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PubMed1990

A short domain of the plant vacuolar protein phytohemagglutinin targets invertase to the yeast vacuole.

Phytohemagglutinin (PHA), the seed lectin of the common bean, accumulates in protein storage vacuoles of storage parenchyma cells in cotyledons. When expressed in yeast, PHA is efficiently targeted to the yeast vacuole [Tague and Chrispeels (1987). J. Cell Biol. 105, 1971-1979]. To identify vacuolar sorting information in PHA, a series of 3' deletions of the PHA gene were fused in-frame to a truncated yeast invertase gene. An amino-terminal portion of PHA composed of a 20-residue signal sequence and 43 residues of the mature protein efficiently targeted invertase to the yeast vacuole. Internal deletions in a short PHA-invertase fusion showed that targeting information exists between residues 14 and 23 of mature PHA. Based on examinations of three-dimensional structures of related lectins, only a portion of these residues would be available on the surface of PHA for interaction with a putative receptor. Amino acid replacements at these positions in a PHA-invertase hybrid caused secretion of the invertase. The results indicate the presence of a vacuolar targeting domain in PHA that is centered at position 19 of the mature protein. This sequence of PHA also shows sequence identity to a vacuolar sorting domain characterized in yeast carboxypeptidase Y. Single amino acid alterations in a short PHA-invertase hybrid protein that caused the highest levels of secretion introduced a glycosylation site at position 21 of PHA. This observation suggests that glycan addition may interfere with recognition of a sorting determinant. These same amino acid changes did not dramatically increase secretion in a long PHA-invertase fusion or in PHA itself. Thus, a second domain of PHA may function in concert with the first one to bring about correct targeting of PHA.

Amino Acid Sequence↗
PubMed1993

Higher plant mitochondria encode an homologue of the nuclear-encoded 30-kDa subunit of bovine mitochondrial complex I.

We describe the structure and expression of a wheat mitochondrial gene, which codes for a subunit of mitochondrial NADH dehydrogenase. The deduced protein sequence has 70% similarity to the 30-kDa subunit of bovine mitochondrial complex I and 65% similarity to the 31-kDa subunit of Neurospora crassa complex I, components of the iron-sulfur-protein fraction, both nuclear-encoded proteins. We named this wheat mitochondrial gene as nad9. The wheat nad9 gene is transcribed in a single mRNA of 0.9 kb that is edited (C-to-U conversions) in 14 positions. Transcript mapping revealed that the first ATG codon is just 20 nucleotides downstream of the mRNA 5' end and that the 3' end is just 23 nucleotides downstream of the nad9 stop codon. The expression of the nad9 gene in plant mitochondria was studied. Polyclonal antibodies prepared against a wheat NAD9 fusion protein specifically recognise the 30-kDa subunit of bovine mitochondrial complex I and a 27.5-kDa protein in the membrane fractions of wheat, maize and common bean mitochondria, whereas the same serum recognizes a 30-kDa protein in the mitochondria of pea, chickpea and lentil.

Amino Acid Sequence↗
PubMed1996

Effect of phosphorus deficiency on growth angle of basal roots in Phaseolus vulgaris.

Root architectural plasticity might be an important factor in the acquisition by plants of immobile nutrients such as phosphorus (P). In this study, we examined the effect of P availability on the orientation of basal roots with respect to gravity, and thereby on the growth angle of these roots of common bean (Phaseolus vulgaris L.). In one set of studies the growth angle of basal roots of bean seedlings was measured over time. Sixteen bean genotypes were examined; six showed a decrease in root orientation with respect to gravity in low P media, one increased orientation, and nine showed no difference within 5 d of basal root emergence. Bean taproots also showed decreased root orientation with respect to gravity in low P. Growth angle after 5 d was correlated with field performance of contrasting genotypes in low P tropical soils. Mineral deficiencies other than P did not cause changes in root angle. In a split pouch system that provided high or low P solution to different parts of the root system, the decrease in root angle in low P was found to be a response to global P availability, and not local to the portion of the root system in low P. Effects of P availability on root angle were associated with reduced shoot P concentration, but preceded effects on plant biomass accumulation and leaf area expansion. Results from growth pouches for genotype G 19833 were confirmed using a solid-phase buffered sand-culture system supplying P at three levels. Pea (Pisum sativum), soybean (Glycine max Williams), chickpea (Cicer arietinum), lima bean (Phaseolus lunatus), and lentil (Lens culinaris) were grown with and without P; soybean and pea also showed decreased basal root angles in low P.

Biomass↗
PubMed2005

The rhizosphere signal molecule lumichrome alters seedling development in both legumes and cereals.

The stimulatory role of lumichrome, a rhizosphere metabolite, was assessed on the growth of legume and cereal seedlings. At a very low nanomolar concentration (5 nm), lumichrome elicited growth promotion in cowpea, soybean, sorghum, millet and maize, but not in common bean, Bambara groundnut and Sudan grass. In soybean and cowpea only, 5 nm lumichrome caused early initiation of trifoliate leaf development, expansion in unifoliate and trifoliate leaves, increased stem elongation and, as a result, an increase in shoot and plant total biomass relative to control. Lumichrome (5 nm) also increased leaf area in maize and sorghum, and thus raised shoot and total biomass but there was no effect on the leaf area of the other cereals. Root growth was also stimulated in sorghum and millet by the supply of 5 nm lumichrome. By contrast, the application of a higher dose of lumichrome (50 nm) depressed development of unifoliate leaves in soybean, the second trifoliate leaf in cowpea, and shoot biomass in soybean. The 50 nm concentration also consistently decreased root development in cowpea and millet, but had no effect on the other species. These data show that lumichrome is a rhizosphere signal molecule that affects seedling development in both monocots and dicots.

Fabaceae↗
PubMed2007

Inactivation of the nodH gene in Sinorhizobium sp. BR816 enhances symbiosis with Phaseolus vulgaris L.

Sulfate modification on Rhizobium Nod factor signaling molecules is not a prerequisite for successful symbiosis with the common bean (Phaseolus vulgaris L.). However, many bean-nodulating rhizobia, including the broad host strain Sinorhizobium sp. BR816, produce sulfated Nod factors. Here, we show that the nodH gene, encoding a sulfotransferase, is responsible for the transfer of sulfate to the Nod factor backbone in Sinorhizobium sp. BR816, as was shown for other rhizobia. Interestingly, inactivation of nodH enables inoculated bean plants to fix significantly more nitrogen under different experimental setups. Our studies show that nodH in the wild-type strain is still expressed during the later stages of symbiosis. This is the first report on enhanced nitrogen fixation by blocking Nod factor sulfation.

Bacterial Proteins↗
PubMed1987

Auxin production by plant-pathogenic pseudomonads and xanthomonads.

Pathogenic strains of Xanthomonas campestris pv. glycines which cause hypertrophy of leaf cells of susceptible soybean cultivars and nonpathogenic strains which do not cause hypertrophy were compared for their ability to produce indole compounds, including the plant hormone indole-3-acetic acid (IAA) in liquid media with or without supplementation with l-tryptophan. Several additional strains of plant-pathogenic xanthomonads and pseudomonads were also tested for IAA production to determine whether in vitro production of IAA is related to the ability to induce hypertrophic growth of host tissues. Indoles present in culture filtrates were identified by thin-layer chromatography, high-performance liquid chromatography, UV spectroscopy, mass spectroscopy, and gas chromatography-mass spectrometry and were quantitated by high-performance liquid chromatography. All strains examined produced IAA when liquid media were supplemented with l-tryptophan. The highest levels of IAA were found in culture filtrates from the common bean pathogen Pseudomonas syringae pv. syringae, and this was the only bacterium tested which produced IAA without addition of tryptophan to the medium. Additional indoles identified in culture filtrates of the various strains included indole-3-lactic acid, indole-3-aldehyde, indole-3-acetamide, and N-acetyltryptophan. Pseudomonads and xanthomonads could be distinguished by the presence of N-acetyltryptophan, which was found only in xanthomonad culture filtrates.

Journal Article↗
PubMed1995

Phylogenetic relationships and host range of Rhizobium spp. that nodulate Phaseolus vulgaris L.

We determined the nucleotide sequences of 16S rRNA gene segments from five Rhizobium strains that have been isolated from tropical legume species. All share the capacity to nodulate Phaseolus vulgaris L., the common bean. Phylogenetic analysis confirmed that these strains are of two different chromosomal lineages. We defined the host ranges of two strains of Rhizobium etli and three strains of R. tropici, comparing them with those of the two most divergently related new strains. Twenty-two of the 43 tested legume species were nodulated by three or more of these strains. All seven strains have broad host ranges that include woody species such as Albizia lebbeck, Gliricidia maculata, and Leucaena leucocephala.

Base Sequence↗
PubMed1999

Requirement for phosphoglucose isomerase of Xanthomonas campestris in pathogenesis of citrus canker.

A mutant (XT906) of Xanthomonas campestris pv. citri, the causal agent of citrus canker, was induced by insertion of the transposon Tn5tac1 and isolated. This mutant did not grow or elicit canker disease in citrus leaves but was still able to induce a hypersensitive response in a nonhost plant (the common bean). The mutant was also unable to grow on minimal medium containing fructose or glycerol as the sole carbon source. A 2.5-kb fragment of wild-type DNA that complemented the mutant phenotype of XT906 was isolated. Sequence analysis revealed that this DNA fragment encoded a protein of 562 amino acids that shows homology to phosphoglucose isomerase (PGI). Enzyme activity assay confirmed that the encoded protein possesses PGI activity. Analysis of the activity of the promoter of the pgi gene revealed that it was inhibited by growth in complex medium but induced by culture in plant extract. These results demonstrate that PGI is required for pathogenicity of X. campestris pv. citri.

Citrus↗
PubMed1999

Isolation and characterization of alfalfa-nodulating rhizobia present in acidic soils of central argentina and uruguay

We describe the isolation and characterization of alfalfa-nodulating rhizobia from acid soils of different locations in Central Argentina and Uruguay. A collection of 465 isolates was assembled, and the rhizobia were characterized for acid tolerance. Growth tests revealed the existence of 15 acid-tolerant (AT) isolates which were able to grow at pH 5.0 and formed nodules in alfalfa with a low rate of nitrogen fixation. Analysis of those isolates, including partial sequencing of the genes encoding 16S rRNA and genomic PCR-fingerprinting with MBOREP1 and BOXC1 primers, demonstrated that the new isolates share a genetic background closely related to that of the previously reported Rhizobium sp. Or191 recovered from an acid soil in Oregon (B. D. Eardly, J. P. Young, and R. K. Selander, Appl. Environ. Microbiol. 58:1809-1815, 1992). Growth curves, melanin production, temperature tolerance, and megaplasmid profiles of the AT isolates were all coincident with these characteristics in strain Or191. In addition to the ability of all of these strains to nodulate alfalfa (Medicago sativa) inefficiently, the AT isolates also nodulated the common bean and Leucaena leucocephala, showing an extended host range for nodulation of legumes. In alfalfa, the time course of nodule formation by the AT isolate LPU 83 showed a continued nodulation restricted to the emerging secondary roots, which was probably related to the low rate of nitrogen fixation by the largely ineffective nodules. Results demonstrate the complexity of the rhizobial populations present in the acidic soils represented by a main group of N2-fixing rhizobia and a second group of ineffective and less-predominant isolates related to the AT strain Or191.

Journal Article↗
PubMed2000

Characterization of two inducible phosphate transport systems in Rhizobium tropici.

Rhizobium tropici forms nitrogen-fixing nodules on the roots of the common bean (Phaseolus vulgaris). Like other legume-Rhizobium symbioses, the bean-R. tropici association is sensitive to the availability of phosphate (P(i)). To better understand phosphorus movement between the bacteroid and the host plant, P(i) transport was characterized in R. tropici. We observed two P(i) transport systems, a high-affinity system and a low-affinity system. To facilitate the study of these transport systems, a Tn5B22 transposon mutant lacking expression of the high-affinity transport system was isolated and used to characterize the low-affinity transport system in the absence of the high-affinity system. The K(m) and V(max) values for the low-affinity system were estimated to be 34 +/- 3 microM P(i) and 118 +/- 8 nmol of P(i) x min(-1) x mg (dry weight) of cells(-1), respectively, and the K(m) and V(max) values for the high-affinity system were 0.45 +/- 0.01 microM P(i) and 86 +/- 5 nmol of P(i) x min(-1) x mg (dry weight) of cells(-1), respectively. Both systems were inducible by P(i) starvation and were also shock sensitive, which indicated that there was a periplasmic binding-protein component. Neither transport system appeared to be sensitive to the proton motive force dissipator carbonyl cyanide m-chlorophenylhydrazone, but P(i) transport through both systems was eliminated by the ATPase inhibitor N,N'-dicyclohexylcarbodiimide; the P(i) transport rate was correlated with the intracellular ATP concentration. Also, P(i) movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the wild-type strain or the mutant. These properties suggest that both P(i) transport systems are ABC type systems. Analysis of the transposon insertion site revealed that the interrupted gene exhibited a high level of homology with kdpE, which in several bacteria encodes a cytoplasmic response regulator that governs responses to low potassium contents and/or changes in medium osmolarity.

ATP-Binding Cassette Transporters↗
PubMed2001

Characterization of rhizobial isolates of Phaseolus vulgaris by staircase electrophoresis of low-molecular-weight RNA.

Low-molecular-weight (LMW) RNA molecules were analyzed to characterize rhizobial isolates that nodulate the common bean growing in Spain. Since LMW RNA profiles, determined by staircase electrophoresis, varied across the rhizobial species nodulating beans, we demonstrated that bean isolates recovered from Spanish soils presumptively could be characterized as Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum bv. viciae and bv. trifolii, and Sinorhizobium fredii.

Electrophoresis↗
PubMed2002

The diversity of Phaseolus-nodulating rhizobial populations is altered by liming of acid soils planted with Phaseolus vulgaris L. in Brazil.

PCR-mediated restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA internally transcribed spacer (ITS) region and the 16S rRNA gene indicated that the rhizobial populations isolated from common bean (Phaseolus vulgaris L.) nodules in the unlimed soil from a series of five lime rates applied 6 years previously to plots of an acidic oxisol had less diversity than those from plots with higher rates of liming. Isolates affiliated with Rhizobium tropici IIB and Rhizobium leguminosarum bv. phaseoli were predominant independent of lime application. An index of richness based on the number of ITS groups increased from 2.2 to 5.7 along the soil liming gradient, and the richness index based on "species" types determined by RFLP analysis of the 16S rRNA gene varied from 0.5 to 1.4. The Shannon index of diversity, based on the number of ITS groups, increased from 1.8 in unlimed soil to 2.8 in limed soil, and, based on RFLP analysis of the 16S rRNA gene, ranged from 0.9 to 1.4. In the limed soil, the subpopulation of R. tropici IIB pattern types contained the largest number of ITS groups. In contrast, there were more R. leguminosarum bv. phaseoli types in the unlimed soil with the lowest pH than in soils with the highest pH. The number of ITS ("strain") groups within R. leguminosarum bv. phaseoli did not change with increased abundance of rhizobia in the soil, while with R. tropici IIB, the number of strain groups increased significantly. Some cultural and biochemical characteristics of Phaseolus-nodulating isolates were significantly related to changes in soil properties caused by liming, largely due to changes in the predominance of the rhizobial species groups.

Brazil↗
PubMed2003

Identification of an emergent and atypical Pseudomonas viridiflava lineage causing bacteriosis in plants of agronomic importance in a Spanish region.

Pseudomonas strains with an atypical LOPAT profile (where LOPAT is a series of determinative tests: L, levan production; O, oxidase production; P, pectinolitic activity; A, arginine dihydrolase production; and T, tobacco hypersensibility) can be regarded as emergent pathogens in the Principality of Asturias (Spain), where they have been causing, since 1999, severe damage in at least three taxonomically unrelated orchard plants of agronomic importance: common bean (Phaseolus vulgaris), kiwifruit (Actinidia deliciosa), and lettuce (Lactuca sativa). These strains are mainly differentiated by production of yellowish mucoid material in hypersucrose medium, used for the levan test, and by a variable pectinolytic activity on different potato varieties. The atypical organisms were identified as Pseudomonas viridiflava based on their 16S rRNA sequences. Among them a certain intraspecies genetic heterogeneity was detected by randomly amplified polymorphic DNA (RAPD) typing. To differentiate between isolates of P. viridiflava and Pseudomonas syringae pathovars, a 16S ribosomal DNA restriction fragment length polymorphism method employing the restriction endonucleases SacI and HinfI was developed. This could be used as a means of reliable species determination after the usual phenotypical characterization, which includes the LOPAT tests.

Genotype↗
PubMed2025

A rhamnose-rich O-antigen of Paraburkholderia phymatum MP20 is required for symbiosis with Mimosa pudica.

Paraburkholderia phymatum, a β-proteobacterium, forms a nitrogen-fixing symbiosis with many species of the large legume genus Mimosa as well as with common bean (Phaseolus vulgaris L.). Paraburkholderia are considered to have evolved nodulation independently from the well-studied α-proteobacteria symbionts of legumes. However, the detailed mechanisms important for β-rhizobia-legume symbiosis have not yet been determined. In this manuscript, we have sequenced the genome of P. phymatum MP20, a strain isolated from Mimosa pudica nodules, and utilized transposon mutagenesis to identify a mutant that showed delayed and ineffective nodulation of M. pudica. Further analysis revealed that the mutant strain produced an altered lipopolysaccharide lacking rhamnose containing O-antigen. Complementation with the wild-type gene restored the symbiosis. Microscopic analysis of the ineffective nodules showed that the mutant strain did not infect the cortical cells but was restricted to the endodermis. The results suggest that the O-antigen of P. phymatum is important for the bacterial infection of cortical cells and for nodule maturation. Further research will unveil the specific involvement of the glycosyltransferase gene in LPS biosynthesis and its impact on successful nodule formation by P. phymatum.IMPORTANCEThe nitrogen-fixing symbiosis between legumes and rhizobia is important for agricultural and environmental sustainability. The mechanisms of the symbiotic interactions are extensively studied using α-rhizobia. In contrast, mechanisms of symbiotic interactions important for β-rhizobia and their Caesalpinioid (mimosoid) legume hosts are not well known. Here, we describe the genome sequence of P. phymatum MP20, a β-rhizobia isolated from the nodules of M. pudica, and isolation and characterization of a transposon mutant defective in symbiosis. We demonstrate that the O-antigen of the LPS is required for nodulation and symbiotic nitrogen fixation. This study broadens our knowledge of symbiotic interactions in β-rhizobia and will lead to a better understanding of the wider rhizobial-legume symbiosis apart from the α-rhizobia.

Symbiosis↗
PubMed1993

Molecular analysis of avirulence gene avrRpt2 and identification of a putative regulatory sequence common to all known Pseudomonas syringae avirulence genes.

The avrRpt2 locus from Pseudomonas syringae pv. tomato causes virulent strains of P. syringae to be avirulent on some, but not all, lines of Arabidopsis thaliana and Glycine max (soybean). We determined the DNA sequence of the avrRpt2 locus and identified the avrRpt2 gene as a 768-bp open reading frame encoding a putative 28.2-kDa protein. Deletion analysis and transcription studies provided further evidence that this open reading frame encodes AvrRpt2. We found that the avrRpt2 gene also has avirulence activity in P. syringae pathogens of Phaseolus vulgaris (common bean), suggesting that disease resistance genes specific to avrRpt2 are functionally conserved among diverse plant species. The predicted AvrRpt2 protein is hydrophilic and contains no obvious membrane-spanning domains or export signal sequences, and there was no significant similarity of AvrRpt2 to sequences in the GenBank, EMBL, or Swiss PIR data bases. A comparison of the avrRpt2 DNA sequence to nine other P. syringae avirulence genes revealed a highly conserved sequence, GGAACCNA-N14-CCACNNA, upstream of the translation initiation codon. This motif is located 6 to 8 nucleotides upstream of the transcription start site in all four P. syringae avirulence genes for which a transcription start site has been determined, suggesting a role as a binding site for a novel form of RNA polymerase. Regulation of avrRpt2 was similar to other P. syringae avirulence genes; expression was high in minimal medium and low in rich medium and depended on the hrpRS locus and an additional locus at the opposite end of the hrp cluster of P. syringae pv. tomato.

Amino Acid Sequence↗
PubMed2005

Effective symbiosis between Rhizobium etli and Phaseolus vulgaris requires the alarmone ppGpp.

The symbiotic interaction between Rhizobium etli and Phaseolus vulgaris, the common bean plant, ultimately results in the formation of nitrogen-fixing nodules. Many aspects of the intermediate and late stages of this interaction are still poorly understood. The R. etli relA gene was identified through a genome-wide screening for R. etli symbiotic mutants. RelA has a pivotal role in cellular physiology, as it catalyzes the synthesis of (p)ppGpp, which mediates the stringent response in bacteria. The synthesis of ppGpp was abolished in an R. etli relA mutant strain under conditions of amino acid starvation. Plants nodulated by an R. etli relA mutant had a strongly reduced nitrogen fixation activity (75% reduction). Also, at the microscopic level, bacteroid morphology was altered, with the size of relA mutant bacteroids being increased compared to that of wild-type bacteroids. The expression of the sigma(N)-dependent nitrogen fixation genes rpoN2 and iscN was considerably reduced in the relA mutant. In addition, the expression of the relA gene was negatively regulated by RpoN2, the symbiosis-specific sigma(N) copy of R. etli. Therefore, an autoregulatory loop controlling the expression of relA and rpoN2 seems operative in bacteroids. The production of long- and short-chain acyl-homoserine-lactones by the cinIR and raiIR systems was decreased in an R. etli relA mutant. Our results suggest that relA may play an important role in the regulation of gene expression in R. etli bacteroids and in the adaptation of bacteroid physiology.

Adaptation, Physiological↗
PubMed2005

Diversification of DNA sequences in the symbiotic genome of Rhizobium etli.

Bacteria of the genus Rhizobium and related genera establish nitrogen-fixing symbioses with the roots of leguminous plants. The genetic elements that participate in the symbiotic process are usually compartmentalized in the genome, either as independent replicons (symbiotic plasmids) or as symbiotic regions or islands in the chromosome. The complete nucleotide sequence of the symbiotic plasmid of Rhizobium etli model strain CFN42, symbiont of the common bean plant, has been reported. To better understand the basis of DNA sequence diversification of this symbiotic compartment, we analyzed the distribution of single-nucleotide polymorphisms in homologous regions from different Rhizobium etli strains. The distribution of polymorphisms is highly asymmetric in each of the different strains, alternating regions containing very few changes with regions harboring an elevated number of substitutions. The regions showing high polymorphism do not correspond with discrete genetic elements and are not the same in the different strains, indicating that they are not hypervariable regions of functional genes. Most interesting, some highly polymorphic regions share exactly the same nucleotide substitutions in more than one strain. Furthermore, in different regions of the symbiotic compartment, different sets of strains share the same substitutions. The data indicate that the majority of nucleotide substitutions are spread in the population by recombination and that the contribution of new mutations to polymorphism is relatively low. We propose that the horizontal transfer of homologous DNA segments among closely related organisms is a major source of genomic diversification.

DNA, Bacterial↗