Nicholas J. Brewin

Plant Cell Wall Remodelling in the Rhizobium–Legume Symbiosis

Colonization of host cells by rhizobium bacteria involves the progressive remodelling of the plant–microbial interface. Following induction of nodulation genes by legume-derived flavonoid signals, rhizobium secretes Nod-factors (lipochitin oligosaccharides) that cause root hair deformations by perturbing the growth of the plant cell wall. The infection thread arises as a tubular ingrowth bounded by plant cell wall. This serves as a conduit for colonizing bacterial cells that grow and divide in its lumen. The transcellular orientation of thread growth is controlled by the cytoskeleton and is coupled to cell cycle reactivation and cell division processes. In response to rhizobium infection, host cells synthesize several new components (early nodulins) that modify the properties of the cell wall and extracellular matrix. Root nodule extensins are a legume-specific family of hydroxyproline-rich glycoproteins targeted into the lumen of the infection thread. They have alternating extensin and arabinogalactan (AGP) glycosylation motifs. The structural characteristics of these glycoproteins suggest that they may serve to regulate fluid-to-solid transitions in the extracellular matrix. Extensibility of the infection thread is apparently controlled by peroxide-driven protein cross-linking and perhaps also by modification of the pectic matrix. Endocytosis of rhizobia from unwalled infection droplets into the host cell cytoplasm depends on physical contact between glycocalyx components of the plant and bacterial membrane surfaces. As endosymbionts, bacteroids remain enclosed within a plant-derived membrane that is topologically equivalent to the plasma membrane. This membrane acquires specialist functions that regulate metabolite exchanges between bacterial cells and the host cytoplasm. Ultimately, however, the fate of the symbiosome is to become a lysosome, causing the eventual senescence of the symbiotic interaction.

Involvement of diamine oxidase and peroxidase in insolubilization of the extracellular matrix: implications for pea nodule initiation by Rhizobium leguminosarum.

Rhizobium leguminosarum colonizes host cells and tissues through infection threads, which are tubular in-growths of the plant cell wall. Monoclonal antibody MAC265 recognizes a plant matrix glycoprotein (MGP) associated with the lumen of these infection threads. This glycoprotein is also released in soluble form from the root tips of pea seedlings. In the presence of hydrogen peroxide, release of glycoprotein from root tips was not observed. Extractability from root tips was therefore used as the basis for investigating the peroxide-driven insolubilization of MGP and the possible involvement of two extracellular enzymes, peroxidase (POD) and diamine oxidase (DAO), was investigated. Release of MGP from root tips was enhanced by application of POD and DAO inhibitors (salicylhydroxamic acid and o-phenanthroline, respectively). Furthermore, release of MGP was inhibited by pretreatment of roots with putrescine (the substrate of DAO) and also by application of a partially purified extract of DAO from pea shoots. Following inoculation of pea roots with R. leguminosarum, elevated levels of DAO transcript were observed by reverse transcriptase-polymerase chain reaction (RT-PCR), but these then dropped to a low level from 4 to 10 days post inoculation, rising again in more mature nodules. In situ hybridization studies indicated that the bulk of the transcription was associated with the infected tissue in the center of the nodule. On the basis of these observations, we postulate that DAO may be involved in the peroxide-driven hardening of MGP in the lumen of infection threads and in the intercellular matrix.

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

SummaryThe 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.

Effects of Boron on Rhizobium-Legume Cell-Surface Interactions and Nodule Development'

Boron (B) is an essential micronutrient for the development of nitrogen-fixing root nodules in pea (Pisum sativum). By using monoclonal antibodies that recognize specific glycoconjugate components implicated in legume root-nodule development, we investigated the effects of low B on the formation of infection threads and the colonization of pea nodules by Rhizobium leguminosarum bv viciae. In B-deficient nodules the proportion of infected host cells was much lower than in nodules from plants supplied with normal quantities of B. Moreover, the host cells often developed enlarged and abnormally shaped infection threads that frequently burst, releasing bacteria into damaged host cells. There was also an over-production of plant matrix material in which the rhizobial cells were embedded during their progression through the infection thread. Furthermore, in a series of in vitro binding studies, we demonstrated that the presence of B can change the affinity with which the bacterial cell surface interacts with the peribacteroid membrane glycocalyx relative to its interaction with intercellular plant matrix glycoprotein. From these observations we suggest that B plays an important role in mediating cell-surface interactions that lead to endocytosis of rhizobia by host cells and hence to the correct establishment of the symbiosis between pea and Rhizobium.

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

SummarySelection 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.

Recombinant Nodulation Plasmids in Rhizobium leguminosarum

SUMMARY: Plasmids pRL 1JI and pRL6JI, which carry determinants essential for symbiosis in Rhizobium leguminosarum field isolates 248 and 128C53, respectively, were both incompatible with two other transmissible plasmids that did not carry symbiotic determinants. When derivatives of these two plasmids (pRL3JI or pRL4JI) were introduced into a strain which already contained pRL 1JI or pRL6JI, recombinant replicons were often obtained: these were of uniform size for each pair of incompatible plasmids. Recombinant nodulation plasmids were also observed following infrequent interactions with pRL10JI, a natural nodulation plasmid that does not belong to the same incompatibility group as pRL3JI or pRL4JI. By the formation of a recombinant nodulation plasmid, it has been possible to transfer the determinants for nodulation ability (Nod+), nitrogen fixation (Fix+) and hydrogen uptake (Hup+) from pRL6JI, a plasmid that was not self-transmissible, on to a replicon that was transmissible at high frequency and carried a selectable drug resistance marker. In the case of pRL10JI, which is also a non-transmissible nodulation plasmid, the formation of a recombinant nodulation plasmid transferred the symbiotic determinants to a transmissible replicon belonging to a different incompatibility group from pRL10JI itself.

Host-plant invasion by Rbizobium: the role of cell-surface components

Rhizobia are soil bacteria that can become endosymbionts, reducing atmospheric nitrogen within nodules formed on the roots of legume plants. During tissue and cell invasion, bacterial cell-surface components adapt the bacterium to survive as an endophyte without eliciting host-defence responses. The structures of many of these components have been established recently, allowing their possible roles in invasion to be defined more clearly.

The Rhizobium--legume symbiosis.

physiological changes involved in the differentiation of bacteroids within the nodule. Certainly the addition of nodules to fixed nitrogen in the form of NH+4 or NO-3 does repress N2-ase, although the concentrations required are greater than for repression in free-living nitrogen-fixing bacteria (Klamberger 1977). Various hypotheses have been put forward to account for the repression of N2-ase activity in the nodule. One hypothesis was that in the presence of exogenous nitrate the plant’s nitrate reductase competed for photosynthate, thereby rendering N2-ase inactive because it was starved of an energy supply (Oghoghorie & Pate 1971). Evidence against this was the finding (Chen & Phillips 1976) that addition of NO-3 to pea leaves did not inhibit N2-ase, and that, if the plants were exposed to an atmosphere enriched with CO2, the extent of N2-ase repression when NO-3 was applied to the roots was the same as in a normal atmosphere despite the increase in the plants’ photosynthetic capability. Thus, if there is any competition for photosynthate between N2-ase and nitrate reductase, it must be localized. This model does not explain why NH+4 is also efficient as a repressor of N2-ase. Recently Bisseling, van den Bos & van Kamman (1978) showed that under conditions where NH4NO3 repressed N2-ase activity in pea nodules, it did not affect synthesis of the enzyme. They proposed that the inhibition of N2-ase in the nodule was indirect, being mediated by an inhibition of the synthesis of leg-haemoglobin.

Two Transmissible Plasmids in Rhizobium leguminosarum strain 300

SUMMARY: The transposon Tn5, which specifies kanamycin resistance, was inserted into transmissible plasmids of Rhizobium leguminosarum strain 1062, a derivative of strain 300. In this way several kanamycin-resistant derivatives of the two smallest plasmids (pRL8JI and pIJ1001) were obtained, but there was no evidence that any of the other plasmids of strain 1062 was transmissible. Kanamycin-sensitive derivatives, each apparently cured of either pRL8JI or pIJ1001, still induced nitrogen-fixing nodules on peas and were not phenotypically distinct from the parental strain. Both plasmids were transmissible at low frequency to other R. leguminosarum strains, but they could be mobilized efficiently by pRLlJI, another transmissible R. leguminosarum plasmid. When Tn5-marked pIJ1001 was transferred to a strain of R. phaseoli, the majority of the transconjugants lost the ability to nodulate Phaseolus beans, the normal host for this species. This was due to the loss from R. phaseoli of a nodulation plasmid which was apparently incompatible with pIJ1001.

Transfer of Symbiotic Genes with Bacteriocinogenic Plasmids in Rhizobium leguminosarum

SUMMARY: Transfer of derivatives of the bacteriocinogenic plasmid pRL1JI into eight symbiotically defective strains of R. leguminosarum resulted in suppression of the mutant phenotype in four cases. These included non-infective, ineffective and temperature-sensitive ineffective phenotypes. However, in none of these strains was the defect suppressible after high frequency transfer of derivatives of two other bacteriocinogenic plasmids, pRL3JI or pRL4JI. Nodulation ability was co-transferred at high frequency (> 95%) with bacteriocin production by the plasmid pRL1JI. The other two plasmids, pRL3JI and pRL4JI, also mediated the transfer of nodulation ability but at much lower frequency (10-3 to 10-4 per plasmid transfer). Sometimes, transconjugants that had acquired nodulation ability after the transfer of derivatives of plasmids pRL3JI or pRL4JI acted subsequently as high frequency donors for nodulation ability in a manner that was apparently similar to strains containing pRL1JI.