NodD binds to target DNA in isologous octamer

ＢａｃｔｅｒｉａｉｎｔｈｅｇｅｎｅｒａｏｆＲｈｉｚｏｂｉｕｍ,Ｂｒａｄｙｒｈｉｚｏｂｉｕｍ,ＡｚｏｒｈｉｚｏｂｉｕｍａｎｄＳｉｎｏｒｈｉｚｏｂｉｕｍｅｌｉｃｉｔｒｏｏｔｏｒｓｔｅｍｎｏｄｕｌｅｓｏｎｔｈｅｉｒｓｐｅｃｉｆｉｃｈｏｓｔｐｌａｎｔｓａｎｄｆｉｘａｔｍｏｓｐｈｅｒｉｃｎｉｔｒｏｇｅｎｔｈｅｒｅｉｎ.Ｎｏｄｕｌａｔｉｏｎｇｅｎｅｓ(ｎｏｄ,ｎｏｌａｎｄｎｏｅ)ｐｌａｙｉｍｐｏｒｔａｎｔｒｏｌｅｓｉｎｔｈｅｓｕｃｃｅｓｓｆｕｌｅｓｔａｂｌｉｓｈｍｅｎｔｏｆｔｈｅｓｙｍｂｉｏｓｉｓ,ｙｅｔｍｏｓｔ…     

在结瘤基因nodA启动子内发现了两个不同功能的结构区域

在豌豆根瘤菌（ＲｈｉｚｏｂｉｕｍＬｅｇｕｍｉｎｏｓａｒｕｍ）结瘤基因ｎｏｄＡ的启动子内发现了具有两个不同功能的结构区域,其一我们称为Ｒｉｐ,在ｎｏｄＡ诱导表达中起着关键作用,可能识别经诱导剂作用而发生构象变化的调控蛋白ＮｏｄＤ,另一为ＲＩＰ缺失后留下的,我们称为ＲＰ区,只要ＲＰ存在,不需要诱导剂,ＮｏｄＤ蛋白即能导致结瘤基因ｎｏｄＡ的表达。因此该区可能识别原始构象的调控蛋白ＮｏｄＤ。     

苜蓿中华根瘤菌042B nodD基因的克隆、序列分析及其表达

苜蓿中华根瘤菌０４２Ｂ是一株能在苜蓿和大豆上结瘤的菌株。将０４２Ｂ的ｎｏｄＳＤ基因克隆到时载体ｐＢＢＲ１ＭＣＳ－５,并在豌豆根瘤菌ＬＲＲ５０４５系统中进行功能分析,发现０４２Ｂ的ＮｏｄＤ蛋白能与大豆的类黄酮化合物ｇｅｎｉｓｔｅｉｎ结合,也怀苜蓿原类黄酮化合物ｌｕｔｅｏｌｉｎ反应。     

Rhizobium leguminosarum exoB mutants are deficient in the synthesis of UDP-glucose 4'-epimerase

Rhizobium leguminosarum bv. viciae Exo- mutant strains RBL5523,exo7::Tn5,RBL5523,exo8::Tn5 and RBL5523,exo52::Tn5 are affected in nodulation and in the syntheses of lipopolysaccharide, capsular polysaccharide, and exocellular polysaccharide. These mutants were complemented for nodulation and for the syntheses of these polysaccharides by plasmid pMP2603. The gene in which these mutants are defective is functionally homologous to the exoB gene of Rhizobium meliloti. The repeating unit of the residual amounts of EPS still made by the exoB mutants of R. leguminosarum bv. viciae lacks galactose and the substituents attached to it. The R. leguminosarum bv. viciae and R. meliloti exoB mutants fail to synthesize active UDP-glucose 4'-epimerase.     

Induction of microbial genes for pathogenesis and symbiosis by chemicals from root border cells.

Reporter strains of soil-borne bacteria were used to test the hypothesis that chemicals released by root border cells can influence the expression of bacterial genes required for the establishment of plant-microbe associations. Promoters from genes known to be activated by plant factors included virE, required for Agrobacterium tumefaciens pathogenesis, and common nod genes from Rhizobium leguminosarum bv viciae and Rhizobium meliloti, required for nodulation of pea (Pisum sativum) and alfalfa (Medicago sativum), respectively. Also included was phzB, an autoinducible gene encoding the biosynthesis of antibiotics by Pseudomonas aureofaciens. The virE and nod genes were activated to different degrees, depending on the source of border cells, whereas phzB activity remained unaffected. The homologous interaction between R. leguminosarum bv viciae and its host, pea, was examined in detail. Nod gene induction by border cells was dosage dependent and responsive to environmental signals. The highest levels of gene induction by pea (but not alfalfa) border cells occurred at low temperatures, when little or no bacterial growth was detected. Detached border cells cultured in distilled water exhibited increased nod gene induction (ini) in response to signals from R. leguminosarum bv viciae.     

Natural variation in host-specific nodulation of pea is associated with a haplotype of the SYM37 LysM-type receptor-like kinase

Rhizobium leguminosarum bv. viciae, which nodulates pea and vetch, makes a mixture of secreted nodulation signals (Nod factors) carrying either a C18:4 or a C18:1 N-linked acyl chain. Mutation of nodE blocks the formation of the C18:4 acyl chain, and nodE mutants, which produce only C18:1-containing Nod factors, are less efficient at nodulating pea. However, there is significant natural variation in the levels of nodulation of different pea cultivars by a nodE mutant of R. leguminosarum bv. viciae. Using recombinant inbred lines from two pea cultivars, one which nodulated relatively well and one very poorly by the nodE mutant, we mapped the nodE-dependent nodulation phenotype to a locus on pea linkage group I. This was close to Sym37 and PsK1, predicted to encode LysM-domain Nod-factor receptor-like proteins; the Sym2 locus that confers Nod-factor-specific nodulation is also in this region. We confirmed the map location using an introgression line carrying this region. Our data indicate that the nodE-dependent nodulation is not determined by the Sym2 locus. We identified several pea lines that are nodulated very poorly by the R. leguminosarum bv. viciae nodE mutant, sequenced the DNA of the predicted LysM-receptor domains of Sym37 and PsK1, and compared the sequences with those derived from pea cultivars that were relatively well nodulated by the nodE mutant. This revealed that one haplotype (encoding six conserved polymorphisms) of Sym37 is associated with very poor nodulation by the nodE mutant. There was no such correlation with polymorphisms at the PsK1 locus. We conclude that the natural variation in nodE-dependent nodulation in pea is most probably determined by the Sym37 haplotype.     

Distribution of O-acetyl groups in the exopolysaccharide synthesized by Rhizobium leguminosarum strains is not determined by the Sym plasmid

The patterns of O-acetylation of the exopolysaccharide (EPS) from the Sym plasmid-cured derivatives of Rhizobium leguminosarum bv. trifolii strain LPR5, R. leguminosarum bv. trifolii strain ANU843 and R. leguminosarum bv. viciae strain 248 were determined by 1H and 13C NMR spectroscopy. Beside a site indicative of the chromosomal background, these strains have one site of O-acetylation in common, namely residue b of the repeating unit. The O-acetyl esterification pattern of EPS of the Sym plasmid-cured derivatives of strains LPR5, ANU843, and 248 was not altered by the introduction of a R. leguminosarum bv. viciae Sym plasmid or a R. leguminosarum bv. trifolii Sym plasmid. The induction of nod gene expression by growth of the bacteria in the presence of Vicia sativa plants or by the presence of the flavonoid naringenin, produced no significant changes in either amount or sites of O-acetyl substitution. Furthermore, no such changes were found in the EPS from a Rhizobium strain in which the nod genes are constitutively expressed. The substitution pattern of the exopolysaccharide from R. leguminosarum is, therefore, determined by the bacterial genome and is not influenced by genes present on the Sym plasmid. This conclusion is inconsistent with the suggestion of Philip-Hollingsworth et al. (Philip-Hollingsworth, S., Hollingsworth, R. I., Dazzo, F. B., Djordjevic, M. A., and Rolfe, B. G. (1989) J. Biol. Chem. 264, 5710-5714) that nod genes of R. leguminosarum bv. trifolii, by influencing the acetylation pattern of EPS, determine the host specificity of nodulation.     

Rhizobium leguminosarum bv. viciae contains a second fnr/fixK-like gene and an unusual fixL homologue

Genes of Rhizobium leguminosarum bv. viciae VF39 coding for the regulatory elements NifA, FixL and FixK were isolated, sequenced and genetically analysed. The fixK–fixL region is located upstream of the fixNOQP operon on the non-nodulation plasmid pRleVF39c. The deduced amino acid sequence of FixL revealed an unusual structure in that it contains a receiver module (homologous to the N-terminal domain of response regulators) fused to its transmitter domain. An oxygen-sensing haem-binding domain, found in other FixL proteins, is conserved in R. leguminosarum bv. viciae FixL. R. leguminosarum bv. viciae possesses a second fnr -like gene, designated fixK , whose encoded gene product is very similar to Rhizobium meliloti and Azorhizobium caulinodans FixK. Individual R. leguminosarum bv. viciae fixK and fixL insertion mutants displayed a Fix⁺ phenotype. A reduced nitrogen-fixation activity was found for a R. leguminosarum bv. viciae fnrN -deletion mutant, whereas no nitrogen-fixation activity was detectable for a fixK / fnrN double mutant. The R. leguminosarum bv. viciae nifA gene is expressed independently of FixL and FixK under aerobic and microaerobic conditions, whereas fixL gene expression is induced under microaerobiosis. Another orf was identified downstream of fixK–fixL and encodes a product which has homology to pseudoazurins from different species. Mutation of this azu gene showed that it is dispensable for nitrogen fixation.     

Molecular characterization of the nodulation gene, nodT, from two biovars of Rhizobium leguminosarum

DNA sequencing of the nodIJ region from Rhizobium leguminosarum biovar trifolii revealed the nodT gene immediately downstream of nodJ. DNA hybridizations using a nodT-specific probe showed that nodT is present in several R. leguminosarum strains. Interestingly, a flavonoid-inducible nodT gene homologue in R. leguminosarum bv. viciae is not in the nodABCIJ operon but is located downstream of nodMN. The sequence of the nodT gene from bv. viciae was determined and a comparison of the predicted amino-acid sequences of the two nodT genes shows them to be conserved; the predicted protein sequences appear to have a potential transit sequence typical of outer-membrane proteins. Mutations affecting nodT in either biovar had no observed effect on nodulation of the legumes tested.     

Sugar-binding activity of pea lectin enhances heterologous infection of transgenic alfalfa plants by Rhizobium leguminosarum biovar viciae

Transgenic alfalfa (Medicago sativa L. cv Regen) roots carrying genes encoding soybean lectin or pea (Pisum sativum) seed lectin (PSL) were inoculated with Bradyrhizobium japonicum or Rhizobium leguminosarum bv viciae, respectively, and their responses were compared with those of comparably inoculated control plants. We found that nodule-like structures formed on alfalfa roots only when the rhizobial strains produced Nod factor from the alfalfa-nodulating strain, Sinorhizobium meliloti. Uninfected nodule-like structures developed on the soybean lectin-transgenic plant roots at very low inoculum concentrations, but bona fide infection threads were not detected even when B. japonicum produced the appropriate S. meliloti Nod factor. In contrast, the PSL-transgenic plants were not only well nodulated but also exhibited infection thread formation in response to R. leguminosarum bv viciae, but only when the bacteria expressed the complete set of S. meliloti nod genes. A few nodules from the PSL-transgenic plant roots were even found to be colonized by R. leguminosarum bv viciae expressing S. meliloti nod genes, but the plants were yellow and senescent, indicating that nitrogen fixation did not take place. Exopolysaccharide appears to be absolutely required for both nodule development and infection thread formation because neither occurred in PSL-transgenic plant roots following inoculation with an Exo(-) R. leguminosarum bv viciae strain that produced S. meliloti Nod factor.     

A cultivar-specific interaction between Rhizobium leguminosarum bv. trifolii and subterranean clover is controlled by nodM, other bacterial cultivar specificity genes, and a single recessive host gene.

Insertion mutagenesis identified two negatively acting gene loci which restrict the ability of Rhizobium leguminosarum bv. trifolii TA1 to infect the homologous host Trifolium subterraneum cv. Woogenellup. One locus was confirmed by DNA sequence analysis as the nodM gene, while the other locus, designated csn-1 (cultivar-specific nodulation), is not located on the symbiosis plasmid. The presence of these cultivar specificity loci could be suppressed by the introduction of the nodT gene from ANU843, a related R. leguminosarum bv. trifolii strain. Other nod genes, present in R. leguminosarum bv. viciae (including nodX) and R. meliloti, were capable of complementing R. leguminosarum bv. trifolii TA1 for nodulation on cultivar Woogenellup. Nodulation studies conducted with F2 seedlings from a cross between cultivar Geraldton and cultivar Woogenellup indicated that a single recessive gene, designated rwt1, is responsible for the Nod- association between strain TA1 and cultivar Woogenellup. Parallels can be drawn between this association and gene-for-gene systems common in interactions between plants and biotrophic pathogens.