The root nodule symbiosis of plants with nitrogen-fixing bacteria affects global nitrogen cycles and food production but is restricted to a subset of genera within a single clade of flowering plants. To explore the genetic basis for this scattered occurrence, we sequenced the genomes of 10 plant species covering the diversity of nodule morphotypes, bacterial symbionts, and infection strategies. In a genome-wide comparative analysis of a total of 37 plant species, we discovered signatures of multiple independent loss-of-function events in the indispensable symbiotic regulator in 10 of 13 genomes of nonnodulating species within this clade. The discovery that multiple independent losses shaped the present-day distribution of nitrogen-fixing root nodule symbiosis in plants reveals a phylogenetically wider distribution in evolutionary history and a so-far-underestimated selection pressure against this symbiosis.

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Azorhizobium caulinodans ORS571 nifA regulation is partially mediated by the nitrogen regulatory gene ntrC. However, the residual nifA expression in ntrC mutant strains is still modulated by the cellular nitrogen and oxygen status. A second ntrC-homologous region, linked to ntrC, was identified and characterized by site-directed insertion mutagenesis and DNA sequencing. Tn5 insertions in this region cause pleiotropic defects in nitrogen metabolism and affect free-living as well as symbiotic nitrogen fixation. DNA sequencing and complementation studies revealed the existence of a bicistronic operon (ntrYX). NtrY is likely to represent the transmembrane 'sensor' protein element in a two-component regulatory system. NtrX shares a high degree of homology with NtrC proteins of other organisms and probably constitutes the regulator protein element. The regulation of the ntrYX and ntrC loci and the effects of ntrYX, ntrY and ntrX mutations on nifA expression were examined using beta-galactosidase gene fusions. NtrY/NtrX were found to modulate nifA expression and ntrYX transcription was shown to be partially under the control of NtrC.

Piriformospora indica has been shown to improve the growth of many plant species including Arabidopsis thaliana, but the mechanism by which this is achieved is still unclear. Arabidopsis root colonization by P. indica was examined in sterile culture on the medium of Murashige and Skoog. P. indica formed intracellular structures in Arabidopsis root epidermal cells and caused changes in root growth, leading to stunted and highly branched root systems. This effect was because of a diffusible factor and could be mimicked by IAA. In addition, P. indica was shown to produce IAA in liquid culture. We suggest that auxin production affecting root growth is responsible for, or at least contributes to, the beneficial effect of P. indica on its host plants.

Filamentous aerobic soil actinobacteria of the genus Frankia can induce the formation of nitrogen-fixing nodules on the roots of a diverse group of plants from eight dicotyledonous families, collectively called actinorhizal plants. Within nodules, Frankia can fix nitrogen while being hosted inside plant cells. Like in legume/rhizobia symbioses, bacteria can enter the plant root either intracellularly through an infection thread formed in a curled root hair, or intercellularly without root hair involvement, and the entry mechanism is determined by the host plant species. Nodule primordium formation is induced in the root pericycle as for lateral root primordia. Mature actinorhizal nodules are coralloid structures consisting of multiple lobes, each of which represents a modified lateral root without a root cap, a superficial periderm and with infected cells in the expanded cortex. In this review, an overview of nodule induction mechanisms and nodule structure is presented including comparisons with the corresponding mechanisms in legume symbioses.

To identify genes specifically expressed during early stages of actinorhizal nodule development, a cDNA library made from poly(A) RNA from root nodules of Alnus glutinosa was screened differentially with nodule and root cDNA, respectively. Seven nodule-enhanced and four nodule-specific cDNA clones were isolated. By using in situ hybridization, two of the nodule-specific cDNAs were shown to be expressed at the highest levels in infected cells before the onset of nitrogen fixation; one of them, ag12 (A. glutinosa), was examined in detail. Sequencing showed that ag12 codes for a serine protease of the subtilisin (EC 3.4.21.14) family. Subtilisins previously appeared to be limited to microorganisms. However, subtilisin-like serine proteases have recently been found in archaebacteria, fungi, and yeasts as well as in mammals; a plant subtilisin has also been sequenced. In yeast and mammals, subtilases are responsible for processing peptide hormones. A homolog of ag12, ara12, was identified in Arabidopsis; it was expressed in all organs, and its expression levels were highest during silique development. Hence, our study shows that subtilases are also involved in both symbiotic and nonsymbiotic processes in plant development.

Cyanobacteria belonging to the genus Nostoc comprise free-living strains and also facultative plant symbionts. Symbiotic strains can enter into symbiosis with taxonomically diverse range of host plants. Little is known about genomic changes associated with evolutionary transition of Nostoc from free-living to plant symbiont. Here, we compared the genomes derived from 11 symbiotic Nostoc strains isolated from different host plants and infer phylogenetic relationships between strains. Phylogenetic reconstructions of 89 Nostocales showed that symbiotic Nostoc strains with a broad host range, entering epiphytic and intracellular or extracellular endophytic interactions, form a monophyletic clade indicating a common evolutionary history. A polyphyletic origin was found for Nostoc strains which enter only extracellular symbioses, and inference of transfer events implied that this trait was likely acquired several times in the evolution of the Nostocales. Symbiotic Nostoc strains showed enriched functions in transport and metabolism of organic sulfur, chemotaxis and motility, as well as the uptake of phosphate, branched-chain amino acids, and ammonium. The genomes of the intracellular clade differ from that of other Nostoc strains, with a gain/enrichment of genes encoding proteins to generate l-methionine from sulfite and pathways for the degradation of the plant metabolites vanillin and vanillate, and of the macromolecule xylan present in plant cell walls. These compounds could function as C-sources for members of the intracellular clade. Molecular clock analysis indicated that the intracellular clade emerged ca. 600 Ma, suggesting that intracellular Nostoc symbioses predate the origin of land plants and the emergence of their extant hosts.

INTRODUCTIONBiosphere nitrogen is subjected to rapid turnover, and because it is eventually lost as nitrogen into the atmosphere, its maintenance requires continuous replenishment with reduced nitrogen from atmospheric nitrogen. Biological reduction of nitrogen to ammonia can be performed only by some prokaryotes and is a highly oxygen-sensitive process. The most efficient nitrogen fixers establish a symbiosis with higher plants in which the energy for nitrogen fixation and, in general, the oxygen protection system are provided by the plant partner. In two groups of symbiotic interactions, the prokaryotic partners are soil bacteria (rhizobia in legume symbioses and Frankia bacteria in actinorhizal symbioses), whereas in the case of symbiosis of Gunnera, nitrogen is fixed by the cyanobacterium Nostoc. In Gunnera, the symbionts reside in already existing stem glands, whereas in legumes and actinorhizal plants, new organs, the root nodules, are formed by the plant upon infection with the symbiont. In all three systems, the prokaryotes fix nitrogen inside the cells of their host, but they are separated from the plant cytoplasm by membranes derived from the plant plasmalemma ( Figure 1).Because research on legume symbiosis is the most advanced of these three symbiotic systems, in this article we concentrate mainly on this system. The interaction of rhizobia and legumes begins with signal exchange and recognition of the symbiotic partners, followed by attachment of the rhizobia to the plant root hairs. The root hair deforms, and the bacteria invade the plant by a newly formed infection thread growing through it. Simultaneously, cortical cells are mitotically activated, giving rise to the nodule primordium. lnfection threads grow toward the primordium, and the bacteria are then released into the cytoplasm of the host cells, surrounded by a plantderived peribacteroid membrane (PBM). The nodule primordium thereupon develops into a mature nodule, while the bacteria differentiate into their endosymbiotic form, which is known as the bacteroid ( Figure 1A). Bacteroids, together with the surrounding PBMs, are called symbiosomes. At this stage, bacteria synthesize nitrogenase, which catalyzes the reduction of nitrogen. The product of nitrogen fixation, ammonia, is then exported to the plant.All of the steps of nodule development involve the expression of nodule-specific plant genes, the socalled nodulin genes (van Kammen, 1984). The early nodulin genes encode products To whom correspondence should be addressed.that are expressed before the onset of nitrogen fixation and are involved in infection and nodule development. The products of the late nodulin genes are involved in the interaction with the endosymbiont and in the metabolic specialization of the nodule (Nap and Bisseling, 1990).In the first part of this review, we describe the early steps of the interaction between rhizobia and legumes that result in the formation of a nitrogen-fixing nodule. We focus on the role of specific lipooligosaccharides secreted by rhizobia in t...