Equilibrium between the "genuine mutualists" and "symbiotic cheaters" in the bacterial population co-evolving with plants in a facultative symbiosis

The mathematical model for evolution of the plant-microbe facultative mutualistic interactions based on the partners’ symbiotic feedbacks is constructed. Using the example of rhizobia-legume symbiosis, we addressed these feedbacks in terms of the metabolic exchange resulting in the parallel improvements of the partners’ fitness (positive feedbacks). These improvements are correlated to the symbiotic efficiency dependent on the ratio of N₂-fixing bacterial strains (“genuine mutualists”) to the non- N₂-fixing strains (“symbiotic cheaters”) in the root nodules. The computer experiments demonstrated that an interplay between the frequency-dependent selection (FDS) and the Darwinian (frequency-independent) selection pressures implemented in the partners’ populations ensures an anchoring or even domination for the newly generated host-specific mutualists (which form N₂-fixing nodules only with one of two available plant genotypes) more successfully than for the non-host-specific mutualists (which form N₂-fixing nodules with both plant genotypes). The created model allows us to consider the mutualistic symbiosis as a finely balanced polymorphic system wherein the equilibrium in bacterial population may be shifted in favor of “genuine mutualists” due to the partner-stipulated selection for an improved symbiotic efficiency implemented in the plant population.     

Simulation of evolution implemented in the mutualistic symbioses towards enhancing their ecological efficiency, functional integrity and genotypic specificity

We created the mathematical model for the evolution of the Efficiency of Mutualistic Symbioses (EMS) which was estimated as the microsymbiont impacts on the host’s reproductive potential. Using the example of rhizobia–legume interaction, the relationships were studied between EMS and Functional Integrity of Symbiosis (FIS) which is represented as a measure for concordance of changes in the partners’ genotypic frequencies under the environmental fluctuations represented by the minor deviations of the systemic model parameters. The FIS indices correlate positively with EMS values suggesting an enhancement of FIS via the natural selection operating in the partners’ populations in favor of high EMS. Due to this selection, nodular habitats may be closed for colonization by the non-beneficial bacterial strains and the Genotypic Specificity of Mutualism (GSM) in partners’ interactions is enhanced: the selective advantage of host-specific vs non-host-specific mutualists is increasing. The novelty of our model is to suggest a selective background for macroevolutionary events reorganizing the structure and functions of symbiotic systems and to present its evolution as a result of shifting the equilibrium between different types of mutualists under the impacts of the symbiosis-stipulated modes of natural selection.     

Effect of Inoculation with Various Rhizobia Strains on the Fatty Acid Composition of Peribacteroid and Bacteroid Membranes of the Nodule Symbiosomes

The fatty acid (FA) composition of bacteroid and peribacteroid membranes was studied in the symbiotic pairs differing in their nitrogen-fixing efficiency; the results are compared with the FA composition of plasmalemma and free-living rhizobia. The experiments involved lupine plants inoculated with strains of Bradyrhizobium lupini359a (Nod⁺Fix⁺) and 400 (Nod⁺Fix L) manifesting high and low nitrogen-fixing efficiency, respectively, and broad bean plants inoculated with strains of Rhizobium leguminosarum97 (Nod⁺Fix⁺) and 87 (Nod⁺Fix L) of high and low nitrogen-fixing efficiency, respectively. We showed that the rhizobia of the strains 359a and 97 were able to form nodules with peribacteroid membranes containing FA mainly or exclusively of plant origin. These strains were able to develop effective symbiotic pairs with legume plants. The use of strains 400 and 87 resulted in the formation of nodules with peribacteroid membranes containing typical bacterial (branched-chain) FAs; these strains were characterized by an ineffective symbiosis.     

Characterization of heat resistant mutant strains of Rhizobium sp. [Cajanus] for growth, survival and symbiotic properties

Fourteen heat resistant mutant strains were isolated from a wild-type strain (PP201, Nod⁺ Fix⁺) of Rhizobium sp. (Cajanus) by giving it a heat shock of 43°C. These mutant strains showed a greater increase in optical density (O.D.) and a higher viable cell count in both rhizospheric and non-rhizospheric soil at high temperature. Symbiotic studies showed that pigeon pea plants inoculated with a few mutant strains had ineffective nodules (Nod⁺ Fix⁻) under controlled temperature (43°C) conditions, but under natural high temperature (40–45°C) conditions, the host plants infected with all the mutant strains showed higher total shoot nitrogen than the plants inoculated with the parent strain. Four mutant strains (HR-3, HR-6, HR-10 and HR-12) were found to be highly efficient for all the symbiotic parameters, and thus have the potential to be used as bioinoculants in the North-Western regions of India during the summer season.     

Simulation of evolution implemented in the mutualistic symbioses towards enhancing their ecological efficiency,functional integrity and genotypic specificity

We created the mathematical model for the evolution of the Efficiency of Mutualistic Symbioses (EMS) which was estimated as the microsymbiont impacts on the host’s reproductive potential. Using the example of rhizobia–legume interaction, the relationships were studied between EMS and Functional Integrity of Symbiosis (FIS) which is represented as a measure for concordance of changes in the partners’ genotypic frequencies under the environmental fluctuations represented by the minor deviations of the systemic model parameters. The FIS indices correlate positively with EMS values suggesting an enhancement of FIS via the natural selection operating in the partners’ populations in favor of high EMS. Due to this selection, nodular habitats may be closed for colonization by the non-beneficial bacterial strains and the Genotypic Specificity of Mutualism (GSM) in partners’ interactions is enhanced: the selective advantage of host-specific vs non-host-specific mutualists is increasing. The novelty of our model is to suggest a selective background for macroevolutionary events reorganizing the structure and functions of symbiotic systems and to present its evolution as a result of shifting the equilibrium between different types of mutualists under the impacts of the symbiosis-stipulated modes of natural selection.     

The pea ( Pisum sativum L.) genes sym33 and sym40 control infection thread formation and root nodule function

Two novel non-allelic mutants that were unable to fix nitrogen (Fix⁻) were obtained after EMS (ethyl methyl sulfonate) mutagenesis of pea (Pisum sativum L.). Both mutants, SGEFix⁻–1 and SGEFix⁻–2, form two types of nodules: SGEFix⁻–1 forms numerous white and some pink nodules, while mutant SGEFix⁻–2 forms white nodules with a dark pit at the distal end and also some pinkish nodules. Both mutations are monogenic and recessive. In both lines the manifestation of the mutant phenotype is associated with the root genotype. White nodules of SGEFix⁻–1 are characterised by hypertrophied infection threads and infection droplets, mass endocytosis of bacteria, abnormal morphological differentiation of bacteroids, and premature degradation of nodule symbiotic structures. The structure of the pink nodules of SGEFix⁻–1 does not differ from that of the parental line, SGE. White nodules of SGEFix⁻–2 are characterised by “locked” infection threads surrounded with abnormally thick plant cell walls. In these nodules there is no endocytosis of bacteria into host-cell cytoplasm. The pinkish nodules of SGEFix⁻–2 are characterised by virtually undifferentiated bacteroids and premature degradation of nodule tissues. Thus, the novel pea symbiotic genes, sym40 and sym33, identified after complementation analysis in SGEFix⁻–1 and SGEFix⁻–2 lines, respectively, control early nodule developmental stages connected with infection thread formation and function. Received: 12 June 1998 / Accepted: 25 June 1998     

Loss‐of‐function of ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen‐fixing symbiosis with specific Mesorhizobium loti strains

The nitrogen‐fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix^– mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix^– mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix^– mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (LjAPN1), encodes a nepenthesin‐type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain‐specific Fix^– phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen‐fixing) symbiosis in a rhizobial strain‐dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.     

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.     

Localized Changes in Flavonoid Biosynthesis in Roots of Lotus pedunculatus after Infection by Rhizobium loti

Two-dimensional paper chromatography in four solvent systems, high-sensitivity spray reagents, and UV absorption spectroscopy were used to separate and characterize flavonoids and isoflavonoids in roots and root nodules of 20-d-old Lotus pedunculatus Cav. Seedlings were grown either under sterile conditions or after inoculation with Fix⁺ or Fix⁻ strains of Rhizobium loti. Flavonoids rather than isoflavonoids predominated in all tissues. Flavonoid profiles in sterile and denodulated root tissues were remarkably similar, both qualitatively and quantitatively. At least 14 partially purified flavonoid aglycones and conjugates were found in root extracts; denodulated root tissues contained no compounds that were not also present in sterile roots. Fix⁺ rhizobia were responsible for major postinfection shifts in plant flavonoid biosynthesis at the sites of nodule morphogenesis. Polymeric flavolans were absent from Fix⁺ nodules but present in all root tissues and in Fix⁻ nodules. Catechin was detected only in Fix⁺ nodules.     

The requirement for exopolysaccharide precedes the requirement for flavolan-binding polysaccharide in nodulation of Leucaena leucocephala by Rhizobium loti

Rhizobium loti strain PN4115 (NZP2213 str-1) ineffectively nodulates Leucaena leucocephala, i.e., strain PN4115 induces nodulation (Nod⁺) and is able to invade these nodules (Inv⁺), but fails to fix nitrogen (Fix^–). Strain PN4115 does not synthesize a flavolan-binding polysaccharide (FBP), which is synthesized by the fully effective (Nod⁺Inv⁺Fix⁺) R. loti strain PN184 (NZP2037 str-1). The FBP may offer protection from prodelphinidin-rich flavolans synthesized by Lc. leucocephala. In this work, we show that exopolysaccharide (EPS)-negative mutants derived from strain PN4115 have a more severe ineffective phenotype (Nod⁺Inv^–Fix^–) on Lc. leucocephala than strain PN4115. This suggests that EPS from strain PN4115 is functional during invasion of Lc. leucocephala and that the requirement for EPS precedes the requirement for FBP. Received: 8 October 1996 / Accepted: 11 December 1996     

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.     

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.     

Narrow- and Broad-Host-Range Symbiotic Plasmids of Rhizobium spp. Strains That Nodulate Phaseolus vulgaris

Agrobacterium transconjugants containing symbiotic plasmids from different Rhizobium spp. strains that nodulate Phaseolus vulgaris were obtained. All transconjugants conserved the parental nodulation host range. Symbiotic (Sym) plasmids of Rhizobium strains isolated originally from P. vulgaris nodules, which had a broad nodulation host range, and single-copy nitrogenase genes conferred a Fix⁺ phenotype to the Agrobacterium transconjugants. A Fix⁻ phenotype was obtained with Sym plasmids of strains isolated from P. vulgaris nodules that had a narrow host range and reiterated nif genes, as well as with Sym plasmids of strains isolated from other legumes that presented single nif genes and a broad nodulation host range. This indicates that different types of Sym plasmids can confer the ability to establish an effective symbiosis with P. vulgaris.     

N2-fixation ability of Brazilian soybean cultivars with Sinorhizobium fredii and Sinorhizobium xinjiangensis

The main N₂-fixing symbiotic associations with soybean (Glycine max (L.) Merrill) plants are realized with bacteria belonging to the species Bradyrhizobium japonicum and B. elkanii. However, in 1982, fast-growing rhizobia were isolated from soybean root nodules collected in The People's Republic of China and these bacteria are today classified as Sinorhizobium fredii and S. xinjiangensis. The fast growing strains formed an effective symbiosis with primitive soybean cultivars such as Peking, but not with most North American cultivars, which are the progenitors of almost all Brazilian cultivars. The main purpose of this study was to evaluate the ability of 80 soybean cultivars from the Brazilian germplasm bank to produce effective nodules when inoculated with S. fredii or S. xinjiangensis strains. Sixty-six percent of the Brazilian genotypes formed effective nodules with both Sinorhizobium species. However, when 20 Fix⁺ genotypes were inoculated with a mixture of B. elkanii and S. fredii, at a ratio of 1:1, most or all nodules were occupied by B. elkanii. Consequently, there was no relationship between the growth rate in vitro and the ability to compete for nodule occupancy. Fast-growing strains have also been isolated from soybean nodules in Brazil, but the ecological importance of these symbiotic associations is still to be determined.     

Anatomy, physiology and biochemistry of root nodules of Sprint-2 Fix-, a symbiotically defective mutant of pea (Pisum sativum L.)

A plant-determined pea mutant Sprint-2 Fix^– and the parentalline Sprint-2 were compared for selected physiological and biochemicalparameters. The Fix^– mutation prevented differentiationof Rhizobium leguminosarum bacteria into bacteroids and producedlarge, white, non-fixing nodules. These lacked nitrogenase-linkedrespiration, but had a background rate of CO₂ evolution similarto the normal Fix⁺ nodules. The cortical structure of the ineffectivenodules suggests the existence of an oxygen diffusion barrierand this was supported by a low oxygen concentration in thecentral region (0.5–3.0%), measured using an O₂ sensitivemicro-electrode. Sucrose and starch contents were similar innormal and ineffective nodules while ononitol content was about15 times lower in the Fix^– nodules. The distribution ofstarch was also different in the two nodule types. The activitiesof glutamine synthetase (GS), sucrose synthase (SS), phosphoenolpyruvatecarboxylase (PEPC) and alanine pyruvate aminotransferase (APAT)were markedly higher in Fix⁺ nodules while the activities ofpyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH) andglutamate dehydrogenase (GDH) were higher in Fix^– nodules.The data from immunodetection of host nodule proteins confirmedthe reduced levels of sucrose synthase and the almost completeabsence of glutamine synthetase and leghaemoglobin in mutantnodules. There was no significant difference in the amount ofnitrogenase component 1 extracted from the microsymbiont ofnormal and ineffective nodules, but component 2 was hardly detectablein the Fix^– mutant. Key words: Pisum sativum, Fix^– mutant, nodules     

Expression of host genes during root nodule development in soybeans

Summary Nine unique nodulin cDNA clones from soybean have been characterized with regard to the size of the RNA and the corresponding protein products. Based on the sequence homology between clones C51 and E27 and the multiple RNA species corresponding to clones D41 and E41, it is suggested that some of the nodulin genes represent members of small gene families. The amino acid sequence deduced from the nucleotide sequence of clones C51 and E27 revealed the presence of a signal peptide and no stop transfer signal, typical of membrane proteins, suggesting that the proteins encoded by these clones are localized in organelles and as such probably involved in ureide biosynthesis (Boland et al. 1982; Schubert and Boland 1984). Based on the timing of appearance of RNA corresponding to the nodulin clones and the pattern of their accumulation, at least three sets of nodulin genes are being represented here. Al1 the nodulin RNAs examined were made in Fix^- nodules formed by strain Ag168 (which does not make Cl component of nitrogenase) at a level comparable to that in Fix⁺ nodules and at a very reduced level in Fix^- nodules formed by strain HS124 (which show very few infected cells). It is concluded that all the nodulin genes examined here are induced independent of nitrogenase activity.     

Nodulation and effective nitrogen fixation of Macroptilium atropurpureum (siratro) by Burkholderia tuberum, a nodulating and plant growth promoting beta-proteobacterium, are influenced by environmental factors

Background and aims

Burkholderia tuberum STM678^T was isolated from a South African legume, but did not renodulate this plant. Until a reliable host is found, studies on this and other interesting beta-rhizobia cannot advance. We investigated B. tuberum STM678^T’s ability to induce Fix⁺ nodules on a small-seeded, easy-to-propagate legume (Macroptilium atropurpureum). Previous studies demonstrated that B. tuberum elicited either Fix^- or Fix⁺ nodules on siratro, but the reasons for this difference were unexplored.

Methods

Experiments to promote effective siratro nodule formation under different environmental conditions were performed. B. tuberum STM678^T’s ability to withstand high temperatures and desiccation was checked as well as its potential for promoting plant growth via mechanisms in addition to nitrogen fixation, e.g., phosphate solubilization and siderophore production. Potential genes for these activities were found in the sequenced genomes.

Results

Higher temperatures and reduced watering resulted in reliable, effective nodulation on siratro. Burkholderia spp. solubilize phosphate and produce siderophores. Genes encoding proteins potentially involved in these growth-promoting activities were detected and are described.

Conclusions

Siratro is an excellent model plant for B. tuberum STM678^T. We identified genes that might be involved in the ability of diazotrophic Burkholderia species to survive harsh conditions, solubilize phosphate, and produce siderophores.     

Rhizobium leguminosarum strains forming nitrogen-rich nodules inPisum sativum

The nodules which developed on the roots ofPisum sativum after inoculation withRhizobium leguminosarum bv.viciae strains showing a Hup⁺ symbiotic phenotype and/or a high relative efficiency of electron transfer to dinitrogen (RE) had a high nitrogen content of their tissue. In comparison with the nodules initiated by a strain possessing the Hup⁻ symbiotic phenotype or by an indigenous soilRhizobium population, the nitrogen-rich root nodules contained up to four times more nitrogen (9.2% of dry mass). In the nitrogen-rich nodules, total amino acid, and especially Ala, Lys and Phe contents were significantly increased. The nitrogen-rich nodule symbiotic phenotype (Nrn) was well expressed, irrespective of pea cultivars and host plant age. If a strain showing the Nrn phenotype was applied in double-strain inocula, a significant correlation was found between increasing rates of the strain and increasing percentage nitrogen content of nodule tissue in the nodulated plants.