Regulation of signal transduction and bacterial infection during root nodule symbiosis

Among plant-microbe interactions, root nodule symbiosis is one of the most important beneficial interactions providing legume plants with nitrogenous compounds. Over the past years a number of genes required for root nodule symbiosis has been identified but most recently great advances have been made to dissect signalling pathways and molecular interactions triggered by a set of receptor-like kinases. Genetic and biochemical approaches have not only provided evidence for the cross talk between bacterial infection of the host plant and organogenesis of a root nodule but also gained insights into dynamic regulation processes underlying successful infection events. Here, we summarise recent progress in the understanding of molecular mechanisms that regulate and trigger cellular signalling cascades during this mutualistic interaction.     

Immunodetection and intracellular localization of caldesmon-like proteins in Amoeba proteus

Summary. Caldesmon immunoanalogues were detected in Amoeba proteus cell homogenates by the Western blot technique. Three immunoreactive bands were recognized by polyclonal antibodies against the whole molecule of chicken gizzard caldesmon as well as by a monoclonal antibody against its C-terminal domain: one major and two minor bands corresponding to proteins with apparent molecular masses of 150, 69, and 60 kDa. The presence of caldesmon-like protein(s) in amoebae was revealed as well in single cells after their fixation, staining with the same antibodies, and recording their total fluorescence in a confocal laser scanning microscope. Proteins recognized by the antibodies bind to filamentous actin. This was established by a cosedimentation assay in cell homogenates and by colocalization of the caldesmon-related immunofluorescence with the fluorescence of filamentous actin stained with rhodamine-labelled phalloidin, demonstrated in optical sections of single cells in a confocal microscope. Caldesmon is colocalized with filamentous actin in the withdrawn cell regions where the cortical actomyosin network contracts and actin is depolymerized, in the frontal zone where actin is polymerized again and the cortical cytoskeleton is reconstructed, inside the nucleus and in the perinuclear cytoskeleton, and probably at the cell-to-substratum adhesion sites. The regulatory role of caldesmon in these functionally different regions of locomoting amoebae is discussed.Correspondence and reprints: Department of Cell Biology, Nencki Institute of Experimental Biology, ulica Pasteura 3, 02-093 Warsaw, Poland.Received October 7, 2002; accepted December 2, 2002; published online August 26, 2003     

Recruitment of novel calcium-binding proteins for root nodule symbiosis in Medicago truncatula

Legume rhizobia symbiotic nitrogen (N2) fixation plays a critical role in sustainable nitrogen management in agriculture and in the Earth's nitrogen cycle. Signaling between rhizobia and legumes initiates development of a unique plant organ, the root nodule, where bacteria undergo endocytosis and become surrounded by a plant membrane to form a symbiosome. Between this membrane and the encased bacteria exists a matrix-filled space (the symbiosome space) that is thought to contain a mixture of plant- and bacteria-derived proteins. Maintenance of the symbiosis state requires continuous communication between the plant and bacterial partners. Here, we show in the model legume Medicago truncatula that a novel family of six calmodulin-like proteins (CaMLs), expressed specifically in root nodules, are localized within the symbiosome space. All six nodule-specific CaML genes are clustered in the M. truncatula genome, along with two other nodule-specific genes, nodulin-22 and nodulin-25. Sequence comparisons and phylogenetic analysis suggest that an unequal recombination event occurred between nodulin-25 and a nearby calmodulin, which gave rise to the first CaML, and the gene family evolved by tandem duplication and divergence. The data provide striking evidence for the recruitment of a ubiquitous Ca(2+)-binding gene for symbiotic purposes.     

Productivity of soybean-rhizobium symbiosis after modification of root nodule bacteria activity with exogenous proteins

Plant growth experiments were conducted to assess symbiotic efficiency, photosynthetic rates, and the development of soybean (Glycine max (L.) Merrill) seedlings after seed inoculation with active and inactive strains of root nodule bacteria Bradyrhizobium japonicum preincubated in the presence homologous and heterologous proteins. The properties of active and inactive symbiotic strains were differentially modulated by homologous soybean lectin, which had a marked influence on plant physiological condition. The incubation of active rhizobia with a homologous lectin, i.e., lectin of the respective plant, increased the nitrogen-fixing activity of nodules and, consequently, elevated photosynthetic rates and weight increments in soybean plants. At the same time, the homologous lectin suppressed the symbiotic properties of inactive strain of nodule bacteria. The preincubation of rhizobia with a heterologous pea lectin had virtually no effect on functioning of symbiotic apparatus and photosynthetic rate, whereas the preincubation of root nodule bacteria with human albumin exerted an effect similar to that induced by a homologous lectin on symbiotic productivity.     

Involvement of Arachis hypogaea Jasmonate ZIM domain/TIFY proteins in root nodule symbiosis

Journal of Plant Research - Jasmonate ZIM domain (JAZ) proteins are the key negative regulators of jasmonate signaling, an important integrator of plant–microbe relationships. Versatility of...     

Lysosomal membrane proteins of Amoeba proteus, as studied with monoclonal antibodies.

Monoclonal antibodies were prepared against lysosomal membrane proteins of amoebae and used to follow lysosome-phagosome fusion after induced phagocytosis. The specificity of antibodies was checked by indirect immunofluorescence microscopy, immunoelectron microscopy, and localization of the antigen in subcellular fractions. The antibody-recognized proteins started to appear on the membranes of phagolysosomes about 5 min after phagocytosis as detected by indirect immunofluorescence, and the intensity of fluorescence increased for up to 1 h. Results of injection experiments in which purified antibodies had been injected into living cells and probed by indirect fluorescence indicated that the antigens were located on the cytoplasmic side of the lysosomal membranes. Lysosomes fuse with phagosomes on the one hand but not with non-fusible vesicles such as symbiosomes on the other. The results support the view that a membrane component(s) of non-fusible vesicles somehow prevents lysosomes from fusing with them.     

Identification of the small nuclear RNAs associated with the mitotic chromosomes of Amoeba proteus

Amebas contain 7 electrophoretically distinct species of small nuclear RNAs (snRNAs), some of which are known to associate in a striking manner with mitotic chromosomes. These RNAs can be divided into 2 classes, one consisting of 4 snRNA species that shuttle in a non-random way between nucleus and cytoplasm during interphase and one consisting of 3 snRNA species that do not leave the nucleus at all during interphase. In the work reported here we sought to determine which class is associated with mitotic chromosomes. Through a series of micromanipulative procedures we arranged for the shuttling snRNAs to be the only radioactive molecules in the cell. Such cells were allowed to enter mitosis, whereupon they were fixed and subjected to autoradiography. In those cells no radioactive snRNAs were found associated with mitotic chromosomes. It is concluded, therefore, that those snRNAs that do associate with mitotic chromosomes must be one or more of the non-shuttling species. — In the Discussion, how the non-shuttling snRNAs may function in cell activities is considered.     

Regulation of the soybean-Rhizobium nodule symbiosis by shoot and root factors

The availability of soybean mutants with altered symbiotic properties allowed an investigation of the shoot or root control of the relevant phenotype. By means of grafts between these mutants and wild-type plants (cultivar Bragg and Williams), we demonstrated that supernodulation as well as hypernodulation (nitrate tolerance in nodulation and lack of autoregulation) is shoot controlled in two mutants (nts382 and nts1116) belonging most likely to two separate complementation groups. The supernodulation phenotype was expressed on roots of the parent cultivar Bragg as well as the roots of cultivar Williams. Likewise it was shown that non-nodulation (resistance to Bradyrhizobium) is root controlled in mutant nod49. The shoot control of nodule initiation is epistatically suppressed by the non-nodulation, root-expressed mutation. These findings suggest that different plant organs can influence the expression of the nodulation phenotype.     

Isolation and characterization of some of the proteins that shuttle between cytoplasm and nucleus in Amoeba proteus

The Rapidly Migrating Proteins (RMP) which shuttle nonrandomly between nucleus and cytoplasm and equilibrate in approximately equal amounts in each compartment, were isolated from Amoeba proteus by implanting ³H-protein containing nuclei into unlabeled cells and some time later extracting the labeled material from the cytoplasms of such cells. The labeled material was subsequently fractionated by gel filtration in Sephadex G-100 columns. The RMP are soluble in dilute salt solutions and appear as a heterogenous group of molecules, one component of which seems to be a single species of protein accounting for ca. one-third of the RMP fraction. Because of its distinctness this component, called the LR fraction, received the major attention in this study. LR was found to comprise ca. 17% of the aqueous-soluble proteins of the nucleus and ca. 3–4% of the total cell protein. LR has a very low molecular weight as determined, e.g., by its elution from a Sephadex G-100 column. Because of its low molecular weight, LR could be purified by taking advantage of the fact that LR is (1) soluble in a saturated Solution of ammonium sulfate and (2) insoluble in butanol, diethyl ether, and 10% trichloroacetic acid. LR migrates toward the anode as a single band when subjected to electrophoresis on “standard disc” and SDS polyacrylamide gels. It does not enter a gel designed to separate basic proteins (at pH 4.0). When subjected to Sephadex G-25 gel filtration LR migrates through the gel as a single band and elutes from the gel at a position in the middle of the linear separation range that indicates its molecular weight is ca. 2300. The only N-terminal amino acid found in the LR fraction is proline. Evidence is presented to show that LR is not the product of a non-specific breakdown of protein produced during its isolation, but the possibility that it results from the cleavage of a single chemical bond of a larger polypeptide, has not been eliminated. When injected into non-labeled amebae, purified radioactive LR concentrates in the nucleus — just as radioactive RMP concentrates in a recipient cell nucleus when an amino acid-labeled nucleus is implanted into an unlabeled cell.     

Preservation and phallotoxin-staining of the microfilament system in Amoeba proteus

The spatial organization of the microfilament system as the main component of the cytoskeleton in Amoeba proteus was preserved by a glutaraldehyde-lysine-fixation and visualized with fluorescent phallotoxins (NBD- phallacidin , R-phalloidin). Results obtained by means of this method coincide exactly with observations gained from immunocytochemical, ultrastructural and molecular cytochemical studies, i.e., the microfilament system is mainly displayed beneath the cell membrane, at the hyalo - granuloplasmic border and around the cell nucleus. The preparation procedure employed is suitable for the rapid demonstration of cytoplasmic microfilaments in cells difficult to preserve by chemical fixation.     

Mechanics and control of the cytoskeleton in Amoeba proteus.

Many models of the cytoskeletal motility of Amoeba proteus can be formulated in terms of the theory of reactive interpenetrating flow (Dembo and Harlow, 1986). We have devised numerical methodology for testing such models against the phenomenon of steady axisymmetric fountain flow. The simplest workable scheme revealed by such tests (the minimal model) is the main preoccupation of this study. All parameters of the minimal model are determined from available data. Using these parameters the model quantitatively accounts for the self assembly of the cytoskeleton of A. proteus: for the formation and detailed morphology of the endoplasmic channel, the ectoplasmic tube, the uropod, the plasma gel sheet, and the hyaline cap. The model accounts for the kinematics of the cytoskeleton: the detailed velocity field of the forward flow of the endoplasm, the contraction of the ectoplasmic tube, and the inversion of the flow in the fountain zone. The model also gives a satisfactory account of measurements of pressure gradients, measurements of heat dissipation, and measurements of the output of useful work by amoeba. Finally, the model suggests a very promising (but still hypothetical) continuum formulation of the free boundary problem of amoeboid motion. by balancing normal forces on the plasma membrane as closely as possible, the minimal model is able to predict the turgor pressure and surface tension of A. proteus. Several dynamical factors are crucial to the success of the minimal model and are likely to be general features of cytoskeletal mechanics and control in amoeboid cells. These are: a constitutive law for the viscosity of the contractile network that includes an automatic process of gelation as the network density gets large; a very vigorous cycle of network polymerization and depolymerization (in the case of A. proteus, the time constant for this reaction is approximately 12 s); control of network contractility by a diffusible factor (probably calcium ion); and control of the adhesive interaction between the cytoskeleton and the inner surface of the plasma membrane.     

Purification and characterization of soybean root nodule ferric leghemoglobin reductase

A ferric leghemoglobin reductase from the cytosol of soybean (Glycine max) root nodules was purified to homogeneity and partially characterized. The enzyme is a flavoprotein with flavin adenine dinucleotide as the prosthetic group and consists of two identical subunits, each having a molecular mass of 54 kilodaltons. The pure enzyme shows a high activity for ferric leghemoglobin reduction with NADH as the reductant in the absence of any exogenous mediators. The enzyme also exhibits NADH-dependent 2,6-dichloroindophenol reductase activity. A sequence of the first 50 N-terminal amino acids of the purified protein was obtained. Comparisons with known protein sequences have shown that the sequence of the ferric leghemoglobin reductase is highly related to those of the flavin-nucleotide disulfide oxido-reductases, especially dihydrolipoamide dehydrogenase of the pyruvate dehydrogenase complex.     

Identification with proteomics of novel proteins associated with the peribacteroid membrane of soybean root nodules

Soybean peribacteroid membrane (PBM) proteins were isolated from nitrogen-fixing root nodules and subjected to N-terminal sequencing. Sequence data from 17 putative PBM proteins were obtained. Six of these proteins are homologous to proteins of known function. These include three chaperones (HSP60, BiP [HSP70], and PDI) and two proteases (a serine and a thiol protease), all of which are involved in some aspect of protein processing in plants. The PBM homologs of these proteins may play roles in protein translocation, folding, maturation, or degradation in symbiosomes. Two proteins are homologous to known, nodule-specific proteins from soybean, nodulin 53b and nodulin 26B. Although the function of these nodulins is unknown, nodulin 53b has independently been shown to be associated with the PBM. All of the eight proteins with identifiable homologs are likely to be peripheral rather than integral membrane proteins. Possible reasons for this apparent bias are discussed. The identification of homologs of HSP70 and HSP60 associated with the PBM is the first evidence that the molecular machinery for co- or post-translational import of cytoplasmic proteins is present in symbiosomes. This has important implications for the biogenesis of this unique, nitrogen-fixing organelle.     

ATP and the autonomy of the contractile vacuole in Amoeba proteus

Contractile vacuole function in amoebae treated with immobilizing (5 mM) and nonimmobilizing (0.125 mM) concentrations of ATP has been studied. In ATP-immobilized amoebae, most vacuolar parameters are accelerated, especially the rate of output which passes from 30 to 70 micron3/sec. This favors the concept of an autonomous vacuole, fully functional in the absence of any bulk contribution to it from remote points of the cell. A lower concentration of ATP (0.125 mM), which does not inhibit movement, causes a still greater acceleration of vacuolar function. Work is in progress to elucidate the site and mode of action of exogenous ATP on Amoeba.