Rhizobium meliloti Host Range nodH Gene Determines Production of ...

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hairs by Rhizobium trifolii, p. 239-245. In H. J. Evans, P. J.. Bottomley, and W. E. Newton (ed.), Nitrogen fixation research progress. Martinus Nijhoff, Amsterdam.
Vol. 170, No. 12

JOURNAL OF BACTERIOLOGY, Dec. 1988, p. 5489-5499

0021-9193/88/125489-11$02.00/0 Copyright © 1988, American Society for Microbiology

Rhizobium meliloti Host Range nodH Gene Determines Production of an Alfalfa-Specific Extracellular Signal CATHERINE FAUCHER,' FABIENNE MAILLET,1 JACQUES VASSE,1 CHARLES ROSENBERG,' ANTON A. N. VAN BRUSSEL,2 GEORGES TRUCHET,1 AND JEAN DItNARIEl* Laboratoire de Biologie Moleculaire des Relations Plantes-Microorganismes, Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, BP 27, 31326 Castanet Tolosan Cedex, France,' and Department of Plant Molecular Biology, University of Leiden, Nonnensteeg 3, 2311 VJ Leiden, The Netherlands2 Received 20 June 1988/Accepted 6 September 1988 The Rhizobium meliloti nodH gene is involved in determining host range specificity. By comparison with the wild-type strain, NodH mutants exhibit a change in host specificity. That is, although NodH mutants lose the ability to elicit root hair curling (Hac-), infection threads (Inf), and nodule meristem formation (Nod-) on the homologous host alfalfa, they gain the ability to be Hac+ Inf+ Nod' on a nonhomologous host such as common vetch. Using root hair deformation (Had) bioassays on alfalfa and vetch, we have demonstrated that sterile supernatant solutions of R. meliloti cultures, in which the nod genes had been induced by the plant flavone luteolin, contained symbiotic extracellular signals. The wild-type strain produced at least one Had signal active on alfalfa (HadA). The NodH- mutants did not produce this signal but produced at least one factor active on vetch (HadV). Mutants altered in the common nodABC genes produced neither of the Had factors. This result suggests that the nodABC operon determines the production of a common symbiotic factor which is modified by the NodH product into an alfalfa-specific signal. An absolute correlation was observed between the specificity of the symbiotic behavior of rhizobial cells and the Had specificity of their sterile filtrates. This indicates that the R. melioti nodH gene determines host range by helping to mediate the production of a specific extraceilular signal.

Soil bacteria of the genus Rhizobium are able to elicit the formation of nitrogen-fixing nodules on the roots of their leguminous hosts. The interaction between fast-growing Rhizobium species and their host plants is highly specific (24). For example, R. meliloti strains nodulate Medicago, Melilotus, and Trigonella species, while R. leguminosarum biovar viciae can form nodules on Pisum and Vicia species. In the symbiosis between R. meliloti and alfalfa (Medicago sativa), the formation of nodules is a complex process involving the following steps: root hair curling (Hac), infection thread formation within root hairs (Inf), and initiation of a nodule meristem and nodule organogenesis (Nod) (5, 36, 37, 40). Genes involved in the control of these steps have been located on the pSym megaplasmid (21, 25, 30). Genetic, cytological, and molecular studies have allowed the identification and characterization of several common and specific nodulation (nod) genes (5, 17, 18, 21, 34). The R. meliloti nodABC genes are referred to as common nod genes: mutations in one of these genes can be complemented by cloned nodulation genes from another Rhizobium species without changing the host range (5, 18, 21). They are required for root hair curling and nodule induction. The nodD gene, of which R. meliloti has three functional copies (13-15), is responsible for activating the expression of other nod genes in the presence of root exudates (14, 28, 29; C. Rosenberg, F. Maillet, M. A. Honma, and J. Denarie, manuscript in preparation). nodDI was previously considered a common nodulation gene (8, 28). However, recent evidence indicates that nodD can be involved in controlling the host range by modulating nod gene expression as a function of the composition of the legume host root exudates (14, 16, 33). *

The R. meliloti nodH and nodFEG genes are also host range determinants (5, 6, 17, 34). A mutation in one of the nodH or nodFEG genes cannot be fully complemented by the cloned nodulation genes from other Rhizobium strains (5, 17, 21). In addition, mutations in nodH or nodFEG genes result in an altered host range for the mutated Rhizobium strains (5, 17, 21); in contrast to the wild type, strains having mutations in these genes exhibit altered infection and nodulation of the homologous host alfalfa and can elicit root hair curling on heterologous hosts such as clover and vetch. Van Brussel et al. (38, 39) have shown that R. 1eguminosarum biovar viciae (called R. leguminosarum in the following text) strains can induce a phenotype called thick and short root (Tsr) on a particular host, Vicia sativa subsp. nigra (common vetch). The Tsr phenotype cannot be provoked by strains carrying a transposon TnS-induced mutation in nodABC or nodD. This Tsr reaction can be induced by a sterile supematant of a culture which has been induced by an appropriate flavonoid (44). Supernatants of induced cultures also elicit marked root hair deformation (Had) on common vetch (44). Thus, the R. leguminosarum nodABC genes are involved in the production of at least one extracellular symbiotic signal which can be detected by Tsr and Had bioassays on vetch. In this paper we present a root hair deformation (Had) bioassay for alfalfa. Then, using Had bioassays on common vetch and alfalfa, we studied the production of extracellular signals by R. meliloti. We show that the R. meliloti nodH host range gene, which determines the specificity of hair curling, mediates the production of a specific extracellular signal which provokes deformation of root hairs of alfalfa and not of vetch. When the wild-type strain, a NodAmutant, and a NodH- mnutant were compared, a complete correlation was observed between the specificity of the

Corresponding author. 5489

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J. BACTERIOL. TABLE 1. Bacteria and plasmids used in this study Relevant characteristicsa

Bacterial strain or plasmid

E. coli K-12 C600 MC1061 ED8767 GM13540 R. meliloti RCR2011 GMI766 GM1255 GM1357 GM15382 GM15384 GM15387 GM15390 GMI5517 GM15513 GM15381 GM15521 GM15378 GM15598 GM15375 GM15430 GM15431 GM15429 GM15376 GMI5619 GMI5618 GM15624 RmlO21 TY9B8 JT314 R. leguminosarum 248

Wild type thr leu rpsL hsdR araDl39 A(ara leu)7697 lacX74 galU galK hsdR rpsL supE supF met hsdS recA56 Spontaneous Nalr derivative of ED8767

19 19 28 19 19

Wild type; Nod' Fix' on M. sativa A(nod-nifA)766 Nod-Spr A(fix-1074 nod nifHDK)7125 (TnS) Nod- Nalr A(nodDJABC)HG.01 Nod- Rif BV Nmr Smr nodA::Tn5-2208 nodB::TnS-154 nodC::Tn5-2217 Region Ila nod::TnS-2412 Region Ila nod::TnS-2202 Region lIb nod::TnS-119 nodE: :Tn5-2309 nodE::TnS-2307 nodF::TnS-2407 nodG::Tn5-314 nodH::TnS-2121 nodH::Tn5-148 nodH::Tn5-2313

30 T. Huguet 35 T. Huguet 5 5 5 5 5 5 5 5 5 This study 5

nodl-::Tn5-2219

5

nodH::Tn5-2212 A(nodH)DEK8 Nmr Bli A(nodEFH)DEKII Nmr Bl' A(nodGEFH)DEKJO Nmr Blr Spontaneous Smr derivative of SU47 Derivative of 1021; nodDl::TnS Derivative of 1021; nodG::TnS Wild type; Nod' Fix' on V. hirsuta and V. sativa subsp. nigra

4 4 4 25 28

IncPl plasmids RP4 pR751-pMG2 pRK290 pGMI515 pRmSL26 pRmJ30

Tcr Apr Kmr Tra+ Gmr Smr Tra+ ori(RK2) Tcr RP4-prime (in vitro), Tcr Apr (Fig. 1) pLAFR1-prime, Tcr (Fig. 1) pRK290-prime, Tcr (Fig. 1) IJRK290-prime, Tcr (Fig. 1) pGMI149 pRK290-prime, Tcr (Fig. 1) pSB2 nodC::lacZ fusion in pRmSL26, Tcr Sp' pRmM57 nodDl::lacZ fusion in pRmJ30, Tcr Sp' pRmM61 nodDl::lacZ fusion in pGMI149, Tcr Spr pGMI929 nodC::lacZ fusion in pGMI149, Tcr'pr pGMI930 nodH::TnS-2121 in pGMI149, BIF Nmr Smr Tcr pGMI387 nodC::lacZ fusion and nodH::Tn5-2121 in pGMI149, Bl' Nmr Smr Spr Tcr pGMI795 nodDI::lacZ fusion and nodH::TnS-2121 in pGMI149, Bli Nmr Smr Spr Tc' pGMI796 nodH::Tn5-2121 in pSB2, BlI Nmr Smr Tcr pGMI794 ColEl plasmid pRK2013 Helper plasmid for mobilizing pRK290 and pLAFR1 derivatives; tra(RK2) ori(ColE1) Kmr a

R. meliloti strains carrying TnS are resistant to bleomycin

34 39 2 5 7 35 25 11 5 S. B. Sharma 28 28 This study This study S This study This study This study 7

(Bl), neomycin (Nm), and streptomycin (Sm).

symbiotic behavior (Hac, Inf, and Nod) of these bacteria and the Had specificity of their sterile filtrates. This indicates that .nodH determines the host range by mediating the production of a specific extracellular signal. MATERIALS AND METHODS

Microbiological techniques. Bacterial strains and plasmids described in Table 1 and Fig. 1. Conditions used for bacterial growth, conjugation, and transduction experiments have been described elsewhere (5, 35). Plasmids containing nod::lac fusions were maintained in Escherichia coli

are

Source or reference

MC1061. The concentration of bleomycin (Bl) in selective plates was 20 ,ug/ml. The nodG: :Tn5-314 mutation was isolated by Swanson et al. (34). To construct strain GMI5598, we introduced the nodG::TnS-314 mutation into strain 2011 by transduction with bacteriophage N3 (26). The induction of R. meliloti nod genes With luteolin and 3-galactosidase activity assays were ',performed as previously described (28). The introduction of nod::TnS insertions and nod::lac fusions into pGMI149 and pSB2 was performed by a series of marker exchange experiments (31) as follows. The mutations, present initially in an Inc P1 plasmid derivative, were

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2 kb FIG. 1. Physical and genetic map of the nodulation (nod) region of R. meliloti 2011. The horizontal line represents the restriction map (E, EcoRI; H, HindIII). The plasmids are shown below the physical map; the dotted line represents the deleted region of plasmid pSB2. Above the physical map, the arrows indicate the direction of transcription of the nod genes (6, 15, 16). The numbers and vertical lines define the locations of nod::Tn5 insertions (solid lines) or nod::lac fusions (dashed lines).

introduced into pSym by transduction with phage N3 (27) into an R. meliloti recipient containing pR751-pMG2 as an incompatible "chasing plasmid" (5). The mutations were then introduced from pSym into the appropriate pRK290prime plasmid by homologous recombination and mobilized into an E. coli recipient (C600 or GM13540) by triparental mating with E. coli K-12(pRK2013) used as a source of mobilizing plasmid (5). The resulting plasmid, carrying the nod mutation, was then introduced by mating into the appropriate Rhizobium strain. DNA biochemistry. The pSym megaplasmid and the other plasmids were visualized after agarose gel electrophoresis (30). The locations of the nod::TnS insertions and nod::lac fusions, after their introduction into the appropriate plasmids, were checked by restriction fragment analysis. Restriction endonucleases were purchased from Boehringer Mannheim and New England Biolabs. Plasmid isolation, digestion by restriction enzymes, and agarose gel electrophoresis were carried out by standard procedures (26). Plant cultivation. Seeds of Medicago sativa cv. Gemini were obtained from Tourneur Freres (Coulommiers, France) and seeds of Vicia sativa subsp. nigra and of Medicago, Melilotus, and Trigonella species were provided by G. Genier (Station d'Amelioration des Plantes Fourrageres, Institut de la Recherche Agronomique, Lusignan, France). Vetch nodulation assays were performed as already described (39) in a controlled environment cabinet at 220C with a 16-h day length. To reisolate the NodH- bacteria from vetch nodules, the nodules were first sterilized for 5 min in calcium hypochlorite (33 mg/ml) and then rinsed five times in sterile water. The sterility of the last rinse was checked by plating a sample on TY agar. The nodules were then individually crushed in 0.5 ml of sterile water, and bacteria were isolated on neomycin-containing (100 p,g/ml) TY plates. Light microscopy of root deformations was performed as described previously (40). Tsr and Had bioassays. The vetch Tsr bioassays were

carried out as described before (39) at 220C with a 16-h day length. When required, luteolin was added to the plant growth medium at a final concentration of 1.5 FM. The search for a Tsr effect in Medicago, Melilotus, and Trigonella species was done under the same conditions. The Tsr phenotype was quantified by measuring the length of the main roots after 9 days. Twenty plants were used for each treatment. The significance of length differences between two sets of plant roots was calculated by the test of comparison of means (32). Sterile supernatant fluids from flavonoid-induced R. leguminosarum cultures were obtained by using low-density cultures (ca. 5 x 105 bacteria per ml) grown in the medium already described (44). Sterile supernatant fluids from R. meliloti were prepared by the same procedure, but bacteria were first grown in the C yeast-mannitol medium (41). Naringenin and luteolin, the flavonoid inducers used for R. leguminosarum and R. meliloti, respectively, were used at a final concentration of 1.5 ,uM. After centrifugation (5, 000 x g), the supernatants were passed through a nitrocellulose filter (0.45 Kum pore size; Millipore) and autoclaved at 110°C for 30 min. Filtrates were checked for sterility before use. The vetch Had bioassay has been described previously (44). For alfalfa Had bioassays, the Fahraeus assembly was used (10): two seedlings, with roots 1 to 1.5 cm long, were aseptically transferred between slide and cover slip in a 30-mm-diameter test tube containing 15 ml of liquid induction medium (44). The root system was checked for sterility and observed at day 6 by light microscopy after methylene blue staining (40). Five replicates of two plants were used for each treatment. RESULTS R. meliloti nodH mutants are Tsr+ on Vicia sativa subsp. nigra. In the search for biological assays allowing the detection of putative R. meliloti symbiotic signals, we first looked for a legume host which would exhibit a Tsr phenotype with

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FIG. 2. Infection phenotypes on vetch root hairs of wild-type R. meliloti RCR2011 (A) and R. meliloti GM15375 (nodH::TnS mutant) (B, C, and D). (A) Root hairs are rare and slightly deformed. (B) Hac+ phenotype. The arrowheads point to the unusually large infection focus of curled root hairs. Note that a much higher number of root hairs were elicited by the NodH- mutant than by wild-type R. meliloti (Hai phenotype, compare A and B). (C) Detail of a root hair, showing the large infection focus (arrowheads) and a developing infection thread (arrow). (D) Inf phenotype. The arrow points to the infection thread. A and B: Bar, 100 ,m. C and D: Bar, 50 ,um.

this bacterium. R. meliloti nodulates Medicago, Melilotus, and Trigonella genera. Eleven species of Medicago were studied, M. aculeata, M. arabica, M. intertexta, M. laciniata, M. littoralis, M. lupulina, M. orbicularis, M. rugosa, M. sativa, M. tornata, and M. truncatula cv. Jemalong, as well as two species of Melilotus (M. alba and M. indica) and two species of Trigonella (T. foenum-graecum and T. suavissima). Since we were looking for a root shortening determined by nod genes, a putative Tsr effect was estimated by comparing the length of the main roots of plants inoculated with either the wild-type RCR2011 strain or a derivative, GMI766, which carries a pSym deletion which removes all the known nodulation genes. Root lengths were measured before the appearance of nodules to avoid the well-known effect of nodulation on root system development (38, 39). No significant Tsr effect could be detected on any R. meliloti host tested. Since we were unsuccessful in our search for a Tsr

phenotype with R. meliloti homologous hosts, we decided to test the nonhomologous host V. sativa subsp. nigra. To examine this possibility, we first studied the symbiotic behavior of various R. meliloti derivatives on V. sativa when grown on agar slants. The wild-type strain RCR2011 did not provoke root hair curling (Hac-) (Fig. 2A), infection thread formation (Inf), or nodule formation (Nod-). In contrast, two mutants, GM15375 and GMI5376, carrying a TnS-induced mutation in the host range gene nodH, elicited strong reactions on vetch root hairs (Hac+ Inf+) (Fig. 2B-D). Methylene blue staining revealed a marked hair curling characterized by an infection focus of unusually large size (Fig. 2B and C). Infection threads were formed within root hairs (Fig. 2C and D); in comparison with infection threads induced on vetch by R. leguminosarum 248, they were much less numerous and often distorted, and their appearance was slightly delayed. Ineffective (Fix-) root deformations could be seen after 3 weeks. Cytological studies revealed that they

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TABLE 2. Bacterium-induced Tsr effects on V. sativ'a subsp. nigria Mean root

Strain characteristics

Strain or control"

Jensen medium +L -L R. leguminosaruim +L -L R. meliloti 2011 +L -L TY9B8 GM15382 GM1357 GM15517

GM15390 GM15513 GM15378 GM15381 GM15521 GM15598 GM15375

Sterile control 10.4 1.8 a 10.2 ± 0.8 'a

Wild type 3.8 ± 0.6 c 4.3±0.6 c

Wild type nodDl::Tn5 nodA::Tn5-2208

A(nodDIABC) nodIla::Tn5-2202 nodlla::Tn5-2412 nodllb::TnS-119 nodF::Tn5-2407 nodE::Tn5-2309 nodE::TnS-2307 nodG::TnS-314 nodH::Tn5-2121

+L -L

GM15430 GM15431 GM15429 GM15619 GM15618 GM15624 GM1766 GM15574 GM1799 GM15627 GM15882 GM1899 GM15170 GM15883 GM15884

GM15585

length (mm) ± SD"

nodH::Tn5-148 nodH::Tn5-2313 nodH::Tn5-2219

A(nodH) A(nodFEH)

A(nodFEGH) Deletion of all nod genes GM1766(pRMJ30) GM1766(pRmSL26)

GM1766(pSB2) GM1766(pGMI149) GM1766(pGMI515) GM1766(pGMI149[nodH::Tn5-2121]) GM1766(pSB2 [nodH::Tn5-2121]) GM1766(pGMI149 [nodH::Tn5-2121 nodC::IacZ]) GM1766(pGMI149[nodH::TnS-2121 nodDl::IacZ])

9.6 9.7 10.1 10.2 10.5 9.7 10.3 9.2 9.6 9.7 9.3 9.6

± 1.0 0.8 ± 0.8 1.1 ± 0.6 ± 0.9 ± 0.6 ± 1.1 ± 1.4 ± 1.2 ± 1.3 ± 0.9

7.6 8.4 8.0 8.5 8.2 7.9 8.6 8.4 10.3 8.8 8.4 9.6 9.6 9.6 7.1 7.0 9.5 7.3

± ± ± ± ±

±

± ±

± ± ± ± ± ± ± ± ± ±

a a a a a a a

ab a a a a

1.6 b 1.0 b 1.8 b 1.2 b 1.1 b 1.1 b 1.2 b 1.4 b 1.0 a 1.1 ab 1.3 b 0.8 a 1.3 a 1.3 a 1.5 b 1.1 b 0.8 a 1.5 b

" Luteolin was added to test medium for all strains except where indicated (- L). For the sterile control and some strains, values both with and without luteolin induction were determined. b Results were analyzed by comparison of means. Values followed by the same letter did not differ significantly (i.e., P > 0.01). Values followed by a did not differ significantly from that for the sterile medium control, and values followed by b did not differ significantly from that for strain R. Ineliloti GM15375 (NodH-).

had the anatomy of nodules: apical meristem, peripheral endodermis, and vascular bundles (not shown). The fact that R. meliloti nodH mutants were found to be infective (Hac+ Inf+ Nod') on vetch prompted us to study their Tsr phenotype on liquid-grown vetch seedlings. Whereas the R. meliloti wild-type strain was Tsr-, four independent NodH- mutants provoked thickening and shortening of vetch roots. Because shortening is easier to measure than thickening, we estimated the Tsr effect by measuring root length (Table 2). The significance of length differences between two sets of plant roots was calculated by the statistical test of comparison of means; the probability level of P = 0.01 was considered significant. The Tsr effect elicited by the R. meliloti NodH mutants was not as strong as that induced by the R. leguminosarum wild-type strain but was statistically significant. Thus, the acquisition by NodH- mutants of the ability to infect vetch is associated with the ability to induce a Tsr reaction on this host. Luteolin is a flavone which activates the transcription of R. meliloti common nodABC genes (29) as well as the host range nodFEG and nodH genes (Rosenberg et al., in prepa-

ration). The addition of luteolin to the vetch growth medium did not change the Tsr- phenotype of the wild-type control but increased the Tsr+ effect of the NodH- mutants (this difference was significant at P = 0.05 but not at P = 0.01). The stimulating effect of luteolin suggested that nod genes might be involved. R. meliloti nod genes required for induction of vetch Tsr. To identify the R. meliloti nod genes required for induction of Tsr, we inoculated vetch seedlings with two series of R. meliloti strains. One series had TnS insertions in various nod genes or small deletions of the pSym megaplasmid that removed various combinations of the nodFEG and nodH host range genes. In the other series, we tested the effect of various combinations of nod genes on pRK290 plasmid derivatives in strain GM1766. This strain has a large deletion (about 300 kilobases [kb]) in pSym, which removes the whole nodulation region as well as the regulatory genes nodD2 and nodD3. The deletions and plasmids are described in Table 1 and Fig. 1, and the results of Tsr experiments are shown in Table 2. Which R. meliloti nod genes play a negative role in Tsr

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TABLE 3. Influence of nodH on the 13-galactosidase activity of nod::IacZ fusions"

nod: :IacZ fusions

nodH background

Control medium

,-Galactosidase sp act Alfalfa Luteolin exudate

(U)"

Naringenin

Vetch exudate

pRmM57 (nodC::IacZ)

Wild type (NodH+) GM15619 (NodH-)

3 4

50 64

41 40

4 3

10 9

pRmM61 (nodDl::IacZ)

Wild type (NodH+) GM15619 (NodH-)

115 134

130 131

128 125

135 139

167 138

a Alfalfa exudates were prepared as described by Mulligan and Long (28); vetch exudates were prepared as described by Zaat et al. (44). b 3-Galactosidase activity was assayed by the method of Mulligan and Long (28); each value represents the mean of three experiments.

induction? Among the series of R. meliloti mutants altered in various nod genes, only nodH mutations resulted in a significant Tsr+ phenotype. Nevertheless, a TnS insertion in nod region IIb resulted in a slight Tsr effect. With this exception, none of the strains having a functional nodH gene induced detectable Tsr. This gene had a clear negative effect on induction of the vetch Tsr reaction. In the absence of an active nodH gene, which nod genes are required for the induction of Tsr? The presence of plasmid pRmJ30, containing the nodDI and nodABC genes, was sufficient to elicit the Tsr response. Nevertheless, the Tsr reaction was more marked with plasmid pRmSL26, which contains the region nod IIa in addition to nodDlABC genes. Inactivation of nodC resulted in a Tsr- phenotype (compare pGMI149 [nodH::Tn5 nodC::lac] and pGMI149 [nodH::TnS]). Inactivation of nodDI did not alter the Tsr effect; this result suggests that the copy of nodD3 present in pGMI149 was controlling the nodABC operon in the vicinity of vetch roots. Comparison of strains carrying deletions of nodH, nodHFE, and nodHFEG indicated that the presence of the nodFE genes might have slightly increased the efficiency of the Tsr effect, but this increase was not statistically significant. This role of nodFE was also suggested by the fact that GM1766(pSB2 [nodH::Tn5-2121]) elicited significantly more marked Tsr than did GMI766(pRmSL26). Thus, the common nodABC operon is absolutely required and the nodH operon has a strong negative effect. It is worth noting that Tsr induction was more pronounced when the nod genes were located on a P1 plasmid instead of on pSym; GMI766 strains with pSB2 or pGMI149 carrying a nodH::TnS mutation induced significantly more marked Tsr than did strains having a nodH::TnS in pSym (statistical analysis not shown). Is nodH involved in nod gene regulation? The nodH gene had been shown earlier to play a positive role in hair curling of the homologous host alfalfa and a negative role in hair curling of nonhomologous hosts such as clover and vetch (5, 17). Recently, the regulatory nodD genes were shown to control host range by modulating the expression of nod operons as a function of the composition of host root exudates (14, 16, 33). Thus, the question arose whether nodH had a regulatory function for other nod genes in response to various hosts. To study the effect of nodH on the expression of other nod operons, translational fusions of the E. coli lacZ gene with nodDI and nodC were used (Table 1 and Fig. 1). The nod::lac fusions, present in derivatives of the broad-host-range plasmid pRK290, were introduced into R. meliloti strains with and without a deletion in nodH located on the resident pSym megaplasmid. These strains contained the three nodD regulatory genes on pSym and nodDI on the vector plasmid. ,-Galactosidase activity was assayed in the presence of root exudates from homologous

(alfalfa) or heterologous hosts (vetch) and also with luteolin or naringenin, flavonoid inducers of nod genes of R. meliloti and R. leguminosarum, respectively (Table 3). Inactivation of nodH did not result in a detectable change in expression of the nod::lac fusions in the presence of homologous or heterologous exudates or flavonoid inducers. These results indicate that the role of nodH in determining host specificity is not controlling gene expression of other nod operons dependent on specific plant exudates. Bioassays for extracellular symbiotic signals of R. meliloti. If it is not acting at the gene expression level, how could the nodH product determine host specificity? Van Brussel et al. (39) have shown that the Tsr effect can be elicited by treating the root system of vetch seedlings with the sterile supernatant of an R. leguminosarum culture grown under conditions of nod gene derepression, for example, in the presence of the flavanone naringenin. We therefore prepared sterile supernatants of cultures of wild-type R. meliloti and a NodHmutant grown in the presence of luteolin. The filtrates of both cultures were Tsr- on vetch (data not shown). The filtrate of an R. leguminosarum control culture was Tsr-'. Recently, Zaat et al. (44) reported a bioassay for symbiotic signals of R. leguminosarum, based on root hair deformation (Had) of vetch, which was more sensitive than the Tsr bioassay. The Had assay consists of adding a sample of the sterile filtrate under study to the liquid medium in which young vetch seedlings are grown. After 6 days, the root hairs are observed microscopically. The plant reaction is recorded as Had' when numerous root hairs are strongly distorted. We checked filtrates of induced R. meliloti cultures with the vetch Had assay. Whereas the filtrate of the wild type was Had- (Fig. 3A), the filtrate of the NodH- mutant was clearly Had' (Fig. 3B). The supernatant of a noninduced culture of the NodH- mutant was Had- (Table 4). To determine whether the R. meliloti nodH gene activity resulted in the lack of production of an extracellular vetchspecific symbiotic signal (Had or Tsr factor) or in the modification of a factor that elicits a Had or Tsr response on vetch into an alfalfa-specific factor, it was important to devise a bioassay for a putative alfalfa-specific rhizobial signal. The sterile filtrate of an induced culture of R. meliloti did not provoke a detectable thickening or shortening of alfalfa roots (Tsr phenotype), but the filtrate resulted in a very strong deformation of alfalfa root hairs (Had' phenotype, Fig. 3D). After 6 days, a large proportion of root hairs were deformed along a large part of the mature hair-covered surface of the root system (Fig. 3D). The root hairs showed very irregular growth, and various types of deformation were observed (Fig. 3D). Bulging and branching were most frequently seen, but undulations and twisting were also observed. Interestingly, various types of deformations such as branching and bulging could frequently be observed on

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x

jjj ,\5\'ti"42~~~~~~~~f

FIG. 3. Effects of sterile supernatants from cultures of wild-type R. meliloti RCR2011 (A and D), R. meliloti GMI5375 (nodH::Tn5) (B and E), and R. leguminosarum 248 (C and F) on root hair deformation (Had) of common vetch (A, B, and C) and alfalfa (D, E, and F). The supernatants were prepared from cultures induced with luteolin. (A, E, and F) Had- phenotype. (B, C, and D) Had' phenotype. The arrowheads point to root hair deformations. Bars, 100 p.m.

the same individual root hair, giving rise to very distorted hairs (Fig. 3D). Control experiments with uninduced cultures and with induction medium alone did not produce root hair deformations (Had- phenotype; Table 4). The supernatants of R. meliloti mutants with either a deletion of the nodDJABC genes or a TnS insertion in nodA, nodB, or nodC were Had- (Table 4). Thus, the alfalfa root hair deformation bioassay allowed the detection of an extracellular factor(s) whose production requires the nodABC genes. nodHf determines production of an extracellular alfalfaspecific signal. To study the specificity of the extracellular symbiotic factors, we used the vetch and alfalfa Had bioassays in parallel. The supernatant of an induced culture of R. leguminosarum was found to be Had' on vetch and Had- on

alfalfa after induction with either naringenin or luteolin (Fig. 3C and F). In contrast, the supernatant of an induced R. meliloti wild-type culture was Had' on alfalfa and Had- on vetch (Table 4 and Fig. 3A and D). This strongly suggested that the symbiotic extracellular signals present in the sterile supernatants were specific and that this specificity was due to the bacteria and not to the inducer. What are the respective roles of nodABC and nodH in the production of the R. meliloti signals? The facts that inactivation of nodABC resulted in suppression of the production of both factors active on vetch (we call this factor HadV) and alfalfa (HadA), whereas NodH- mutants were HadA- and HadV+ (Table 4 and Fig. 3), suggest that the nodABC operon acts earlier than nodH in a linear pathway. This was

5496

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J. BACTERIOL.

TABLE 4. Comparison of infection behavior of bacterial cells with the activity of sterile supernatants obtained after flavonoid induction of cultures" Phenotype on:

Alfalfa

Strain Bacterial cells Hac

R. leguminosarum wild type R. meliloti Wild type

-

Inf

-

Supernatants Nod

-

(Had)

-

Bacterial cells Hac

+

Inf

+

Supernatants Nod

+

(Had)

+ -

+

+

+

+ - (-F)

-

-

-

-

-

-

-

-

+

+

+

+

(AnodDIABC)b nodH::TnS-2121

Vetch

nodA::Tn5-2208 nodB::TnS-154 nodC::TnS-2217 Mixture of wild type and nodH::TnS-2121 strainsc

(-F)

-(-F)

+

+

a Supernatants were obtained from cultures induced by appropriate flavonoids (luteolin for R. meliloti and luteolin or naringenin for R. leguminosarum) except when indicated (-F). Supernatants were autoclaved at 110°C for 30 min. Results for the infection phenotype of R. meliloti strains on alfalfa are taken from Debelld et al. (5). b The phenotypes of cells of this strain on vetch were not determined. Supernatant phenotype only determined.

also supported by the following results. The plasmid pRmJ30 (nodDI nodABC) conferred to R. meliloti GMI766 the ability to produce a signal which induced a Had' phenotype on vetch. The same strain carrying the pSB2 plasmid (containing, in addition to the common nod genes, the host range genes), now produced a filtrate which was Had' on alfalfa but Had- on vetch, suggesting that a signal active on alfalfa (HadA factor) was now present and that the HadV factor (active on vetch) was absent. The absence of a Had reaction on vetch with R. meliloti strains having an active nodH gene could be due either (i) to the lack of sufficient HadV factor or (ii) to inhibition of the action of HadV on vetch in the presence of HadA. A mixture of filtrates from the wild-type strain and the NodH- mutant was Had' on vetch and Had' on alfalfa (Table 4), suggesting that HadA does not inhibit the action of HadV on vetch. These results indicate that nodH might transform the "common" (because encoded by the common nodABC genes) HadV signal into an alfalfaspecific signal. Correlation between symbiotic and Had specificities. In NodA- and NodH- mutants of R. meliloti, loss of the ability to infect and nodulate alfalfa was associated with inability to produce the HadA signal (Table 4). The specificity in Had bioassays of the sterile filtrates of wild-type R. leguminosarum and R. meliloti strains was similar to the specificity that the bacteria themselves exhibited during infection (Table 4). This correlation was further demonstrated by comparison of two strains differing only in the nodH gene and having very different host ranges. The wild-type R. meliloti bacteria were Hac+ Inf+ Nod' on aifalfa and Hac- Inf Nod- on vetch, and their supernatant was Had' on alfalfa and Hadon vetch (Table 4 and Fig. 3A and D), whereas the NodHbacteria were Hac- Inf- Nod- on alfalfa and Hac+ Inf+ Nod' on vetch (Fig. 2) and their supernatant was Had- on alfalfa and Had' on vetch (Table 4 and Fig. 3B and E). DISCUSSION The vetch Tsr is an early plant reaction and is detectable before nodules can be seen when the plant is inoculated with

a Nod' strain. It was previously reported that the Tsr phenotype elicited on vetch by R. leguminosarum requires the regulatory nodD gene and the common nodABC genes (39, 44). We have shown that R. meliloti derivatives lacking an active nodH gene also elicit a Tsr reaction on vetch and thus that, in addition to nodABC, the host-specific R. meliloti nodH gene is involved in determining the Tsr phenotype. Whereas the wild-type R. meliloti is Hac- Inf- Nod- Tsron vetch, the NodH- derivatives are Hac+ Inf+ Nod' Tsr+ on this host; thus, the same gene seems to control the host specificity of infection and nodulation and of the Tsr reaction. That the nodulation genes nodABC and nodH are controlling both early steps in nodulation and the Tsr phenotype indicates that these two different early host reactions might involve some common plant genes, activated (or repressed) by the same Rhizobium nod signal(s). The vetch Tsr phenotype can be elicited by R. leguminosarum either with bacterial cultures or with the sterile filtrate of a culture grown in conditions in which nod genes are induced (39, 44). In contrast, a bacterial suspension of an R. meliloti NodH- mutant is Tsr+, whereas the filtrate of the corresponding induced culture is Tsr-. Zaat et al. (44) reported that the sterile filtrate of a flavonoid-induced culture of R. leguminosarum is able to elicit on vetch root not only the Tsr phenotype but also the development of a very large number of root hairs (Hai phenotype, for hair induction) which are heavily deformed (Had phenotype). The Hai and Had assays are much more sensitive than the Tsr assay (44). Indeed, we have found that the filtrate of R. meliloti NodH- mutants, which is not significantly Tsr+, is clearly Hai' and Had' on vetch. This indicates that a symbiotic signal is present in the R. meliloti NodH- filtrate but that the vetch Tsr assay is not sensitive enough to allow its detection. This might be because either (i) the HadV (or Tsr) factor produced by R. meliloti NodH- is chemically different (and less active on vetch) from the HadV factor produced by R. leguminosarum or (ii) both HadV factors are identical but are produced in different quantities by the two strains or (iii)

VOL. 170, 1988

R. meliloti produces a compound(s) which partly antago-

nizes the HadV factor. A clear and reproducible Tsr phenotype- could not be observed on alfalfa and various Medicago species. However, it was possible to devise a Had bioassay with alfalfa which allowed the detection of a Had factor in the sterile supernatant of a luteolin-induced culture of R. meliloti. Whereas the Tsr reaction seems to be restricted to a limited number of legumes, such as V. sativa subsp. nigra, the Had bioassay appears to be more generally applicable. This assay is sensitive, since supernatants from cultures having as few as 10' bacteria per ml were clearly Had'. Production of the Had factor by R. meliloti required the nodDIABC genes and the presence of a nod gene flavone inducer, as is the case for the Had factor produced by R. leguminosarum (39, 44). The Had factors produced by R. leguminosarum and R. meliloti may be related to the R. trifolii soluble factors which have been reported to cause branching and other deformations on clover root hairs (1, 9, 42, 43), but these reports did not establish whether the production of such factors is dependent on the nodABC genes. The use of Had bioassays on both alfalfa and vetch has revealed that R. meliloti and R. leguminosarum can produce specific Had factors active on alfalfa and vetch, respectively. R. meliloti can produce at least two different Had factors. The wild-type R. meliloti supernatant is Had' on alfalfa and Had- on vetch and probably contains a HadA signal that is active on alfalfa and not on vetch. The supernatant of a NodH- mutant is Had- on alfalfa and Had' on vetch and probably contains a HadV signal that is active on vetch and not on alfalfa. All these signals of different specificity were produced by rhizobia grown in the same synthetic medium containing luteolin. The cause of the specificity is thus clearly the bacteria and not the inducer. Several recent reports have shown that nodD genes control the host range of Rhizobium spp. by regulating the expression of other nod operons as a function of the flavonoid composition of the root exudates of legume hosts (14, 16, 33). Several lines of evidence indicate that the nodH gene does not determine host range by the type of mechanism described for nodD genes. The filtrates used for Had assays were prepared in the absence of plant root exudates from Rhizobium cultures which were simply induced with luteolin. Under these conditions, the nodH product could not act by selectively modifying other nod gene activity after first distinguishing between homologous and heterologous hosts. Furthermore, studies of nod::lac fusions did not reveal an influence of nodH on expression of the nodABC and nodDi operons in the presence of luteolin, naringenin, or root exudates of alfalfa or vetch. Thus, the specificity of infection and nodulation in R. meliloti is determined at at least two levels: (i) nodD genes activate the expression of other nod operons as a function of specific plant flavonoid signals, and (ii) the nodH gene, when activated, determines the production of an extracellular signal(s) which allows the recognition of specific legumes. In addition to nodH, other R. meliloti loci, such as nodFEG and nod region Ilb, have been shown to determine the host range (5, 6, 17; E. Cervantes, S. B. Sharma, and C. Rosenberg, manuscript in preparation). We are presently investigating whether these genes are, like nodH, involved in the production of symbiotic extracellular

signals. Although it does not act by regulating nod gene expression, the nodH product could act directly at a metabolic level and influence HadA production. Whereas a NodH- mutant filtrate was HadA- HadV+, NodA- and NodC- filtrates

R. MELILOTI ALFALFA-SPECIFIC SIGNAL

5497

were HadA- HadV-. This suggests, if we hypothesize that the nodABC and nodH operons determine the production of the HadV and HadA signals, that nodABC would mediate an earlier step than nodH. We thus propose the following working hypothesis: the common nodABC operon determines the production of a precursor symbiotic signal (HadV) which is modified by the product of the host range nodH into an alfalfa-specific signal (HadA). In a NodH- mutant, a precursor HadV factor accumulates in the supernatant, whereas in a NodH+ wild-type strain, the precursor is not detectable and the HadA signal accumulates. It is also possible that the HadA factor could block the production of HadV, perhaps by inhibiting the activities of the NodABC products, assuming that they are enzymes coding for steps in a biochemical pathway. R. meliloti mutants altered in the nodABC or in the nodH operon cannot complement each other, when coinoculated onto alfalfa, to restore infection and nodule formation (F. M. Ausubel and C. Rosenberg, personal communication). This is consistent with the hypothesis that nodABC and nodH genes contribute to the production of a "combined" symbiotic signal. As already reported for R. leguminosarum (39, 44), mutations in the R. meliloti nodABC operon cause both loss of infection and nodulation and loss of detectable production of Had factors. These results indicate that in Rhizobium spp., nodABC-encoded extracellular factors might be involved in controlling the symbiotic process. A complete correlation exists for wild-type R. meliloti and its NodH- derivatives between the specificity of the symbiotic behavior of bacterial cells (marked hair curling, infection thread formation, and nodulation) and the specificity of the supernatant activity (root hair deformation). This indicates that nodil, the major R. meliloti host range gene, might determine the host specificity of infection and nodulation by mediating the production of an alfalfa-specific extracellular HadA signal. Current hypotheses concerning the molecular basis of Rhizobium host specificity propose specific bacterial attachment to root hairs and a role for bacterial surface components, such as exopolysaccharides and lipopolysaccharides (8). Dazzo and co-workers reported that nod genes of R. trifolii affect both the number and location of noncarbohydrate (acetate, pyruvate, and P-hydroxybutyrate) substitutions of R. trifolii capsular polysaccharides (3; S. PhilipHollingsworth, R. I. Hollingsworth, F. B. Dazzo, M. A. Djordjevic, and B. G. Rolfe, submitted for publication). In R. meliloti, mutations affecting the production of acidic exopolysaccharides (and the substitution of succinate or pyruvate) or ,B-1-2 glucans result in an abnormal intercellular infection, eliciting the formation of bacteria-free nodules, but the host range seems to be unaffected (12, 20, 22, 23). We are presently using the Had bioassays to purify the Had signals. Determination of the chemical structure of Had signals should reveal whether they can be derived from bacterial surface components and should contribute to our understanding of the molecular mechanisms by which rhizobia infect and nodulate their host legumes in a specific manner. ACKNOWLEDGMENTS We thank Laboratoires Bristol for providing us with kanamycin, Laboratoires Diamant for neomycin, and Laboratoires Lepetit for rifampicin. We are grateful to Sharon R. Long and Thierry Huguet for providing bacterial strains. We are particularly grateful to Shashi B. Sharma for providing the plasmid pSB2 prior to publication. We

5498

FAUCHER ET AL.

thank David Barker for reviewing the manuscript. Statistical analysis was performed by Jer6me Kaan (Station de Biometrie, INRA Toulouse). This work was supported by a Biological Nitrogen Fixation grant from Soci6td Nationale Elf Aquitaine, Entreprise Miniere et Chimique, Rh6ne Poulenc, and Charbonnages de France-Chimie to Laboratoire de Biologie Moleculaire des Relations Plantes-Microorganismes.

1. 2. 3.

4.

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tion of R. leguminosarum and other rhizobia and agrobacteria harbouring a leguminosarum Sym plasmid. Plant Sci. Lett. 27: 317-325. 39.

van Brussel, A. A. N., S. A. J. Zaat, H. C. J. Canter Cremers, C. A. WQfel1man, E. Pees, T. Tak, and B. J. J. Lugtenberg. 1986. Role of plant root exudate and Sym plasmnid-localized nodulation genes in the synthesis by R. leguminosarum of Tsr factor, which causes thick and short roots on common vetch. J. Bacteriol. 165:517-522. 40. Vasse, J. M., and G. L. Truchet. 1984. The Rhizobium-legume symbiosis: observation of root infection by bright-field microscopy after staining with methylene blue. Planta 161:487-489. 41. Vincent, J. M. 1970. A manual for the practical study of

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root-nodule bacteria. IBP Handbook 15. Blackwell Scientific Publications, Oxford. 42. Yao, P. Y., and J. M. Vincent. 1976. Factors responsible for the curling and branching of clover root hairs by Rhizobium. Plant Soil 45:1-16. 43. Yao, P. Y., and J. M. Vincent. 1969. Host-specificity in the root hair "curling factor" of Rhizobium spp. Aust. J. Biol. Sci. 22: 413-423. 44. Zaat, S. A.. J., A. A. N. van Brussel, T. Tak, E. Pees, and B. J. J. Lugtenberg. 1987. Flavonoids induce Rhizobium leguminosarum to produce nodABC gene-related factors that cause thick, short roots and root hair responses on common vetch. J. Bacteriol. 169:3388-3391.