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Conidial anastomosis fusion between Colletotrichum species.

Colletotrichum lindemuthianum is a pathogen of the common bean plant (Phaseolus vulgaris) causing anthracnose. Large numbers of isolates can rapidly arise with different genetic and chromosomal compositions but their origin is unknown since sexual fruit bodies have only been found in the laboratory. We have recently described the occurrence of special kinds of hyphae that create anastomoses directly between conidia. In this work we show that conidial anastomoses can occur between two different Colletotrichum species. The implications of this observation on the generation of genetic diversity in these species are discussed.

Colletotrichum↗

Nodulation factors from Rhizobium tropici are sulfated or nonsulfated chitopentasaccharides containing an N-methyl-N-acylglucosaminyl terminus.

Phaseolus vulgaris (common bean) can be nodulated by several Rhizobium species. Among them, Rhizobium tropici has a relatively broad host range, as it is able to infect beans, Leucaena trees, and several other legumes. This work describes the isolation and the characterization of extracellular factors (Nod factors) whose production from R. tropici was triggered by the transcriptional activation of its nod genes. These factors consist of a chitopentaose backbone in which the N-acetyl group of the nonreducing end glucosaminyl residue is replaced by an N-methyl-N-vaccenoyl one. Some of these molecules are sulfated on position 6 of the terminal reducing glucosamine.

Acetylglucosamine↗

Heat-induced aggregation of Phaseolus vulgaris L. Proteins: an electron spin resonance study.

The mechanism of heat-induced aggregation of Phaseolus vulgaris L. proteins and of subunit interactions of importance for susceptibility of proteins to proteolysis was studied by electron spin resonance (ESR) spectroscopy. The mobility of a spin label bound to lysine residues was monitored at two different pH-induced (neutral and alkaline) association states of proteins extracted from raw and cooked common bean. The molecular weight of the protein complexes was assessed by size exclusion-high performance liquid chromatography (SE-HPLC) of labeled proteins. Upon alkaline dissociation, both native and denatured protein subunits underwent a reassociation process to form soluble complexes of molecular weight higher than the species originally present at neutral pH. However, unlike native proteins, impaired mobility of the spin label was observed in the aggregates that are formed after dissociation of subunits of denatured proteins, indicating a reduced accessibility of lysine residues. Trapping of lysine residues inside protein aggregates may explain limited digestibility in the small intestine of proteins in cooked legumes.

Cooking↗

Structure and evolution of Cyclops: a novel giant retrotransposon of the Ty3/Gypsy family highly amplified in pea and other legume species.

We characterized a novel giant Gypsy-like retrotransposon, Cyclops, present in about 5000 copies in the genome of Pisum sativum. The individual element Cyclops-2 measures 12 314 bp including long terminal repeats (LTRs) of 1504 bp and 1594 bp, respectively, showing 4.1% sequence divergence between one another. Cyclops-2 carries a polypurine tract (PPT) and an unusual primer binding site (PBS) complementary to tRNA-Glu. The element is bounded by 5 bp target site duplications and harbors three successive internal regions with homology to retroviral genes gag (424 codons) and pol (1382 codons) and an additional open reading frame (423 codons) of unknown function indicating the element's potential capacity for gene transduction. The pol region contains sequence motifs related to the enzymes protease, reverse transcriptase, RNAse H and integrase in the same typical order (5'-PR-RT-RH-IN-3') known for retroviruses and Gypsy-like retrotransposons. The reading frame of the pol region is disrupted by several mutations suggesting that Cyclops-2 does not encode functional enzymes. A phylogenetic analysis of the reverse transcriptase domain confirms our differential genetic assessment that Cyclops from pea is a novel element with no specific relationship to the previously described Gypsy-like elements from plants. Genomic Southern hybridizations show that Cyclops is abundant not only in pea but also in common bean, mung bean, broad bean, soybean and the pea nut suggesting that Cyclops may be an useful genetic tool for analyzing the genomes of agronomically important legumes.

Amino Acid Sequence↗

The Tpv2 family of retrotransposons of Phaseolus vulgaris: structure, integration characteristics, and use for genotype classification.

The Tpv2 family of transposable elements of common bean, Phaseolus vulgaris, belongs to the Ty1/copia group of long terminal repeat (LTR) containing retrotransposons. By reverse transcriptase (RT)-PCR and by analysis of genomic clones, we characterized four of the approximately 40 copies present in the Phaseolus genome, and the genomic environment of their integration sites. Tpv2 integrated preferentially into actively transcribed regions. While none of the isolated elements had all the functional domains necessary for transposition, analysis of bean cultivars suggested that some members of the Tpv2 family transposed in recent breeding history. Probes derived from Tpv2, as well as flanking genomic sequences, may be useful for classifying Phaseolus cultivars.

Amino Acid Sequence↗

Phylogenetic analysis of two potyvirus pathogens of commercial cowpea lines: implications for obtaining pathogen-derived resistance.

As a prelude to developing engineered resistance to two important potyvirus pathogens of cowpea, a phylogenetic analysis of strains of Cowpea aphid-borne mosaic virus (CAbMV) and Bean common mosaic virus--blackeye cowpea strain (BCMV-B1C) was undertaken. Nucleotide sequences for the coat protein genes and 3'-untranslated regions of four CAbMV and one BCMV-B1C strains were determined and included in an analysis with published sequences. While all the newly sequenced viruses showed strong homology with the existing respective sequences in the database, the CAbMV group showed a divergence into two subgroups. These groups differed from each other by more than some CAbMV strains differed from the South African Passiflora virus (CAbMV-SAP), which has distinct biological characteristics. The implications of the sequence analyses are discussed with respect to a strategy for the generation of engineered resistance to both groups of viruses.

3' Untranslated Regions↗

Alternaria brasiliensis sp. nov., a leaf pathogen on Phaseolus vulgaris.

Alternaria brasiliensis sp. nov., was isolated from leaves of common bean, Phaseolus vulgaris L., showing punctiform non concentric leaf spots of brown color. Besides the symptomatological differences, the new Alternaria species presents distinct type of arrangement of conidia chain, body and beak size. The disease was observed in Montanha county, State of Espirito Santo, Brazil, but not yet reported in the literature. The causal agent of the disease is now described by the first time. The epithet used here is referring to the country where the species has been found.

Alternaria↗

The importance of root gravitropism for inter-root competition and phosphorus acquisition efficiency: results from a geometric simulation model.

We have observed that low soil phosphorus availability alters the gravitropic response of basal roots in common bean (Phaseolus vulgaris L.), resulting in a shallower root system. In this study we use a geometric model to test the hypotheses that a shallower root system is a positive adaptive response to low soil P availability by (1) concentrating root foraging in surface soil horizons, which generally have the highest P availability, and (2) reducing spatial competition for P among roots of the same plant. The growth of nine root systems contrasting in gravitropic response over 320 h was simulated in SimRoot, a dynamic three-dimensional geometric model of root growth and architecture. Phosphorus acquisition and inter-root competition were estimated with Depzone, a program that dynamically models nutrient diffusion to roots. Shallower root systems had greater P acquisition per unit carbon cost than deeper root systems, especially in older root systems. This was due to greater inter-root competition in deeper root systems, as measured by the volume of overlapping P depletion zones. Inter-root competition for P was a significant fraction of total soil P depletion, and increased with increasing values of the P diffusion coefficient (De), with root age, and with increasing root gravitropism. In heterogenous soil having greater P availability in surface horizons, shallower root systems had greater P acquisition than deeper root systems, because of less inter-root competition as well as increased root foraging in the topsoil. Root P acquisition predicted by SimRoot was validated against values for bean P uptake in the field, with an r2 between observed and predicted values of 0.75. Our results support the hypothesis that altered gravitropic sensitivity in P-stressed roots, resulting in a shallower root system, is a positive adaptive response to low P availability by reducing inter-root competition within the same plant and by concentrating root activity in soil domains with the greatest P availability.

Computer Simulation↗

Vacuolar sorting determinants within a plant storage protein trimer act cumulatively.

The mechanism for vacuolar sorting of seed storage proteins is as yet poorly understood and no receptor has been identified to date. The homotrimeric glycoprotein phaseolin, which is the major storage protein of the common bean, requires a transient tetrapeptide at the C-terminus for its vacuolar sorting. A mutated construct without the tetrapeptide is secreted. We show here that coexpression of wild-type phaseolin and the mutated, secreted form in transgenic tobacco results in the formation of mixed trimers and partial vacuolar delivery of the mutated polypeptides and partial secretion of wild-type polypeptides. This indicates that the sorting signal has a cumulative effect within a phaseolin trimer. The result is discussed in the light of the hypothesized mechanisms for vacuolar sorting of seed storage proteins.

Mutation↗

Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences.

Over the past decade, several high value proteins have been produced in different transgenic plant tissues such as leaves, tubers, and seeds. Despite recent advances, many heterologous proteins accumulate to low concentrations, and the optimization of expression cassettes to make in planta production and purification economically feasible remains critical. Here, the regulatory sequences of the seed storage protein gene arcelin 5-I (arc5-I) of common bean (Phaseolus vulgaris) were evaluated for producing heterologous proteins in dicotyledonous seeds. The murine single chain variable fragment (scFv) G4 (ref. 4) was chosen as model protein because of the current industrial interest in producing antibodies and derived fragments in crops. In transgenic Arabidopsis thaliana seed stocks, the scFv under control of the 35S promoter of the cauliflower mosaic virus (CaMV) accumulated to approximately 1% of total soluble protein (TSP). However, a set of seed storage promoter constructs boosted the scFv accumulation to exceptionally high concentrations, reaching no less than 36.5% of TSP in homozygous seeds. Even at these high concentrations, the scFv proteins had antigen-binding activity and affinity similar to those produced in Escherichia coli. The feasibility of heterologous protein production under control of arc5-I regulatory sequences was also demonstrated in Phaseolus acutifolius, a promising crop for large scale production.

Arabidopsis↗

Colonization strategies and conjugal gene transfer of inoculated Pseudomonas syringae on the leaf surface.

Survival, colonization and activity of Pseudomonas syringae bacteria inoculated onto the leaf surface of the common bean (Phaseolus vulgaris) was studied. Inoculated Ps. syringae cells shortened by half their size in 100% humidity and by an average of one fifth in 40-60% humidity. The respiring portion of the population, measured by the formation of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC)-formazan crystals, decreased more in 40-60% humidity than in 100% humidity. In scanning electron micrographs, the bacterial cells on leaf surfaces were seen embedded in a mucoid matrix. Intraspecies conjugation of plasmid RP1 also occurred in 40-60% humidity conditions. The portion of transconjugants temporally rose higher than the same portion in 100% humidity conditions. Therefore, although only a small proportion of the inoculated cells remained active on the leaf surface in 40-60% humidity, a relatively high rate of conjugation was still seen. Gene spreading was thus efficient on the leaf surface also when conditions did not allow bacterial population growth.

Biofilms↗

CLNR1, the AREA/NIT2-like global nitrogen regulator of the plant fungal pathogen Colletotrichum lindemuthianum is required for the infection cycle.

Nitrogen starvation is generally assumed to be encountered by biotrophic and hemibiotrophic plant fungal pathogens at the beginning of their infection cycle. We tested whether nitrogen starvation constitutes a cue regulating genes that are required for pathogenicity of Colletotrichum lindemuthianum, a fungal pathogen of common bean. The clnr1 (C. lindemuthianumnitrogen regulator 1) gene, the areA/nit-2 orthologue of C. lindemuthianum, was isolated. The predicted CLNR1 protein exhibits high amino acid sequence similarities with the AREA and NIT2 global fungal nitrogen regulators. Targeted clnr1- mutants are unable to use a wide array of nitrogen sources, indicating that clnr1 is the C. lindemuthianum major nitrogen regulatory gene. The clnr1- mutants are non-pathogenic, although few anthracnose lesions seldom occur on whole plantlets. Surprisingly, cytological analysis reveals that the clnr1- mutants are not disturbed from the penetration stage until the end of the biotrophic phase, but that they are impaired during the setting up of the necrotrophic phase. Thus, through CLNR1, nitrogen starvation constitutes a cue for the regulation of genes that are compulsory for this stage of the C. lindemuthianum infection process. Additionally, clnr1- mutants complemented with the Aspergillus nidulans areA gene are fully pathogenic, indicating that areA is able to activate the C. lindemuthianum suited genes, normally under the control of clnr1.

Amino Acid Sequence↗

Quorum signal molecules as biosurfactants affecting swarming in Rhizobium etli.

Swarming motility is suggested to be a social phenomenon that enables groups of bacteria to coordinately and rapidly move atop solid surfaces. This multicellular behavior, during which the apparently organized bacterial populations are embedded in an extracellular slime layer, has previously been linked with biofilm formation and virulence. Many population density-controlled activities involve the activation of complex signaling pathways using small diffusible molecules, also known as autoinducers. In Gram-negative bacteria, quorum sensing (QS) is achieved primarily by means of N-acylhomoserine lactones (AHLs). Here, we report on a dual function of AHL molecules in controlling swarming behavior of Rhizobium etli, the bacterial symbiotic partner of the common bean plant. The major swarming regulator of R. etli is the cinIR QS system, which is specifically activated in swarming cells by its cognate AHL and other long-chain AHLs. This signaling role of long-chain AHLs is required for high-level expression of the cin and rai QS systems. Besides this signaling function, the long-chain AHLs also have a direct role in surface movement of swarmer cells as these molecules possess significant surface activity and induce liquid flows, known as Marangoni flows, as a result of gradients in surface tension at biologically relevant concentrations. These results point to an as-yet-undisclosed direct role of long-chain AHL molecules as biosurfactants.

4-Butyrolactone↗

Crystal structure of arcelin-5, a lectin-like defense protein from Phaseolus vulgaris.

In the seeds of the legume plants, a class of sugar-binding proteins with high structural and sequential identity is found, generally called the legume lectins. The seeds of the common bean (Phaseolus vulgaris) contain, besides two such lectins, a lectin-like defense protein called arcelin, in which one sugar binding loop is absent. Here we report the crystal structure of arcelin-5 (Arc5), one of the electrophoretic variants of arcelin, solved at a resolution of 2.7 A. The R factor of the refined structure is 20.6%, and the free R factor is 27.1%. The main difference between Arc5 and the legume lectins is the absence of the metal binding loop. The bound metals are necessary for the sugar binding capabilities of the legume lectins and stabilize an Ala-Asp cis-peptide bond. Surprisingly, despite the absence of the metal binding site in Arc5, this cis-peptide bond found in all legume lectin structures is still present, although the Asp residue has been replaced by a Tyr residue. Despite the high identity between the different legume lectin sequences, they show a broad range of quaternary structures. The structures of three different dimers and three different tetramers have been solved. Arc5 crystallized as a monomer, bringing the number of known quaternary structures to seven.

Anti-Infective Agents↗

A cDNA for nuclear-encoded chloroplast translational initiation factor 2 from a higher plant is able to complement an infB Escherichia coli null mutant.

Formation of the initiation translation complex containing the three initiation factors, IF1, IF2, and IF3, tRNA(fMet), and GTP constitutes the earliest event in the protein synthesis. IF2, a GTP-binding protein, is the principal factor involved in selecting and binding fMet-tRNA(fMet) to the 30 S ribosomal subunit. Although some chloroplast initiation translational factors have been identified and purified from algae, none of these factors have been characterized from plants. In this work, we report the molecular characterization of a nuclear-encoded chloroplastic IF2 gene from common bean (PvIF2cp). We show that the PvIF2cp gene encodes a protein containing a chloroplast translocation signal peptide, able to target a green fluorescent protein fusion protein to chloroplasts. A high accumulation of PvIF2cp transcript was found in photosynthetic tissues, whereas low mRNA levels were detected in etiolated plants and in nonphotosynthetic organs. Additional data indicate that the PvIF2cp transcript accumulation is modulated by light. The PvIF2cp gene encodes a functional factor, since the PvIF2cp conserved region, containing the G-domain and the C-terminal end, complements an Escherichia coli infB null mutation. Phylogenetic analysis using the PvIF2cp conserved region suggests that the PvIF2cp gene originated via endosymbiotic gene transfer to the nucleus and that it may be a useful marker for phylogeny reconstruction.

Active Transport, Cell Nucleus↗

Nitrogenase and antioxidant enzyme activities in Phaseolus vulgaris nodules formed by Rhizobium tropici isogenic strains with varying tolerance to salt stress.

Common bean plants inoculated with salt-tolerant Rhizobium tropici wild-type strain CIAT899 formed a more active symbiosis than did its decreased salt-tolerance (DST) mutant derivatives (HB8, HB10, HB12 and HB13). The mutants formed partially effective (HB10, HB12) or almost ineffective (HB8, HB13) nodules (Fix(d)) under non-saline conditions. The DST mutant formed nodules that accumulated more proline than did the wild-type nodules, while soluble sugars were accumulated mainly in ineffective nodules. Under salt stress, plant growth, nitrogen fixation, and the activities of the antioxidant defense enzymes of nodules were affected in all symbioses tested. Overall, mutant nodules showed lower antioxidant enzyme activities than wild-type nodules. Levels of nodule catalase appeared to correlate with symbiotic nitrogen-fixing efficiency. Superoxide dismutase and dehydroascorbate reductase seem to function in the molecular mechanisms underlying the tolerance of nodules to salinity.

Antioxidants↗

How host larval age, and nutrition and density of the parasitoid Dinarmus basalis (Hymenoptera: Pteromalidae) influence control of Acanthoscelides obtectus (Coleoptera: Bruchidae).

Choice of the targeted host developmental stage, regulation of parasitoid numbers released and introduction of food supplements are operational factors with a potential to influence the level of biological control. In a closed laboratory storage system maintained over two generations of the host, the impact of these three parameters on the control potential of the parasitoid Dinarmus basalis Rondani was investigated for high populations of larvae of Acanthoscelides obtectus (Say) feeding inside dry common bean seeds Phaseolus vulgaris. The beans were already infested with immature bruchids at the beginning of the storage period to simulate harvest conditions, characterized in a previous study. Treatments resulted in a reduction of 48-75% of the bruchid population within 16 weeks of storage. The best timing of parasitoid release was at the simulated harvest, as later releases reduced the bruchid population only by about half this percentage. Host feeding is postulated to be the key factor involved in the observed difference. The effect of increasing the number of parasitoids strongly depended on host age and food supplement. Addition of vials with honey had no direct effect on the bruchid population or on the parasitoid progeny. The ecological significance of these findings and implications for biological control are discussed.

Age Factors↗

Factors influencing pulse consumption in Latin America.

In Latin America, pulse consumption ranges from 1 kg/capita per year (Argentina) to 25 kg/capita per year (Nicaragua). Common beans account for 87 % of the total. The differences between countries, regions or groups of population within the same country can be explained by the following factors: (1) beans are very nutritious; (2) beans and maize are traditional foods and the habit of consuming them is deeply rooted in many people and communities; (3) the rural population eats more pulses than the urban population, due to geographical constraints that limit exchanges and favour consumption of locally produced foods; (4) income level, beans are still the poor man's meat; and (5) other factors, such as consumers' taste, the constraints on cooking beans, etc. The evolution of the consumption level in the future will depend on the urbanization of the population, access to processed foods and income level. Health issues would be an argument for maintaining or increasing the current consumption level.

Cooking↗