Gene expression and localization of a ß-1,3-glucanase of Lotus japonicus.

Phytohormone abscisic acid (ABA) inhibits root nodule formation of leguminous plants. LjGlu1, a ß-1,3-glucanase gene of Lotus japonicus, has been identified as an ABA responsive gene. RNA interference of LjGlu1 increased nodule number. This suggests that LjGlu1 is involved in the regulation of nodule formation. Host legumes control nodule number by autoregulation of nodulation (AON), in which the presence of existing root nodules inhibits further nodulation. For further characterization of LjGlu1, we focused on the expression of LjGlu1 in relation to AON. In a split-root system, LjGlu1 expression peaked when AON was fully induced. Hairy roots transformed with LjCLE-RS1, a gene that induces AON, were generated. Expression of LjGlu1 was greater in the transgenic roots than in untransformed roots. LjGlu1 was not induced in a hypernodulating mutant inoculated with Mesorhizobium loti. These results suggest that the expression of LjGlu1 is involved in the system of AON. However, neither hypernodulation nor enlarged nodulation zone was observed on the transgenic hairy roots carrying LjGlu1-RNAi, suggesting that LjGlu1 is not a key player of AON. Recombinant LjGlu1 showed endo-ß-1,3-glucanase activity. LjGlu1-mOrange fusion protein suggested that LjGlu1 associated with M. loti on the root hairs. Exogenous ß-1,3-glucanase inhibited infection thread formation by both the wild type and the mutant, and nodule numbers were reduced. These results suggest that LjGlu1 is expressed in response to M. loti infection and functions outside root tissues, resulting in the inhibition of infection.

Characteristics of bacteroids in indeterminate nodules of the leguminous tree Leucaena glauca.

Rhizobia establish symbiosis with legumes. Bacteroids in indeterminate nodules of Inverted Repeat Lacking Clade (IRLC) legumes undergo terminal differentiation caused by Nodule-specific Cysteine-Rich peptides (NCRs). Microscopic observations of bacteroids and the detection of NCRs in indeterminate nodules of the non-IRLC legume Leucaena glauca were performed. A portion of the bacteroids showed moderate cell elongation, loss of membrane integrity, and multiple nucleoids. The symbiosome contained multiple bacteroids and NCR-like peptides were not detectable. These results indicate that bacteroid differentiation in L. glauca is different from that in IRLC legumes although both hosts form indeterminate nodules.

Nitric oxide production induced in roots of Lotus japonicus by lipopolysaccharide from Mesorhizobium loti.

Lipopolysaccharide (LPS) is a bacterial molecule that induces nitric oxide (NO) production and triggers defense systems in plant-pathogen interactions. NO production is induced in the roots of Lotus japonicus after inoculation of the roots with its microsymbiont Mesorhizobium loti. However, the rhizobial molecule that induces NO production has not yet been identified. We investigated NO production in the roots of L. japonicus by treatment with LPS of M. loti. LPS was prepared by phenol-hot water extraction and separated into several fractions: polysaccharide, lipooligosaccharide, oligosaccharide and lipid A. In the roots of L. japonicus, NO production was observed with an NO-specific fluorescent dye 4, 10 and 24 h after treatment with each fraction of LPS. NO production was detected 4 h after treatment with all fractions. NO production was also detectable 24 h after treatment, except after treatment with the polysaccharide and oligosaccharide fractions. Expression of a class 1 hemoglobin gene and application of an NO scavenger showed that the treatment with LPS and LOS induced a similar response to inoculation with M. loti. These data suggest that LPS of M. loti induces NO production after inoculation with M. loti.

Identification by suppression subtractive hybridization of Frankia genes induced under nitrogen-fixing conditions.

Frankia is an actinobacterium that fixes nitrogen under both symbiotic and free-living conditions. We identified genes upregulated in free-living nitrogen-fixing cells by using suppression subtractive hybridization. They included genes with predicted functions related to nitrogen fixation, as well as with unknown function. Their upregulation was a novel finding in Frankia.

Overexpression of class 1 plant hemoglobin genes enhances symbiotic nitrogen fixation activity between Mesorhizobium loti and Lotus japonicus.

Plant hemoglobins (Hbs) have been divided into three groups: class 1, class 2, and truncated Hbs. The various physiological functions of class 1 Hb include its role as a modulator of nitric oxide (NO) levels in plants. To gain more insight into the functions of class 1 Hbs, we investigated the physical properties of LjHb1 and AfHb1, class 1 Hbs of a model legume Lotus japonicus and an actinorhizal plant Alnus firma, respectively. Spectrophotometric analysis showed that the recombinant form of the LjHb1 and AfHb1 proteins reacted with NO. The localization of LjHb1 expression was correlated with the site of NO production. Overexpression of LjHb1 and AfHb1 by transformed hairy roots caused changes in symbiosis with rhizobia. The number of nodules formed on hairy roots overexpressing LjHb1 or AfHb1 increased compared with that on untransformed hairy roots. Furthermore, nitrogenase activity as acetylene-reduction activity (ARA) of LjHb1- or AfHb1-overexpressing nodules was higher than that of the vector control nodules. Microscopic observation with a NO-specific fluorescent dye suggested that the NO level in LjHb1- and AfHb1-overexpressing nodules was lower than that of control nodules. Exogenous application of a NO scavenger enhanced ARA in L. japonicus nodules, whereas a NO donor inhibited ARA. These results suggest that the basal level of NO in nodules inhibits nitrogen fixation, and overexpression of class 1 Hbs enhances symbiotic nitrogen fixation activity by removing NO as an inhibitor of nitrogenase.

Transient transformation of frankia by fusion marker genes in liquid culture.

Frankia is a nitrogen-fixing actinobacterium that establishes root nodule symbiosis with actinorhizal plants. The molecular basis of the symbiosis is largely unknown because genetic manipulation of Frankia has not been feasible. In this study we made novel technical attempts to transform Frankia strain CcI3. We generated fusion marker genes consisting of a tetracycline resistance gene with a high codon usage similarity to Frankia's and promoters of the strain's translation initiation factor 3 gene. We flanked the fusion genes with genomic sequences from strain CcI3 in the expectation that they would be integrated into the targeted site by homologous recombination. We introduced the transformation constructs into Frankia cells by electroporation and selected transformants in liquid media. The growth of antibiotic resistant cells was dependent on the presence of construct DNA. PCR analysis of the genome and reverse transcription-PCR analysis confirmed that the marker genes were introduced into the cells. Integration of the marker genes into the chromosome by homologous recombination did occur, but at a low frequency. Most of the constructs were not integrated into the chromosome and existed as degraded molecules in the cells. Marker genes declined in the transformant population during maintenance, showing that the transformation was unstable.

Expression of a class 1 hemoglobin gene and production of nitric oxide in response to symbiotic and pathogenic bacteria in Lotus japonicus.

Symbiotic nitrogen fixation by the collaboration between leguminous plants and rhizobia is an important system in the global nitrogen cycle, and some molecular aspects during the early stage of host-symbiont recognition have been revealed. To understand the responses of a host plant against various bacteria, we examined expression of hemoglobin (Hb) genes and production of nitric oxide (NO) in Lotus japonicus after inoculation with rhizobia or plant pathogens. When the symbiotic rhizobium Mesorhizobium loti was inoculated, expression of LjHb1 and NO production were induced transiently in the roots at 4 h after inoculation. In contrast, inoculation with the nonsymbiotic rhizobia Sinorhizobium meliloti and Bradyrhizobium japonicum induced neither expression of LjHb1 nor NO production. When L. japonicus was inoculated with plant pathogens (Ralstonia solanacearum or Pseudomonas syringae), continuous NO production was observed in roots but induction of LjHb1 did not occur. These results suggest that modulation of NO levels and expression of class 1 Hb are involved in the establishment of the symbiosis.

Transposon mediation allows a symbiotic plasmid of Rhizobium leguminosarum bv. trifolii to become a symbiosis island in Agrobacterium and Rhizobium.

The symbiotic plasmid (pSym) of Rhizobium leguminosarum bv. trifolii 4S5, which carries Tn5-mob, was successfully transferred into Agrobacterium tumefaciens A136 by using a conjugation method. The resulting transconjugants induced the development of ineffective nitrogen-fixing nodules on the roots of white clover seedlings. Depending on the manner in which the pSym was retained, the transconjugants were divided into two groups of strains, Afp and Afcs. pSym was retained as a plasmid in the Afp strains but was integrated into the int gene encoding a phage-related integrase on the linear chromosome of A. tumefaciens A136 in strain Afcs1 (one of the Afcs strains) to form a symbiosis island. Conjugation was performed between strain Afcs1 and R. leguminosarum bv. trifolii H1 (a pSym-cured derivative of wild-type strain 4S), and the Rhizobium H1tr strains were screened as transconjugants. Eighteen of the H1tr strains induced effective nitrogen-fixing nodules on the roots of the host plants. pSym was transferred into all of the transconjugants, except for strain H1tr1, at the same size as pSym of strain 4S5. In strain H1tr1, pSym was integrated into the chromosome as a symbiosis island. These data suggest that pSym can exist among Rhizobium and Agrobacterium strains both as a plasmid and as a symbiosis island with transposon mediation.

Split-root study of autoregulation of nodulation in the model legume Lotus japonicus.

We used a split-root system to determine the timing for induction of the autoregulation of nodulation (AUT) in Lotus japonicus (Regel) Larsen after inoculation with Mesorhizobium loti. The signal took at least five days for full induction of AUT and inhibition of infection thread formation. Strain ML108 (able to nodulate but unable to fix nitrogen) induced full AUT, but ML101 (unable to nodulate or to fix nitrogen) did not induce autoregulation. These results indicate that Nod factor-producing strains induce AUT, but that the nitrogen fixed by rhizobia and supplied to the plant as ammonia does not elicit the AUT in L. japonicus.

Transcriptome profiling of Lotus japonicus roots during arbuscular mycorrhiza development and comparison with that of nodulation.

To better understand the molecular responses of plants to arbuscular mycorrhizal (AM) fungi, we analyzed the differential gene expression patterns of Lotus japonicus, a model legume, with the aid of a large-scale cDNA macroarray. Experiments were carried out considering the effects of contaminating microorganisms in the soil inoculants. When the colonization by AM fungi, i.e. Glomus mosseae and Gigaspora margarita, was well established, four cysteine protease genes were induced. In situ hybridization revealed that these cysteine protease genes were specifically expressed in arbuscule-containing inner cortical cells of AM roots. On the other hand, phenylpropanoid biosynthesis-related genes for phenylalanine ammonia-lyase (PAL), chalcone synthase, etc. were repressed in the later stage, although they were moderately up-regulated on the initial association with the AM fungus. Real-time RT-PCR experiments supported the array experiments. To further confirm the characteristic expression, a PAL promoter was fused with a reporter gene and introduced into L. japonicus, and then the transformants were grown with a commercial inoculum of G. mosseae. The reporter activity was augmented throughout the roots due to the presence of contaminating microorganisms in the inoculum. Interestingly, G. mosseae only colonized where the reporter activity was low. Comparison of the transcriptome profiles of AM roots and nitrogen-fixing root nodules formed with Mesorhizobium loti indicated that the PAL genes and other phenylpropanoid biosynthesis-related genes were similarly repressed in the two organs.

A class 1 hemoglobin gene from Alnus firma functions in symbiotic and nonsymbiotic tissues to detoxify nitric oxide.

Actinorhizal symbiosis is as important in biological nitrogen fixation as legume-rhizobium symbiosis in the global nitrogen cycle. To understand the function of hemoglobin (Hb) in actinorhizal symbiosis, we characterized a Hb of Alnus firma, AfHb1. A cDNA that encodes nonsymbiotic Hb (nonsym-Hb) was isolated from a cDNA library of A. firma nodules probed with LjHb1, a nonsym-Hb of Lotus japonicus. No homolog of symbiotic Hb (sym-Hb) could be identified by screening in the cDNA library or by polymerase chain reaction (PCR) using degenerate primers for other sym-Hb genes. The deduced amino acid sequence of AfHb1 showed 92% sequence similarity with a class 1 nonsym-Hb of Casuarina glauca. Quantitative reverse transcriptase-PCR analysis showed that AfHb1 was expressed strongly in the nodules and enhanced expression was detected under cold stress but not under hypoxia or osmotic stress. Moreover, AfHfb1 was strongly induced by the application of nitric oxide (NO) donors, and the application of a NO scavenger suppressed the effect of NO donors. Acetylene reduction was strongly inhibited by the addition of NO donors. AfHb1 may support the nitrogen fixation ability of members of the genus Frankia as a NO scavenger.

Suppression of root nodule formation by artificial expression of the TrEnodDR1 (coat protein of White clover cryptic virus 1) gene in Lotus japonicus.

TrEnodDR1 (Trifolium repens early nodulin downregulation 1) encodes a coat protein of White clover cryptic virus 1. Its expression in white clover was down-regulated at the time when root nodules formed. We surmised that its artificial expression would interfere with root nodulation. Therefore, we investigated the effects of its artificial expression on the growth and root nodulation of Lotus japonicus (a model legume). Transformants were prepared by Agrobacterium spp.-mediated transformation. The growth of transformants was reduced and the number of root nodules per unit root length was greatly decreased relative to control. The concentration of endogenous abscisic acid (ABA), which controls nodulation, increased in plants containing TrEnodDR1. These phenotypes clearly were canceled by treatment with abamine, a specific inhibitor of ABA biosynthesis. The increase in endogenous ABA concentration explained the reduced stomatal aperture and the deformation of root hairs in response to inoculation of transgenic L. japonicus with Mesorhizobium loti. Transcriptome comparison between TrEnodDR1 transformants and control plants showed clearly enhanced expression levels of various defense response genes in transformants. These findings suggest that TrEnodDR1 suppresses nodulation by increasing the endogenous ABA concentration, perhaps by activating the plant's innate immune response. This is the first report of the suppression of nodulation by the artificial expression of a virus coat protein gene.

Symbiotic rhizobium and nitric oxide induce gene expression of non-symbiotic hemoglobin in Lotus japonicus.

We characterized the expression profiles of LjHb1 and LjHb2, non-symbiotic hemoglobin (non-sym-Hb) genes of Lotus japonicus. Although LjHb1 and LjHb2 showed 77% homology in their cDNA sequences, LjHb2 is located in a unique position in the phylogenetic tree of plant Hbs. The 5'-upstream regions of both genes contain the motif AAAGGG at a position similar to that in promoters of other non-sym-Hb genes. Expression profiles obtained by using quantitative RT-PCR showed that LjHb1 and LjHb2 were expressed in all tissues of mature plants, and expression was enhanced in mature root nodules. LjHb1 was strongly induced under both hypoxic and cold conditions, and by the application of nitric oxide (NO) donor, whereas LjHb2 was induced only by the application of sucrose. LjHb1 was also induced transiently by the inoculation with the symbiotic rhizobium Mesorhizobium loti MAFF303099. Observations using fluorescence microscopy revealed the induction of LjHb1 expression corresponded to the generation of NO. These results suggest that non-sym-Hb and NO have important roles in stress adaptation and in the early stage of legume-rhizobium symbiosis.

Control of nodule number by the phytohormone abscisic Acid in the roots of two leguminous species.

The effects of the phytohormone abscisic acid (ABA) on plant growth and root nodule formation were analyzed in Trifolium repense (white clover) and Lotus japonicus, which form indeterminate and determinate nodules, respectively. In T. repense, although the number of nodules formed after inoculation with Rhizobium leguminosarum bv. trifolii strain 4S (wild type) was slightly affected by exogenous ABA, those formed by strain H1(pC4S8), which forms ineffective nodules, were dramatically reduced 28 days after inoculation (DAI). At 14 and 21 DAI, the number of nodules formed with the wild-type strain was decreased by exogenous ABA. In L. japonicus, the number of nodules was also reduced by ABA treatment. Thus, exogenous ABA inhibits root nodule formation after inoculation with rhizobia. Observation of root hair deformation revealed that ABA blocked the step between root hair swelling and curling. When the ABA concentration in plants was decreased by using abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase, the number of nodules on lateral roots of abamine-treated L. japonicus increased dramatically, indicating that lower-than-normal concentrations of endogenous ABA enhance nodule formation. We hypothesize that the ABA concentration controls the number of root nodules.

A novel symbiotic nitrogen-fixing member of the Ochrobactrum clade isolated from root nodules of Acacia mangium.

Ten strains of root nodule bacteria were isolated from the nodules of Acacia mangium grown in the Philippines and Thailand. Partial sequences (approx. 300 bp) of the 16S rRNA gene of each isolate were analyzed. The nucleotide sequences of strain DASA 35030 indicated high homology (>99%) with members of the genus Ochrobactrum in Brucellaceae, although the sequences of other isolates were homologous to those of two distinct genera Bradyrhizobium and Rhizobium. The strain DASA 35030 was strongly suggested to be a strain of Ochrobactrum by full length sequences of the 16S rRNA gene, fatty acids composition, G+C contents of the DNA, and other physiological characteristics. Strain DASA 35030 induced root nodules on A. mangium, A. albida and Paraserianthes falcataria. The nodules formed by strain DASA 35030 fixed nitrogen and the morphology of the nodules is the same as those of nodules formed by the other isolates. This is the first report that the strain of Ochrobactrum possesses complete symbiotic ability with Acacia.

Expression of symbiotic and nonsymbiotic globin genes responding to microsymbionts on Lotus japonicus.

Leguminous plants have both symbiotic and nonsymbiotic hemoglobin (sym- and nonsym-Hb) genes. Three symbiotic (LjLb1, 2, 3) and one nonsymbiotic (LjNSG1) Hb genes were isolated from a genomic library of Lotus japonicus MG20 Miyakojima. Two motif sequences (AAAGAT and CTCTT) critical for nodule specific expression were conserved on the 5'-upstream sequence of LjLb1, 2 and 3. The 5'-upstream region of LjNSG1 contained the sequence consensus for nonsym-Hb. RT-PCR with specific primer sets for each LjLb gene showed that all the sym-Hb genes (LjLb1, 2, 3) were expressed specifically and strongly in root nodules. The expression of LjLb1, 2 and 3 could not be detected in root, leaf or stem of a mature plant, whereas low level expression was detected in seedlings by RT-PCR. This suggests that sym-Hbs may have another unknown function besides being oxygen transporter for the microsymbiont in symbiotic nitrogen fixation. The expression of LjNSG1, examined with RT-PCR, was detected at low level in root, leaf and stem. The expression of LjNSG1 was enhanced in root nodules, whereas it was repressed in roots colonized by mycorrhizal fungi Glomus sp. R10. The repression of the nonsym-Hb gene was also observed in the roots of Medicago sativa colonized by Glomus sp. R10.

Symbiotic root nodule bacteria isolated from yam bean (Pachyrhizus erosus).

A total of 25 isolates from root nodules of yam bean (Pachyrhizus erosus L. Urban), a tuber-producing leguminous plant, were characterized. All isolates formed effective nodules mainly on lateral roots while edible tubers were developed on the taproot. The root nodules formed were identified as the typical determinate type. By an analysis of the partial sequences of the 16S rRNA gene (approximately 300 bp) of 10 strains which were selected randomly, the isolated root nodule bacteria of yam bean were classified into two different genera, Rhizobium and Bradyrhizobium. Two strains, YB2 (Bradyrhizobium group) and YB4 (Rhizobium group) were selected and used for further analyses. The generation time of each strain was shown to be 22.5 h for strain YB2 and 0.8 h for strain YB4, respectively. Differences between strains YB2 and YB4 were also reflected in the bacteroid state in the symbiosome. Symbiosome in nodule cells for the strain YB4 contained one bacteroid cell in a peribacteroid membrane, whereas a symbiosome for strain YB2 contained several bacteroid cells.

Transfer of the symbiotic plasmid from Rhizobium leguminosarum biovar trifolii to Agrobacterium tumefaciens.

This study examined the symbiotic properties of Agrobacterium transconjugants isolated by transferring a Tn5-mob-marked derivative of the 315 kb megaplasmid pRt4Sa from Rhizobium leguminosarum bv. trifolii 4S (wild-type strain) to Agrobacterium tumefaciens A136 as the recipient. The genetic characteristics of the AT4S transconjugant strains were ascertained by random amplified polymorphic DNA (RAPD) analyses and Southern hybridization using Tn5-mob and nod genes as probes. Several of these AT4S transconjugants carrying pRt4Sa were able to nodulate roots of the normal legume host, white clover. In addition, some AT4S transconjugant strains were able to induce nodules on other leguminous plants, including alfalfa and hairy vetch. A characteristic bacteroid differentiation was observed in clover and alfalfa nodules induced by the AT4S-series strains, although nitrogen-fixing activity (acetylene reduction) was not found. Furthermore, strain H1R1, obtained by retracing transfer of the pRt4Sa::Tn5-mob from strain AT4Sa to strain H1 (pRt4Sa cured derivative of 4S), induced Fix(+) nodules on clover roots. These results indicate the evidence that only nod genes can be expressed in the Agrobacterium background.

Integration of the temperate phage phiU into the putative tRNA gene on the chromosome of its host Rhizobium leguminosarum biovar trifolii.

The plasmid pCI6, carrying the attP site of the temperate phage phiU, integrates into the attB site on the chromosome of Rhizobium leguminosarum biovar trifolii strain 4S. The 4 kb EcoRI-HindIII region of pCI6 involved in site-specific integration was subcloned as the attP fragment of phage phiU and sequenced. The attL fragment, one of the new DNA junctions generated from the insertion of pCI6 into the chromosome of the host Rhizobium, was used as a hybridization probe for isolation of the attB fragment of strain 4S. The nucleotide sequence of the 2 kb PstI fragment of strain 4S, which hybridized with the attL fragment, was decided and compared with that of the attP fragment. A 53 bp common sequence was expected to be the core sequence of site-specific integration between phage phiU and strain 4S. One of the ORFs on the attP fragment, which was located adjacent to the core sequence, had structural homology to the integrase family. However, the attB fragment showed high homology with the tRNA genes of Agrobacterium tumefaciens and E. coli. A 47 bp sequence of the 53 bp core sequence overlapped with this tRNA-like sequence. This indicates that the target site of phage phiU integration is the putative tRNA gene on the chromosome of the Rhizobium host.