Nodules are induced on alfalfa roots by Agrobacterium tumefaciens and Rhizobium trifolii containing small segments of the Rhizobium meliloti nodulation region.

Regions of the Rhizobium meliloti nodulation genes from the symbiotic plasmid were transferred to Agrobacterium tumefaciens and Rhizobium trifolii by conjugation. The A. tumefaciens and R. trifolii transconjugants were unable to elicit curling of alfalfa root hairs, but were able to induce nodule development at a low frequency. These were judged to be genuine nodules on the basis of cytological and developmental criteria. Like genuine alfalfa nodules, the nodules were initiated from divisions of the inner root cortical cells. They developed a distally positioned meristem and several peripheral vascular bundles. An endodermis separated the inner tissues of the nodule from the surrounding cortex. No infection threads were found to penetrate either root hairs or the nodule cells. Bacteria were found only in intercellular spaces. Thus, alfalfa nodules induced by A. tumefaciens and R. trifolii transconjugants carrying small nodulation clones of R. meliloti were completely devoid of intracellular bacteria. When these strains were inoculated onto white clover roots, small nodule-like protrusions developed that, when examined cytologically, were found to more closely resemble roots than nodules. Although the meristem was broadened and lacked a root cap, the protrusions had a central vascular bundle and other rootlike features. Our results suggest that morphogenesis of alfalfa root nodules can be uncoupled from infection thread formation. The genes encoded in the 8.7-kilobase nodulation fragment are sufficient in A. tumefaciens or R. trifolii backgrounds for nodule morphogenesis.     

Transfer of Rhizobium meliloti pSym genes into Agrobacterium tumefaciens: host-specific nodulation by atypical infection

The pSym megaplasmid of Rhizobium meliloti 2011 mobilized by plasmid RP4, or plasmid pGMI42, an RP4-prime derivative which carries a 290-kilobase pSym fragment including nitrogenase and nod genes, was introduced into Agrobacterium tumefaciens. The resulting transconjugants induced root deformations specifically on the homologous hosts Medicago sativa and Melilotus alba and not on the heterologous hosts Trifolium pratense and Trifolium repens. The root deformations were shown to be genuine nodules by physiological and cytological studies. Thus, host specificity nodulation genes are located on the pSym megaplasmid. Host nodulation specificity did not seem to require recognition at the root hair level since no infection threads could be detected in the root hairs. Cytological observations indicated that bacteria penetrated only the superficial layers of the host root tissue by an atypical infection process. The submeristematic zone and the central tissue of the nodules were bacteria free. Thus, nodule organogenesis was probably triggered from a distance by the bacteria. Agrobacterium transconjugants carrying pSym induced the formation of more numerous and larger nodules than those carrying the RP4-prime plasmid pGMI42, suggesting that some genes influencing nodule organogenesis are located in a pSym region(s) outside that which has been cloned into pGMI42.     

Nucleotide sequence of Rhizobium meliloti nodulation genes

A Rhizobium meliloti DNA region, determining nodulation functions common in different Rhizobium species, has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined. The sequence data indicates three large open reading frames with the same polarity coding for three proteins of 196, 217 and 402 (or 426) amino acid residues, respectively. We suggest the existence of three nod genes on this region, which were designated as nodA, B and C, respectively. Comparison of the R. meliloti nodA, B, C nucleotide and amino acid sequences with those from R. leguminosarum, as reported in the accompanying paper, shows 69-72% homology, clearly demonstrating the high degree of conservation of common nod genes in these Rhizobium species.     

All nod genes of Rhizobium meliloti are involved in alfalfa nodulation by exo mutants.

Nodulation of alfalfa by exoB mutants of Rhizobium meliloti occurred without root hair curling or infection thread formation. nod exoB double mutants had the same nodulation deficiency as single nod mutants. Therefore, all the known nod genes are involved in nodule induction by exoB mutants, which apparently occurs via intercellular invasion.     

Interaction of nod and exo Rhizobium meliloti in alfalfa nodulation

Among the genes of Rhizobium meliloti SU47 that affect nitrogen-fixing symbiosis with alfalfa are nod genes, in which mutations block nodule induction, and exo genes, in which mutations allow nodule formation but block rhizobial exopolysaccharide production as well as nodule invasion and nitrogen fixation. To investigate whether an exo+ bacterium can "help" (that is, reverse the symbiotic defect of) an exo mutant in trans, we have coinoculated alfalfa with pairs of rhizobia of different genotypes. Coinoculant genotypes were chosen so that the exo+ helper strain was nif while the exo "indicator" strain was nif+, and thus any fixation observed was carried out by the exo coinoculant. We find that a nod exo+ coinoculant can help an exo mutant both to invade nodules and to fix nitrogen. However, a nod+ exo+ coinoculant cannot help an exo mutant: Few exo bacteria are recovered from nodules, some bacteroids differentiate into bizarre aberrant forms, and the nodules fail to fix nitrogen. In a triple coinoculation, the effect of nod+ helper supersedes that of nod helper. Implications of these results for interaction of nod and exo gene products are discussed.     

Expression of Rhizobium meliloti nod genes in Rhizobium and Agrobacterium backgrounds.

Rhizobium meliloti nod genes are required for the infection of alfalfa. Induction of the nodC gene depends on a chemical signal from alfalfa and on nodD gene expression. By using a nodC-lacZ fusion, we have shown that the induction of the R. meliloti nodC gene and the expression of nodD occur at almost normal levels in other Rhizobium backgrounds and in Agrobacterium tumefaciens, but not in Escherichia coli. Xanthomonas campestris, or Pseudomonas savastanoi. Our results suggest that bacterial genes in addition to nodDABC are required for nod gene response to plant cells. We have found that inducing activity is present in other plant species besides alfalfa. Acetosyringone, the A. tumefaciens vir gene inducer, does not induce nodC.     

Interference between Rhizobium meliloti and Rhizobium trifolii nodulation genes: genetic basis of R. meliloti dominance.

Transfer of an IncP plasmid carrying the Rhizobium meliloti nodFE, nodG, and nodH genes to Rhizobium trifolii enabled R. trifolii to nodulate alfalfa (Medicago sativa), the normal host of R. meliloti. Using transposon Tn5-linked mutations and in vitro-constructed deletions of the R. meliloti nodFE, nodG, and nodH genes, we showed that R. meliloti nodH was required for R. trifolii to elicit both root hair curling and nodule initiation on alfalfa and that nodH, nodFE, and nodG were required for R. trifolii to elicit infection threads in alfalfa root hairs. Interestingly, the transfer of the R. meliloti nodFE, nodG, and nodH genes to R. trifolii prevented R. trifolii from infecting and nodulating its normal host, white clover (Trifolium repens). Experiments with the mutated R. meliloti nodH, nodF, nodE, and nodG genes demonstrated that nodH, nodF, nodE, and possibly nodG have an additive effect in blocking infection and nodulation of clover.     

Agrobacterium tumefaciens virulence locus pscA is related to the Rhizobium meliloti exoC locus.

Agrobacterium tumefaciens and Rhizobium meliloti carry related genetic loci which have important roles in virulence and symbiosis. Previously, it was shown that two virulence loci of A. tumefaciens, chvA and chvB, are related to two R. meliloti symbiosis loci, ndvA and ndvB, respectively (T. Dylan, L. Ielpi, S. Stanfield, L. Kashyap, C. Douglas, M. Yanofsky, E. Nester, D. R. Helinski, and G. Ditta, Proc. Natl. Acad. Sci. USA 83:4403-4407, 1986). Here we show that these two phytobacteria possess additional related virulence/symbiosis genes. Results of genetic complementation and DNA hybridization experiments indicate that the pscA virulence locus of A. tumefaciens is structurally and functionally related to the exoC symbiosis locus of R. meliloti. Thus, A. tumefaciens and R. meliloti bear at least three related genetic loci that have crucial roles in establishing the interactions that each bacterium has with its respective host plants.     

Nucleotide sequence of Rhizobium meliloti insertion sequence ISRm1: homology to IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens.

Nucleotide sequencing of Rhizobium meliloti insertion sequence ISRm1 showed that it is 1319 nucleotides long and includes 32/31 nucleotide terminal inverted repeats. Analysis of five different insertion sites using sequencing primers complementary to sequences within the left and right ends demonstrated that ISRm1 generates five bp direct repeats at the sites of insertion. Although ISRm1 has shown a target preference for certain short regions (hot spots), there was no apparent similarity in the DNA sequences near the insertion sites. On one strand ISRm1 contains two contiguous open reading frames (ORFs) spanning most of its length. ISRm1 was found to have over 50% sequence homology to insertion sequences IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens. Their sizes, the sequences of their inverted repeats, and the characteristics of their insertion sites are also comparable, indicating that ISRm1, IS2 and IS426 belong to a class of related insertion sequences. Comparison of the proteins potentially encoded by these insertion sequences showed that the two ORFs found in ISRm1 are also present in IS2 and IS426, suggesting that they may be functional genes.     

At least two nodD genes are necessary for efficient nodulation of alfalfa by Rhizobium meliloti

A Rhizobium meliloti DNA region (nodD1) involved in the regulation of other early nodulation genes has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined. The sequence data indicate a large open reading frame with opposite polarity to nodA, -B and -C, coding for a protein of 308 (or 311) amino acid residues. Tn5 insertion within the gene caused a delay in nodulation of Medicago sativa from four to seven days. Hybridization of nodD1 to total DNA of Rhizobium meliloti revealed two additional nodD sequences (nodD2 and nodD3) and both were localized on the megaplasmid pRme41b in the vicinity of the other nod genes. Genetic and DNA hybridization data, combined with nucleotide sequencing showed that nodD2 is a functional gene, while requirement of nodD3 for efficient nodulation of M. sativa could not be detected under our experimental conditions. The nodD2 gene product consists of 310 amino acid residues and shares 86.4% homology with the nodD1 protein. Single nodD2 mutants had the same nodulation phenotype as the nodD1 mutants, while a double nodD1-nodD2 mutant exhibited a more severe delay in nodulation. These results indicate that at least two functional copies of the regulatory gene nodD are necessary for the optimal expression of nodulation genes in R. meliloti.     

A Rhizobium meliloti lipopolysaccharide mutant altered in competitiveness for nodulation of alfalfa.

A transposon Tn5-induced mutant of Rhizobium meliloti Rm2011, designated Rm6963, showed a rough colony morphology on rich and minimal media and an altered lipopolysaccharide (LPS). Major differences from the wild-type LPS were observed in (i) hexose and 2-keto-3-deoxyoctonate elution profiles of crude phenol extracts chromatographed in Sepharose CL-4B, (ii) silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis patterns of crude and purified LPS fractions, and (iii) immunoreactivities otherwise present in purified LPS of the parental strain Rm2011. In addition, Rm6963 lost the ability to grow in Luria-Bertani medium containing the hydrophobic compounds sodium deoxycholate or SDS and showed a decrease in survival in TY medium supplemented with high calcium concentrations. The mutant also had altered symbiotic properties. Rm6963 formed nodules that fixed nitrogen but showed a delayed or even reduced ability to nodulate the primary root of alfalfa without showing changes in the position of nodule distribution profiles along the roots. Furthermore, 2 to 3 weeks after inoculation, plants nodulated by Rm6963 were smaller than control plants inoculated with wild-type bacteria in correlation with a transient decrease in nitrogen fixation. In most experiments, the plants recovered later by expressing a full nitrogen-fixing phenotype and developing an abnormally high number of small nodules in lateral roots after 1 month. Rm6963 was also deficient in the ability to compete for nodulation. In coinoculation experiments with equal bacterial numbers of both mutant and wild-type rhizobia, only the parent was recovered from the uppermost root nodules. A strain ratio of approximately 100 to 1 favoring the mutant was necessary to obtain an equal ratio (1:1) of nodule occupancy. These results show that alterations in Rm6963 which include LPS changes lead to an altered symbiotic phenotype during the association with alfalfa that affects the timing of nodule emergence, the progress of nitrogen fixation, and the strain competitiveness for nodulation.     

A Rhizobium meliloti mutant that forms ineffective pseudonodules in alfalfa produces exopolysaccharide but fails to form beta-(1----2) glucan.

A mutant of Rhizobium meliloti that elicited the formation of inactive nodules in alfalfa was found not to form beta-(1----2) glucan in vivo or in vitro. It was nonmotile because it lacks flagella. The 235-kilodalton protein which acts as an intermediate in beta-(1----2) glucan synthesis was undetectable in the mutant. These properties of the mutant are common to those of chvB mutants of Agrobacterium tumefaciens. Exopolysaccharide formation by the R. meliloti mutant was about double that by the wild type.     

Expression of an agrocin-encoding plasmid of Agrobacterium tumefaciens in Rhizobium meliloti

Plasmid RP4 was used to mobilize the agrocin 84-encoding plasmid, pAg396, from Agrobacterium tumefaciens strain 396 to A. tumefaciens C58 and C58CI as well as Rhizobium meliloti. It was transferred to, but not stably maintained in, R. leguminosarum. It could not be transferred to R. lupini, R. japonicum or R. trifolii. Plasmid pAg396 did not segregate in R. meliloti and produced levels of agrocin comparable to the parental strain A. tumefaciens 396. The potential of agrocin producing R. meliloti in biological control of crown gall is being investigated.     

Chemotaxis of Rhizobium meliloti to the plant flavone luteolin requires functional nodulation genes.

Luteolin is a phenolic compound from plants that acts as a potent and specific inducer of nodABC gene expression in Rhizobium meliloti. We have found that R. meliloti RCR2011 exhibits positive chemotaxis towards luteolin. A maximum chemotactic response was observed at 10(-8) M. Two closely related flavonoids, naringenin and apigenin, were not chemoattractants. The presence of naringenin but not apigenin abolished chemotaxis of R. meliloti towards luteolin. A large deletion in the nif-nod region of the symbiotic megaplasmid eliminated all chemotactic response to luteolin but did not affect general chemotaxis, as indicated by swarm size on semisoft agar plates and chemotaxis towards proline in capillary tubes. Transposon Tn5 mutations in nodD, nodA, or nodC selectively abolished the chemotactic response of R. meliloti to luteolin. Agrobacterium tumefaciens GMI9050, a derivative of the C58 wild type lacking a Ti plasmid, responded chemotactically to 10(-8) M luteolin. The introduction of a 290-kilobase nif-nod-containing sequence of DNA from R. meliloti into A. tumefaciens GMI9050 enabled the recipient to respond to luteolin at concentrations peaking at 10(-6) M as well as at concentrations peaking at 10(-8) M. The response of A. tumefaciens GMI9050 to luteolin was also abolished by the presence of naringenin.     

Erwinia herbicola isolates from alfalfa plants may play a role in nodulation of alfalfa by Rhizobium meliloti.

Erwinia herbicola was isolated from roots of plants derived from surface-sterilized seeds of all alfalfa varieties that were tested. Some of these E. herbicola strains affected nodulation by certain strains of Rhizobium meliloti. In previously published work we presented the isolation of slow-and fast-nodulating variants from a single culture of R. meliloti 102F51. In the absence of E. herbicola, the slow-nodulating variant induced the formation of nodules on alfalfa as rapidly as the faster-nodulating strain. The rates of nodulation by the faster-nodulating variant were the same in the presence and absence of E. herbicola. All of the previously reported slower-nodulating strains derived from R. meliloti 102F51 nodulated more rapidly on sterilized plants than in the presence of certain E. herbicola isolates.