Rapid inactivation of the maize transposable element En/Spm in Medicago truncatula.

Transposable elements have been widely used as mutagens in many organisms. Among them, the maize transposable element En/Spm has been shown to transpose efficiently in several plant species including the model plant Arabidopsis, where it has been used for large-scale mutagenesis. To determine whether we could use this transposon as a mutagen in the model legume plant Medicago truncatula, we tested the activity of the autonomous element, as well as two defective elements, in this plant, and in Arabidopsis as a positive control. In agreement with previous reports, we observed efficient excision of the autonomous En/Spm element in A. thaliana. This element was also active in M. truncatula, but the transposition activity was low and was apparently restricted to the tissue culture step necessary for the production of transgenic plants. The activity of one of the defective transposable elements, dSpm, was very low in A. thaliana and even lower in M. truncatula. The use of different sources of transposases suggested that this defect in transposition was associated with the dSpm element itself. Transposition of the other defective element, I6078, was also detected in M. truncatula, but, as observed with the autonomous element, transposition events were very rare and occurred during tissue culture. These results suggest that the En/Spm element is rapidly inactivated in the regenerated plants and their progeny, and therefore is not suitable for routine insertion mutagenesis in M. truncatula.

Efficient transposition of the Tnt1 tobacco retrotransposon in the model legume Medicago truncatula.

The tobacco element, Tnt1, is one of the few active retrotransposons in plants. Its transposition is activated during protoplast culture in tobacco and tissue culture in the heterologous host Arabidopsis thaliana. Here, we report its transposition in the R108 line of Medicago truncatula during the early steps of the in vitro transformation-regeneration process. Two hundred and twenty-five primary transformants containing Tnt1 were obtained. Among them, 11.2% contained only transposed copies of the element, indicating that Tnt1 transposed very early and efficiently during the in vitro transformation process, possibly even before the T-DNA integration. The average number of insertions per transgenic line was estimated to be about 15. These insertions were stable in the progeny and could be separated by segregation. Inspection of the sequences flanking the insertion sites revealed that Tnt1 had no insertion site specificity and often inserted in genes (one out of three insertions). Thus, our work demonstrates the functioning of an efficient transposable element in leguminous plants. These results indicate that Tnt1 can be used as a powerful tool for insertion mutagenesis in M. truncatula.

Transformation of floral organs with GFP in Medicago truncatula.

A high frequency of embryogenesis and transformation from all parts of flowers of two lines of Medicago truncatula R-108-1 and Jemalong J5 were obtained. Using this flower system, we obtained transgenic plants expressing promoter-uidA gene fusions as well as the gfp living cell color reporter gene. Moreover, this method allows us to save time and to use a smaller greenhouse surface for the culture of donor plants. Southern hybridization showed that the internal gfp fragment had the expected size and the number of T-DNA copies integrated in the plant genome varied between one and three. These data suggest that the presence of the GFP protein has no toxic effects, since no rearrangement of the gfp reporter gene was detected in the regenerated plants.

Bigfoot. a new family of MITE elements characterized from the Medicago genus.

We have characterized from the legume plant Medicago a new family of miniature inverted-repeat transposable elements (MITE), called the Bigfoot transposable elements. Two of these insertion elements are present only in a single allele of two different M. sativa genes. Using a PCR strategy we have isolated 19 other Bigfoot elements from the M. sativa and M. truncatula genomes. They differ from the previously characterized MITEs by their sequence, a target site of 9 bp and a partially clustered genomic distribution. In addition, we show that they exhibit a significantly stable secondary structure. These elements may represent up to 0.1% of the genome of the outcrossing Medicago sativa but are present at a reduced copy number in the genome of the autogamous M. truncatula plant, revealing major differences in the genome organization of these two plants.

MsPG3, a Medicago sativa polygalacturonase gene expressed during the alfalfa-Rhizobium meliloti interaction.

Polygalacturonase (PG) is one of the most important enzymes associated with plant cell wall degradation. It has been proposed to participate in the early steps of the Rhizobium-legume interaction. We have identified two classes of cDNA fragments corresponding to two classes of PG genes in the Medicago genome. One of this class, represented by E2 in M. truncatula and Pl1 in M. sativa, seems to be related to previously characterized plant PG genes expressed in pollen. We have isolated the genomic clone containing the entire gene corresponding to the second class (E3). We showed that MsPG3 is a single gene in the Medicago genome coding for PG. By reverse transcription-PCR, MsPG3 expression was detected in roots 1 day after Rhizobium inoculation. The early induction of the MsPG3, as also seen by in situ hybridization experiments, supports its involvement in the early stages of the Rhizobium-legume infection process. In addition, by analyzing the expression of a MsPG3 promoter-gus construct in Vicia hirsuta-transgenic root nodules, we showed that MsPG3 was expressed in all cells of nodule primordia and in the cells of the invasion zone. By Northern blot, MsPG3 transcripts are not detected in various Medicago tissues, indicating that the function of this gene is related closely to symbiosis. Thus, our results strongly suggest the involvement of MsPG3 gene during meristem formation and/or in the infection process, probably by facilitating cell wall rearrangement, penetration of the bacteria through the root hair wall, or infection thread formation and release of bacteria in plant cells. MsPG3 represents a class of PG genes, distinct from the pollen-specific genes, and it is the first pectic encoded enzyme demonstrated to be involved in Rhizobium-legume symbiosis.

Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssp. falcata lines improved in somatic embryogenesis.

We describe a simple and efficient protocol for regeneration-transformation of two diploid Medicago lines: the annual M. truncatula R108-1(c3) and the perennial M. sativa ssp. falcata (L.) Arcangeli PI.564263 selected previously as highly embryogenic genotypes. Here, embryo regeneration of R108-1 to complete plants was further improved by three successive in vitro regeneration cycles resulting in the line R108-1(c3). Agrobacterium tumefaciens-mediated transformation of leaf explants was carried out with promoter-gus constructs of two early nodulins (MsEnod12A and MsEnod12B) and one late nodulin (Srglb3). The transgenic plants thus produced on all explants within 3-4 months remained diploid and were fertile. This protocol appears to be the most efficient and fastest reported so far for leguminous plants.

The expression pattern of alfalfa flavanone 3-hydroxylase promoter-gus fusion in Nicotiana benthamiana correlates with the presence of flavonoids detected in situ.

Flavanone 3-hydroxylase is an enzyme acting in the central part of the flavonoid biosynthesis pathway. It is generally encoded by a single gene and seems to have a key position for the regulation in this pathway. These two features make a single f3h promoter-gus fusion a suitable tool to study both the f3h expression and the regulation of this pathway. We present here the spatial and temporal analysis of the expression of an alfalfa flavanone 3-hydroxylase (f3h) promoter-gus fusion introduced into Nicotiana benthamiana. The Medicago sativa (alfalfa) f3h promoter directed gus expression in flowers, stems, leaves and roots. In flowers, GUS activity was observed in pollen grains, in ovules, in ovary placenta and in the epidermis, medullary parenchyma, trichomes and second cortical cellular layer surrounding the vascular bundles of the peduncle. In stems, GUS activity was detected at the same places as in the peduncle except for the medullary parenchyma. In roots, we found GUS staining in root hairs, epidermis and in the vascular bundles of the elongated zone. Finally, in leaves, the f3h promoter expressed essentially in the stalk cells of the multicellular trichomes. The expression pattern of the f3h-gus fusion was correlated to the presence of flavonoids in situ. These data indicate that this construct can be very useful to study factors controlling the production of flavonoids.

Plant genes induced in the Rhizobium-legume symbiosis.

Rhizobium, Bradyrhizobium and Azorhizobium can elicit the formation of N2-fixing nodules on the roots or stems of their leguminous host plants. The nodule formation involves several developmental steps determined by different sets of genes from both partners, the gene expression being temporally and spatially coordinated. The plant proteins that are specifically synthesised during the formation and function of the nodule are called nodulins. The nodulins that are expressed before the onset of N2 fixation are termed early nodulins. These proteins are probably involved in the infection process as well as in nodule morphogenesis rather than in nodule function. The nodulins expressed just before or during N2 fixation are termed late nodulins and they participate in the function of the nodule by creating the physiological conditions required for nitrogen fixation, ammonium assimilation and transport. In this review we will describe nodulins, nodulin genes and the relationship between nodulin gene expression and nodule development. The study of nodulin gene expression may provide insight into root-nodule development and the mechanism of communication between bacteria and host plant.

Molecular characterization and expression of alfalfa (Medicago sativa L.) flavanone-3-hydroxylase and dihydroflavonol-4-reductase encoding genes.

Flavonoids are plant phenolic compounds involved in leguminous plant-microbe interactions. Genes implied in the central branch (chalcone synthase (CHS), chalcone isomerase (CHI)) or in the isoflavonoid branch of the flavonoid biosynthesis pathway have been characterized in Medicago sativa. No information is available to date, however, on genes whose products are involved in the synthesis of other types of flavonoids. In this paper we present the genomic organization as well as the nucleotide sequence of one flavanone-3-hydroxylase (F3H) encoding gene of M. sativa, containing two introns and exhibiting 82-89% similarity at the amino acid level to other F3H proteins. This is the first report on the genomic organization of a f3h gene so far. We present also the sequence of a partial dihydroflavonol-4-reductase (DFR) M. sativa cDNA clone. Southern blot experiments indicated that f3h and dfr genes are each represented by a single gene within the tetraploid genome of M. sativa. By a combination of Northern blot and RT-PCR analysis, we showed that both f3h and dfr genes are expressed in flowers, nodules and roots, with a pattern distinct from chs expression. Finally, we show that dfr is expressed in M. sativa leaves whereas f3h is not. The role played by these two genes in organs other than flowers remains to be determined.

Alfalfa Root Flavonoid Production Is Nitrogen Regulated.

Flavonoids produced by legume roots are signal molecules acting both as chemoattractants and nod gene inducers for the symbiotic Rhizobium partner. Combined nitrogen inhibits the establishment of the symbiosis. To know whether nitrogen nutrition could act at the level of signal production, we have studied the expression of flavonoid biosynthetic genes as well as the production of flavonoids in the roots of plants grown under nitrogen-limiting or nonlimiting conditions. We show here that growth of the plant under nitrogen-limiting conditions results in the enhancement of expression of the flavonoid biosynthesis genes chalcone synthase and isoflavone reductase and in an increase of root flavonoid and isoflavonoid production as well as in the Rhizobium meliloti nod gene-inducing activity of the root extract. These results indicate that in alfalfa (Medicago sativa L.) roots, the production of flavonoids can be influenced by the nitrogen nutrition of the plant.

Alfalfa Enod12 genes are differentially regulated during nodule development by Nod factors and Rhizobium invasion.

MsEnod12A and MsEnod12B are two early nodulin genes from alfalfa (Medicago sativa). Differential expression of these genes was demonstrated using a reverse transcription-polymerase chain reaction approach. MsEnod12A RNA was detected only in nodules and not in other plant tissues. In contrast, MsEnod12B transcripts were found in nodules and also at low levels in roots, flowers, stems, and leaves. MsEnod12B expression was enhanced in the root early after inoculation with the microsymbiont Rhizobium meliloti and after treatment with purified Nod factors, whereas MsEnod12A induction was detected only when developing nodules were visible. In situ hybridization showed that in nodules, MsEnod12 expression occurred in the infection zone. In empty Fix- nodules the MsEnod12A transcript level was much reduced, and in spontaneous nodules it was not detectable. These data indicate that MsEnod12B expression in roots is related to the action of Nod factors, whereas MsEnod12A expression is associated with the invasion process in nodules. Therefore, alfalfa possesses different mechanisms regulating MsEnod12A and MsEnod12B expression.

Functional analysis of the Sesbania rostrata leghemoglobin glb3 gene 5'-upstream region in transgenic Lotus corniculatus and Nicotiana tabacum plants.

Expression of the Sesbania rostrata leghemoglobin glb3 gene was analyzed in transgenic Lotus corniculatus and tobacco plants harboring chimeric glb3-uidA (gus) gene fusions to identify cis-acting elements involved in nodule-specific gene expression and general transcriptional control. A 1.9-kilobase fragment of the glb3 5'-upstream region was found to direct a high level of nodule-specific beta-glucuronidase (GUS) activity in L. corniculatus, restricted to the Rhizobium-infected cells of the nodules. The same fragment directed a low level of GUS activity in tobacco, restricted primarily to the roots and to phloem cells of the stem and petiole vascular system. A deletion analysis revealed that the region between coordinates -429 and -48 relative to the ATG was sufficient for nodule-specific expression. Replacement of the -161 to -48 region, containing the glb3 CAAT and TATA boxes, with the heterologous truncated promoters delta-p35S and delta-pnos resulted in a loss of nodule specificity and reduction of GUS activity in L. corniculatus but a significant increase in tobacco, primarily in the roots. The same fragment could not direct nodule-specific expression when fused to a heterologous enhancer in cis. This region contains DNA sequences required, but not sufficient, for nodule-specific expression in L. corniculatus that function poorly or may be involved in promoter silencing in tobacco. By fusing further upstream fragments to the delta-p35S and delta-pnos promoters, two positive regulatory regions were delimited between coordinates -1601 and -670, as well as -429 and -162. The former region appears to function as a general enhancer because it significantly increased promoter activity in both orientations in L. corniculatus and tobacco. The latter region could enhance gene expression in both orientations in tobacco, but only in the correct orientation in L. corniculatus. These results show that efficient expression of the S. rostrata glb3 gene in nodules is mediated by an ATG-proximal, tissue-specific element, as well as further 5'-upstream positive elements; that the S. rostrata glb3 promoter is induced in a nodule-specific fashion in the heterologous legume L. corniculatus, suggesting a high degree of conservation of the relevant regulatory signals; and that the S. rostrata lb promoter is not silent in the nonlegume tobacco, but is expressed primarily in the roots.

Regulation of plant genes specifically induced in nitrogen-fixing nodules: role of cis-acting elements and trans-acting factors in leghemoglobin gene expression.

Transgenic alfalfa plants harboring a gene fusion between the soybean leghemoglobin (lbc3) promoter region and the chloramphenicol acetyl transferase (cat) gene were used to determine the influence of rhizobial mutants on lb gene expression in nodules. The promoter region of the Sesbania rostrata glb3 (Srglb3) leghemoglobin gene was examined for the presence of conserved motifs homologous to binding site 1 and 2 of the soybean lbc3 promoter region, found to interact with a trans-acting factor present in soybean nodule nuclear extracts (Jensen EO, Marcker KA, Schell J, de Bruijn FJ, EMBO J 7:1265-1271, 1988). Subfragments of the S. rostrata glb3 (Srglb3) promoter region were examined for binding to trans-acting factors from nodule nuclear extracts. In addition to the binding sites previously identified (Metz BA, Welters P, Hoffmann HJ, Jensen EO, Schell J, de Bruijn FJ, Mol Gen Genet 214: 181-191), several other sites were found to interact with trans-acting factors. In most cases the same trans-acting factor(s) were shown to be involved. One fragment (202) was found to bind specifically to a different factor (protein) which was extremely heat-resistant (100 degrees C). The appearance of this factor was shown to be developmentally regulated since the expected protein-DNA complexes were first observed around 12 days after infection, concomitant with the production of leghemoglobin proteins. Fragments of the Srglb3 5' upstream region were fused to the beta-glucuronidase reporter gene with its own CAAT and TATA box region or those of the cauliflower mosaic virus 35S and nopaline synthase (nos) promoters.(ABSTRACT TRUNCATED AT 250 WORDS)

The Azorhizobium caulinodans nitrogen-fixation regulatory gene, nifA, is controlled by the cellular nitrogen and oxygen status.

The nucleotide sequence of the Azorhizobium caulinodans ORS571 nifA locus was determined and the deduced NifA amino acid sequence compared with that of NifA from other nitrogen-fixing species. Highly conserved domains, including helix-turn-helix and ATP-binding motifs, and specific conserved residues, such as a cluster of cysteines, were identified. The nifA 5' upstream region was found to contain DNA sequence motifs highly homologous to promoter elements involved in nifA/ntr-mediated control and a consensus element found in the 5' upstream region of the Bradyrhizobium japonicum 5-aminolevulinic acid synthase (hemA) gene and of Escherichia coli genes activated during anaerobiosis via the fnr (fumarate nitrate reduction) control system. A nifA-lac fusion was constructed using miniMu-lac and its activity measured in different genetic backgrounds and under various physiological conditions (in culture and in planta). NifA expression was found to be negatively autoregulated, repressed by rich nitrogen sources and high oxygen concentrations, and controlled (partially) by the ntrC gene, both in culture and in planta. DNA supercoiling was also implicated in nifA regulation, since DNA gyrase inhibitors severely repressed nifA-lac expression.

Rhizobium meliloti 1021 has three differentially regulated loci involved in glutamine biosynthesis, none of which is essential for symbiotic nitrogen fixation.

We have cloned and characterized three distinct Rhizobium meliloti loci involved in glutamine biosynthesis (glnA, glnII, and glnT). The glnA locus shares DNA homology with the glnA gene of Klebsiella pneumoniae, encodes a 55,000-dalton monomer subunit of the heat-stable glutamine synthetase (GS) protein (GSI), and complemented an Escherichia coli glnA mutation. The glnII locus shares DNA homology with the glnII gene of Bradyrhizobium japonicum and encodes a 36,000-dalton monomer subunit of the heat-labile GS protein (GSII). The glnT locus shares no DNA homology with either the glnA or glnII gene and complemented a glnA E. coli strain. The glnT locus codes for an operon encoding polypeptides of 57,000, 48,000, 35,000, 29,000, and 28,000 daltons. glnA and glnII insertion mutants were glutamine prototrophs, lacked the respective GS form (GSI or GSII), grew normally on different nitrogen sources (Asm+), and induced normal, nitrogen-fixing nodules on Medicago sativa plants (Nod+ Fix+). A glnA glnII double mutant was a glutamine auxotroph (Gln-), lacked both GSI and GSII forms, but nevertheless induced normal Fix+ nodules. glnT insertion mutants were prototrophs, contained both GSI and GSII forms, grew normally on different N sources, and induced normal Fix+ nodules. glnII and glnT, but not glnA, expression in R. meliloti was regulated by the nitrogen-regulatory genes ntrA and ntrC and was repressed by rich N sources such as ammonium and glutamine.

Synthesis of an opine-like compound, a rhizopine, in alfalfa nodules is symbiotically regulated.

We show that the promoter of the mos locus, which encodes genes required for the synthesis of a nodule-specific, opine-like compound, a rhizopine, in alfalfa nodules is regulated by the symbiotic nitrogen-fixation regulatory gene nifA. The 5'-regulatory region and amino-terminal end of the first open reading frame of the mos locus are highly homologous to the 5'-regulatory region and amino-terminal portion of the Rhizobium meliloti nifH gene. The coordinate regulation of mos and nif genes suggests that the mos locus plays a symbiotic role. We propose that the rhizopine enhances the survival of the bacterial partner in the symbiosis.

Mini-Mulac transposons with broad-host-range origins of conjugal transfer and replication designed for gene regulation studies in Rhizobiaceae.

Novel mini-Mu derivatives were constructed, carrying a truncated lacZYA operon fused to the terminal 117 bp of the Mu S-end, for the isolation of translational lac fusions by mini-Mu-mediated insertion mutagenesis. Different selectable markers (chloramphenicol resistance; gentamycin resistance) were introduced to allow selection for mini-Mu insertions in different replicons and bacterial strains. A mini-Mulac derivative carrying the site for conjugal transfer of plasmid RP4 (oriT) and the origin of replication of the Agrobacterium rhizogenes Ri plasmid (oriRiHRI) was constructed to enable one-step lac-fusion mutagenesis of cloned (plasmid-borne) regions in Escherichia coli and efficient conjugal transfer of gene fusions to to a variety of Gram-negative bacteria. The conjugation frequency, stability and copy number of replicons carrying mini-Mulac derivatives with oriT and oriRiHRI in members of the Rhizobiaceae such as Rhizobium meliloti, Azorhizobium caulinodans ORS571 and Agrobacterium tumefaciens C58 was examined.

Multiple mutations in the transferred regions of the Agrobacterium rhizogenes root-inducing plasmids.

Agrobacterium rhizogenes induces root formation at the site of inoculation in plants and inserts fragments of its Ri plasmid into the plant nuclear DNA. The transferred region (T-DNA) of the Ri plasmid of the A. rhizogenes strain A4 is made of two fragments, namely TL and TR; the latter harbors a sequence homology with the tms loci (responsible for auxin synthesis) of A. tumefaciens. On Daucus carota slices, single insertion mutations on the TL region of A. rhizogenes do not confer a mutant phenotype while an insertion-deletion in the TR region do confer a Basatt phenotype. Six double mutants with a single insertion in the TL region and the same deletion-insertion of the TR region were constructed. Three of these double mutants were avirulent on D. carota which indicates that in A. rhizogenes A4 the TL and the TR regions cooperate to confer a full infectious phenotype.

Chromosomal location and nucleotide sequence of the Escherichia coli dapA gene.

In Escherichia coli, the first enzyme of the diaminopimelate and lysine pathway is dihydrodipicolinate synthetase, which is feedback-inhibited by lysine and encoded by the dapA gene. The location of the dapA gene on the bacterial chromosome has been determined accurately with respect to the neighboring purC and dapE genes. The complete nucleotide sequence and the transcriptional start of the dapA gene were determined. The results show that dapA consists of a single cistron encoding a 292-amino acid polypeptide of 31,372 daltons.

Construction and uses of a new transposable element whose insertion is able to produce gene fusions with the neomycin-phosphotransferase-coding region of Tn903.

We describe the construction of a transposable element derived from the Mu phage that upon insertion is able to create a gene fusion between the region of Tn903 coding for neomycin phosphotransferase (NPT I), which confers resistance to aminoglycosides including kanamycin (KmR), neomycin and G418, and the control elements of the gene where the insertion occurs. A chloramphenicol (Cm) transacetylase gene (cat) that confers resistance to Cm is present in the transposon so that transposition events can be monitored even when no active fusions with the nptI coding region occur. The transposase gene is deleted and, therefore, this transposon is perfectly stable upon insertion. The properties of this new transposable element were studied by obtaining gene fusions between the Escherichia coli L-arabinose operon and 'nptI gene. In some of them the KmR phenotype is induced by arabinose. Insertions of this element in cloned fragments of the T-DNA region of Agrobacterium rhizogenes were also isolated. Some of them confer a KmR phenotype upon its E. coli carriers, which indicates that portions of the T-DNA are expressed in these cells.