findings like role of lectin in deciding the host range property, presence of chitinases in healthy plants, presence of ENOD genes in non legumes and LCO as a plant morphogen suggests that role of LCO is not limited to symbiosis alone. In plants either they themselves act as an endogenous hormone or act via some secondary messenger. All these reports indicate universal distribution of LCO and their derivatives, and their role in development growth regulation in diverse range of organisms. In light of these findings nod genes become even more important in crop improvement than the role they are known to play in legume-rhizobial symbiosis.

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