Increased Bean (Phaseolus vulgaris L.) Nodulation Competitiveness of Genetically Modified Rhizobium Strains

Rhizobium leguminosarum bv. phaseoli strain collections harbor heterogeneous groups of bacteria in which two main types of strains may be distinguished, differing both in the symbiotic plasmid and in the chromosome. We have analyzed under laboratory conditions the competitive abilities of the different types of Rhizobium strains capable of nodulating Phaseolus vulgaris L. bean. R. leguminosarum bv. phaseoli type I strains (characterized by nif gene reiterations and a narrow host range) are more competitive than type II strains (that have a broad host range), and both types are more competitive than the promiscuous rhizobia isolated from other tropical legumes able to nodulate beans. Type I strains become even more competitive by the transfer of a non-Sym, 225-kilobase plasmid from type II strain CFN299. This plasmid has been previously shown to enhance the nodulation and nitrogen fixation capabilities of Agrobacterium tumefaciens transconjugants carrying the Sym plasmid of strain CFN299. Other type I R. leguminosarum bv. phaseoli transconjugants carrying two symbiotic plasmids (type I and type II) have been constructed. These strains have a diminished competitive ability. The increase of competitiveness obtained in some transconjugants seems to be a transient property.     

Rhizobium nodulation genes involved in root hair curling (Hac) are functionally conserved

Summary Five specific transposon-induced nodulation defective (Nod⁻) mutants from different fast-growing species ofRhizobium were used as the recipients for the transfer of each of several endogenous Sym(biosis) plasmids or for recombinant plasmids that encode early nodulation and host-specificity functions. The Nod⁻ mutants were derived fromR. trifolii, R. meliloti and from a broad-host-rangeRhizobium strain which is able to nodulate both cowpea (tropical) legumes and the non-legumeParasponia. These mutants had several common features (a), they were Nod⁻ on all their known plant hosts, (b), they could not induce root hair curling (Hac⁻) and (c), the mutations were all located on the endogenous Sym-plasmid of the respective strain. Transfer to these mutants of Sym plasmids (or recombinant plasmids) encoding heterologous information for clover nodulation (pBR1AN, pRt032, pRt038), for pea nodulation (pJB5JI, pRL1JI::Tn1831), for lucerne nodulation (pRmSL26), or for the nodulation of both tropical legumes and non-legumes (pNM4AN), was able to restore root hair curling capacity and in most cases, nodulation capacity of the original plant host(s). This demonstrated a functional conservation of at least some genes involved in root hair curling. Positive hybridization between Nod DNA sequences fromR. trifolii and from a broad-host-rangeRhizobium strain (ANU240) was obtained to other fast-growingRhizobium strains. These results indicate that at least some of the early nodulation functions are common in a broad spectrum ofRhizobium strains.     

Host-specific nodulation is encoded on a 14kb DNA fragment in Rhizobium trifolii

Summary The Rhizobium trifolii genes necessary for nodule induction and development have been isolated on a 14.0kb fragment of symbiotic (Sym) plasmid DNA. When cloned into a broad-host-range plasmid vector, these sequences confer a clover nodulation phenotype on a derivative of R. trifolii which has been cured of its endogenous Sym plasmid. Furthermore, these sequences encode both host specificity and nodulation functions since they confer the ability to recognize and nodulate clover plants on Agrobacterium and a fast-growing cowpea Rhizobium. This indicates that the bacterial genes essential for the initial, highly-specific interaction with plants are closely linked.     

Plasmid-determined nodulation and nitrogen-fixation abilities inRhizobium phaseoli

Summary InRhizobium phaseoli strain 8002, the 190 Md plasmid pRP2JI which determines the ability to produce nitrogen-fixing nodules onPhaseolus beans (Nod⁺ Fix⁺) and the production of melanin on L-tyrosine-containing media (Mel⁺), was shown to be transmissible by conjugation to otherRhizobium strains. When pRP2JI was transferred to Nod^- strains ofR. leguminosarum (which normally nodulates peas) the transconjugants gained the ability to nodulatePhaseolus beans and to make melanin.Out of 187 derivatives of strain 8002 carrying pRP2JI plasmids into which the transposon Tn5 had been inserted, six were Fix^- Nod⁺ Mel⁺, one was Fix^- Nod⁺ Mel^- and four were Fix⁺ Nod⁺ Mel^-. Three other derivatives of strain 8002 were Nod^- Mel^-; these had suffered deletions of c 30 Md in pRP2JI. Thus the genes for melanin production and nodulation appear to be closely linked, but melanin production is not necessary for the induction of nitrogen-fixing nodules onPhaseolus beans.     

Melanin production encoded by a cryptic plasmid in a Rhizobium leguminosarum strain

Rhizobium leguminosarum strain VF39, isolated from nodules of field-grown faba beans in the Federal Republic of Germany, was shown to contain six plasmids ranging in molecular weight from 90 to 400 Md. Hybridisation to nif gene probes, plasmid curing, and mobilisation to other strains of Rhizobium and to Agrobacterium showed that the third largest plasmid, pRleVF39d (220 Md), carried genes for nodulation and nitrogen fixation. This plasmid was incompatible with pRL10JI, the Sym plasmid of R. leguminosarum strain JB300. Of the other plasmids, the two smallest (pRleVF39a and pRleVF39b, 90 and 160 Md respectively) were shown to be self-transmissible at a low frequency. Although melanin production is as yet unreported in strains of R. leguminosarum biovar viceae, strain VF39 produced a dark pigment, which, since it was not produced on minimal media and its production was greatly enhanced by the presence of tyrosine in the media, is probably melanin-like. Derivatives of VF39 cured of pRleVF39a no longer produced this pigment, but regained the ability to produce it when this plasmid was transferred into them. Strains of Agrobacterium tumefaciens, R. meliloti, and some strains of R. leguminosarum carrying pRleVF39a did not produce this pigment, indicating perhaps that some genes elsewhere on the VF39 genome are also involved in pigment production. Plasmid pRleVF39a appeared to be incompatible with the cryptic Rhizobium plasmids pRle336b and pRL8JI (both ca. 100 Md), but was compatible with the R. leguminosarum biovar phaseoli Sym plasmids pRP1JI, pRP2JI and pRph51a, all of which also code for melanin production. The absence of pRleVF39a in cured derivatives of VF39 had no effect on the symbiotic performance or competitive ability of this strain.     

Analysis of R-primes demonstrates that genes for broad host range nodulation of Rhizobium strain NGR234 are dispersed on the Sym plasmid

Summary R-prime plasmids carrying regions of the symbiotic (Sym) plasmid of the broad host range Rhizobium strain NGR234 were isolated in intergeneric matings with Escherichia coli K12. Three R-primes carrying approximately 180 kb (pMN23), 220 kb (pMN31) and 330 kb (pMN49) of Sym DNA were characterized in more detail. Restriction enzyme analysis and hybridization studies showed that these R-primes carried large overlapping regions of the Sym plasmid, and had the symbiotic genes (two copies of nifH, D and K; nodA, B, C and D; region II; host specific nodulation (hsn) genes) located over half of the 470 kb Sym plasmid. Only the largest of these R-primes (pMN49) contained the complete nodulation host range of the original parent strain NGR234. This broad host range was shown to be present on plasmid pMN49 by being expressed in Agrobacterium tumefaciens strain A136. Furthermore the R-prime plasmids were shown to contain different regions of distinctive host specific nodulation (hsn) for tropical legume infection and for the nodulation of the non-legume Parasponia. Nodulation of soybeans, however, required an additional region that was not essential for the nodulation of other tropical legumes. Strain NGR234 was also found to nodulate the stem and roots of the tropical legume Sesbania rostrata at a very low efficiency. However, the R-prime mini Sym plasmid constructions enabled a greater efficiency of nodulation of Sesbania rostrata to occur.     

Isolation of symbiotically defective mutants in Rhizobium leguminosarum by insertion of the transposon Tn5 into a transmissible plasmid

Summary Selection was made for the transposition of the kanamycin resistance transposon Tn5 from a location on the chromosome of R. leguminosarum into a transmissible, bacteriocinogenic plasmid that also carries genes required for the induction of nitrogen-fixing nodules on peas.One hundred and sixty independent insertions into transmissible plasmids were isolated. When these plasmids were transferred by conjugation into a non-nodulating strain, which carries a deletion in one of its resident plasmids, of the 160 isolates tested 14 yielded transconjugants that formed nodules that did not fix nitrogen (Fix^-) and in a further 15 cases the transconjugants were unable to form nodules (were Nod^-). When transferred to a symbiotically proficient strain (i.e. Nod⁺ Fix⁺) none of the transconjugants was symbiotically defective; thus the mutations were not dominant.When kan was transduced from the clones that generated Fix^- transconjugants into a Fix⁺ recipient the majority of transductants inherited Fix^- indicating that the insertion of Tn5 had induced the symbiotic mutations. Transduction of kan, from the clones that failed to donate Nod⁺ by conjugation to strain 6015, occurred at barely detectable frequencies and it was not possible to demonstrate transduction of Nod^-. kan was co-transduced with Nod⁺ from some of the clones and some of these transductants also inherited the ability to produce medium bacteriocin and to transfer at high frequency by conjugation. Thus the genes for all these characters are closely linked.     

Regulation of Nodulation by Rhizobium meliloti 102F15 on Its Mutant Which Forms an Unusually High Number of Nodules on Alfalfa

A mutant (WL3A150) of Rhizobium meliloti 102F51 that elicits an unusually high number of nodules on its host, alfalfa (Medicago sativa), supports the idea that the host may rely on early bacteroid development in the nodule or on metabolites produced in the infection thread as one of the signals to control further nodulation. This mutant was initially isolated because of its Fix⁻ phenotype. It consistently formed many more nodules than all the other Fix⁻ mutants isolated from strain 102F51 (a total of 11 mutants). Nodules formed by this mutant were small and white and were indistinguishable in appearance from nodules formed by the other Fix⁻ mutants. An ultrastructural study of the nodules, however, showed that this mutant, although forming numerous infection threads, failed to develop into bacteroids. The ability of the mutant to form an unusually high number of nodules coulde be suppressed in a time-dependent manner by the presence of the wild type.     

Rhizobium phaseoli: A molecular genetics view

We have used molecular genetics techniques to analyze the structural and functional organization of genetic information ofRhizobium phaseoli, the symbiont of the common bean plantPhaseolus vulgaris. As in otherRhizobium species, the genome consists of the chromosome and plasmids of high molecular weight. Symbiotic determinants, nitrogen fixation genes as well as nodulation genes, are localized on a single replicon, the symbiotic (sym) plasmid. Thesym plasmid of differentR. phaseoli strains was transferred to anAgrobacterium tumefaciens strain cured of its native plasmids. In all cases, Agrobacterium transconjugants able to nodulate bean plants were obtained. Some of the transconjugants had the capacity to elicit an effective symbiosis. The genome ofR. phaseoli is complex, containing a large amount of reiterated DNA sequences. In mostR. pahseoli strains one of such reiterated DNA families corresponds to the nitrogenase structural genes (nif genes). A functional analysis of these genes suggested that the presence of reiteratednif genesis is related to the capacity of fixing atmospheric nitrogen in the symbiotic state. The presence of several repeated sequences in the genome might provide sites for recombination, resulting in genomic rearrangements. By analyzing direct descendants of a single cell in the laboratory, evidence of frequent genomic rearrangements inR. phaseoli was found. We propose that genomic rearrangements constitute the molecular basis of the frequent variability and loss of symbiotic properties in different Rhizobium strains.     

Genomic basis of symbiovar mimosae in Rhizobium etli

Background

Symbiosis genes (nod and nif) involved in nodulation and nitrogen fixation in legumes are plasmid-borne in Rhizobium. Rhizobial symbiotic variants (symbiovars) with distinct host specificity would depend on the type of symbiosis plasmid. In Rhizobium etli or in Rhizobium phaseoli, symbiovar phaseoli strains have the capacity to form nodules in Phaseolus vulgaris while symbiovar mimosae confers a broad host range including different mimosa trees.

Results

We report on the genome of R. etli symbiovar mimosae strain Mim1 and its comparison to that from R. etli symbiovar phaseoli strain CFN42. Differences were found in plasmids especially in the symbiosis plasmid, not only in nod gene sequences but in nod gene content. Differences in Nod factors deduced from the presence of nod genes, in secretion systems or ACC-deaminase could help explain the distinct host specificity. Genes involved in P. vulgaris exudate uptake were not found in symbiovar mimosae but hup genes (involved in hydrogen uptake) were found. Plasmid pRetCFN42a was partially contained in Mim1 and a plasmid (pRetMim1c) was found only in Mim1. Chromids were well conserved.

Conclusions

The genomic differences between the two symbiovars, mimosae and phaseoli may explain different host specificity. With the genomic analysis presented, the term symbiovar is validated. Furthermore, our data support that the generalist symbiovar mimosae may be older than the specialist symbiovar phaseoli.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-575) contains supplementary material, which is available to authorized users.     

A molecular linkage map of nitrogenase and nodulation genes in Rhizobium trifolii

Summary A molecular map was constructed linking the nitrogenase structural genes (nif) and nodulation genes (nod) in the white clover symbiont, Rhizobium trifolii. In R. trifolii strain ANU843 these two genetic regions are located some 16 kilobases (kb) apart on the 180 kb symbiotic (Sym) plasmid. The molecular linkage of nod and nif genetic regions was established by hybridization analysis using recombinant plasmids containing overlapping cloned sequences. Nodulation genes were located by means of a Tn5-induced nodulation-defective mutant that failed to induce clover root hair curling (Hac^- phenotype). A cloned wild-type DNA fragment was shown to phenotypically correct the Hac^- mutation by complementation. The nifHDK genes were cloned by positive hybridization to another R. trifolii nif-specific probe. Location of the nif genes relative to the nod genes was established by analysis of a Sym plasmid deletion derivative.     

Selection and symbiotic properties ofRhizobium leguminosarum biovarphaseoli strains harboring pRtr5a

TheRhizobium leguminosarum biovartrifolii symbiotic plasmid pRtr5a has been transferred toR. leguminosarum biovarphaseoli RCR 3644-S1. The transconjugant selection had been done byTrifolium pratense plants. All transconjugants lacked the resident pSym, but had complete pRtr5a, and were Fix⁺ onT. repens andT. alexandrinum, Fix^– onT. subterraneum, and formed a few small white and Fix^– nodules onPhaseolus vulgaris. It is shown that this nodulation onP. vulgaris is due to pRtr5a. The presence of pRtr5a and/or the passage throughTrifolium pratense nodules provoke(s) the recipient strain symbiotic plasmid loss.     

Genetic Analysis of Rhizobium leguminosarum bv. Phaseoli Mutants Defective in Nodulation and Nodulation Suppression

Nodulation-defective rhizobia and their nodule-forming derivatives containing cloned DNA from the wild type were used to study nodulation suppression in Phaseolus vulgaris L. Non-nitrogen-fixing derivatives which formed rhizobia-containing white nodules induced partial suppression. Comparison of this with the complete suppression by Fix⁺ derivatives and a Fix^- mutant which formed rhizobia-containing pink nodules suggests that the extent of suppression may be related to successive stages of nodule development.     

Linkage of genes for nitrogenase and nodulation ability on plasmids in Rhizobium leguminosarum and R. phaseoli

Summary The ability to identify genes that specify nitrogenase (nif genes) in Rhizobium depends on the close homology between then and the corresponding nif genes of Klebsiella pneumoniae (Nuti et al. 1979; Ruvkun and Ausubel 1980). Rhizobium plasmids of high molecular weight (>100 Md) were separated on agarose gels, transferred to nitrocellulose filters and tested for their ability to hybridise with radioactively labelled pSA30, containing the nifKDH region of K. pneumoniae. Five large plasmids, each present in different strains of R. leguminosarum or R. phaseoli, were found to hybridise. Each of these plasmids had previously been shown to determine other symbiotic functions such as nodulation ability. The nif genes on three different plasmids appeared to be in conserved DNA regions since they were within an EcoRI restriction fragment of the same size.     

Symbiotic mutants of USDA191, a fast-growing Rhizobium that nodulates soybeans

Summary Symbiotic and auxotrophic mutants of Rhizobium japonicum strain USDA191 were isolated using Tn5 mutagenesis and techniques that cause plasmid deletions and plasmid curing. Characterization of several mutants that are unable to nodulate (Nod^-) or unable to fix nitrogen (Fix^_) showed that nod and nif genes are located within one regions of a 200 MD plasmid (pSym191). Blot hybridization analysis of plasmids in other fast-growing R. japonicum strains showed that nod as well as nif sequences are located on plasmids in eight strains but are apparently carried in the chromosome in two strains.     

Symbiotic diversity of Ensifer meliloti strains recovered from various legume species in Tunisia

Ensifer meliloti (formerly Sinorhizobium meliloti) was first considered as a specific microsymbiont of Medicago, Melilotus and Trigonella. However, strains of E. meliloti were recovered from root nodules of various legume species and their symbiotic status still remains unclear. Here, we further investigate the specificity of these strains. A collection of 47 E. meliloti strains isolated in Tunisia from root nodules of Medicago truncatula, Medicago sativa, Medicago ciliaris, Medicago laciniata, Medicago marina, Medicago scutellata, Phaseolus vulgaris, Cicer arietinum, Argyrolobium uniflorum, Lotus creticus, Lotus roudairei, Ononis natrix, Retama raetam, Genista saharae, Acacia tortilis, Hedysarum carnosum and Hippocrepis bicontorta were examined by REP-PCR fingerprinting, PCR-RFLPs of the 16S-23S rDNA IGS, the nifH gene and nifD-K intergenic spacer, and sequencing of 16S rRNA and nodA genes. Their nodulation range was also assessed by cross-inoculation experiments. No clear correlation was found between chromosomal backgrounds and host plants of origin. The nodulation polyvalence of the species E. meliloti was associated with a high symbiotic heterogeneity. On the basis of PCR-RFLP data from the nifH gene and nifD-K intergenic spacer, E. meliloti strains isolated from non-Medicago legumes harboured distinct genes and possessed wider host ranges. Some strains did not nodulate Medicago species. On the basis of nodA phylogeny, the majority of the Tunisian strains, including strains from Medicago, harboured distinct nodA alleles more related to those found in E. medicae than those found in E. meliloti. However, more work is still needed to characterize this group further. The diversity observed among M. laciniata isolates, which was supported by nodA phylogeny, nifH typing and the efficiency profile on M. ciliaris, indicated that what was thought to be bv. medicaginis is certainly heterogeneous.     

Nitrogen-fixing nodules induced by Agrobacterium tumefaciens harboring Rhizobium phaseoli plasmids.

Rhizobium phaseoli CFN299 forms nitrogen-fixing nodules in Phaseolus vulgaris (bean) and in Leucaena esculenta. It has three plasmids of 185, 225, and 410 kilobases. The 410-kilobase plasmid contains the nitrogenase structural genes. We have transferred these plasmids to the plasmid-free strain Agrobacterium tumefaciens GMI9023. Transconjugants containing different combinations of the R. phaseoli plasmids were obtained, and they were exhaustively purified before nodulation was assayed. Only transconjugants harboring the 410-kilobase plasmid nodulate P. vulgaris and L. esculenta. Nodules formed by all such transconjugants are able to reduce acetylene. Transconjugants containing the whole set of plasmids from CFN299 nodulate better and fix more nitrogen than the transconjugants carrying only the Sym plasmid. Microscopic analysis of nodules induced by A. tumefaciens transconjugants reveals infected cells and vascular bundles. None of the A. tumefaciens transconjugants, not even the one with the whole set of plasmids from CFN299, behaves in symbiosis like the original R. phaseoli strain; the transconjugants produce fewer nodules and have lower acetylene reduction (25% as compared to the original R. phaseoli strain) and more amyloplasts per nodule. More than 2,000 bacterial isolates from nodules of P. vulgaris and L. esculenta formed by the transconjugants were analyzed by different criteria. Not a single rhizobium could be detected. Our results show that R. phaseoli plasmids may be expressed in the A. tumefaciens background and direct the formation of effective, differentiated nodules.     

Endophytic Occupation of Root Nodules and Roots of Melilotus dentatus by Agrobacterium tumefaciens

Agrobacterium strains have been frequently isolated from the root nodules of different legumes. Various possible mechanisms have been proposed to explain the existence of these bacteria in nodules, but there is no sufficient experimental evidence to support the estimations. In this work, we proved that the Agrobacterium strain CCBAU 81181, which was originally isolated from the root nodules of Onobrychis viciaefolia, and a symbiotic strain of Sinorhizobium meliloti CCBAU 10062 could coinhabit the root nodules of Melilotus dentatus. Analyses were performed by using a fluorescence marker, reisolation of bacteria from nodules, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of whole cellular proteins, and polymerase chain reaction amplification of symbiotic genes. The inoculation of A. tumefaciens CCBAU 81181 did not affect the growth and nodulation of plants. CCBAU 81181 and 24 other Agrobacterium strains isolated from nodules were incapable of nodulating on their original or alternative host and 22 strains of these strains were endophytes in the roots and stems of their hosts. Also, the tumor-inducing A. tumefaciens strains IAM 13129^T and C58 were found capable of entering the roots of Glycyrrhiza pallidiflora, but did not cause pathogenic symptoms. With these results, we conclude that A. tumefaciens strains could be endophytic bacteria in the roots, stems, and root nodules. This finding partially explains why Agrobacterium strains were frequently isolated from the surface-sterilized nodules.     

Enhancement of nodulation by some arid climate strains of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> biovar <Emphasis Type="Italic">trifolii</Emphasis> using protoplast fusion

Fourteen randomly clover indigenous nodulated Rhizobium strains were isolated from different locations in Saudi Arabia. They were identified as different strains of the genus Rhizobium leguminosarum biovar trifolii and characterized for their intrinsic antibiotic resistance against a range of antibiotics, nodulation capability and plasmid profiles. Results revealed the presence of high molecular weight plasmids (megaplasmids) in all the selected strains. Based on the ability for nodulation production, two weak strains (RtI1 and RtI2) and one efficient strain (RtA1) were selected for protoplast fusion and the numbers of nodules produced by the intra-specific protoplast fusion strains were investigated. Results clearly confirmed the effective role of the protoplast fusion in enhancing both nodulation production capacity of Rhizobium species and their range of antibiotic resistance. Protoplast fusion of the local Rhizobium species resulted in 1.93- to 5.67-fold increase in nodulation number compared to their parental strains, which was considered an excellent result concerning agricultural practices, especially the formation of nitrogen-fixing root nodules on legume crop plants. Protoplast fusion also produced fusants with a wide range of antibiotic resistance, another advantage added to the new strains against environmental stresses. In conclusion, protoplast fusion proved its efficiency as a tool for constructing a second generation of Rhizobia with much better characteristics for efficient applications in arid land.