Ann M. Hirsch

Legume-Nodulating Betaproteobacteria: Diversity, Host Range, and Future Prospects

Rhizobia form specialized nodules on the roots of legumes (family Fabaceae) and fix nitrogen in exchange for carbon from the host plant. Although the majority of legumes form symbioses with members of genus Rhizobium and its relatives in class Alphaproteobacteria, some legumes, such as those in the large genus Mimosa, are nodulated predominantly by betaproteobacteria in the genera Burkholderia and Cupriavidus. The principal centers of diversity of these bacteria are in central Brazil and South Africa. Molecular phylogenetic studies have shown that betaproteobacteria have existed as legume symbionts for approximately 50 million years, and that, although they have a common origin, the symbiosis genes in both subclasses have evolved separately since then. Additionally, some species of genus Burkholderia, such as B. phymatum, are highly promiscuous, effectively nodulating several important legumes, including common bean (Phaseolus vulgaris). In contrast to genus Burkholderia, only one species of genus Cupriavidus (C. taiwanensis) has so far been shown to nodulate legumes. The recent availability of the genome sequences of C. taiwanensis, B. phymatum, and B. tuberum has paved the way for a more detailed analysis of the evolutionary and mechanistic differences between nodulating strains of alpha- and betaproteobacteria. Initial analyses of genome sequences have suggested that plant-associated Burkholderia spp. have lower G+C contents than Burkholderia spp. that are opportunistic human pathogens, thus supporting previous suggestions that the plant- and human-associated groups of Burkholderia actually belong in separate genera.

Role of lectins (and rhizobial exopolysaccharides) in legume nodulation

The lectin recognition hypothesis proposes that plant lectins mediate specificity in the Rhizobium-legume symbiosis. Although the hypothesis was developed eight years before nod genes were identified in rhizobia and sixteen years before Nod factor was shown to be a major determinant of host specificity, experiments performed recently using transgenic lectin plants support its main tenets.

PLANT HORMONES AND NODULATION : WHAT'S THE CONNECTION ?

Ever since Thimann [20] proposed that auxin plays a role in nodule development, considerable effort has been expended to show whether phyto- hormones are involved. Because the hormones are involved in other types of organogenesis, it seems likely that they have a role in nodule de- velopment. However, important questions remain unanswered:

Plant lectins: the ties that bind in root symbiosis and plant defense

Lectins are a diverse group of carbohydrate-binding proteins that are found within and associated with organisms from all kingdoms of life. Several different classes of plant lectins serve a diverse array of functions. The most prominent of these include participation in plant defense against predators and pathogens and involvement in symbiotic interactions between host plants and symbiotic microbes, including mycorrhizal fungi and nitrogen-fixing rhizobia. Extensive biological, biochemical, and molecular studies have shed light on the functions of plant lectins, and a plethora of uncharacterized lectin genes are being revealed at the genomic scale, suggesting unexplored and novel diversity in plant lectin structure and function. Integration of the results from these different types of research is beginning to yield a more detailed understanding of the function of lectins in symbiosis, defense, and plant biology in general.

MOLECULAR SIGNALS AND RECEPTORS: CONTROLLING RHIZOSPHERE INTERACTIONS BETWEEN PLANTS AND OTHER ORGANISMS

Rhizosphere interactions are affected by many different regulatory signals. As yet, however, only a few have been identified. Signals, by definition, contain information, react with a receptor, and elicit a response. Signals may thus represent the highest level of evolved response in rhizosphere communities and, in that sense, occupy a supreme control point. At the same time, some signals may function as modulators of downstream responses, rather than on/off switches. To assess these possibilities, several interactions between plants and soil organisms are described, starting with the molecular interactions between legu- minous plants and symbiotic bacteria of the family Rhizobiaceae, one of the best-charac- terized plant-microbe associations in the rhizosphere. We then examine other interactions between plants and soil organisms for overlap and/or connections with the rhizosphere signals utilized in the legume-Rhizobium symbiosis. Whether information currently avail- able reflects the interaction of the organisms in nature or only in the laboratory has not always been determined. Thus, the key ecological issue of how important some of the signals are under field conditions remains to be addressed. Molecular tools now available make this task less daunting than in the past, and thus a new age of experimental field ecology may soon burst forth in rhizosphere studies. By identifying the signals, receptors, and the critical control points, we can better understand the organismal dynamics in this key belowground ecosystem.

The role of phytohormones in plant-microbe symbioses

Plant hormones, especially auxin, cytokinin, and ethylene, have long been implicated in nodule development. In addition, plant hormones have been shown to have increased concentrations in mycorrhizal associations. We show that the early nodulin (ENOD) genes can be used as indicators for the status of endogenous hormones in symbiotic root tissues. Transcripts for ENOD2 and ENOD40 genes are shown to accumulate in uninoculated, cytokinin-treated alfalfa roots, even in roots of the non-nodulating alfalfa mutant MN1008, which is unresponsive to Rhizobium meliloti inoculation and to Nod factor treatment. Transcripts for these ENOD genes also accumulate in mycorrhizal roots of alfalfa. A model describing the involvement of cytokinin and auxin in stimulating cell divisions in the inner cortex which leads to nodule formation is presented.

Lotus corniculatus Nodulation Specificity Is Changed by the Presence of a Soybean Lectin Gene

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.

Plant-associated symbiotic Burkholderia species lack hallmark strategies required in mammalian pathogenesis

Burkholderia is a diverse and dynamic genus, containing pathogenic species as well as species that form complex interactions with plants. Pathogenic strains, such as B. pseudomallei and B. mallei, can cause serious disease in mammals, while other Burkholderia strains are opportunistic pathogens, infecting humans or animals with a compromised immune system. Although some of the opportunistic Burkholderia pathogens are known to promote plant growth and even fix nitrogen, the risk of infection to infants, the elderly, and people who are immunocompromised has not only resulted in a restriction on their use, but has also limited the application of non-pathogenic, symbiotic species, several of which nodulate legume roots or have positive effects on plant growth. However, recent phylogenetic analyses have demonstrated that Burkholderia species separate into distinct lineages, suggesting the possibility for safe use of certain symbiotic species in agricultural contexts. A number of environmental strains that promote plant growth or degrade xenobiotics are also included in the symbiotic lineage. Many of these species have the potential to enhance agriculture in areas where fertilizers are not readily available and may serve in the future as inocula for crops growing in soils impacted by climate change. Here we address the pathogenic potential of several of the symbiotic Burkholderia strains using bioinformatics and functional tests. A series of infection experiments using Caenorhabditis elegans and HeLa cells, as well as genomic characterization of pathogenic loci, show that the risk of opportunistic infection by symbiotic strains such as B. tuberum is extremely low.

Isolation of chalcone synthase and chalcone isomerase cDNAs from alfalfa (Medicago sativa L.): highest transcript levels occur in young roots and root tips

Flavonoids are involved in several different interactions between plants and microorganisms. In the Rhizobium-legume symbiosis, they play an important role as inducers of rhizobial nodulation (nod) genes. We have identified from an alfalfa cDNA library four clones for chalcone synthase (CHS) and two clones for chalcone isomerase (CHI); CHS and CHI are key enzymes in flavonoid biosynthesis. In Medicago sp., CHS is encoded by 8–12 genes, and CHI is encoded by 1–2 genes. Here we report the DNA sequence of these clones as well as their relatedness to other legume CHS and CHI clones. In addition, we report on the expression patterns of two CHS gene family members as well as the CHI gene in M. sativa cv. Iroquois. While CHS and CHI transcript levels are high in root tips and entire young roots, they are low in effective nodules elicited by wild-type strains of Rhizobium meliloti and very low in aerial portions of the plant (stems, leaves, flowers). However, wounding the cotyledons results in a rapid increase in transcript levels of both chalcone synthase and chalcone isomerase genes in these organs.

Isolation and characterization of cDNA and genomic clones of MsENOD40; transcripts are detected in meristematic cells of alfalfa

SummaryAn alfalfa genomic clone and three cDNA clones for ENOD40 (MsENOD40) have been isolated and characterized. At the nucleotide level, the MsENOD40 clones exhibit ca. 79% identity to a soybean (GmENOD40) cDNA clone. The alfalfa cDNA clones lack an AUG translational start codon and potentially encode a polypeptide no longer than 46 amino acids. There is only 39% homology between the putative polypeptides of GmENOD40 and MsENOD40, suggesting that these two proteins may have different functions. However, MsENOD40 transcripts showed a pattern of localization similar to that of GmENOD40 transcripts in that mRNAs were detected by in situ hybridization in the pericycle of inoculated roots, in the nodule primordium, and in stem cells adjacent to the secondary phloem, i.e., the cells of the procambium. In addition, we detected MsENOD40 transcripts in other meristematic cells of the plant, including the nodule meristem, pre-emergent lateral root tips, and the margins of young leaf primordia. The location of MsENOD40 transcripts in dividing cells suggests that the ENOD40 gene product may play a role in mitosis or in associated processes, such as protein synthesis. However, because transcripts were associated with monosomes rather than polysomes, it is likely that MsENOD40 RNA is not translated into protein. Therefore, the RNA itself may have a function in developmental processes.