Sinorhizobium meliloti nifA gene exerts a pleiotropic effect on nodulation through the enhanced plant defense response

Sinorhizobium meliloti nifA gene is required for the expression of a bunch of nif and fix genes. Here, we report its pleiotropic effects on the nodule formation. Compared with wild type strain, nifA mutant sig- nificantly reduced nodule suppression rate in split-root system. The plants inoculated with mutant strain produced lower amount of daidzein and less necrotic cells on their roots. In addition, the defense genes failed to be evoked by nifA mutant at the early nodulation stage. These findings indicated that host defense response was one of the mechanisms mediated by nifA gene to regulate nodule formation during symbiosis. Even though nifA mutant could increase the number of nodules in host plant, it synthesized lower Nod factors than wild type. This suggested that nifA gene mediated multiple and diverse instances in nodulation formation.     

The enoyl-ACP reductase gene, <Emphasis Type="Italic">fabI1</Emphasis>, of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> is involved in salt tolerance,swarming mobility and nodulation efficiency

Our previous work showed that an enoyl-ACP reductase gene fabI1 of Sinorhizobium meliloti was down-regulated in the nifA mutant nodule bacteria. To gain a better understanding of fabI1 gene, a single site insertion mutant was constructed in this study. The fabI1 mutant was retarded in cell growth, and its ability to grow on media with high concentration of NaCl was reduced. In addition, the mutant was completely defective in swarming phenotype. During symbiosis, the fabI1 mutant had delayed nodule formation...     

Screening of the interacting proteins with NifA in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7 by the yeast two-hybrid system

NifA in AzospiriUum brasilense plays a key role in regulating the synthesis and activity of nitrogenase in response to ammonia and oxygen available. In this work we used the yeast two-hybrid system to identify the proteins that interact with NifA. The nifA gene was fused to the yeast two-hybrid vector pGBD-C2, and three A. brasilense Sp7 genomic libraries for use in yeast two-hybrid studies were constructed. Screening of the libraries identified four clones encoding proteins that interact with NifA. The confirmation of the interactions of each gene product of the four clones and NifA were carried out by exchanging the vectors for nifA and the four clones and by mutageneses of the four clones with shift reading frame experiments in yeast two-hybrid studies. DNA sequence analyses showed that two clones encode proteins containing PAS domains that play an important role in signal transduction. One clone has high similarity with the fhuE gene of Escherichia coli, whose gene product is involved in iron uptake and transportation, and the other clone encodes an unknown protein.     

Regulation of nitrogen metabolism genes by nifA gene product in Klebsiella pneumoniae

The Klebsiella pneumoniae nifA gene product, which is known to activate expression of the nitrogen fixation (nif) structural genes, is shown here also to be able to substitute for the product of the gene glnG (ntrC) in the regulation of other nitrogen metabolism genes. An evolutionary relationship between the nifA and glnG genes is suggested.     

Identification of genes induced during Medicago sativa nodule development by using the cDNA-AFLP technique

Under limited nitrogen conditions, rhizobia are ableto induce the formation of nitrogen-fixing root nodules on their leguminous plant host. This organogenetic process is triggered by a complex exchange of molecu- lar signals between the host plant and bac…     

PAS domain of the deduced Org35 protein mediates the interaction with NifA

PAS domains are sensory input domains and pro- tein-protein interaction sites that have been identified recently in a family of sensory proteins from all king-doms of life[1,2]. A variety of environmental stimuli such as light, oxygen, redox potential, an…     

Upstream CRP-binding site is not essential for CRP-cAMP-mediated inhibition on the nifU promoter

When the NifA-mediated activation of Klebsiella pneumoniae nifU promoter is recreated in Escherichia coli, it has been observed that CRP-cAMP has an inhibitory effect on the nifU promoter. Sequence analysis indicates that there is a strong CRP-binding site located upstream of the nifU promoter, overlapping completely with a previously identified NifA-binding site. In vitro gel retardation analysis indicates that this putative CRP-binding site has similar affinity for CRP, when compared with that at the lac promoter, suggesting that CRP could effectively compete with NifA for such a binding site under physiological conditions. When this putative CRP-binding site on nifU was mutated, in vitro gel retardation analysis indicates that CRP can no longer bind to the mutant promoter. However, when constitutively expressed NifA is used as the activator, CRP-cAMP-mediated inhibitory effect on this mutant nifU promoter has no significant difference when compared with that obtained from its wild-type promoter. These results suggest that direct interaction between CRP and Eσ54, other than the DNA binding site(s) competition between CRP and NifA, plays the principal role in the CRP-cAMP-mediated inhibitory effect on nifU.     

Deregulation of a Ca2+/calmodulin-dependent kinase leads to spontaneous nodule development

Induced development of a new plant organ in response to rhizobia is the most prominent manifestation of legume root-nodule symbiosis with nitrogen-fixing bacteria. Here we show that the complex root-nodule organogenic programme can be genetically deregulated to trigger de novo nodule formation in the absence of rhizobia or exogenous rhizobial signals. In an ethylmethane sulphonate-induced snf1 (spontaneous nodule formation) mutant of Lotus japonicus, a single amino-acid replacement in a Ca2+/calmodulin-dependent protein kinase (CCaMK) is sufficient to turn fully differentiated root cortical cells into meristematic founder cells of root nodule primordia. These spontaneous nodules are genuine nodules with an ontogeny similar to that of rhizobial-induced root nodules, corroborating previous physiological studies. Using two receptor-deficient genetic backgrounds we provide evidence for a developmentally integrated spontaneous nodulation process that is independent of lipochitin-oligosaccharide signal perception and oscillations in Ca2+ second messenger levels. Our results reveal a key regulatory position of CCaMK upstream of all components required for cell-cycle activation, and a phenotypically divergent series of mutant alleles demonstrates positive and negative regulation of the process.     

Hg2+对银合欢和蚕豆根瘤菌生长影响的初步研究

用液体培养,通过测定培养液中的光密度、细菌数量和氨氮,测定了不同浓度Hg2 对蚕豆和银合欢两种根瘤菌的生长的影响,结果表明:两种根瘤菌对不同浓度的Hg2 表现出不同的耐受性,蚕豆根瘤菌可以耐受10ppm浓度的Hg2 ,而银合欢根瘤菌只能耐受4ppm以下的Hg2 ,但蚕豆根瘤菌的延滞期比银合欢根瘤菌相对要长.两种根瘤菌总的生长趋势是:Hg2 浓度越高,延滞期越长.蚕豆根瘤菌在10ppm以下的Hg2 浓度范围内,表现出较高的固氮活性,而银合欢根瘤菌在Hg2 浓度大于4ppm以后,固氮活性呈现较低的水平.     

Sinorhizobium meliloti lsrB is involved in alfalfa root nodule development and nitrogen-fixing bacteroid differentiation

Rhizobia interact with host legumes to induce the formation of nitrogen-fixing nodules, which is very important in agriculture and ecology. The development of nitrogen-fixing nodules is stringently regulated by host plants and rhizobial symbionts. In our previous work, a new Sinorhizobium meliloti LysR regulator gene (lsrB) was identified to be essential for alfalfa nodulation. However, how this gene is involved in alfalfa nodulation was not yet understood. Here, we found that this gene was associated with prevention of premature nodule senescence and abortive bacteroid formation. Heterogeneous deficient alfalfa root nodules were induced by the in-frame deletion mutant of lsrB (lsrB1-2), which was similar to the plasmid-insertion mutant, lsrB1. Irregular senescence zones earlier appeared in these nodules where bacteroid differentiation was blocked at different stages from microscopy observations. Interestingly, oxidative bursts were observed in these nodules by DAB staining. The decreased expression of lipopolysaccharide core genes (lpsCDE) was correspondingly determined in these nodules. S. meliloti lipopolysaccharide is required for suppression of oxidative bursts or host cell defense. These findings demonstrate that the S. meliloti lsrB gene is involved in alfalfa root nodule development and bacteroid differentiation by suppressing oxidative bursts or defense responses in host cells.     

Extra-copy nifA enhances the nodulation efficiency of Sinorhizobium fredii

Previous investigations have shown that nifA gene is involved in nodulation and symbiotic nitrogen fixation regulation of Rhizobium. We study the role of nifA on nodulation of leguminous plants. We found that Sinorhizobium fredii harboring multi-copy plasmid carrying the constitutively expressed Klebsiella pneumoniae nifA exhibited an increase of noduiation activity and nodulation competitiveness on soybean plants. The Nod-factor secreted by the rhizobia cells containing the multi-copied nifA was assayed,and preliminary results showed that S. fredii containing the multi-copy plasmid carrying nifA produced higher strength of Nod-factor than the rhizobia containing the same plasmid carrying the vector did.     

A plant receptor-like kinase required for both bacterial and fungal symbiosis

Most higher plant species can enter a root symbiosis with arbuscular mycorrhizal fungi, in which plant carbon is traded for fungal phosphate. This is an ancient symbiosis, which has been detected in fossils of early land plants. In contrast, the nitrogen-fixing root nodule symbioses of plants with bacteria evolved more recently, and are phylogenetically restricted to the rosid I clade of plants. Both symbioses rely on partially overlapping genetic programmes. We have identified the molecular basis for this convergence by cloning orthologous SYMRK ('symbiosis receptor-like kinase') genes from Lotus and pea, which are required for both fungal and bacterial recognition. SYMRK is predicted to have a signal peptide, an extracellular domain comprising leucine-rich repeats, a transmembrane and an intracellular protein kinase domain. Lotus SYMRK is required for a symbiotic signal transduction pathway leading from the perception of microbial signal molecules to rapid symbiosis-related gene activation. The perception of symbiotic fungi and bacteria is mediated by at least one common signalling component, which could have been recruited during the evolution of root nodule symbioses from the already existing arbuscular mycorrhiza symbiosis.     

Regulation of the Drosophila segmentation gene hunchback by two maternal morphogenetic centres

Segmentation in the inset embryo is initiated by maternally provided information, which is stored in the developing oocyte. In Drosophila, the genes necessary for this process have been genetically characterized. The anterior segmented region is organized by the bicoid (bcd) gene product. The posterior segmented region is organized by several interacting gene products, among them the oskar (osk) gene product. The first zygotic group of genes, which are thought to respond to the spatial cues provided by the maternal genes, are the gap genes, whose members include hunchback (hb), Krüppel (Kr) and knirps (kni). To elucidate the role played by the maternal genes in expression of the gap gene hb, antibodies were raised against a fusion protein and were used for the cytological localization of the hb gene product in wild-type and mutant embryos. The hb protein is predominantly located in the nucleus. Its spatial expression includes the formation of an anterior-posterior gradient during the early cleavage stages and a strong zygotic expression in the anterior half of the embryo. Analysis of embryos mutant for the maternal genes affecting the anterior-posterior segmentation pattern shows that the formation of the early gradient is controlled by the osk group of genes, whereas efficient activation of the zygotic anterior expression domain is dependent on bcd activity.     

Interaction between PⅡ and NifA in Azospirillum brasilense Sp7

The interaction between PⅡ and NifA in A.brasilense Sp7 was investigated by using the yeast two-hybrid system.Our experimental results showed that PⅡ directly interacted with the entire NifA protein and its N-terminal domain,but did not interact with the central domin and the C-terminal domain of NifA.No interaction happened if glnB coding for PⅡ was frame-shift mutated.Pz,a homolog of PⅡ,had no interation with NifA.