Plant Molecular Biology 4:147-160 (1985). �9 Martinus N(jhoJf/Dr W. Junk Publishers, Dordrecht. Printed in the Netherlands.

Rhizobium nodulation genes involved inroot hair curling (Hac) are functionally conserved

M. A. Djordjevic, P. R. Schofield 1, R. W. Ridge, N. A. Morrison, B. J. Bassam, J. Plazinski, J. M. Watson I & B. G. Rolfe Department of Genetics and ICentre for Recombinant DNA Research, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra City, 2601, Australia

Keywords: Rhizobium Nod genes, functional conservation

Summary

Five specific transposon-induced nodulation defective ( N o d ) mutants from different fast-growing species of Rhizobium were used as the recipients for the transfer of each of several endogenous Sym(biosis) plasmids or for recombinant plasmids that encode early nodulation and host-specificity functions. The Nod- mutants were derived from R. trifoliL R. meliloti and from a broad-host-range Rhizobium strain which is able to nodulate both cowpea (tropical) legumes and the non-legume Parasponia. These mutants had several common features (a), they were Nod on all their known plant hosts, (b), they could not induce root hair curling ( H a c ) and (c), the mutations were all located on the endogenous Sym-plasmid of the respective strain. Transfer to these mutants of Sym plasmids (or recombinant plasmids) encoding heterologous information for clover nodulation (pBR1AN, pRt032, pRt038), for pea nodulation (pJB5JI, pRLIJI: :Tn1831), for lucerne nodulation (pRmSL26), or for the nodulation of both tropical legumes and non-legumes (pNM4AN), was able to restore root hair curling capacity and in most cases, nodulation capacity of the original plant host(s). This demonstrated a functional conservation of at least some. genes involved in root hair curling. Positive hybridization between Nod DNA sequences from R. trifolii and from a br~ad-host-range Rhizobium strain (ANU240) was obtained to other fast-growing Rhizobium strains. These results indicate that at least some of the early nodulation functions are common in a broad spectrum of Rhizobium strains.

Introduction

The Rhizobium-legume symbiosis involves a complex interaction between procaryotic and euca- ryotic organisms. Root nodules form as a result of a multi-step process in which the Rhizobium strain may fix atmospheric nitrogen and reduce it to am- monia (32, 24). Rhizobium mutants defective in var- ious steps of the symbiotic interaction have been sought by using transposon mutagenesis, usually with the drug-resistance transposon Tn5 (4, 25, 28). The results of various research groups indicate that many nodulation(Nod) and nitrogen-fixation genes (Nif and Fix) occur on a single plasmid species in a broad range of fast-growing Rhizobium strains. This plasmid has been designated a Sym(biosis)

plasmid (12). Mapping and hybridization studies, using different Rhizobium derivatives which are defective in their nodulation ability and their Sym plasmid content, have shown that Nod, Nif, and Fix genes are linked in one region of the Sym plas- mid and are separated by about 20 kb of DNA. This occurs in R. trif olii ( 26, 33), R. leguminosarum (11, 8), and 'R , meliloti (2, 17) strains. Conjugation experiments using either self-transmissible Sym plasmids or recombinant plasmids which contain segments o fSym plasmid DNA have indicated that genes which influence the host-specificity of the bacterium may also reside in the Nod region and that they could be closely linked (9,27).

Recently, the resident Sym plasmid in a fast- growing Rhizobium strain (ANU240) was identi-

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fled (19). This strain is able to nodulate a variety of tropical legumes and the non-legume Parasponia. Mobilization of a derivative of the ANU240 Sym plasmid, designated pNM4AN, to temperate fast- growing Rh&obium strains and to Agrobacterium tumefaciens resulted in the transfer of the host range properties of strain ANU240 to these strains (20).

In previous reports we have shown that transpo- son mutagenesis could be used to identify Nod genes in R. trifolii which occur on the Sym plasmid (28, 26). All Nod mutants isolated to date map in a small region of the Sym plasmid in R. trifolii strain ANU843 (P. R. Schofield, M. A. Djordjevic unpub- lished). A 14 kb DNA fragment which encompasses the sites of the Nod ::Tn5 mutations in R. trifolii was cloned (33, 27). Transfer of this 14 kb segment (plasmid pRt032) to other Rhizobium strains and to A. tumefaciens, resulted in these transconjugants being able to nodulate clovers suggesting genes which determine the clover host-specificity are also located on this segment of DNA (33, 27). Similar experiments with cloned DNA fragments from the R. leguminosarum Sym plasmid, pRL1JI , have shown that nodulation and host-range genes occur on a 10 kb segment of DNA on this plasmid (9). Derivatives of plasmid p R L I J I and plasmid pRmSL26 (17), the latter of which contains many of the early Nod genes from the R. meliloti strain 1021, are also used in this study.

The possibility that some genes involved in nodu- lation are common was first reported by Banfalvi et al. (1). They showed that an R. leguminosarum Sym plasmid, pJB5JI, could restore lucerne nodu- lation ability to a R. meliloti mutant (ZB 157) which possessed a Sym plasmid deletion. The significance of this result was later enhanced by the more precise mapping of the deletion in strain ZB157 (16). Kon- dorosi et al. (16) have also shown that the nodula- tion ability of several Nod mutants can be restored by the transfer to these strains of homologous Nod information (pKSK5) and by heterologous Nod information (pJB5J1). In addition, cross hybridiza- tion between DNA from the R. melilotiNod region and Sym plasmid sequences from a broad-host- range Rhizobium strain MPIK3030 (apparently identical to ANU240) was also demonstrated (2l). These fast-growing strains (MPIK3030 and ANU240) are able to nodulate a wide range of tropical legumes and soybeans (14, 19, 20, 21).

Via the introduction of heterologous Rhizobium nodulation genes, we describe the correction of spe- cific Nod mutants in a variety of Rhizobium strains. Since positive hybridization was also ob- tained between the Nod DNA segments from var- ious sources, this functional conservation of Nod genes probably reflects conservation at the DNA sequence level. Thus this study provides further support for the notion that there is a functional conservation of at least some early Nod genes in various fast-growing Rhizobium strains.

Materials and methods

Strains used. Lists of donor and recipient strains used are given in Tables 1 and 2 respectively. Fig. 4 lists the plasmids used for DNA hybridization analysis.

Plasmid transfers. Plasmids were transferred to the recipient strains using a patch mating technique (7). When no direct selection for plasmid transfer was possible (for example when transferring a Kin- resistance plasmid to a Km r strain) a plant selection method was employed (3). These matings were ar- ranged so that neither the donor nor the recipient strain was able to nodulate the inoculated plant host. Only the transconjugant strain possessing the transferred plasmid could nodulate if correction of the Nod defect occurred.

Transfer of plasmid pNM4AN (Cm r, Km r) to other Rhizobium strains was mediated by the Inc P-1 plasmid, pJB3JI (14) which was present in the donor strain used (see Table 1). Since the expres- sion of Cm r was sometimes unreliable, transfer of Tc r (located on pJB3JI) was selected instead. High mobilization rates ofplasmid pNM4AN by pJB3JI have been demonstrated (20) so that the majority of Tc r transconjugants possessed plasmid pNM4AN.

Plasmid pRmSL26 (Tc9 was mobilized using a triparental mating with E. coli strain H B I 01, carry- ing plasmid pRK2013 (5) as described by Long et al. (17). Plasmid pBR1AN (Km") (7) and pJB5JI (Kin ~) (13) are self-transmissible among the Rhizo- biaceae.

Plasmid pRt032 (Cb9 (26, 27) contains a 14 kb HindIII segment of DNA from the Nod region of the R. trifoliistrain ANU843. This DNA segment is cloned into a self-transmissible derivative of

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Table 1. A list of donor strains used in this study.

Strain Plasmid characteristics Ref. or source

ANU618 I

ANU987

ANU 11092

ANU6171

ANUl122

ANUI072

ANU 11263

pBR 1 AN (K m r, Tra +) R. trifolii pSym (230 kb) 7

pRt032 (Cb r, Tra +) cloned R. trifolii Nod genes (14 kb insert) 27

pRt038 (Km r, Cb r, Tc r, Tra +) carries some R. trifolii Nod genes 26

pJB5Jl (Km r, Tra +) R. leguminosarum pSym (210 kb) 13

pRLIJI::TnI831 (SpL Tra +) R. leguminosarum pSym (210 kb)

pRmSL26 (Tc r, Mob +) R. meliloti Nod plasmid

pNM4AN (Cm r, Km r, Mob +) ANU240 pSym + pJB3JI 20

C. A. Wijffelman

17

i The background strain for these plasmids is the Nod (zxpSym) R. leguminosarum strain, ANU615, (Sm r, Rif r, trp, phe).

2 pRt038 is an RP4 derivative containing a 7.2 kb EcoR1 R. trifolii Nod DNA insert.

3 Plasmid pNM4AN possesses a cointergrate of plasmid pSUPI011 (Cm r, Km r Mob + ) (29). Plasmid pJB3Jl (a Km s R68.45 derivative) mobilizes plasmid pNM4AN at high rates (20).

p K T 2 4 0 (M. B a g d a s a r i a n , p e r s o n a l c o m m u n i c a -

t ion) . P l a s m i d p R t 0 3 2 is se l f - t ransmiss ib le at h igh

rates to R h i z o b i u m . P l a s m i d pRt038 (Tc r, K m r,

C b r) c o n t a i n s a 7.2 kb E c o R 1 D N A f r a g m e n t

c l o n e d in to p l a s m i d R P 4 (26). Th is 7.2 kb E c o R 1 f r a g m e n t lies w h o l l y w i th in t he 14 kb R. tr i fo l i i H i n d l I I f r a g m e n t o f p l a s m i d pRt032 , but it does

no t ca r ry all the R. tr i fol i i N o d genes f r o m s t ra in

A N U 8 4 3 .

M i n i m a l m e d i u m (23) s u p p l e m e n t e d wi th an t ib i -

ot ics was used to se lect R h i z o b i u r n t r a n s c o n j u g a n t

s t ra ins as mos t d o n o r s t ra ins used were a u x o t r o p h -

ic. A n t i b i o t i c c o n c e n t r a t i o n s ( # g / m l ) used were:

Km-150 , Sp 150, T c l ( fo r A N U 7 9 4 der iva t ives ) Tc4

( for A N U 8 4 3 d e r i v a t i v e s ) , C m 15, Cb 75.

Plant tests. N o d u l a t i o n o f wh i t e c lovers ( T r i f o l i u m repens, N Z whi t e c love r 5826), s u b t e r r a n e a n c lo-

vers (T. s u b t e r r a n e u m , M t B a r k e r var ie ty) , s i ra t ro

( M a c r o p t i l i u m a t r o p u r p u r e u m ) and luce rne ( M e - d icago sat iva, H u n t e r R i v e r va r ie ty ) were d o n e us-

ing the r a p i d p l a t e assay sys t em o f R o l f e et al. (23)

on m o d i f i e d F~trhaeus m e d i u m (31). Tes ts fo r pea

n o d u t a t i o n and the g r o w t h c o n d i t i o n s used fo r the

v a r i o u s test p lan ts were desc r ibed p rev ious ly (23).

R o o t ha i r cu r l ing assays were as dec r ibed p r e v i o u s -

ly (17). T h e c r i te r ia used to j u d g e the ex ten t o f r o o t

ha i r cu r l i ng on the v a r i o u s p lan t s used were the

Table2. Characteristics of Rhizobium recipients and other strains used.

Strain Relevant characteristics ,~ Ref. or source

R. trifolii ANU851 ANU891 ANU453 ANU845 ANU871

R. meliloti ZBI57 1126

cowpea Rhizobium ANU240 ANUI255 ANUI256 ANU265

Other strains ANUI046 ANU 100

Nod ::Tn5 (Hac-) from ANU843 l Nod ::Tn5 (Hac) from ANU8431 Nod ::Tn5 (Hac) from ANU794 pSym Nod (Hac) from ANU843 ApSym Nod- (Hac) from ANU851

(=ANUI000) ApSym Nod (Hac-) from Rm41 (=ANU1071) Nod ::Mu-Tn5 (Hac) from 1021

Smr NGR234 Nod-::Tn5 (Hac'-) from ANU240 Spectinomycin-resistant ANU 1255 pSym- Nod-(Hac) from ANU240

(=USDA205) Nod + (soybeans) (=IHP100) Nod + (pigeon peas)

28 P. R. Schofield 7

26 26

1 17

19 20 N. A. Morrison 19

H. H. Keyser P. J. Dart

I The Tn5 insertions in strains ANU851 and ANU891 are approximately 1.7 kb apart. Preliminary data indicates that these Tn5 insertion lie in different genes (M. A. Djordjevic and P. R. Schofield, unpublished).

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same as those used by Yao and Vincent (34, 35). A marked hair curling response indicates that highly distorted root hairs were prominent on the plants used and were easily found. M i n o r (infrequent) distortions or waving of the root hairs which oc- curred in some cases after inoculation of a Sym plasmid-cured Rhizobium strain on its original host plant were not regarded as being significant as these effects could occasionally be seen on uninocu- lated control plants.

Filter hybridizations. All hybridizations were done at 65 ~ to restriction endonuclease digests of pRmSL26 (which contains the nodulation region of R. meliloti) and pRt572 (which contains a substan- tial port ion of the R. trifolii nodulation genes). The probe DNA sources were either from the R. trifolii nodulation region or from part of the nodulefion region of strain ANU240 (pRs23). Primed DNA synthesis with DNA polymerase I (Klenow frag- ment) was used to make hybridization probes (28). Probes were hybridized for 18 h at 65 ~ washed at least 4 times in 2 X SSC, and were exposed for 2-6 days at 80 ~ with a Du Pont Lightning plus inten- sifying screen to X-ray film (Kodak type XAR-5).

Plasmid visualizations. The method of Eckhardt (10), with modifications, (6) was used to examine the plasmid profiles of Rhizobium strains.

Plasmid isolation. Recombinant plasmid DNA and genomic DNA were isolated as previously de- scribed (26). Plasmids were digested with various restriction enzymes which were purchased from Boehringer-Mannheim and used according to the manufacturer 's recommendations. DNA samples were separated by gel electrophoresis and where applicable these DNAs were transferred to nitrocel- lulose filters according to the method of Southern (30).

Preparation of nodule sections for light microsco- py. Specimens for light microscopy were fixed in 2.5% glutaraldehyde/2.0% paraformaldehyde in 25 mM phosphate buffer (pH 6.8) for 6 h, post-fixed (after 3 rinses in buffer) in osmium tetroxide in distilled water for 2 h, rinsed and dehydrated in a gradual acetone series. They were then embedded in Spurr 's resin gradually over 3 days and polymerised in fresh resin overnight at 60 ~ Sections were cut

with a glass knife at 0.5 #m, stained with toluidine blue (pH 11.1), and viewed with Nikon optiphot and planapo objectives.

Results

Functional complementation of R. trifolii Nod mutants

Three non-nodulating ( N o d ) mutants ofR. trifolii induced by transposon Tn5 mutagenesis were used to show whether both homologous and heterolo- gous nodulation information could restore the abil- ity of these strains to infect their original host (clo- vers): The mutants have the common phenotype of being unable to curl clover root hairs (Hac- pheno- type, 32, 24) and represent insertions into at least two separate nodulation genes as determined by com- plementation and DNA sequence data (P. R. Scho- field, M. A. Djordjevic, unpublished). Plasmids pRmSL26 (R. meliloti), pJB5JI (R. leguminosa- rum), pBR1AN, pRt032, pRt038, (R. trifolii) were transferred to each of these mutants, and the resul- tant transconjugants were found to nodulate both white and subterranean clover plants (Table 3). Examination of the infected plants showed that the ability to markedly curl clover root hairs (Hac +) and to induce infection thread formation was re- stored (as exemplified by Fig. 1) thus indicating that infection had proceeded in a normal manner. Isolation of bacteria from the resulting nodules showed that about 90% of single colony isolates retained the antibiotic resistance characteristics of the transconjugants. When single colony isolates from nodules were reinoculated onto white clovers, 85-95% of the colonies tested retained their ability to nodulate this host. While there was no delay in the appearance of nodules induced by Nod mu- tants complemented with homologous (pRt032, pRt038, or pBR1AN) information, there was a slight delay in the onset of nodulation by the same Nod- mutants complemented by heterologous in- formation (2 days for pRmSL26 transconjugants and 4-7 days delay for pJB5JI transconjugants). Collectively, these results indicated that there was little instability of the Nod + phenotype in the trans- conjugants tested. Plasmid profiles of bacterial iso- lates f rom nodules showed that the introduced plasmid was present in the appropriate Nod +

Table 3. Restoration of nodulation ability in specific Rhizobium mutants by heterologous plasmids.

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R. trifolii R. meliloti Rhizobium mutants I mutants 2 ANU240 mutant 3 (white clover (lucerne (siratro nodulation) nodulation) nodulation)

R. trifolii plasmids pBRI AN + (Hac +) + (Hac +) N.D.4 pRt032 § (Hac +) + (Hac +) + ( Hac+ ) pRt038 + (Hac +) (Hac) +(Hac +)

R. leguminosarurn plasmids pJ B5J 1 + (Hac +) + (Hac +) N.D.4 pR L 1Jl::Tn1831 + (Hac +) N.D. 4 ( Hac+ )

R. meliloti pRmSL26 + (Hac +) + (Hac +) -(Hac )

ANU240 sym plasmid pNM4AN N.D. (Hac) +

i R. trifolii mutants include ANU851, ANU453, and ANU891. 2 R. meliloti mutants include strains ZB157 and 1126. 3 Strain ANUI256 or ANUI255 was used here. 4 N D - not done.

strains (data not presented). The wild-type strain, ANU843, induces the pro-

duct ion of ni t rogen-f ixing nodules on clovers after 4 weeks. As expected, the R . t r i f o l i i mutants that had been complemented with homologous nodula- t ion in format ion could also induce ni t rogen-f ixing clover nodules after 4 5 weeks post inoculat ion. In addit ion, ni trogen-fixing nodules were produced by the R . t r i f o l i i t ransconjugant strains possessing plasmid pJB5JI after 4 5 weeks. In contrast , the corresponding Nod t ransconjugants possessing

plasmid pRmSL26 did not produce ni t rogen-f ixing nodules on clovers unt i l after 6-9 weeks had elapsed. At this time, be tween40 70% of the plants inoculated with these part icular t ransconjugants produced a ni t rogen-f ixing response. There was al- so a considerable delay (15-30 days) in the forma- t ion of the typically elongated nodules produced on white clovers by the t ransconjugant conta in ing pRmSL26 when compared to wild-type controls.

Because there was a delay in the induc t ion of a ni t rogen-f ixing response by white clover plants in-

Fig. 1. The response of white clover root hair cells exposed to R. trifolii strain ANU851 and its derivatives. Fig. la Uncurled root hairs inoculated with strain ANU851. Fig. 1 b - White clover root hairs have been inoculated with ANU851 (pRmSL26) cells. Fig. lc - Infection threads (arrowed) induced by ANU851 (pRmSL26) cells.

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ocu la ted with ANU891, ANU851, and ANU453 ca r ry ing p R m S L 2 6 , several (28 day old) nodules were sect ioned and examined under the light micro- scope (Fig. 2). These show the p roduc t ion of an extens ive area of p lant cells con ta in ing bacter ia , and there was no evidence of the occurrence of p r e m a t u r e senesence. No exp lana t ion can be of- fered for the slow deve lopment of the n i t rogen-f ix- ing nodules.

Trans fe r of the two foreign p lasmids ( p R m S L 2 6 and pJB5JI ) to two other ANU843 derivatives, s t ra in ANU845 (which is cured of the resident Sym

plasmid) and s t ra in ANU871 (a S y m plasmid delet- ed mutant) , was also done to de te rmine whether white c lover nodu la t ion or root hair cur l ing capaci- ty could be res tored to these strains. The results show that s trains ANU871 (pJB5JI ) and ANU845 (pJB5JI ) were able to cause cons ide rab le d is tor t ion of white clover roo t hairs, but a Nod- phenotype resulted. However , the co r r e spond ing Sym plasmid cured or deleted strains con ta in ing pRmSL26 , did not cause marked d i s to r t ion to c lover roo t hairs. Some roo t hair d i s to r t ion was observed in the ma- ture roo t hairs on plants inocula ted with ANU871

Fig. 2. Light micrograph sections of a white clover nodule induced by a reconstructed R. triJolii mutant. Fig. 2a shows the overall morphology of a longitudinal section of the slow-developing nodule induced by ANU891 (pRmSL26) cells, while Fig. 2b and 2e show higher magnifications of the bacteria-filled cells. Bacteroid forms are clearly seen. The bars are equivalent to 100 micrometers.

(pRmSL26) cells, but this was not extensive and did not affect any other area of the plant. The presence of plasmid pJB5JI in all the R. trifolii mutant strains used, was found to confer the ability to nodulate peas, as expected. In contrast, the pres- ence of plasmid pRmSL26, in either of the R. trifo- lii recipients, failed to confer lucerne nodulation ability to these strains. Examinat ion of the root hairs of lucerne plants inoculated with strains ANU851, ANU891, or ANU453 carrying plasmid pRmSL26 showed tha t no detectable Hac + re- sponse could be observed. Furthermore, lucerne root hairs examined after the addition of strains ANU871 (ApSym) and ANU845 ( p S y m ) carrying plasmid pRmSL26 also showed no root hair curling response.

The transfer of the cowpea Sym plasmid pNM4AN to the same recipient strains was attempt- ed. However no detectable transfer of this plasmid to any of the ANU843 derivatives or strain ANU453 could be demonstrated. A possible expla- nation for this observation is that bacteriocin pro- duction influenced by the presence of plasmid pNM4AN in the various strains involved resulted in the unsuccessful transfer of plasmid pNM4AN (M. A. Djordjevic, unpublished). In addition, plasmid pNM4AN could not be transferred from any background to derivatives of the R. trifolii strains used in this study.

Functional complementation o f R. meliloti N o d recipients

In previous reports (17, 7) it was shown that the transfer of plasmid pRmSL26 to R. meliloti strain 1126 (ANU1071) and of plasmids p B R I A N and pJB5JI to R. meliloti strain ZB157 (ANU1000) which possess a Sym plasmid deletion, was able to restore the abilty of these recipient strains to nodu- late the original host plant, lucerne. The ability to restore lucerne nodulation to these mutants was investigated further by the transfer to both of these strains of the following heterologous nodulation plasmids: pRt032, pRt038, pBR1AN, pJB5JI, and the broad-host-range plasmid pNM4AN (Table 3).

As a control test, plasmid pRmSL26 was trans- ferred to R. meliloti strain ZB157 to determine whether lucerne nodulation could be restored to this strain. As expected, ZBI57 ' (pRmSL26) cells were able to nodulate lucerne. Similarly, the

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transfer to both R. meliloti recipients (strains ZB157 and 1126) of plasmids pRt032, pBR1AN, and pJB5JI was found to restore the ability of these strains to nodulate lucerne plants (Table 3). The nodules produced by strain 1126 transconjugants were normal in appearance although the pro- nounced elongation of the nodules produced did not occur with ZB157 carrying pRt032, pBR1AN or pJB5JI. The number of nodules produced by the strain 1126 (pRt032) transconjugants were most abundant at about 20-25 per plant. These nodules possessed darkened areas at their bases, and they were slow to develop an elongated cigar-shape. Production of a nitrogen-fixing nodule was also delayed by 14-28 days. Light micrograph sections of the slow-developing nitrogen-fixing lucerne nod- ules showed that there was extensive senesence of the bacteroid-containing cells (Fig. 3).

The transfer of plasmid pRt038, which carries some but not all of the R. trifoliiNod genes (26, 27), did not restore lucerne nodulation to either R. meli- loti Nod mutant. Since plasmid pRt032 restores a Nod + phenotype to the insertion mutant 1126, this result suggests that the equivalent Nod gene inter- rupted by bacteriophage Mu insertion in strain 1126 does not occur on plasmid pRt038, but does occur on plasmid pRt032.

There was a slight delay in the appearance of lucerne nodules induced by the R. rneliloti Nod + transconjugants possessing the heterologous plas- mids pRt032, pBRIAN, or pJB5J1. This ranges from 5 days in the case of pRt032 transconjugants to 7-9 days in the case of transconjugants possess- ing either plasmid pBR1AN or pJB5JI. Transcon- jugants containing either plasmid pRt032, pBR 1 AN, or pJB5JI were unable to nodulate their respective plant hosts (clovers and peas). However, examina- tion of the root hairs of clover and lucerne plants infected with the various transconjugants showed that pronounced root hair curling (the Hac + pheno- type) occurred on both host plants, but only lucerne plants showed the ability to form nodules.

In contrast to the ability of the R. trifolii and R. leguminosarum plasmids to restore the ability of the two R. meliloti Nod recipients to nodulate lucerne, transfer of the cowpea Sym plasmid pNM4AN to either R. meliloti mutant did not re- sult in the ability of these strains to nodutate lucerne or cause root hair curling.(Hac phenotype). While strains ZB157 (pNM4AN) and 1126 (pNM4AN)

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Fig. 3. Light micrograph section of lucerne nodules induced by a reconstructed R. meliloti mutant. Fig. 3a shows the overall morphology of a longitudinal section through a lucerne nodule induced by strain 1126 (pRt032). An

extensive zone of senesence is indicated by the area between the arrows. Fig. 3b and 3c show higher magnifications of the bacteria-filled late symbiotic cells and senescent cells, respectively. ES: early symbiotic; LS: late symbiotic; S: senescent cells. Bars indicated are 100 micrometers.

were unable to nodulate lucerne, they were both able to nodulate the tropical legume siratro and induce nitrogen-fixing nodules. The siratro nodules produced were normal in appearance and number, and there was no delay in the onset of nodulation when compared with control tests. The root hairs of the siratro plants were markedly distorted (Hac +) and infection threads were observed.

Since the introduction of plasmid pNM4AN did

not complement either of the R. meliloti Nod mu- tants, the effect of the transfer of this plasmid to strain Rm41 (parent strain of ZB157) and to strain 1021 (parent strain of 1126) was investigated. Inhi- bition of nodulation ability on lucerne plants oc- curred when Rm41 (pNM4AN) was inoculated on these plants. Instead of lucerne nodules forming, callus-like pseudo-nodules resulted. Strain Rm41 (pNM4AN), however, was able to form nodules on

siratro plants which were normal in appearance. In contrast, strain 1021 (pNM4AN) was able to induce nodule formation on both lucerne and siratro, al- though the nodulation response was not as exten- sive as either strain 1021 on lucerne plants or strain ANU240 on siratro plants.

Transfer to a Nod mutant o f a fast-growing cow- pea Rhizobium

Transfer of the Sym plasmid pNM4AN to the Tn5-induced Nod mutant derivative of the broad- host-range strain ANU240 (ANU1256) and to a Sym plasmid-cured derivative, ANU265 (20), re- sulted in the restoration of the ability of this strain to nodulate siratro and to produce nitrogen-fixing nodules (see also Table 3).

Siratro nodulation ability could also be restored to this Tn5-induced mutant by the transfer of the R. trifolii plasmids pRt032 and pRt038. While the nod- ules were of normal appearance, there was both a delay in the onset of nodulation (10 14 days), and the number of nodules produced was low, with only 10-20% of the siratro plants producing nodules. Nodulation usually occurred on the main tap root. Bacteria isolated from these nodules possessed the antibiotic resistance pattern of the transconjugants. Significantly, nodule isolates of strain ANU1256 carrying plasmid pRt032 or pRt038 did not display any enhanced nodulation ability when reinoculated onto siratro, as only 10-20% of plants produced nodules as before. As expected, strain ANU265 ( p S y m - ANU240 derivative) carrying the R. trifolii plasmid pBR1AN or pRt032 were Hac Nod- on siratro but were Hac + Nod + on clovers. According- ly, strain ANU1256 (Nod::Tn5) carrying either of the R. trifolii plasmids (pBR1AN or pRt032) was Nod + on clovers.

In contrast to the ability of the R. trifolii plas- raids to correct the siratro nodulation ability of strain ANU1256, the transfer of the R. meliloti plasmid pRmSL26 or the R. leguminosarum plas- mid pRL 1J 1 ::Tn1831 to strain ANU 1256 (or to the pSym strain ANU265) did not result in restoration of the ability to nodulate or cause root hair curling on siratro. The transfer o fpRmSL26 to these recip- ients was repeated in 4 separate experiments with over 100 plants tested. The transconjugant strains containing pRmSL26 were also unable to induce root hair curling or nodulation on lucerne plants.

155

Plasmid gels of the transconjugants ANU 1256 and ANU265 carrying plasmid pRmSL26 showed that the introduced plasmid was present and had no detectable alteration (data not shown).

While the transfer of the R. leguminosarum plasmid pRL1JI: :Tn1831 to strain ANU1256 (Tn5::Nod) did not restore siratro nodulation abili- ty (40 plants tested), a marked root hair curling response occurred on all siratro plants inoculated with this transconjugant type. As expected, both ANU1256 and ANU265 (pSym-) carrying pRLIJI : :TnI831 were able to induce pea nodula- tion.

Hybridization data

The functional correction of the Nod phenotype of various Rhizobium strains by heterologous nod- ulation information suggested that there could be substantial structural gene conservation of at least some early nodulation genes in fast-growing Rhi- zobium species. Hybridization probes (see Fig. 4) made with DNA subfragments of plasmid pRt032, which carries the nodulation region of R. trifolii strain ANU843, were used to determine if there was DNA homology with restriction fragments of R. meliloti plasmid pRmSL26. Some of the nodula- tion genes of pRmSL26 have been shown to reside on large (8.7 kb) EcoRI and (5.5 kb) PstI subfrag- merits of this plasmid (17, S. R. Long, personal communication). The genes involved in root hair curling of R. trifolii strain ANU843 are located on

252 851

I I - ] I Ir

H p R t 5 9 0 E I I

E pRtOll E I I

E pRt572 E 1 I

H pRt587 H I r

Fig, 4. Restriction map showing the subclones used as probes from the R. tri foli i ANU843 Nod region. The map positions of the Tn5-induced Nod mutants 851 and 252 are shown. The 7.2 kb E c o R I insert of pRt572 and the 14 kb H i n d l l l insert of pRt587 are the same as the inserts in plasmids pRt038 and pRt032, respectively. H = H i n d l I l , E = EcoR! and B = B a m H I

156

the 7.2 kb E c o R I fragment and the adjacent 4.1 kb E c o R I H i n d l I l fragment of plasmid pRt032 (M. A. Djordjevic and P. R. Schofield, unpublished).

The results of this hybridizat ion experiment are shown in Fig. 5. This clearly shows that the 7.2 kb R. tr(folii Nod probe hybridized specifically to the large (8.7 kb) E c o R I fragment of pRmSL26. D N A fragments f lanking the 7.2 kb R. trifolii Nod frag- ment hybridized to the ends of 8.7 kb R. rneliloti

fragment as well as to corresponding adjacent R. mel i lo t i fragments (Fig. 5b, c, d). These results sug- gest that substantial D N A homology occurs in the nodula t ion regions of both R. trifolii and R. meli lo-

ti. To determine whether there was any cross-hy-

bridization of part of the nodulat ion region of the broad-host - range strain ANU240 to the nodula t ion regions of other rhizobia, plasmid pRs23 (derived f rom D N A sequences surrounding the Tn5 inser- t ion of strain ANU1256) was hybridized to restric- t ion digests o f plasmids containing most of the nodula t ion regions of R. tri foli i (pRt572) and R. mel i lo t i (pRmSL26) . Plasmid pRs23 hybridized poor ly to a 5.5 kb PstI subfragment of p R m S L 2 6

(Fig. 5e). In addition, weak cross-hybridization of pRs23 to digests of the 7.2 kb Nod fragment of R. tr(folii also occurred (data not shown).

Hybridizat ion of Nod D N A sequences from strain ANU240 (pRs23) to representatives of other fast-growing rhizobia was examined. Southern blots containing plasmid D N A from diverse fast- growing R h i z o b i u m strains including ANU240 (control), USDA205 (a R h i z o b i u m strain able to nodulate Peking variety soybeans) and ANU 100 (a R h i z o b i u m strain able to nodulate pigeon peas), were hybridized to plasmid pRs23. Specific plasmid species hybridized in all cases (Fig. 6). This indicat- ed that homology to Sym plasmid-derived D N A sequences f rom ANU240 occurred on plasmid spe- cies in other diverse fast-growing rhizobia.

Discussion

The transfer of homologous and heterologous nodulat ion information to Tn5-induced Nod mu- tants of R. trifolii resulted in restorat ion of the ability of these strains to induce root hair curling

Fig. 5. Autoradiographs of the response of restriction enzyme digests of the R. meBloti plasmid pRmS L26 to radioactively labelled Nod DNA fragments from the R. trifolii strain, ANUS43 and from strain ANU240.

Fig. 5a shows the migration ofh Hindlll size markers (lane 1) compared to an EcoRl digest of plasmid pRmSL26. A small 2.2 kb EcoR1 band of plasmid pRmSL26 (lane 2) reported by Long et al. (17) clearly migrates further than the 2.0 kb * size marker.

Fig. 5 b, c and d show the response of EcoR1 digest of pRmSL26 to various R. trifolii Nod DNA fragments (which together comprise the 14 kb Hindlll fragment of plasmid pRt032). Plasmid pRt572 (Fig. 5b) hybridizes specifically to the 8.7 kb EcoRl pRmSL26 fragment while plasrnids pRt011 (Fig. 5c) and pRt590 (Fig. 5d) both hybridize to the 8.7 kb EcoRl fragment and to corresponding adjacent pRmSL26 EcoRl DNA fragments.

Fig. 5e shows the response of plasmid pRmSL26 to radioactively-labelled pRs23 (derived from the strain ANU240 Nod region). A 5.5 kb PstI fragment of pRmSL26 hybridizes to this probe. Additional (uppermost) hybridizing bands (indicated by arrows) are due to plasmid (vector-vector) cross-hybridization between pLAFRI and pBR325. All DNA fragment sizes are given in kilobases (kb).

157

Fig. 6. The plasmid profiles and corresponding autoradiograms of various Rhizobium strains after exposure to radioactively-labelled ANU240 Nod DNA sequences. The plasmid profiles of strains ANU240 (control) (lane 1), AN U 1046 (US DA205) (lane 3), and ANU 100 ( H P 100) (lane 5) were hybridized against radioactively-labelled pRs23 plasmid DNA. The response to this probe (shown in lanes 2, 4 and 6, respectively) indicates that DNA sequences homologous to ANU240 Nod DNA occur on large indigenous plasmids in other diverse fast-growing rhizobia.

and nodule formation on white clovers. This result indicates that pRmSL26 and pJB5JI encode func- tionally-equivalent nodulation gene products to those ofR. trifolii. Furthermore, the Tn5 insertions in R. trifolii mutants ANU453, ANU851, and ANU891 must be located in genes which do not airectly determine the host-specific interaction be- tween R. trifolii and clover.

Similarly, the transfer of homologous and hete- rologous information to specific Nod mutants of R. meli lot i also resulted in the restoration of Hac + phenotype and the initiation of nodules on lucerne plants. The correction of a Nod mutant ofR. meli- loti with heterologous information was first report- ed by Banfalvi et al., (1) when they found that the transconjugant strain ZB 157 (pJB5JI) could nodu- late lucerne plants. Since strain ZB157 has been reported to possess a deletion of all the genes in- volved in the hair curling step and possibly other Nod genes (15, 16), these overall results imply that substantial functional conservation exists for at least some of the gene products involved in the early steps of plant infection for the R h i z o b i u m species R. trifolii, R. leguminosarum and R. meliloti. Con- sistent with this finding is that cross-hybridization can be demonstrated between R. trifolii Nod DNA fragments and DNA fragments of the R. meliloti

Nod plasmid pRmSL26. This has also been shown recently by using heteroduplex mapping (22) with R. trifolii and R. me l i l o t iNod DNA fragments. The R. trifolii Nod fragment used by Ptihler et al. (22) is the same as the one used in this study (pRt572), except the fragment used by these workers pos- sesses a Tn5 insertion. Collectively these results indicate that one can discount the possibility that the cross hybridization obtained was due to DNA sequence homology from areas adjacent to the Nod genes and hence was due to Nod gene homology between R. trifolii and R. meliloti.

Transfer of the R. trifolii plasmid pRt032, which contains a 14 kb DNA insert, restores white clover nodutation ability to a Sym plasmid deletion mutant ANU871 and a Sym plasmid-cured mutant ANU845 (27). In contrast, strains ANU845 ( p S y m ) and ANU871 (ApSym) carrying either of the heterologous plasmids pRmSL26 or pJB5JI were unable to nodulate white clovers. This shows that while both these heterologous plasmids can suppress the hair curling defects of the Tn5-induced R. trifolii mutants, they lack the requirements for clover host specificity. Consequently, the recombi- nant plasmid pRt032 which confers white clover nodulation to a variety of rhizobia, must carry genes for clover host specificity, root hair curling

158

and nodulation as concluded by Schofield et al. (27). The ability to nodulate a host plant obviously requires a combination of both non-specific and specific gene products. This is also supported by the finding that plasmid pRmSL26 is unable to confer white clover or lucerne nodulation ability on R. trifolii strains ANU845 (pSym-) and ANU871 (ApSym) and plasmids pBR1AN, pJB5JI and pRmSL26 are unable to restore lucerne nodulation ability to R. meliloti strains possessing more sub- stantial Sym plasmid deletions than strain ZB157 (7, 16; M. A. Djordjevic, unpublished).

I f all the genes involved, either directly or indi- rectly, in root hair curling were identical, then wild- type R. meliloti strains would be able to distort the root hairs of clovers and possibly form nodules on these plants. However, wild-type R. rneliloti strains have been reported to do neither when exposed to white clover plants (34, 35). Moreover, the transfer of plasmid pNM4AN to the R. meliloti mutants used in this study (ZB157 and 1126) did not.result in the restoration of lucerne nodulation. This result cannot be due to a lack of expression of the nodula- tion genes encoded on this plasmid in R. meliloti, as both R. meliloti Nod- derivatives possessing plus- mid pNM4AN were able to nodulate siratro plants. In addition, hybridization of the nodulation re- gions of pRmSL26 and Nod DNA sequences de- rived f rom plasmid pNM4AN was found to occur. A possible role for the plant host in determining the expression of the bacterial Nod genes involved in root hair curling could explain this result or that some adverse interaction occurs between the Nod genes on plasmid pNM4AN and the Sym plasmids of R. meliloti strains Rm41 and 1021.

Transfer of the R. trifolii plasmids pRt032 and pRt038 to the Nod- mutant (ANU1256) of the strain ANU240, resulted in restoration of the ability of this strain to nodulate legumes although the efficiency of nodule formation was low. This result suggests that there is some similarity in the genetic information required to invade tropical legumes and temperate legumes. Consistent with this im- plied conservation of nodulation information is the result which shows that cross-hybridization occurs between DNA sequences isolated from the nodula- tion region of R. trifolii and DNA sequences from the Sym plasmid of the ANU240 Rhizobium. These results may indicate that there is conservation of Nod gene function and possibly of DNA sequence in

at least some of the genes in'c,olved in the early nodulation steps in a broad spectrum of fast-grow- ing Rhizobium strains with diverse host ranges. It is also important to note that the AN U240 Nod strain ANUI256 is unable to nodulate all tested host plants. However, the inability ofplasmid pRmS L26 and pRL1JI: :TnI831 to restore nodulation ability to the Nod mutant ANU1256 indicates that the functional conservation of some early Nod genes may not apply in all cases or that these introduced genes are not expressed in the appropriate manner. The ability of the R. leguminosarum plasmid pRL1JI : : Tn1831 to restore a siratro root hair curl- ing response to ANUI256 indicates that some correction of the H a ~ defect occurred. However, no nodules resulted after screening at least 40 sira- tro plants for 6 weeks. No explanation can be of- fered for this result.

Plasmid pRmSL26 has been reported to carry a substantial amount of nodulation information (17), and in accordance with this claim it is able to restore lucerne nodulation ability to a Sym plasmid dele- tion mutant of R. meliloti strain ZB 157. This result indicates that pRmSL126 carries all the genes in- volved in the hair curling step since strain ZB157 is reported to have its Hac genes deleted (15, 16). However, since there is an apparent inability of plasmid pRmSL26 to show effective Hac + expres- sion in many foreign backgrounds including R. trifolii [and A. tumefaciens (S. R. Long, personal communication)], this suggests that the presence of genes only involved in the hair curling phenotype may not be sufficient for nodulation to occur and that other additional or regulatory genes are re- quired for the phenotypic expression of these Hac genes on plasmid pRmSL26. Consistent with this hypothesis is the identification of a host specificity region separate from the Nod genes in R. meliloti strain 41 (16). I f plasmid pRmSL26 does not con- tain this region then this may explain why this plasmid cannot restore nodulation ability to the ANU240 Nod derivative. On the other hand, the recombinant R. trifolii plasmid pRt032 is able to (a) correct the inability of many specific Nod mutants (ANU851, ANU453, ANU891, ANUI071 and ANUI256) in a variety of foreign backgrounds; (b) restore lucerne nodulation ability to a R. meliloti mutant (ZB157) which has sustained a substantial deletion of many Nod genes and (c) confer clover nodulation ability to A. tumefaciens (26) and to a

S y m p l a s m i d - c u r e d de r i va t i ve o f s t ra in A N U 2 4 0

( A N U 2 6 5 ) . Th is sugges ts tha t this p l a s m i d ' m a y

possess add i t i ona l genes necessary fo r the expres -

s ion o f the N o d genes e n c o d e d on this p l a smid and

tha t the o r g a n i s a t i o n o f the genes i n v o l v e d in p lan t

i n f e c t i o n may be d i f fe ren t in R. t r i fo l i i and R. me l i -

loti .

Recen t ly , there have been repor t s tha t s egmen t s

o f D N A ca r ry ing N o d genes de r i ved f o m a s low-

g r o w i n g P a r a s p o n i a R h i z o b i u m s t ra in can re s to re

luce rne n o d u l a t i o n abi l i ty to specif ic N o d R. mel i -

lo t i s t ra ins w h e n i n t r o d u c e d to these s t ra ins (18).

C o n s i s t e n t wi th these resul ts is the f i nd ing t h a t a

h y b r i d i z a t i o n p robe , de r ived f r o m the S y m p l a s m i d

of s t ra in A N U 2 4 0 (a b r o a d - h o s t - r a n g e R h i z o b i -

u m ) , are able to c ros s -hybr id i ze to e n d o g e n o u s

p l a smid D N A sequences f r o m a wide va r i e ty o f

f a s t - g r o w i n g R h i z o b i u m s t ra ins i n c l u d i n g t hose

s t ra ins ab le to n o d u l a t e s o y b e a n s and p i g e o n peas.

Thus , it seems l ikely tha t the f u n c t i o n a l c o n s e r v a -

t i on o f s o m e genes i n v o l v e d in ear ly p lan t i n fec t ion

steps d e m o n s t r a t e d here for f a s t - g r o w i n g R h i z o b i -

u m s t ra ins , m a y e x t e n d to the s l o w - g r o w i n g rh i zo -

bia.

Acknowledgements

, M A D , R W R and B J B are rec ip ien t s o f C o m -

m o n w e a l t h P o s t G r a d u a t e R e s e a r c h A w a r d s . P R S

is the rec ip ient o f a F a r r e r M e m o r i a l R e s e a r c h S c h o l -

a rsh ip . W e w o u l d l ike to a c k n o w l e d g e the t echn ica l

ass i s tance o f J a n M c I v e r and E l e n a G ~ r t n e r . S h a r o n

L o n g is t h a n k e d fo r p r o v i d i n g p l a s m i d p R m S L 2 6 ,

M. B a g d a s a r i a n fo r p r o v i d i n g the T r a + d e r i v a t i v e o f

p l a s m i d p K T 2 4 0 and C. A. Wij f f e lman fo r p r o v i d i n g

p l a smid p R L 1 J l : : T n 1 8 3 1 .

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Received 9 May 1984; in revised form 15 August 1984; accepted 25 August 1984.