Transcripts for two genes expressed early in alfalfa nodule development (MsENOD40 and MsENOD2) are found in mycorrhizal roots, but not in noncolonized roots or in roots infected with the fungal pathogen Rhizoctonia solani. These same two early nodulin genes are expressed in uninoculated roots upon application of the cytokinin 6-benzylaminopurine. Correlated with the expression of the two early nodulin genes, we found that mycorrhizal roots contain higher levels of trans-zeatin riboside than nonmycorrhizal roots. These data suggest that there may be conservation of signal transduction pathways between the two symbioses-nitrogen-fixing nodules and phosphate-acquiring mycorrhizae.Of the two commonly described symbioses of roots-nitrogenfixing nodules in response to members of Rhizobiaceae or Frankiaceae and phosphate-acquiring arbuscular mycorrhizae (AM) between roots and endophytic fungi-the more ancient of the two is AM. Structures identified as arbuscules have been found in Aglaophyton major, an early Devonian land plant, providing evidence for at least a Ͼ400-million-year-old association between terrestrial plants and fungi (1). In contrast, the Rhizobium-legume symbiosis is much younger, having been established no more than 65 to 136 million years ago, when the angiosperms evolved. However, unlike the AM symbiosis, which is found in almost all angiosperm families, Rhizobiuminduced nitrogen-fixing symbioses appear to be restricted to a single subclade of a larger nitrogen-fixing clade (2).LaRue and Weeden (3) proposed that nodulation evolved from the more ancient AM association because of certain similarities between the two symbioses. For example, some legumes are Nod Ϫ (absence of nodule formation) as well as Myc Ϫ

Transgenic alfalfa (Medicago sativa L. cv Regen) roots carrying genes encoding soybean lectin or pea (Pisum sativum) seed lectin (PSL) were inoculated with Bradyrhizobium japonicum or Rhizobium leguminosarum bv viciae, respectively, and their responses were compared with those of comparably inoculated control plants. We found that nodule-like structures formed on alfalfa roots only when the rhizobial strains produced Nod factor from the alfalfa-nodulating strain, Sinorhizobium meliloti. Uninfected nodule-like structures developed on the soybean lectin-transgenic plant roots at very low inoculum concentrations, but bona fide infection threads were not detected even when B. japonicum produced the appropriate S. meliloti Nod factor. In contrast, the PSL-transgenic plants were not only well nodulated but also exhibited infection thread formation in response to R. leguminosarum bv viciae, but only when the bacteria expressed the complete set of S. meliloti nod genes. A few nodules from the PSL-transgenic plant roots were even found to be colonized by R. leguminosarum bv viciae expressing S. meliloti nod genes, but the plants were yellow and senescent, indicating that nitrogen fixation did not take place. Exopolysaccharide appears to be absolutely required for both nodule development and infection thread formation because neither occurred in PSL-transgenic plant roots following inoculation with an Exo Ϫ R. leguminosarum bv viciae strain that produced S. meliloti Nod factor. Bacteria belonging to the family Rhizobiaceae (Rhizobium, Bradyrhizobium, Azorhizobium, Mesorhizobium, and Sinorhizobium) induce the formation of nitrogenfixing nodules on their leguminous hosts. This symbiotic interaction, which is governed by sequential signal exchange between rhizobia and their symbiotic partners, exhibits a high degree of specificity, and a number of signal molecules involved in the initial stages of this specificity have been extensively studied. Nod factors are synthesized by the products of rhizobial nod genes, which are induced by plantsecreted molecules such as flavonoids (Hirsch, 1992; Long, 1996). The Sinorhizobium meliloti Nod factor consists of a variable-length N-acetylglucosamine oligomer with a C-16 acyl tail at the non-reducing end and a sulfate at the reducing end, whereas the Rhizobium leguminosarum bv viciae Nod factor has a C-18 fatty acyl residue and no sulfate (for review, see Schultze and Kondorosi, 1998). Nod factors are considered the main rhizobial inducËer molecules for nodulation because the purified molecules elicit, in a host-specific way, many of the plant responses observed in the early stages of nodule formation. These responses include changes in free calcium levels and ion balance, alterations in cytoskeletal organization and morphology of root hairs, the initiation of cortical cell divisions (Ccd), and the triggering of nodule development (Spaink et al., 1991; Truchet et al., 1991;

Plant lectins have been implicated as playing an important role in mediating recognition and specificity in the Rhizobium-legume nitrogen-fixing symbiosis. To test this hypothesis, we introduced the soybean lectin gene Le1 either behind its own promoter or behind the cauliflower mosaic virus 35S promoter into Lotus corniculatus, which is nodulated by R. loti. We found that nodulelike outgrowths developed on transgenic L. corniculatus plant roots in response to Bradyrhizobium japonicum, which nodulates soybean and not Lotus spp. Soybean lectin was properly targeted to L. corniculatus root hairs, and although infection threads formed, they aborted in epidermal or hypodermal cells. Mutation of the lectin sugar binding site abolished infection thread formation and nodulation. Incubation of bradyrhizobia in the nodulation (nod) gene-inducing flavonoid genistein increased the number of nodulelike outgrowths on transgenic L. corniculatus roots. Studies of bacterial mutants, however, suggest that a component of the exopolysaccharide surface of B. japonicum, rather than Nod factor, is required for extension of host range to the transgenic L. corniculatus plants.

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