Search

Rhizobium species strain NGR234 is the most promiscuous known rhizobium. In addition to the non-legume Parasponia andersonii, it modulates at least 70 genera of legumes. Here we show that the modulation genes of this bacterium determine the production of a large family of Nod-factors which are N-acylated chitin pentamers carrying a variety of substituents. The terminal non-reducing glucosamine is N-acylated with vaccenic or palmitic acids, is N-methylated, and carries varying numbers of carbamoyi groups. The reducing N-acetyl-glucosamine residue is substituted on position 6 with 2-O-methyl-L-fucose which may be acetylated or sulphated or non-substituted. All three internal residues are N-acetylated. At pico- to nano-molar concentrations, these signal molecules exhibit biological activities on the tropical legumes Macroptilium and Vigna (Phaseoleae), as well as on both the temperate genera Medicago (Trifotiae) and Vicia (Viciae). These data strongly suggest that the uniquely broad host range of NGR234 is mediated by the synthesis of a family of varied sulphated and non-sulphated lipo-oligosaccharide signals. [ABSTRACT FROM AUTHOR]

Transfer of a cosmid containing nodSU from Rhizobium sp. NGR234 to Rhizobium fredii USDA257 expands the host range for nodulation to include the perennial tropical legumes, Leucaena leucocephala and Leucaena diversifolia. Complementation experiments with a series of subclones established that nodS and its associated nod-box promoter from NGR234 are sufficient to confer this extended host-range phenotype to L. Ieucocephala. Strain USDA257 contains its own copy of nodSU, including upstream nod-box sequences. Although both nucleotide and deduced amino acid sequences of the reading frames are homologous between the two strains, there are gaps within the promoter region and the 5'-end of nodS of USDA257. Consequently, the deduced NodS protein of USDA257 is shorter than its counterpart from NGR234, and the distance between the nod-box and the initiation codon is greater. A 36 bp deletion encompasses the extreme right border of the USDA257 nod-box and extends into the upstream leader sequence, Transcriptional fusions with both nod-boxes confirmed that the promoter from NGR234 is flavonoid-inducible, and that the nod-box from USDA257 is not. These observations were corroborated by Northern analysis with a nodS-containing Xhoi fragment as hybridization probe. Flavonoid-induced cells of NGR234 gave an intense signal, but those of USDA257 yielded only a weak trace of hybridization. EcoRI fragments with homology to nodSU of USDA257 are present in 17 of 35 tested strains, including several representatives of Bradyrhizobium japonicum, Rhizobium sp., R. loti, and R. fredii. Two wild-type, leucaena-nodulating strains of Rhizobium sp. lack this homology. We conclude that a genetic defect in expression of nodS accounts for the inability of USDA257 to nodulate leucaena and that diverse rhizobia may have evolved alternative mechanisms to nodulate this legume species. [ABSTRACT FROM AUTHOR]

Haynaldia villosa is a wild grass belonging to the tribe Triticeae, which includes important crops such as wheat, barley, and rye. The alcohol-soluble proteins of H. villosa have extensive immunological relatedness with wheat prolamines as visualized by Western blot analysis. Amorphous protein inclusions surrounded by a limiting membrane are commonly found in the vacuoles of endosperm and subaleurone layers of H. villosa seeds. A layer of cells just beneath the aleurone layer is rich in ER. Unlike that in other cell types, the ER in these cells is highly dilated and contains materials at its swollen distal ends. These materials are structurally similar to substances found in the protein bodies. Protein A-gold immunocytochemical localization studies employing antibodies against wheat prolamine confirmed that the inclusions found in the lumen of the ER do not contain prolamines. This observation indicates that the ER does not act as the site of prolamine accumulation in H. villosa. Protein bodies found in the vacuoles and the vesicles associated with the Golgi complexes were specifically labeled. This suggests that Golgi complexes mediate the transport of prolamines into vacuoles of H. villosa endosperm cells, in a fashion analogous to that of other vacuolar proteins of dicotyledonous plants. [ABSTRACT FROM AUTHOR]

Water is a scarce resource in Central Asia, and many catchments span international boundaries, among them that of the Ili River, which is shared by China and Kazakhstan. Since 1970, the natural hydrological regime of the Ili River, both absolute flow rates and cycles, has changed due to construction of reservoirs such as that at Kapchagai, as well as natural climatic cycles and the growth of water consumption in the basin. Using data from Kazhydromet, we calculated that flow rates below Kapchagai dam averaged 468 m3/sec before construction of the dam, 366 m3/sec while the reservoir was being filled, and 489 m3/sec between 1988 and 2013. The dam has profoundly altered the annual cycle of flows in the river, with reductions in the summer and increases in the winter, when water is released to produce hydropower. The effects of these changes are being heightened by China’s increasing diversion of the river’s water. The sustainable use of decreasing water resources to conserve the biodiversity of the Ili-Balkhash basin’s ecosystems mandates a solution to the water allocation challenge between China and Kazakhstan. This will require a basin-wide approach that includes modernization of water distribution systems and careful consideration to relative priority needs for food, hydropower, and communal uses in both countries.

Public-sector scientists have a mandate to independently evaluate agricultural products available to American farmers on the open market, whereas the companies that sell the products must protect their intellectual property.  However, as a consequence of the latter concern, public scientists currently are prohibited by industry-imposed restrictions from conducting research on commercialized transgenic seed without permission of the company.  Industry acknowledged the seriousness of the problem after public warnings by a large group of entomologists to EPA and scientific advisory panels that the assumption of independence of public-sector studies on these products is no longer valid under current restrictions.  Both industry and public scientists are working to find an amicable, mutually-acceptable solution.  Recently, the American Seed Trade Association brokered a draft set of principles designed to protect the legitimate property rights of companies while allowing public scientists independence to conduct most types of research on their commercialized products without the need for case-by-case agreements.  While there are a number of potential pitfalls in implementation of the principles across companies, this effort represents a major step forward, and there is reason for optimism that this approach can be made to work to the benefit of industry, public scientists, and the American public.

Genetically, Rhizobium sp. strain NGR234 and R. fredii USDA257 are closely related. Small differences in their nodulation genes result in NGR234 secreting larger amounts of more diverse lipo-oligosaccharidic Nod factors than USDA257. What effects these differences have on nodulation were analyzed by inoculating 452 species of legumes, representing all three subfamilies of the Leguminosae, as well as the nonlegume Parasponia andersonii, with both strains. The two bacteria nodulated P. andersonii, induced ineffective outgrowths on Delonix regia, and nodulated Chamaecrista fasciculata, a member of the only nodulating genus of the Caesalpinieae tested. Both strains nodulated a range of mimosoid legumes, especially the Australian species of Acacia, and the tribe Ingeae. Highest compatibilities were found with the papilionoid tribes Phaseoleae and Desmodieae. On Vigna spp. (Phaseoleae), both bacteria formed more effective symbioses than rhizobia of the "cowpea" (V. unguiculata) miscellany. USDA257 nodulated an exact subset (79 genera) of the NGR234 hosts (112 genera). If only one of the bacteria formed effective, nitrogen-fixing nodules it was usually NGR234. The only exceptions were with Apios americana, Glycine max, and G. soja. Few correlations can be drawn between Nod-factor substituents and the ability to nodulate specific legumes. Relationships between the ability to nodulate and the origin of the host were not apparent. As both P. andersonii and NGR234 originate from Indonesia/Malaysia/Papua New Guinea, and NGR234's preferred hosts (Desmodiinae/Phaseoleae) are largely Asian, we suggest that broad host range originated in Southeast Asia and spread outward.

We mutagenized Sinorhizobium fredii HH103-1 with Tn5-B20 and screened about 2,000 colonies for increased beta-galactosidase activity in the presence of the flavonoid naringenin. One mutant, designated SVQ287, produces lipochitooligosaccharide Nod factors (LCOs) that differ from those of the parental strain. The nonreducing N-acetylglucosamine residues of all of the LCOs of mutant SVQ287 lack fucose and 2-O-methylfucose substituents. In addition, SVQ287 synthesizes an LCO with an unusually long, C20:1 fatty acyl side chain. The transposon insertion of mutant SVQ287 lies within a 1.1-kb HindIII fragment. This and an adjacent 2.4-kb HindIII fragment were sequenced. The sequence contains the 3' end of noeK, nodZ, and noeL (the gene interrupted by Tn5-B20), and the 5' end of nolK, all in the same orientation. Although each of these genes has a similarly oriented counterpart on the symbiosis plasmid of the broad-host-range Rhizobium sp. strain NGR234, there are significant differences in the noeK/nodZ intergenic region. Based on amino acid sequence homology, noeL encodes GDP-D-mannose dehydratase, an enzyme involved in the synthesis of GDP-L-fucose, and nolK encodes a NAD-dependent nucleotide sugar epimerase/dehydrogenase. We show that expression of the noeL gene is under the control of NodD1 in S. fredii and is most probably mediated by the nod box that precedes nodZ. Transposon insertion into neoL has two impacts on symbiosis with Williams soybean: nodulation rate is reduced slightly and competitiveness for nodulation is decreased significantly. Mutant SVQ287 retains its ability to form nitrogen-fixing nodules on other legumes, but final nodule number is attenuated on Cajanus cajan.

Lipopolysaccharides (LPS) and capsular polysaccharides (K antigens) may influence the interaction of rhizobia with their specific hosts; therefore, we conducted a comparative analysis of Sinorhizobium fredii and Sinorhizobium meliloti, which are genetically related, yet symbiotically distinct, nitrogen-fixing microsymbionts of legumes. We found that both species typically produce strain-specific K antigens that consist of 3-deoxy-D-manno-2-octulosonic acid (Kdo), or other 1-carboxy-2-keto-3-deoxy sugars (such as sialic acid), and hexoses. The K antigens of each strain are distinguished by glycosyl composition, anomeric configuration, acetylation, and molecular weight distribution. One consistent difference between the K antigens of S. fredii and those of S. meliloti is the presence of N-acetyl groups in the polysaccharides of the latter. In contrast to the K antigens, the LPS of Sinorhizobium spp. are major common antigens. Rough (R) LPS is the predominant form of LPS produced by cultured cells, and some strains release almost no detectable smooth (S) LPS upon extraction. Sinorhizobium spp. are delineated into two major RLPS core serogroups, which do not correspond to species (i.e., host range). The O antigens of the SLPS, when present, have similar degrees of polymerization and appear to be structurally conserved throughout the genus. Interestingly, one strain was found to be distinct from all others: S. fredii HH303 produces a unique K antigen, which contains galacturonic acid and rhamnose, and the RLPS did not fall into either of the RLPS core serogroups. The results of this study indicate that the conserved S- and RLPS of Sinorhizobium spp. lack the structural information necessary to influence host specificity, whereas the variable K antigens may affect strain-cultivar interactions.

Sinorhizobium fredii strain USDA208 is a nitrogen-fixing bacterium that forms nodules on roots of soybean and other legume plants. We previously found that the Tn5-containing mutant 208T3, which was derived from strain USDA208, is both deficient in production of exopolysaccharides and more competitive than the wild-type strain in competing against other rhizobia for nodulation of soybean. We now demonstrate that the transposon insertion of the mutant lies in a locus that is highly homologous to a portion of the exo region, which functions in exopolysaccharide biosynthesis by Sinorhizobium meliloti. We sequenced 2906 bp surrounding the insertion site and identified three genes: exoA, exoM, and exoO. The transposon lies within exoM, a glucosyl transferase. A cosmid containing exoHKLAMONP of S. meliloti restores exopolysaccharide production by mutant 208T3 to wild-type levels. Although exo mutants of S. meliloti are defective in their abilities to form indeterminate nodules, the capacities of mutant 208T3 and its wild-type parent to form such nodules on five legume species are indistinguishable. Thus the symbiotic function of exopolysaccharide in S. fredii appears to differ fundamentally from that in S. meliloti.

Flavonoid cues from the plant host cause symbiotic, nitrogen-fixing rhizobia to synthesize lipochitooligosaccharides (LCOs), which act as return signals to initiate the nodulation process. Rhizobium fredii strain USDA257 is known to produce four LCOs, all substituted with vaccenic acid (C18:1). We show here that a mutant strain can replace vaccenic acid with a C16:0 side chain, and that strain USDA191 synthesizes additional LCOs that differ from one another in the length and unsaturation of their fatty acyl substituents. USADA191 and 257DH4 both produce a novel LCO with glucose substituted for N-acetyl-D-glucosamine in the backbone of the molecule.

Rhizobium fredii strains USDA257 and USDA191 both contain a set of nodulation genes termed nolXWBTUV. In the USDA257 background, nolX prevents infection of soybean cultivars such as McCall, and in both backgrounds, it blocks nodulation of Erythrina spp. We report here that expression of nolX is differentially responsive to a panel of flavonoids, and that the most potent inducers are also the most active inducers of nodC, a conventional, nod box-associated gene. Cell-free protein extracts from uninduced and flavonoid-induced cells of strains USDA191 and USDA257 retard the electrophoretic mobility of DNA sequences that lie upstream of nolX. Binding is dependent both on nodD1 and nodD2, and it is abolished by the presence of a double-stranded, 23-bp oligonucleotide that lies within a 114-bp TaqI/SacII restriction fragment. This oligomer has significant sequence homology to A3, a putative negative regulatory element from R.leguminosarum bv. viciae. Deletion of the A3-homologous sequences elevates the basal and flavonoid-inducible expression of nolX by about 50%.

Soil bacteria of the genera Azorhizobium, Bradyrhizobium, and Rhizobium are collectively termed rhizobia. They share the ability to penetrate legume roots and elicit morphological responses that lead to the appearance of nodules. Bacteria within these symbiotic structures fix atmosphere nitrogen and thus are of immense ecological and agricultural significance. Although modern genetic analysis of rhizobia began less than 20 years ago, dozens of nodulation genes have now been identified, some in multiple species of rhizobia. These genetic advances have led to the discovery of a host surveillance system encoded by nodD and to the identification of Nod factor signals. These derivatives of oligochitin are synthesized by the protein products of nodABC, nodFE, NodPQ, and other nodulation genes; they provoke symbiotic responses on the part of the host and have generated immense interest in recent years. The symbiotic functions of other nodulation genes are nonetheless uncertain, and there remain significant gaps in our knowledge of several large groups of rhizobia with interesting biological properties. This review focuses on the nodulation genes of rhizobia, with particular emphasis on the concept of biological specificity of symbiosis with legume host plants.

Rhizobium fredii is a nitrogen-fixing bacterial symbiont of soybean and a number of other legume species. We have studied the transcriptional organization of a Sym plasmid locus that restricts the host range of R. fredii USDA257 at both the host species and cultivar level. The genes of this host-specificity locus, noIXWBTUV, are transcribed from three promoters. Two of these, which are upstream of noIW and noIBTUV, are oriented face to face and initiate transcription at sites that are 14 bp apart. The third lies upstream from noIX. The noIW promoter is constitutive, whereas the noIB and noIX promoters are inducible by flavonoid signals. We have attempted to express genes from this locus in Escherichia coli systems, both in vivo and in vitro. We detected the insert- and orientation-specific expression of two genes, noIX and noIW, but we were unable to obtain expression of noIBTUV. Antiserum raised against NoIT nevertheless detected an abundantly expressed polypeptide of the predicted size in protein extracts of USDA257. This observation, as well as RNA dot blot data from a series of mutants, indicates that noIBTUV is expressed as a single transcriptional unit in R. fredii. Immunological detection of NoIT, and of a second protein, NoIX, was strictly dependent on flavonoid induction. The NoIX protein was larger than the size predicted from the previously published nucleotide sequence, and this led to resequencing and revision of the open reading frame.

We have isolated and characterized the extracellular Nod factors of Rhizobium fredii USDA257, a nitrogen-fixing symbiont of soybean [Glycine max (L.) Merr.] and several other legume species. These signals are produced upon exposure to the isoflavone genistein and consist of a series of substituted, beta 1,4-linked tri-, tetra-, and pentamers of N-acetylglucosamine. N-Vaccenic acid replaces acetate on the nonreducing residue, and the reducing residue contains alpha-linked 2-O-methylfucose on carbon 6. Small amounts of a fucose-containing tetramer also were present. The Nod factors elicit root-hair deformations on soybean and two other plants at concentrations ranging from 10(-6) to 10(-12) M.

Agrobacterium tumefaciens Chry5, a wild-type strain originally isolated from chrysanthemum, is unusually tumorigenic, particularly on soybean. We have mapped the Chry5 Ti plasmid by genomic walking and restriction endonuclease analysis, and have located its virulence, T-DNA, plasmid incompatibility, and L,L-succinamopine utilization loci. Southern analysis has revealed that about 85% of the Chry5 Ti plasmid is highly homologous to another Ti plasmid, pTiBo542. Although all the functions that we have located on pTiChry5 are encoded by pTiBo542-homologous regions, the two Ti plasmids differ in their genetic organization. The overall patterns of restriction sites in the plasmids also differ, with the exception of an approximately 12 kb segment of the virulence region, where the BamHI sites appear to be conserved. Complementation analysis has shown that deletion of a DNA segment which flanks the oncogenic T-DNA results in severe attenuation of virulence. This region also contains a sequence that is repeated in the Chry5 genome outside the Ti plasmid, and that is widely distributed in the Rhizobiaceae.

A cDNA clone corresponding to a 19 kD salt-soluble globulin of rice (Oryza sativa L.) was isolated by screening a lambda gt11 expression library of endosperm mRNA with antibodies raised against the purified rice seed alpha-globulin. The cDNA contained a single large open reading frame encoding a putative globulin precursor of molecular weight of 21 kD. The polypeptide consists of 182 amino acids and is devoid of lysine residues. Computer analysis of the NH2-terminal sequence of the globulin precursor protein indicated the presence of an 18 amino acid signal peptide. Segments of the amino acid sequence of the rice globulin were homologous with sequences in high molecular weight glutelins of wheat. Antibodies specific for rice seed globulin also cross-reacted with seed proteins from wheat, rye and triticale.