Expression of LjENOD40 genes in response to symbiotic and non-symbiotic signals: LjENOD40-1 and LjENOD40-2 are differentially regulated in Lotus japonicus

Nitrogen fixation in nodules provides leguminous plants with an ability to grow in nitrogen-starved soil. Infection of the host plants by microsymbionts triggers various physiological and morphological changes during nodule formation. In Lotus japonicus, expression of early nodulin (ENOD) genes is triggered by perception of bacterial signal molecules, nodulation factors (Nod factors). We examined the expression patterns of ENOD40 genes during the nodule formation process. Two ENOD40 genes of L. japonicus were specifically expressed in the nodule formation process, but they showed different expression patterns upon infection. Each ENOD40 gene demonstrates an individual specificity and regulation with regard to rhizobial infection.     

Genomics insights into symbiotic nitrogen fixation

Following an interaction with rhizobial soil bacteria, legume plants are able to form a novel organ, termed the root nodule. This organ houses the rhizobial microsymbionts, which perform the biological nitrogen fixation process resulting in the incorporation of ammonia into plant organic molecules. Recent advances in genomics have opened exciting new perspectives in this field by providing the complete gene inventory of two rhizobial microsymbionts. The complete genome sequences of Mesorhizobium loti, the symbiont of several Lotus species, and Sinorhizobium meliloti, the symbiont of alfalfa, were determined and annotated in detail. For legume macrosymbionts, expressed sequence tag projects and expression analyses using DNA arrays in conjunction with proteomics approaches have identified numerous genes involved in root nodule formation and nitrogen fixation. The isolation of legume genes by tagging or positional cloning recently allowed the identification of genes that control the very early steps of root nodule organogenesis.     

Controlling the expression of rhizobial genes during nodule development with elements and an inducer of the lac operon

A simple strategy was tested for imposing artificial regulation of rhizobial genes during nodule development. Isopropyl-β-d-1-thiogalactoside (IPTG) was added to liquid root media to sustain expression of rhizobial genes controlled by Escherichia coli lac promoter/operators and repressor gene lacI. Conversely, a rinsing protocol was devised to remove IPTG sufficiently that genes could be repressed after having been induced. gusA under this control exhibited clearly delineated expression and repression in both the determinate Rhizobium etli-Phaseolus vulgaris and the indeterminate Sinorhizobium meliloti-Medicago sativa symbioses. Apparently, IPTG was taken up in sufficiently undegraded concentrations that gene expression was derepressed even in interior portions of the nodule. Moreover, the rinsing protocol led to obvious repression of gusA. Importantly, no deleterious effects of IPTG on nodule development, infection, or nitrogen fixation were observed. An R. etli CE3 gene required for lipopolysaccharide O antigen and infection on bean was put under this control by means of a two-plasmid construct. When this construct was added to a strain with a null mutation in this gene, infection, nodule development, and nitrogenase activity all depended on the length of time before IPTG was rinsed from the roots after inoculation.     

Spontaneous root-nodule formation in the model legume Lotus japonicus: a novel class of mutants nodulates in the absence of rhizobia

Root-nodule development in legumes is an inducible developmental process initially triggered by perception of lipochitin-oligosaccharide signals secreted by the bacterial microsymbiont. In nature, rhizobial colonization and invasion of the legume root is therefore a prerequisite for formation of nitrogen-fixing root nodules. Here, we report isolation and characterization of chemically induced spontaneously nodulating mutants in a model legume amenable to molecular genetics. Six mutant lines of Lotus japonicus were identified in a screen for spontaneous nodule development under axenic conditions, i.e., in the absence of rhizobia. Spontaneous nodules do not contain rhizobia, bacteroids, or infection threads. Phenotypically, they resemble ineffective white nodules formed by some bacterial mutants on wild-type plants or certain plant mutants inoculated with wild-type Mesorhizobium loti. Spontaneous nodules formed on mutant lines show the ontogeny and characteristic histological features described for rhizobia-induced nodules on wild-type plants. Physiological responses to nitrate and ethylene are also maintained, as elevated levels inhibit spontaneous nodulation. Activation of the nodule developmental program in spontaneous nodules was shown for the early nodulin genes Enod2 and Nin, which are both upregulated in spontaneous nodules as well as in rhizobial nodules. Both monogenic recessive and dominant spontaneous nodule formation (snf) mutations were isolated in this mutant screen, and map positions were determined for three loci. We suggest that future molecular characterization of these mutants will identify key plant determinants involved in regulating nodulation and provide new insight into plant organ development.     

Pseudomonas fluorescens CHA0 can kill subterranean termite Odontotermes obesus by inhibiting cytochrome c oxidase of the termite respiratory chain

In the rhizosphere and their interaction with plants rhizobia encounter many different plant compounds, including phytohormones like auxins. Moreover, some rhizobial strains are capable of producing the auxin, indole-3-acetic acid (IAA). However, the role of IAA for the bacterial partner in the legume– Rhizobium symbiosis is not known. To identify the effect of IAA on rhizobial gene expression, a transposon (mTn 5gusA - oriV ) mutant library of Rhizobium etli , enriched for mutants that show differential gene expression under microaerobiosis and/or addition of nodule extracts as compared with control conditions, was screened for altered gene expression upon IAA addition. Four genes were found to be regulated by IAA. These genes appear to be involved in plant signal processing, motility or attachment to plant roots, clearly demonstrating a distinct role for IAA in legume– Rhizobium interactions.     

The coexistence of symbiosis and pathogenicity-determining genes in Rhizobium rhizogenes strains enables them to induce nodules and tumors or hairy roots in plants

Bacteria belonging to the family Rhizobiaceae may establish beneficial or harmful relationships with plants. The legume endosymbionts contain nod and nif genes responsible for nodule formation and nitrogen fixation, respectively, whereas the pathogenic strains carry vir genes responsible for the formation of tumors or hairy roots. The symbiotic and pathogenic strains currently belong to different species of the genus Rhizobium and, until now, no strains able to establish symbiosis with legumes and also to induce tumors or hairy roots in plants have been reported. Here, we report for the first time the occurrence of two rhizobial strains (163C and ATCC11325T) belonging to Rhizobium rhizogenes able to induce hairy roots or tumors in plants and also to nodulate Phaseolus vulgaris under natural environmental conditions. Symbiotic plasmids (pSym) containing nod and nif genes and pTi- or pRi-type plasmids containing vir genes were found in these strains. The nodD and nifH genes of the strains from this study are phylogenetically related to those of Sinorhizobium strains nodulating P. vulgaris. The virA and virB4 genes from strain 163C are phylogenetically related to those of R. tumefaciens C58, whereas the same genes from strain ATCC 11325T are related to those of hairy root-inducing strains. These findings may be of high relevance for the better understanding of plant-microbe interactions and knowledge of rhizobial phylogenetic history.     

The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation

Establishment of the Rhizobium-legume symbiosis depends on a molecular dialogue, in which rhizobial nodulation (Nod) factors act as symbiotic signals, playing a key role in the control of specificity of infection and nodule formation. Using nodulation-defective (Nod-) mutants of Medicago truncatula to study the mechanisms controlling Nod factor perception and signalling, we have previously identified five genes that control components of a Nod factor-activated signal transduction pathway. Characterisation of a new M. truncatula Nod- mutant led to the identification of the Nod Factor Perception (NFP) locus. The nfp mutant has a novel phenotype among Nod- mutants of M. truncatula, as it does not respond to Nod factors by any of the responses tested. The nfp mutant thus shows no rapid calcium flux, the earliest detectable Nod factor response of wild-type plants, and no root hair deformation. The nfp mutant is also deficient in Nod factor-induced calcium spiking and early nodulin gene expression. While certain genes controlling Nod factor signal transduction also control the establishment of an arbuscular mycorrhizal symbiosis, the nfp mutant shows a wild-type mycorrhizal phenotype. These data indicate that the NFP locus controls an early step of Nod factor signal transduction, upstream of previously identified genes and specific to nodulation.     

根瘤菌生物地理学的研究进展

根瘤菌是一类革兰氏阴性菌,能与特定的宿主植物共生形成根瘤,将空气中的分子态氮转变为植物可以利用的氨态氮。研究根瘤菌的生物地理分布格局及形成机制,不仅具有理论上的意义,还对根瘤菌接种剂的选择具有指导意义。目前,随着分子生物学技术的发展,以及根瘤菌多样性研究数据的积累,根瘤菌生物地理学取得了较大的进展。本文综述了根瘤菌生物地理学的研究方法及现状,并对今后的重点研究方向作了展望。     

Use of marker genes in competition studies of Rhizobium

Use of marker genes has several advantages in studying rhizobial competition compared to traditional approaches. Reporter genes such as the ß-glucuronidase gene (gusA) or a thermostable ß-glucosidase gene (celB) allow detection of rhizobial strains in nodules when they are still attached to the root system. Analysis is extremely simple, fast and permits a high data throughput. This detection technique is therefore highly suitable for the study of rhizobial competition and studies using gusA-marked strains of Rhizobium are presented. By making use of gusA and celB, differentially marked strains can be produced and distinguished easily on roots. The availability of two marker genes permits competition studies of two or more than two strains and analysis of dual nodule occupancy. As this methodology does not require sophisticated equipment, a GUS Gene Marking Kit was developed.     

Genetic resources of nodule bacteria

Nodule bacteria (rhizobia) form highly specific symbiosis with leguminous plants. The efficiency of accumulation of biological nitrogen depends on molecular-genetic interaction between the host plant and rhizobia. Genetic characteristics of microsymbiotic strains are crucial in developing highly productive and stress-resistant symbiotic pairs: rhizobium strain-host plant cultivar (species). The present review considers the issue of studying genetic resources of nodule bacteria to identify genes and their blocks, responsible for the ability of rhizobia to form highly effective symbiosis in various agroecological conditions. The main approaches to investigate of intraspecific and interspecific genetic and genomic diversity of nodule bacteria are considered, from MLEE analysis to the recent methods of genomic DNA analysis using biochips. The data are presented showing that gene centers of host plants are centers of genetic diversification of nodule bacteria, because the intraspecific polymorphism of genetic markers of the core and the accessory rhizobial genomes is extremely high in them. Genotypic features of trapped and nodule subpopulations of alfalfa nodule bacteria are discussed. A survey of literature showed that the genomes of natural strains in alfalfa gene centers exhibit significant differences in genes involved in control of metabolism, replication, recombination, and the formation of defense response (hsd genes). Natural populations of rhizobia are regarded as a huge gene pool serving as a source of evolutionary innovations.     

Symbiosis specificity in the legume: rhizobial mutualism

Legume plants are able to engage in root nodule symbiosis with nitrogen-fixing soil bacteria, collectively called rhizobia. This mutualistic association is highly specific, such that each rhizobial species/strain interacts with only a specific group of legumes, and vice versa. Symbiosis specificity can occur at multiple phases of the interaction, ranging from initial bacterial attachment and infection to late nodule development associated with nitrogen fixation. Genetic control of symbiosis specificity is complex, involving fine-tuned signal communication between the symbiotic partners. Here we review our current understanding of the mechanisms used by the host and bacteria to choose their symbiotic partners, with a special focus on the role that the host immunity plays in controlling the specificity of the legume - rhizobial symbiosis.     

Rhizobium common nod genes are required for biofilm formation

In legume nitrogen-fixing symbioses, rhizobial nod genes are obligatory for initiating infection thread formation and root nodule development. Here we show that the common nod genes, nodD1ABC , whose products synthesize core Nod factor, a chitin-like oligomer, are also required for the establishment of the three-dimensional architecture of the biofilm of Sinorhizobium meliloti . Common nod gene mutants form a biofilm that is a monolayer. Moreover, adding Nod Factor antibody to S. meliloti cells inhibits biofilm formation, while chitinase treatment disrupts pre-formed biofilms. These results attest to the involvement of core Nod factor in rhizobial biofilm establishment. However, luteolin, the plant-derived inducer of S. meliloti 's nod genes, is not required for mature biofilm formation, although biofilm establishment is enhanced in the presence of this flavonoid inducer. Because biofilm formation is plant-inducer-independent and because all nodulating rhizobia, both alpha- and beta-proteobacteria have common nod genes, the role of core Nod factor in biofilm formation is likely to be an ancestral and evolutionarily conserved function of these genes.     

Nod genes and Nod signals and the evolution of the Rhizobium legume symbiosis.

The establishment of the nitrogen-fixing symbiosis between rhizobia and legumes requires an exchange of signals between the two partners. In response to flavonoids excreted by the host plant, rhizobia synthesize Nod factors (NFs) which elicit, at very low concentrations and in a specific manner, various symbiotic responses on the roots of the legume hosts. NFs from several rhizobial species have been characterized. They all are lipo-chitooligosaccharides, consisting of a backbone of generally four or five glucosamine residues N-acylated at the non-reducing end, and carrying various O-substituents. The N-acyl chain and the other substituents are important determinants of the rhizobial host specificity. A number of nodulation genes which specify the synthesis of NFs have been identified. All rhizobia, in spite of their diversity, possess conserved nodABC genes responsible for the synthesis of the N-acylated oligosaccharide core of NFs, which suggests that these genes are of a monophyletic origin. Other genes, the host specific nod genes, specify the substitutions of NFs. The central role of NFs and nod genes in the Rhizobium-legume symbiosis suggests that these factors could be used as molecular markers to study the evolution of this symbiosis. We have studied a number of NFs which are N-acylated by alpha,beta-unsaturated fatty acids. We found that the ability to synthesize such NFs does not correlate with taxonomic position of the rhizobia. However, all rhizobia that produce NFs such nodulate plants belonging to related tribes of legumes, the Trifolieae, Vicieae, and Galegeae, all of them being members of the so-called galegoid group. This suggests that the ability to recognize the NFs with alpha-beta-unsaturated fatty acids is limited to this group of legumes, and thus might have appeared only once in the course of legume evolution, in the galegoid phylum.     

Differential regulation of a family of apyrase genes from Medicago truncatula

Four putative apyrase genes were identified from the model legume Medicago truncatula. Two of the genes identified from M. truncatula (Mtapy1 and Mtapy4) are expressed in roots and are inducible within 3 h after inoculation with Sinorhizobium meliloti. The level of mRNA expression of the other two putative apyrases, Mtapy2 and Mtapy3, was unaffected by rhizobial inoculation. Screening of a bacterial artificial chromosome library of M. truncatula genomic DNA showed that Mtapy1, Mtapy3, and Mtapy4 are present on a single bacterial artificial chromosome clone. This apyrase cluster was mapped to linkage group seven. A syntenic region on soybean linkage group J was found to contain at least two apyrase genes. Screening of nodulation deficient mutants of M. truncatula revealed that two such mutants do not express apyrases to any detectable level. The data suggest a role for apyrases early in the nodulation response before the involvement of root cortical cell division leading to the nodule structure.     

A dominant function of CCaMK in intracellular accommodation of bacterial and fungal endosymbionts

In legumes, Ca²⁺/calmodulin‐dependent protein kinase (CCaMK) is a component of the common symbiosis genes that are required for both root nodule (RN) and arbuscular mycorrhiza (AM) symbioses and is thought to be a decoder of Ca²⁺ spiking, one of the earliest cellular responses to microbial signals. A gain‐of‐function mutation of CCaMK has been shown to induce spontaneous nodulation without rhizobia, but the significance of CCaMK activation in bacterial and/or fungal infection processes is not fully understood. Here we show that a gain‐of‐function CCaMK^T265D suppresses loss‐of‐function mutations of common symbiosis genes required for the generation of Ca²⁺ spiking, not only for nodule organogenesis but also for successful infection of rhizobia and AM fungi, demonstrating that the common symbiosis genes upstream of Ca²⁺ spiking are required solely to activate CCaMK. In RN symbiosis, however, CCaMK^T265D induced nodule organogenesis, but not rhizobial infection, on Nod factor receptor (NFRs) mutants. We propose a model of symbiotic signaling in host legume plants, in which CCaMK plays a key role in the coordinated induction of infection thread formation and nodule organogenesis.     

Nonlegume hemoglobin genes retain organ-specific expression in heterologous transgenic plants.

Hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa have been isolated [Landsmann et al. (1986). Nature 324, 166-168; Bogusz et al. (1988). Nature 331, 178-180]. The promoters of these genes have been linked to a beta-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus. Both promoters directed root-specific expression in transgenic tobacco. When transgenic Lotus plants were nodulated by Rhizobium loti, both promoter constructs showed a high level of nodule-specific expression confined to the central bacteroid-containing portion of the nodule corresponding to the expression seen for the endogenous Lotus leghemoglobin gene. The T. tomentosa promoter was also expressed at a low level in the vascular tissue of the Lotus roots. The hemoglobin promoters from both nonlegumes, including the non-nodulating species, must contain conserved cis-acting DNA signals that are responsible for nodule-specific expression in legumes. We have identified sequence motifs postulated previously as the nodule-specific regulatory elements of the soybean leghemoglobin genes [Stougaard et al. (1987). EMBO J. 6, 3565-3569].     

Root nodule development: origin, function and regulation of nodulin genes

The symbiotic root nodule, an organ formed on leguminous plants, is a product of successful interactions between the host plant and the soil bacteria, Rhizobium spp. Plant hormones play an important role in the genesis of this organ. The hormonal balance appears to be modulated by the signals produced by bacteria. Many host genes induced during nodule organogenesis and the symbiotic state have been identified and characterized from several legumes. These genes encode nodule-specific proteins (nodulins) which perform diverse functions in root nodule development and metabolism. Formation of a subcellular compartment housing the bacteria is essential to sustain the symbiotic state, and several nodulins are involved in maintaining the integrity and function of this compartment. The bacteroid enclosed in the perbacteroid membrane behaves as an 'organelle,'completely dependent on the host for all its requirements for carbon, nitrogen and other essential elements. Thus it seems likely that the nodulins in the peribacteroid membrane perform specific transport functions. While the function of a few other nodulins is known (e.g. nodulin-100, nodulin-35), a group of uncharacterized nodulins exists in soybean root nodules. These nodulins share structural similarities and seem to have been derived from a common ancestor. Induction of nodulin genes occurs prior to and independent of nitrogen fixation, and thus is a prelude to symbiosis. Although some of the early nodulin genes are induced prior to or during infection, induction of late nodulins requires endocytotic release of bacteria.