Rhizobium meliloti nodulation genes allow Agrobacterium tumefaciens and Escherichia coli to form pseudonodules on alfalfa.

Regions of the Rhizobium meliloti symbiotic plasmid (20 to 40 kilobase pairs long) containing nodulation (nod) genes were transferred to Agrobacterium tumefaciens or Escherichia coli by conjugation. The A. tumefaciens and E. coli transconjugants elicited root hair curling and the formation of ineffective pseudonodules on inoculated alfalfa plants. A tumefaciens elicited pseudonodules formed at a variable frequency, ranging from 15 to 45%, irrespective of the presence of the Ti plasmid. These pseudonodules developed characteristic nodule meristems, and in some nodules, infection threads were found within the interior of nodules. Infrequently, infection threads penetrated deformed root hairs, but these threads were found only in a minority of nodules. There was no evidence of bacterial release from the infection threads. In addition to being found within threads, agrobacteria were also found in intercellular spaces and within nodule cells that had senesced . In the latter case, the bacteria appeared to invade the nodule cells independently of infection threads and degenerated at the same time as the senescing host cells. No peribacteroid membranes enclosed any agrobacteria , and no bacteroid differentiation was observed. In contrast to the A. tumefaciens-induced pseudonodules , the E. coli-induced pseudonodules were completely devoid of bacteria; infection threads were not found to penetrate root hairs or within nodules. Our results suggest that relatively few Rhizobium genes are involved in the earliest stages of nodulation, and that curling of root hairs and penetration of bacteria via root hair infection threads are not prerequisites for nodule meristem formation in alfalfa.     

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.     

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.     

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.     

Microscopic studies of cell divisions induced in alfalfa roots by Rhizobium meliloti

We have used spot-inoculation and new cytological procedures to observe the earliest events stimulated in alfalfa (Medicago sativa L.) roots by Rhizobium meliloti. Roots were inoculated with 1–10 nl of concentrated bacteria, fixed in paraformaldehyde, and after embedding and sectioning stained with a combination of acridine orange and DAPI (4-6-diamidino-2-phenylindole hydrochloride). Normal R. meliloti provoke cell dedifferentiation and mitosis in the inner cortex of the root within 21–24 h after inoculation. This activation of root cells spreads progressively, leading to nodule formation. In contrast, the R. meliloti nodA and nodC mutants do not stimulate any activation or mitosis. Thus the primary and earliest effect of Rhizobium nod gene action is plant cellular activation. A rapid, whole-mount visualization by lactic acid shows that the pattern of nodule form varies widely. Some R. meliloti strains were found to be capable of stimulating on alfalfa roots both normal nodules and a hybrid structure intermediate between a nodule and a lateral root.     

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.     

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.     

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.     

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.     

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.     

Infection and nodulation of clover by nonmotile Rhizobium trifolii.

Nonmotile mutants of Rhizobium trifolii were isolated to determine whether bacterial motility is required for the infection and nodulation of clover. The nonmotile mutants were screened for their ability to infect and nodulate clover seedlings in Fahraeus glass slide assemblies, plastic growth pouches, and vermiculite-sand-filled clay pots. In each system, the nonmotile mutants were able to infect and nodulate 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.     

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.     

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.     

Effect of a bacteriophage on the colonisation and nodulation of clover roots by a strain of Rhizobium trifolii

The presence of a virulent bacteriophage in the root zone of clover growing in seedling agar under controlled environments (14--17 and 19--23 degrees C) produced changes in the persistence and symbiotic effectiveness of a susceptible strain of Rhizobium trifolii. The phage reduced the rhizoplane population of rhizobia and led to the appearance of variant substrains which were less susceptible to the bacteriophage and mostly ineffective in symbiotic nitrogen fixation. Some were also changed in colonial morphology and nutritional requirements. At the higher temperature, the frequency of bacterial variants increased and the number of nodules due to the parent strain decreased. A large initial population of bacteriophage was able to reduce, but generally did not completely suppress, nodulation.     

Effect of a bacteriophage on colonisation and nodulation of clover roots by paired strains of Rhizobium trifolii

The effect of a virulent bacteriophage on the competitive behaviour of paired strains of Rhizobium trifolii in the root zone of clover plants was examined. The presence of bacteriophage reduced the population density of susceptible strains on the root surface and, in nodulation, favoured resistant or even partially resistant strains which were otherwise less able to form nodules. These effects occurred with different growth temperatures, host species, and ratios of strains in the inoculum.     

The viable-but-nonculturable condition is induced by copper in Agrobacterium tumefaciens and Rhizobium leguminosarum.

Many bacteria respond to changes in environmental conditions by entering the viable-but-nonculturable state. We have determined that copper can induce nutrient-starved Agrobacterium tumefaciens and Rhizobium leguminosarum cells to become viable but nonculturable. This is the first report of a chemical inducer of this condition.     

Expression of a Rhizobium phaseoli Sym plasmid in R. trifolii and Agrobacterium tumefaciens: incompatibility with a R. trifolii Sym plasmid

Identification of the Sym plasmid in Rhizobium phaseoli strain RCC3622 is described. Introduction of this plasmid into R. trifolii or Agrobacterium tumefaciens strains resulted in bacteria capable of forming characteristic spherical root nodules on beans. This Sym plasmid, designated pSym9, was characterized as 275 MDa and nonconjugative. pSym9 was incompatible with the R. trifolii Sym plasmid pSym5, and carries genes determining a melanin-like black pigment. A second plasmid of 135 MDa, pRph3622a, was also transferred from R. phaseoli to R. trifolii and A. tumefaciens. Transconjugants carrying this plasmid did not form root nodules on beans. In contrast to other Rhizobium plasmids, pRph3622a was unstable in A. tumefaciens.     

Morphology of root nodules and nodule-like structures formed by Rhizobium and Agrobacterium strains containing a Rhizobium meliloti megaplasmid

We examined expression of the megaplasmid pRme41b of Rhizobium meliloti in two different Rhizobium sp. Strains and in Agrobacterium tumefaciens. Transfer of pRme41b into these bacteria was facilitated by insertion of a recombinant plasmid coding for mobilization functions of RP4 into the nif region (Kondorosi, A., E. Kondorosi, C.E. Pankhurst, W. J. Broughton, and Z. Banfalvi, 1982, Mol. Gen. Genet., 188:433-439). In all cases, transconjugants formed nodule-like structures on the roots of Medicago sativa. These structures were largely composed of meristematic cells but they were not invaded by bacteria. Bacteria were found only within infection threads in root hairs, and within intercellular spaces of the outermost cells of the structures. The donor strain of R. meliloti containing pAK11 or pAK12 in pRme41b initially produced nodules on M. sativa that did not fix nitrogen (Fix- ). In these nodules, bacteria were released from infection threads into the host cells but they did not multiply appreciably. Any bacteroids formed degenerated prematurely. In some cases, however, reversion to a Fix+ phenotype occurred after 4 to 6 wk. Bacteria released into newly infected cells in these nodules showed normal development into bacteriods.