© 2002 by The Society for Integrative and Comparative Biology
Investigation of Four Classes of Non-nodulating White Sweetclover (Melilotus alba annua Desr.) Mutants and Their Responses to Arbuscular-Mycorrhizal Fungi1
1 Department of Molecular, Cell and Developmental Biology
2 Molecular Biology Institute, University of California, Los Angeles, California 90095-1606
3 
Boyce Thompson Institute, Tower Road, Ithaca, New York 14853-1801
4 Agronomy and Natural Resources Department, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel
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The nitrogen-fixing symbiosis between Rhizobiaceae and legumes is one of the best-studied interactions established between prokaryotes and eukaryotes. The plant develops root nodules in which the bacteria are housed, and atmospheric nitrogen is fixed into ammonia by the rhizobia and made available to the plant in exchange for carbon compounds. It has been hypothesized that this symbiosis evolved from the more ancient arbuscular mycorrhizal (AM) symbiosis, in which the fungus associates with roots and aids the plant in the absorption of mineral nutrients, particularly phosphate. Support comes from several fronts: 1) legume mutants where Nod– and Myc– co-segregate, and 2) the fact that various early nodulin (ENOD) genes are expressed in legume AM. Both strongly argue for the idea that the signal transduction pathways between the two symbioses are conserved. We have analyzed the responses of four classes of non-nodulating Melilotus alba (white sweetclover) mutants to Glomus intraradices (the mycorrhizal symbiont) to investigate how Nod– mutations affect the establishment of this symbiosis. We also re-examined the root hair responses of the non-nodulating mutants to Sinorhizobium meliloti (the nitrogen-fixing symbiont). Of the four classes, several sweetclover sym mutants are both Nod– and Myc–. In an attempt to decipher the relationship between nodulation and mycorrhiza formation, we also performed co-inoculation experiments with mutant rhizobia and Glomus intraradices on Medicago sativa, a close relative of M. alba. Even though sulfated Nod factor was supplied by some of the bacterial mutants, the fungus did not complement symbiotically defective rhizobia for nodulation.
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INTRODUCTION |
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Plants undergo two different symbioses to assist them in the acquisition of nutrients from soil solution: the mycorrhizal symbiosis between fungi and roots results in the assimilation of phosphorous and other nutrients, whereas the nitrogen-fixing symbiosis generates ammonia from atmospheric nitrogen by the action of rhizobial bacteria living within nodules, specialized structures formed on plant roots. The arbuscular-mycorrhizal (AM) symbiosis is ancient; it most likely evolved in the Early Devonian (398 million years ago). Fossils of Aglaophyton major, a leafless and rootless plant from the Rhynie chert, have been verified as containing arbuscules (Remy et al., 1994
). However, fungal spores resembling extant Glomus sp. were already present in the mid-Ordovician (476 million years ago) (Redecker et al., 2000), which implies that the AM symbiosis may have evolved as early as the late Silurian, during which the initial diversification of vascular plants occurred (Kendrick and Crane, 1997). It has been proposed that the evolution of the mycorrhizal symbiosis led to the colonization of the land (Pirozynski and Malloch, 1975). In contrast, fossils of the earliest angiosperms have been found only in the Cretaceous (110 million years ago), making it unlikely that the legume-Rhizobium symbiosis would have been established earlier.
There are several common elements observed in the symbiosis between members of the Rhizobiaceae and species of the Fabaceae (legumes) and the association between AM fungi and more than 80% of green land plants (see reviews by Harrison, 1997; Hirsch and Kapulnik, 1998; Barker and Tagu, 2000; Peterson and Guinel, 2000). Flavonoids, molecules secreted by plant roots and seeds, induce rhizobial nod genes as well as AM fungal spore germination and hyphal colonization. Upon induction of their nod genes, rhizobia synthesize Nod factor, a lipochitooligosaccharide signal molecule that is more typical of fungal cell walls than of Gram-negative bacteria. These similarities, together with the ancient nature of the association between AM fungi and terrestrial plants, have led to the proposal that the Rhizobium-legume symbiosis is derived from the mycorrhizal interaction (LaRue and Weeden, 1994). In partial support of this hypothesis, van Rhijn et al. (1997) found that two early nodulin (ENOD) genes, ENOD40 and ENOD2, which serve as markers of the plant's response to rhizobial inoculation, are also expressed in mycorrhizal roots of Medicago sativa, demonstrating that steps in the downstream signal transduction pathways are conserved. These genes are also induced by the plant hormone cytokinin and, as cytokinin levels are enhanced in mycorrhizal roots (van Rhijn et al., 1997), it is likely that the downstream stages in mycorrhiza establishment include an increase in endogenous cytokinin concentrations. Additionally, the genes ENOD5 and ENOD12A are expressed in Pisum sativum mycorrhizal roots (Albrecht et al., 1998), and VfLb29, which encodes a leghemoglobin, is expressed in mycorrhizal roots of Vicia faba (Frühling et al., 1997).
Mutations that result in a non-nodulating (Nod–) condition in several legumes often produce a non-mycorrhizal (Myc–) phenotype as well (Duc et al., 1989; Bradbury et al., 1991; Sagan et al., 1995; Peterson and Guinel, 2000). Resendes et al. (2001) described a low-nodulating pea mutant that shows a corresponding reduction in mycorrizal association. However, the sym10 mutant of pea is Nod–, but Myc+ (Duc et al., 1989), and the Glycine max non-nodulating mutants nod49 and nod139 are also Myc+ (Wyss et al., 1990). Likewise, the Nod– Phaseolus vulgaris mutant R99 is Myc+, but mutant R69, which forms uninfected nodules that lack peripheral vascular bundles, is Myc– (Shirtliffe and Vessey, 1996). Senoo et al. (2000) found that several mycorrhizal mutants of Lotus japonicus (designated Ljsym72, Ljsym71-1 and Ljsym71-2) co-segregate with Nod–. Mutations in the LjSym2, LjSym3, and LjSym4-1 genes, which result in a Nod– phenotype, still allow the formation of a few arbuscules (Myc+) (Wegel et al., 1998), but Ljsym4-2 mutants do not form arbuscules (Bonfante et al., 2000). So far, the identity of only one gene, which when mutated gives rise to a non-nodulating phenotype, has been revealed (Schauser et al., 1999). However, this L. japonicus non-nodulating mutant, described as nin (non-infective), exhibits root hair deformation in response to rhizobial inoculation and is also Myc+ (J. Stougaard, personal communication).
Five different complementation groups, sym1-sym5, of non-nodulating white sweetclover (Melilotus alba annua Desr.) have already been described (Miller et al., 1991; Utrup et al., 1993). Of the five different Nod– complementation groups, earlier studies showed that the sym2 and sym4 mutants underwent root hair curling (Hac) and infection thread formation (Inf) in response to Sinorhizobium meliloti inoculation, and some nodules developed (Utrup et al., 1993). The sym4 mutant (BT68) has been excluded from further studies because it is not a prolific seed producer. Although the sym1 and sym5 mutants exhibited marked root hair deformation (Had), but not Hac, following inoculation with S. meliloti, no infection threads were observed in the Utrup et al. (1993) study. However, one of the sym1 mutant alleles, BT62, represents a weak allele because Wu et al. (1996) found that 12% of the plants formed ineffective nodules. Small, white ineffective nodules were also found on occasion on roots of the sym5 mutant plants.
Of the five different complementation groups, sym3 (represented by mutants BT61, BT69, and BT70) is the least responsive to S. meliloti inoculation. According to Utrup et al. (1993), sym3 root hairs branched or became bulbous at their tips, but infection threads, cortical cell divisions, or nodule primordia were not initiated. In this report, we re-examined the root hair responses of the three different sym3 mutant alleles to S. meliloti at earlier time points than that analyzed by Utrup et al. (1993). Our investigations indicate that the root hair deformation response for the sym3 mutants is significantly less at these earlier time points than at 3 days post-inoculation (dpi), but can be augmented by inoculation with wild-type S. meliloti.
This report gives a first description of the symbiotic phenotypes of the different white sweetclover non-nodulating mutants in response to inoculation with Glomus intraradices. The complexity of responses among the various legumes to inoculation with either rhizobia or AM fungi also led us to examine whether functional complementation could occur between rhizobial Nod– or Exo– mutants and mycorrhizal fungi with the restoration of the bacteria's ability to nodulate or infect nodules, respectively.
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Plant material
The wild-type white sweetclover (Melilotus alba Desr.) U389 and its various non-nodulating mutants were utilized (Miller et al., 1991
). The mutants sym2 (BT59), generated by EMS, and sym5 (BT71), generated by neutron radiation, are represented by mutations at a single locus whereas four different mutant alleles exist for sym1 and three for sym3 (Miller et al., 1991). For sym3, two different mutagens, EMS and neutron radiation, produced mutations at the same locus; BT61 is derived from EMS mutagenesis, and BT69 and BT70 from neutron radiation (Miller et al., 1991). The four different sym1 mutants result from EMS mutagenesis; in this report, we analyzed BT35, BT58, and BT62. Alfalfa (Medicago sativa L. cv. Iroquois) was used for the co-inoculation studies. Sweetclover seeds were scarified with a scarifier or sandpaper. Both sweetclover and alfalfa seeds were surface-sterilized briefly in 95% ethanol, followed by immersion in commercial bleach for 45 min. The seeds were then washed five times with sterile water, placed on 0.8% Phytagar (GiboBRL), and grown in the dark for 48 hr.
Strains
The S. meliloti strains used in this study were Rm1021 (wild-type), Rm5612 (nodC::Tn5), Rm2212 (nodH::Tn5), RJW14 ((nodF)nodL::Tn5), Rm7225 (exoH::Tn5), and Rm7210 (exoY::Tn5). GFP-labeled Nod– strains were generated by introducing the plasmid pHC60 (Cheng and Walker, 1998) into each strain by triparental mating using the helper plasmid pRK2013 (Ditta et al., 1980). The Exo– strains were a generous gift from G. C. Walker. Liquid cultures were grown shaking in an incubator at 30°C, washed once with sterile water, and re-suspended to approximately 108 cells per ml.
Aseptic spores of G. intraradices (Premier Tech, Quebec) were used for inoculating the roots.
Root hair assay
Germinated wild-type and mutant sweetclover seeds were transferred to Petri dishes containing 1/4 strength Hoagland's medium (Machlis and Torrey, 1956) minus nitrogen. Each plant was inoculated with 10 µl of Rm1021 (108 cells/ml). A qualitative assessment of root hairs along the entire length of the root was made at 5, 9.5 and 24 hr post-inoculation (hpi).
Mycorrhizal association
Wild-type and mutant sweetclover seedlings were transferred to autoclaved sand in enclosed Magenta jars (Magenta Corp., Chicago, IL) that were modified with holes in the bottom for drainage and holes in the top covered with MicroporeTM tape (3M Health Care) to prevent ethylene accumulation. Each jar contained 9 plants and was watered with 1/4 strength Hoagland's medium containing limiting phosphate (0.2 mM KH2PO4). Thirty to 50 spores of G. intraradices were supplied to each plant by layering them 4 cm below the sand surface. Mycorrhizal association was assessed for intracellular hyphae and arbuscules 3 weeks after inoculation.
Co-inoculation studies
Alfalfa plants were grown as described for assessing mycorrhizal association except that the plants were watered with 1/4 strength Hoagland's medium minus nitrogen, or minus nitrogen and with limiting phosphate, or with limiting phosphate. Each plant was inoculated with 50 µl of rhizobia (108 cells/ml). For each rhizobial strain, treatments were set up for inoculation with rhizobia alone, co-inoculation with rhizobia and G. intraradices simultaneously, or inoculation of rhizobia one week after inoculation with G. intraradices. Roots were harvested and assessed for root hair deformation, infection thread formation, and nodule development 2 and 4 wk after inoculation with the bacteria.
Growth conditions
The plants were grown in a growth cabinet where they were maintained under a 16-hr light, 8-hr dark cycle at 24°C.
Staining and microscopy
Whole roots were harvested for the assessment of mycorrhizal association. Roots were fixed in FAA (formalin, acetic acid, alcohol) overnight, cleared in 10% KOH for 2 hr at 50°C, and then incubated overnight in a 10 µg/ml solution of wheat germ agglutinin-Alexa Fluor® 488 conjugate (Molecular Probes), which is a fluorescent lectin that binds to N-acetyl-glucosamine and will thus stain fungal structures. For confocal microscopy, counterstaining of the root was done by subsequently incubating the roots in 10 µg/ml acid fuchsin in phosphate-buffered saline for 5 minutes to take advantage of this stain's fluorescent properties. Some roots were cleared and stained with chlorazole black E (Brundrett et al., 1984) or with 1% HCl followed by 0.01% acid fuchsin-lactic acid. The fungi were initially observed and assessed for mycorrhizal association with a Zeiss Axiophot light microscope using fluorescence and a 488 nm excitation filter. Confocal images were taken with a Bio-Rad MRC1024ES (krypton/argon) confocal laser scanning microscope associated with a Nikon Eclipse E800 light microscope using settings for FITC (488 nm) for observation of the fungus and TRITC (568 nm) for observation of the counterstained roots.
Of the three different staining techniques, chlorazole black E gave the best definition of fungal structures at the light microscope level. However, because it is not fluorescent, this stain cannot be used for confocal microscopy, which more clearly illustrates the relationship of the fungus to the inside or outside of the root. Acid fuchsin, which is commonly used as a stain for mycorrhizal fungi, can be used for confocal microscopy because it is fluorescent. However, the time required for destaining the roots to eliminate background fluorescence was inordinately long. Wheat-germ agglutinin conjugated to Alexa Fluor® 488 (Molecular Probes, Inc.) provided the best specific staining to the fungus with no background. Low concentrations of acid fuchsin provided an effective means for staining the root itself.
Sweetclover and alfalfa roots that had been inoculated by rhizobia were harvested and assessed using a Zeiss Axiophot light microscope under bright-field or Nomarski optics in conjunction with fluorescence microscopy for observation of GFP-labeled rhizobia.
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Root hair deformation
We re-examined root hair curling in the sweetclover sym mutants because Utrup et al. (1993)
, who had studied root hair deformation for a number of sym mutants, did not report observations earlier than 3 dpi. In addition, they examined only one of four sym1 mutant alleles (BT62), and only one of three sym3 mutant alleles (BT70). We detected root hair deformation in response to Rm1021 as early as 5 hpi (Fig. 1A). For example, the root hairs of wild-type line U389 and the sym2 mutant (BT59) (Fig. 1C) showed significant deformation (Had+) 5.5 hpi whereas the sym1 (Fig. 1B) and sym5 (data not shown) mutants did not show a significant response until 9 hpi, at which time sym2 and wild-type sweetclover root hairs were extensively deformed (data not shown).
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Utrup et al. (1993)
described a "rippley" or "arcing" root hair response for the sym3 mutant BT70 3 dpi. We did not observe such a response in the three different sym3 mutant alleles when examined within hours of inoculation. At 5.5 hpi, we observed some swelling (Has+; Catoira et al., 2000) of the root hair tip (Fig. 1D), but a comparable amount of swelling was observed in uninoculated sym3 mutant root tips (data not shown). By 24 hpi, uninoculated wild-type root hairs were slightly Has+ (Fig. 1E) whereas inoculated U389 root hairs exhibited extensive Had (Fig. 1F) and the formation of shepherd's crooks. All sym3 mutants at this time had root hairs that were bulging at the tips (Fig. 1G, I); the Has response was somewhat augmented in the Rm1021-inoculated roots (Fig. 1H, J). To test whether the sym3 response was related to the presence of Nod factor, we inoculated sym3 roots with Rm5612, a nodC mutant. We found no difference in root hair response between Rm5612-inoculated roots and the uninoculated control roots (data not shown), indicating that the augmented Has response was most likely due to the presence of Nod factor. There was no obvious root hair response in any of the sweetclover roots following AM-fungal inoculation.
AM formation
We examined the responses of wild-type sweetclover roots and the non-nodulating sym mutants to inoculation with G. intraradices. Roots of U389, the wild-type sweetclover, formed well-established mycorrhizal associations within three weeks after inoculation. Appressoria were present, fungal hyphae penetrated the root cells, and both vesicles and arbuscules were detected within cortical cells (Fig. 2A). The sym2 mutant exhibited a Myc+ phenotype (Fig. 2B), appearing to have even more extensive mycorrhizal colonization than that of the U389 roots. We do not know the reason for this difference in response, particularly because U389 normally develops nitrogen-fixing nodules whereas the sym2 mutant produces ineffective nodules (Utrup et al., 1993).
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The sym1 mutant responses were somewhat variable, depending on the allele studied. BT62, described as "leaky" by Wu et al. (1996)
, was Myc+ (Fig. 2D) whereas BT58 and BT35 were Myc–. External hyphae ramified over the surface of the BT58 roots, but there was no evidence for hyphal penetration (Fig. 2C). The single mutant at the sym5 locus was also Myc– even though sym5 mutants on occasion form very small, white, ineffective nodules. There was widespread hyphal proliferation over the surface of the root with no penetration of the root cells, although appressoria were observed (Fig. 2H).
Similarly, there was no indication of hyphal penetration, or arbuscule or vesicle development in any of the sym3 mutant roots (Fig. 2E–G). The hyphae were extensively branched all over the root surface and appressoria were formed, but there was no penetration into the root tissue (Fig. 2F). These results are summarized in Table 1.
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Complementation experiments
Complementation experiments were done using M. sativa because its response to various rhizobial mutants is well documented. Alfalfa seedlings were co-inoculated with G. intraradices spores and wild-type or nodC, nodH, nodFL, exoH, or exoY mutants of S. meliloti. Root systems were assessed for whether or not the mycorrhizal fungus was able to complement the mutants and allow an increased progression of the rhizobial symbiosis.
At 2 and 4 wk after inoculation, Had, Hac, Inf, and pink nodules were observed on plants that had been inoculated with Rm1021, whether or not plants had also been co-inoculated with G. intraradices. However, signs of nodule development could not be detected on plants inoculated with any of the Nod– mutants, with or without the fungus. The nodC mutant, which is completely deficient in Nod factor production, caused no visible root response with or without co-inoculation. The nodH mutant, which produces Nod factor lacking the sulfate at the reducing end, and the nodFL mutant, which produces sulfated but non-O-acetylated Nod factor, both triggered root hair deformation, similar to that reported previously (Debellé et al., 1986; Ardourel et al., 1994). However, co-inoculation with G. intraradices did not enhance the response. Cytological observations of roots aided by the observation of GFP-labeled bacteria did not reveal infection thread formation after co-inoculation of alfalfa with G. intraradices and any of the Nod– mutants.
The exoH mutant lacks succinyl transferase (Leigh et al., 1987), which is required for the addition of the succinyl modification to the exopolysaccharide, and the exoY mutant lacks a galactosyl transferase (Leigh et al., 1985), which is required for the first step of succinoglycan biosynthesis. Inoculation with either of these mutants resulted in aborted infection threads and uninfected nodules as reported previously (Cheng and Walker, 1998). As with the Nod– mutants, co-inoculation with G. intraradices did not promote further progression of the nodulation phenotype. These results are summarized in Table 2.
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We found that several sym mutants of sweetclover are both Nod– and Myc–: two mutant alleles of sym1 (BT35 and BT58), all three mutant alleles of sym3 (BT61, BG69, BT70), and the single mutant of sym5 (BT71). There are two distinct Myc– phenotypes, Myc–1 and Myc–2, which have been described (Gianinazzi-Pearson, 1996
; Harrison, 1997; Peterson and Guinel, 2000). These designations refer to a breakdown in internal colonization of the plant (Myc–1) or of arbuscule formation (Myc–2). Of the two, the Myc–1 phenotype is the more commonly described. We did not observe arbuscule formation in response to G. intraradices in roots of two of the sym1 mutants (BT35 and BT58), the sym3 mutants, or of the single sym5 (BT71) mutant of sweetclover; all are thus designated Myc–1. Some preliminary experiments with G. mosseae, however, indicate that the sym1 mutant (BT58) may be leaky with respect to mycorrhiza formation (unpublished data, Y.K.). The sweetclover mutants, sym1 (BT62) and sym2 (BT59) are colonized by G. intraradices, and arbuscules are formed within the root cells; thus, they are Myc+. Preliminary results also indicate that there are differences in the expression of the early nodulin gene, ENOD40, in the various Myc– roots versus those that are Myc+(M. R. Lum and A. M. Hirsch, unpublished results). Hence, as observed for other non-nodulating legumes, there is a link between being blocked in the earliest stages of nodulation (Nod–) and Myc–.
If Rhizobium's ability to establish a symbiotic association with plants was derived from arbuscular mycorrhizal fungi, then co-inoculation of G. intraradices and Nod– rhizobia might rescue the non-nodulation phenotype and moreover, co-inoculation of G. intraradices and Exo– rhizobia might produce infected nodules. However, these results did not occur even when a nodFL mutant or Exo– mutant, each of which makes sulfated Nod factors, was utilized. Nod factor sulfation appears to be absolutely required for S. meliloti hosts to respond to inoculation (van Rhijn et al., 2001). The fact that AM-fungal co-inoculation does not rescue Nod– S. meliloti contrasts with our earlier results whereby Exo– and Nod– S. meliloti functionally complemented each other resulting in infected nitrogen-fixing nodules (Klein et al., 1988). However, there are many more profound differences between the potential signal molecule(s) produced by the fungal versus bacterial symbionts than there are between the two different types of rhizobial mutants. For one, S. meliloti Nod factor is both sulfated and acetylated whereas the likely comparable signal molecule in G. intraradices is not. In addition, the host may respond quite differently to the various microbial molecules it encounters. For example, some legume hosts such as alfalfa are known to respond by undergoing cortical cell divisions to treatment with Nod factor alone (Truchet et al., 1991), but others do not. Moreover, there is little published evidence for plant hosts responding to a secreted molecule from AM-fungi although there are reports about a host-produced molecule affecting branching of the AM-fungus (see references in Hirsch and Kapulnik, 1998).
At least two possible mechanisms could be invoked to explain the overlap between the two symbioses: 1) genes were laterally transferred from the fungus to the bacteria; or 2) the plant's long history of establishing mycorrhizal associations with phosphate-acquiring fungi led to the convergence of signal molecules produced by rhizobial cells with those produced by AM fungi. Our data do not allow us to differentiate between these two possibilities. It does enable us to suggest that multiple steps in the symbiotic process are conserved, and not just a single step as evidenced by the fact that several loci (Sym1, Sym3, and Sym5) when mutated are both Nod– and Myc–. However, mutations in the Sym3 locus exhibit the tightest correspondence between Nod– and Myc– because sym3 mutants show the least response to rhizobial inoculation, and all three mutant alleles show the same phenotype in response to S. meliloti and G. intraradices. This suggests that Sym3 is upstream of Sym1 and probably of Sym5 also.
Even though it was originally classed as non-nodulating (Miller et al., 1991), the sweetclover sym2 mutant develops ineffective nodules approximately 25% of the time (Utrup et al., 1993). This may explain why sym2 is Myc+, proceeding beyond the Myc–2 block. However, we do not know why sym2 roots appear to be colonized even more efficiently than the wild-type U389 roots. At this time, only one allele of sym2 is available for study, and thus, any conclusions about its position in a developmental pathway are premature.
To our knowledge, there are no examples of legumes that are both Myc– and capable of producing infected nodules. Those legume Myc– mutants that are Nod+ are generally uninfected (Bradbury et al., 1991; Shirtliffe and Vessey, 1996). The correlation between Myc– and the inability to produce infected nodules suggests that many of the initial stages in the interaction between Rhizobium and its legume host are upstream of those leading to mycorrhiza formation. The lack of functional complementation of rhizobial mutants by co-inoculation with a mycorrhizal fungus further supports this hypothesis.
How do we explain the overlap in gene products involved in both nodulation and mycorrhiza formation? Could these gene products represent upstream components in a common signal transduction pathway such as a receptor or signal transducers? The fact that a number of legume mutations exist, some of which are Nod– and Myc– and others which are Nod– and Myc+, suggests that the two symbiotic pathways share a number of steps at the initial stages of the interaction, or alternatively, that several proteins are grouped together in a receptor complex, some of which are used for both plant-microbe associations. Elucidation of Sym3 and equivalent genes in other legumes may help us sort out the relationship between these two important plant symbioses. The fact that Sym3 is absolutely required for both symbiotic interactions points to the pivotal role of its gene product in serving as an initial "gatekeeper."
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ACKNOWLEDGMENTS |
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J. H. Norris (U. Rhode Island) is thanked for communicating her unpublished data. We also thank L. Iruela-Arispe (UCLA) for use of the confocal microscope and M. Kowalczyk (UCLA) for her help with the illustrations. We are grateful to S. R. Long and R. J. Wais (Stanford U.) and G. C. Walker (MIT) for the rhizobial strains used in the study. P. Gensel (U. North Carolina) is thanked for helpful discussions on Devonian mycorrhizae, and we thank our lab members for their valuable comments on the manuscript. We apologize to authors whose work has not been cited because of space constraints.
This research was supported in part by National Science Foundation grants 96-30842 and 97-23982, UC Mexus grant 017451, and a grant from the Council on Research from the UCLA Academic Senate to AMH. A USPHS National Research Service Award GM07185 and BioStar grant S98-86 supported MRL.
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FOOTNOTES |
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1 From the Symposium Living Together: The Dynamics of Symbiotic Interactions presented at the Annual Meeting of the Society of Integrative and Comparative Biology, 3–7 January 2001, at Chicago, Illinois.
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References |
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