Summary The regulatory NodD proteins of Rhizobium bacteria mediate the activation of a gene set responsible for symbiotic nodule formation by plant signal molecules. Here we discuss the signal recognition and gene activation properties of NodD and present a model summarizing the current knowledge on NodD action. Introduction The processing of extracellular signals in living cells involves the perception of the signal, the transduction of the message and the generation of a cellular response. These events are frequently attained by sensory and regulatory proteins whose action results in switching on specific effector genes. In some cases. both the environmental receptor and the gene activator functions are harbored by a single polypeptide. Such sensor-activator proteins arc present in all types of organisms, from humans to the soil bacteria of the genus ~h izob i ~ m ( l - ~ ) . Rhizobia establish endosymbiosis with a number of dicotyledonous plants, mainly with legumes. The bacteria develop and invade soot nodules on appropriate plants. In the nodule, rhizobia differentiate and fix atmospheric nitrogen which is utilized by the host as a source of combined nitrogen. In turn, the host partner provides metabolites for the bacteria. A set of coordinately regulated Rhizobium genes organized into several transcriptional units control nodule induct i ~ n ( ~(Fig. - ~ ) 1). This regulatory circuit, the so-called nod regulon, is located either on large indigcnous plasmids (in Rhizobiurn) or on the chromosome (in

Hmdyrhizobium and Azorhizobium). One gene set, the ‘common’ nod genes (e.g. nodABC), is essential for nodulation and is conserved among different rhizobia with respect to thcir function and DNA structure. Other nod genes determine the host specificity of nodulation (e.g. nodFEGPQ, nodH) and generally can not be interchanged between species. Recent studies(’”) revealed that the common nod gene products are responsible for the synthesis of an oligosaccharide which is further modified by the products of the host specificity genes(’*)).The completed molecule serves as a return signal for the plant to elicit the first noticeable symbiotic responses, root hair curling and induction of cortical cell division(5). The expression of nod genes requires the regulatory nodD gene and plant-derived signal moleculcs(’1-14). The NodD protein has a crucial role both in plant signal recognition and nodulation gene activation. Here we survey the structural and functional characteristics of NodD that probably enable it to act as the key regulator of the conversion of Rhizobium into its symbiotic form. Organization, Regulation and Function of the nodD Gene Family Various rhizobia infecting a diverse range of host plants contain one to three copies of the regulatory nodD gene(”,11-15) (Table 1). The majority of the nodD alleles are located within the nod gene cluster (albeit some are more distantly linked; Fig. 1) and share 70-90 % homology with each other. Mutants lacking a functional nodD gene are unable to switch on the nod regulon and consequently do not nodulate the host plant(y3.12.13) The level of nodD expression is under the control of a complex set of environmental and intracellular factors. On the whole, nodD is constitutively expressed(”-’3). Beyond this, certain nodD alleles are diverged with respect to the regulation of their transcription br plant signal ~ o m p o u n d s ( l ~ -combined ~~), nitrogen(’7, 9, and regulator} proteins including the activator protein SyrM(’*),the nod repressor protein(16)and NodD itself (autoreg~lation)(’~’~”~~~.~~) (Table 1). The NodD protein is classified by homology as a member of the LysR bacterial gene activator protein family(’). NodD switches on nod genes in the presence

Rm

Fig. 1. Organization of the nod regulon in Rhizobizim meliloti (Rm), R. leguminosarum biovar vicirie (Rlv) and Bradyrhizobium juponicuna (Bj)(4-5’. All genes are nod unless otherwise iiidicated (newly described nod genes are designated as no1 genes). The position of the nod,!, gcne of R . meliloti is from N. Baev (unpublished work from our laboratory). Genes are not drawn to scale: broken vertical lines indicate larger interruptions in the map. Full triangles stand for cis-regulatory sequences (rtod-box).

nudD regulation nodD

Species Rhizobiurn rneliloti leguminorarum bv. rrijiolii bv. viciae hv. phaseoli

fredii NGR234, MPIK3030 Bradyrhizobiirm japoniciirn

Host plants

allele

positive

negative

Natural nod inducers

Medicugo, 1liZelilotus. Trigonella

1

-

2

R R N

L,OF,ON,CR,CH CH L,OF

-

ND ND

OF,GR OM,30M,A

3

Trifdiirrn

Glyririe etc. tropical legumes

1 1* 1 2 3 1" 1*

Glyrirze etc.

1"

Vicia, Pisurn

Pliaseolus

ND,M,S -

-

ND,S -

~

? ? ?

-

-

?

'?

-

-

D,GN D,GN,N,CM

ND,S

N

D.GN

No relevant data are available on riodD genes from rhizobia infecting Lotus (Khizobium loti), Lupims ( R . lupini), Galega (R. gulegae), Paraspotzia (Bradyrhizobiunz paraspoiziae) and Sesbunia (Azorhizobium caulitzoduns) plants. *, One additional nodD copy with unknown function is detcctcd. Abbrcviations for rcgulatory factors are: ND, NodD protcin ( a u t o r c g ~ l a t i o n ) ' ~ ' ~; R. ~ ~ nod ~ ' ~ ~repressor ~"~ protein""): M, SyrM protein('*); N. combined nitrogen(".19); S, plant signal compounds("-'-'); no known regulation. Abbreviations for nod gene inducers isolated from host plant^('^-*^) are: A: apigenin-7-0-glucoside; CH, 4.4'-hydroxy-2'-methoxychalcone:CM, coumestrol: CK. chrysoeriol; D. daidzein: GN, genistein: GR, geraldone; L. luteolin; N, naringenin; OF, 7,4'-hydroxyflavone: ON, 7,4'-hydroxyflavaiione: OM, 7.3'-hydroxy-4'-methoxyflavanone; 3 0 M . 3,5.7,3'-hydroxy-~'-methox~fla~~anone. ?. n o data available.

-.

of plant-derived signal molecules, usually flavono id^("-^^). The nodD genes can exhibit the characteristics of both the common and host specific nodulation genes. In certain cases nodD genes of different origin can be interchanged without loss of function(" 18). In other examples interspecific complementation with nodD genes was not detectable(2s). Extension of the nodulation ability to non-host plants could. however, be achieved bv the transfer of foreign or mutated nodD It was shown that different NodD proteins are diverged in plant signal specificity but have the common ability to activate nod As plants release different sets of signal compounds (Table 1). NodD proteins can function only when invading certain hosts(" &'l) . Th'IS feature explains why the interchangeability of different nodD genes depends on the actual combination of the nndD alleles and the host plant. Also, the differential signal recognition provides the NodD homologs with the the primary level oi host

(Table 1) were various flavonoids belonging to the rubclasses of flavones, flavanones, isoflavones and chalcones (Fig. 2). Compounds of the minor flavonoid subclass, aurones, also have the ability to stimulate nod gene expression('4). The nod gene induction process can also be inhibited by substances having a structure similar to the inducer^(^"^^^",^^). Different flavonoid molecules c,aq act synergistically to enhance nod gene i n d u ~ t i o n ( ' ~ ~ Flavonoids ~~). exert their nod gene regulatory effects at nanomolar concentrations(z4). Extensive genetic studies in recent years have not identified any additional gene which contributes to the In) . flavonoid transduction in Rhizobium except n ~ d D ( ~ spite of this, the binding of flavonoids to the NodD protein has not been formally proven yet. However, much genetic evidence suggests that NodD is the primary site of the flavonoid effect on nod gene Single amino acid substie~pression(~3~ tutions can severely perturb the nod gene induction ability of NodD either positively or ne atively and can alter both signal and host specificity(3, 3 6 ) . Moreover, signal specificity can be transferred between different rhizobia by exchanging parts of NodD(11,29,37). Signal specificity of NodD means that a definite range of substances promotes or supprcsses its nod gene induction ability. Signal compounds can act either as inducer. anti-inducer (inhibitor) or can be ineffective depending on the NodD rotein homolog present in the tested ce11(".'?,1",15-21-?8%.3j.3~) (Fig. 2). NodD homologs can be classified according to their responses to signal molecules. The NodD proteins of rhizobia with broad host range (e.g. Rhizobium spp. NGR234, MPIK3030, R . fredii and Bradyrhizobiurn 1131331h,2X,33).

8

Specificity of NodD Proteins with Signal Compounds Flavonoids are a broad class of plant secondary metabolites exhibiting versatile biolo ical activities in microbial, plant and animal systems(32. Their similarity to the structure of numerous biological1 active molecules, for example steroid hormonesg'), can provide flavonoids with the ability to occupy binding sites of molecules which are crucial in the communication networks of various cell types. The natural inducers of nod genes isolated from host

?

@Qa yJ 0

0

f lavone

f lavanone

i s o f lavone

Nod rcgulatory cffcct in the prcwnce of NodD of Rhizobirinz

leguminosarum 3

5

I

3'

-

-

OH

-

~

-

-

OH ~

OH

OH

OH

OH

bv .

meliloti

Substitution pattern

OH

OH

OH

OH

~

-

-

-

3' ~

-

OH

OH

OH

Ring type

1

2

3

trifdii

viciae

frudii

MPTK3030

flavone isoflavone

A

A Iw

15

Is

?

IS

E

Iw A

E

?

Is

Is

flavone isoflavone flavone iwflavone

A A

A A

E

Iw E

Iw E

IS

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Iw E

Iw A

Iw

Is

1

Is

A

A

A

Is

flavone isoflavone flavanone

Is A A

Iw A A

I\ A Iw

I\ E Is

Is A Is

Is

flavone flavanone

A Iw

A A

E

A

A

Iw-

A

IS

TW

?

Is Is

Iw

Is

Is Is

Is Is Is Is Is

OH

OH

OH

OH

tlavone

Is

E

Is

IS

Is

TS

Is

OH

OH

OH

OH

flavone

E

E

E

E

E

Iw

IS

Fig. 2. Specificit of NodD proteins with flavonoids. The effects of flavonoids 011 riod gene expression are dcrived from in vivo stUd~es(".'2,'4.15.~-~ 18,34,35,38) where strains were monitored for the activity of nod-lucZ reporter gene fusions in the presence of the

specified NodD proleins and flavonoids. Abbreviations for regulatory effects are: Is. inducer (strong); Iw, inducer (w-eak; less than 25 Yo of maximum cffcct): A , anti-inducer; E, ineffective; ? , no data available. OH, hydroxyl substitution at the given ring position.

japonicum) are res onsive to a wide range of as well. We considered that the divergences between corn pound^(^^^'^,^^,^^,P4,38) (Fig. 2). The presence of a NodD proteins with respect to their flavonoid specihydroxyl moiety at the C7 atom of the flavonoid ficity (Fig. 2) might have an imprint on the structure of skeleton is sufficient for activating NodD of their signal binding domain. Dendrograms'") expressMPIK3030(34).Even monocyclic aromatic compounds ing the relationships among seven NodD homologs are (e. g. vanilline) are inducers in conjunction with the presented in Fig. 3A. The dendrogram based on signal NodD of strain NGR234(3s). specificity shows more similarity to the dendrogram By contrast, the NodD proteins of the narrow host established for the C-terminal sequences than to that range bacterium R . meliloii need substitutions also at for the N-terminal ones (Fig. 3A). The maximum ,~~) the C5 and C4' positions for their a c t i ~ i t y ( ~ ' , ~ " , " , ~ ~correlation between signal specificity and protein (Fig. 2). Isoflavones act as inducers in conjunction with sequence delimits a segment of NodD between amino the promiscuous NodD type while they are inactive or acids 154 and 217 (Fig. 3A). inhibitory with the R . nzeliloti NodD homoSeveral other lines of evidence similarly suggest that logs(" 21,24-27,34,35 38) . As an exception, NodD3 of R . the C-terminal part of NodD takes part in the meliloti can accept C7-hydroxylated isoflavones which interaction with the signal molecules. The vast majority lack a 4' hydroxyl group for induction(34)(Fig. 2). The of the positions where amino acids are strongly variable NodD proteins of the R. leguminosarum biovars viciae between vanow NodD species are located in the and trifolii have similar specificities and are intcrmedi(Fig. 3B). ReplaceC-terminal half of the ates between the extreme broad and narrow speciment of the C-terminal 234 amino acids of the ficities mentioned above(1422-24) (Fig. 2). MPIK3030 nodD gene with the corresponding part of the R . meliloti nodDl allele resulted in R . meliloti-type flavonoid recognition and host range properties of the Domain Topology of NodD strain carrying the chimaeric NodD protein("). Most Multifunctional proteins usually have se arate domains alterations that affect the nod gene inducing ability of responsible for their distinct activitiesP','). This situNodD are located downstream of amino acid ation is expected for the sensor-activator NodD protein 132('" 3h,37). Some of these mutant NodD proteins were

A

amino acids 1-100

amino acids 101-300

R.I. bv. trifolii R. I. bv. viciae

f Iovonoid spec i f icit y

R. meliloti 3 R . meliloti 2 R. meliloti 1

R. meliloti 1 R . sp. MPIKM30

R . sp. MPIK3030

I

I

-0.32

I

0.35

1

0.67

0.45

I

I

0.21

B 1

C

-

Binding of DNA Homoloav to .v.

.

- - - - - - -- - - - _ _ _ LysR f a m i l y

150

100

200

-- - - - - - - - --_ - -- ----nuclear

r e c e i v e r modules

-

300 amino acids

Symbols used in panel

membrane

- - - - - - _ _ c - - - - - - _ _ _ _ _ _ _

is0

receptors

B

Variable features : r---l L--. length of C-terminus 7 amino acid Conformations: helical extended 0coil o turn Mutations affecting: V autoregulation V nod gene induction

Fig. 3. Functioiial dissectioii of the h’odD rotein. A. Relationship between lhe divergences observed in the flavonoid specificity and tlic sequences of NodD proteins. Seven NodD8’:’.“’) types were compared pairwise and the numbers 01 the identical types of interactions with flavonoids (as listed in Fig. 2) or the degree of homology bctwceii the aligned‘qu”sequences in various regions wcrc calculated. The resulting similarity matrices were used to creatc relationship dendrograms by the hierarchical clustcring method(39)or to calculate correlation coefficients (numbers) between flavonoid specificity and the sequence of five regions as shown along the line representing NodD. R. A map of NodD. The broad bar represents the NodD protein with its consensus secondary structure as predicted(“) for Seven NodD homologs(ll.m). Symbols used arc defined in the Figure. The mutations indicated by triangles are described in refs. 30, 36, 37. C. Functio~ialmotifs in NodD and its rcgions sharing homology with other regulatory proteii~s(~,””.”~.”),

defective in flavonoid-mediated nod gene induction while others were able to stimulate expression even in the absence of inducers(”’.37). Secondary structure prediction (Fig. 3B) revealed that two NodD regions with a beta-turn-beta conformation (amino acids 172-188 and 222-245) might form a ligand binding crcvicc, as reported for various proteins(42).The presence of a membrane associated hydrophobic alpha helix is predicted between the NodD residues 101 and 117(”) (Fig. 3C). In accordance with this inference, the NodD protein was found to be localized in the inner membrane fraction of the cell(4’). Two segments in the middle part of NodD (amino acids 109-171 and 180-216; Fig. 3C) share 54% homology with conserved parts of the vertebrate nuclear receptors(33).The nuclear receptors transduce extracellular signals in a way analogous to NodDc2’and one of them, the estrogen receptor, can accept some flavonoids as ligands. Conversely, estradiol and phytoestrogens can interact with the NodD of MP1K3030(3’). The regions of nuclear receptors homologous with NodD were shown to take part in ligand A region in the N-terminal half of NodD (Fig. 3C) is similar in structure to the so-called receiver module found in many bacterial regulatory proteins(’”). This

motif is thought to be involved in protein-protein communications(“). The hypothesis that NodD contains a receiver module raises the possibility that NodD interacts with unidentified protein factor(s) or forms multimeric associations with other NodD molecules. Mutations affecting the autoregulatory ability of NodD were localized in the N-terminal part of the protein(30.3”37) (Fig. 3B). A helix-turn-helix motif is predicted in the same segment of NodD (Fig. 3C) that might be involved in DNA-binding and is also present in other LysR-type protein^(^.^'). Although the above mentioned experimental and deductive evidence assigns the flavonoid- and DNA-binding functions of NodD to its C- and N-terminal parts, respectively, some determinants of these activities seem to be dispersed along the sequence of the pr~tein(~’,~’) (Fig. 3B). The segments not closely linked in the primary sequence of NodD could. however, interact physically by protein folding to participate in common functions. A Model for Induction of nod Genes by NodD and Plant Signals A model describing the signaling and nod gene regulatory action of PJodD is presented in Fig. 4. The model incorporates assumptions based on published experimental results, but many details of the model

nod01

nodABC

nodD2

nod03

. I I -

1

ci] [11

Plant signal set non binding f L avo no id

I:

inhibitor for NodDl inducer for Nod03 inducer for NodDl

NodD p o o l

or

a

NodD non-activated

+

nod gene switched off

nod

repressor

0

RNA

or

nod gene induced

NodD activated

- -1 _-_--__-__-___---

I i

&

r---

I I

-- --- --

I-_

protection by

[NodDI

I I I I

--- -- ---

---1

r _ _ _ _ _ _ _ _ _ _

hod repressor' I_ _ _ _ _ _ _ _ - - I

IWWATCCATAGWGTGZATGATTGCN]ATCCAAA (CAATCGATTTTACCAATCTTNU."]

f I

--__----------A

p.hEN

LAIT_nGAGAACC_CT~A~l

Fig. 4. A model for Nodl) action. The case of R. meliloti stmin 41 is depicted('6!. At the bottom the consensus nod-hox s e q ~ e n c e ( ~ and ~ ~ 'the ) R . meliloti nod repressor binding site('" are shown. See text for further explanation.

need further support. Below we discuss the three major steps of nod gene induction.

DNA binding NodD binds to a conserved DNA sequence 47 basepairs long, the so-called nod-box, which is found in front of all known inducible nod operons(16,4hp4x) (Fig. 1). In R. meliloti, the nod-box sequences are located 26-28 basepairs upstream of the nod transcriptional units(49). In vitvo studies suggest that NodD can also bind to the nod-box in the absence of an ind~cer(~""~")but this finding was not confirmed in viuo. In most R . meliloti strains, a negative regulatory factor, the nod repressor protein binds to the transcriptional start sites of the nodDZ and nodD2 genes(16)(Fig. 4). The nod repressor weakens the binding of NodD to the nod-box presumably by competition with RNA polymerase, hence decreasing the expression of nodD and inhibiting nod gene induction(16).

Interaction with signal molecules The expression of nodD gene(s) results in a homo- or heterogeneous pool of NodD proteins de ending on the number of nodD copies in the strain(' ,21,31) (Fig. 4). NodD proteins can presumably be conceived as guards standing sentry in the inner membrane for the appearance of signal molecules(43).Flavonoids accumulate in the inner membrane(45)that can be the site of their interaction with N o ~ D ( " ~Depending ). on the available set of signal molecules. the putative ligand binding site of NodD proteins could be occupied by either inducers or anti-inducers and could also remain unoccupied if there are no available signal molecules ~ ~ " ~ ~of~nod ) transcription able to bind to the NodD form in q ~ e s t i o n ( ~ ~ ~Induction (Fig. 4). NodD molecules failing to meet a signal It has been suggested that upon the putative binding of compound or interacting with an anti-inducer are an inducer molecule to NodD a conformational shift in supposed to retain their original c o n f ~ r m a t i o n ( l ~ , ~ the ~ ) . tertiary structure of the protein might take This conformation would not stimulate transcription of place(14316)). The hypothetical activation event might nod but presumably competes with the exclude the bindin of the nod repressor and promote flavonoid-activated form of NodD for binding to the RNA polymerasefL6) to start up the coordinate inducible nod prornoters(16)(Fig. 4). transcription of the nod r e g ~ l o n ( (Fig. ~ ~ ) 4).

2

Conclusions Transcriptional regulation of nod genes is delicately balanced by a number of factors. Among these the NodD protein is a key element which decides whether the situation is appropriate to convert the developmental status of the bacterial cell for establishing an endosymbiosis with a plant. Regulation of nod genes by NodD and plant signals is a useful model system in studying the transduction of external signals in the developmental control of living cells. As an example, the functional and structural relationship detected between NodD and vertebrale nuclear receptors(33) might help to provide a better understanding of the general mechanism of signal transduction. Future studies will certainly extend and modify our current views on NodD function but will not shake our conviction that the NodD protein will prove attractive to many molecular biologists. Acknowledgements We thank J. Gausz for discussions on the manuscript, and G. Nolienburg, B. Dusha and A. Borka for preparing the illustrations. This work was supported by grants OTKA553, OKKFT(Tt)/1986 and OMFB. and by thc "Dr. Janos BBstyai Holczer" Fund. References 1 SIULK, J. B.: STUCK, A . M . ANDMoTTONFN,J. M. (1990). Signal transduction in bacteria. Nu:we 344. 395-400. 2 EWNS, R . M. (1988). The steroid and thyroid hormone receptor , C ~ t i v oJ,. M. AND WALLACL, J . C. (1988). A large family of bacterial activator proteins. Proc. , k ? l Acnd. Sci. U S A 85, 6602-6606. 4 KoNDOROSt, E. A N D KoNDoRosl, A. (1986). Nodule induction on plant roots by Hhizobium. Trends Riochem. Sci. 11, 296-299. 5 LONG,S. R. (1989). Rhizobium-legume nodulation: life together i n the

underground. Cell 5 6 , 203-214. 6 WiNSoR. B. A. T. (1989). A nod at differentiation: the rzodD gene product and inititation of Rhi?obium nodulation. Trend.7 Biochem. Sci. 5, 199-201. 7 KODRIGUEZ-QL-INONES. F., BANFALVI. Z. . MURPHY.P. AND K~NDOROSI, A. (1987). Interspecies homology of nodulation gene5 in Rhizobium. Plant izZul. Bid. 8, 61-75. 8 G~TTFERT. M.. HORVATH, B.. KOYD~ROSI, E., PLWNOKY. P.. RODRIGUEZF. ~ X DKONDOROSI. A. (1986). At least two nodD genes are QUINONES, necessary for efficient nodulation on alfalfa by Rhizobium melilori. J . Mol. B i d 191. 411-420. 9 HONMA, M. A.

AND AUSCBLL, 1:. M. (1987). Rhizohium nzeliloti has three functional copies of the nodD symbiotic regulatory gene. Pro