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PubMed2005

Structural characterization of a flavonoid-inducible Pseudomonas aeruginosa A-band-like O antigen of Rhizobium sp. strain NGR234, required for the formation of nitrogen-fixing nodules.

Rhizobium (Sinorhizobium) sp. strain NGR234 contains three replicons, the smallest of which (pNGR234a) carries most symbiotic genes, including those required for nodulation and lipo-chito-oligosaccharide (Nod factor) biosynthesis. Activation of nod gene expression depends on plant-derived flavonoids, NodD transcriptional activators, and nod box promoter elements. Nod boxes NB6 and NB7 delimit six different types of genes, one of which (fixF) is essential for the formation of effective nodules on Vigna unguiculata. In vegetative culture, wild-type NGR234 produces a distinct, flavonoid-inducible lipopolysaccharide (LPS) that is not produced by the mutant (NGRomegafixF); this LPS is also found in nitrogen-fixing bacteroids isolated from V. unguiculata infected with NGR234. Electron microscopy showed that peribacteroid membrane formation is perturbed in nodule cells infected by the fixF mutant. LPSs were purified from free-living NGR234 cultured in the presence of apigenin. Structural analyses showed that the polysaccharide portions of these LPSs are specialized, rhamnose-containing O antigens attached to a modified core-lipid A carrier. The primary sequence of the O antigen is [-3)-alpha-L-Rhap-(1,3)-alpha-L-Rhap-(1,2)-alpha-L-Rhap-(1-]n, and the LPS core region lacks the acidic sugars commonly associated with the antigenic outer core of LPS from noninduced cells. This rhamnan O antigen, which is absent from noninduced cells, has the same primary sequence as the A-band O antigen of Pseudomonas aeruginosa, except that it is composed of L-rhamnose rather than the D-rhamnose characteristic of the latter. It is noteworthy that A-band LPS is selectively maintained on the P. aeruginosa cell surface during chronic cystic fibrosis lung infection, where it is associated with an increased duration of infection.

Flavonoids↗
PubMed1964

QUICK SEROLOGICAL METHOD OF CLASSIFYING STRAINS OF RHIZOBIUM JAPONICUM IN NODULES.

Means, Ura M. (U.S. Department of Agriculture, Beltsville, Md.), Herbert W. Johnson, and R. A. Date. Quick serological method of classifying strains of Rhizobium japonicum in nodules. J. Bacteriol. 87:547-553. 1964.-A new method of classifying strains of Rhizobium japonicum serologically by utilizing the contents of nodules as antigens is described. The method differs from standard procedures only in the preparation of antigens, but it is less time-consuming. Agglutination reactions of culture and nodule antigens were identical for 15 of the 17 strains investigated. Nodule antigens of ineffective strain 24 failed to agglutinate with any of the 17 antisera employed, and nodule antigens of strain 46 reacted with two antisera in addition to the two with which the culture antigens reacted. Host genotypes affected the reactions of nodule antigens of strain 46, with the reactions ranging from those typical for culture antigens for some soybean (Glycine max) varieties to reactions with heterologous antisera only for other soybean varieties and six cowpea (Vigna sinensis) varieties. Although nodule antigens of most strains of R. japonicum reacted the same as culture antigens, the inconsistent reactions of nodule antigens of strain 46 suggest precautions in the general use of nodule antigens. Indicated precautions are discussed.

Agglutination Tests↗
PubMed1979

In Vitro Replication of Cowpea Mosaic Virus RNA III. Template Recognition by Cowpea Mosaic Virus RNA Replicase.

Cowpea mosaic virus (CPMV) RNA replicase has been purified about 200-fold from CPMV-infected Vigna unguiculata leaves. Optimal reaction conditions for replicase activity have been established that allow RNA synthesis to proceed for at least 15 h. Using a polymerase assay under conditions optimal for CPMV RNA-directed RNA synthesis, all natural RNA species tested appeared to be able to direct the incorporation of labeled ribonucleotides, whereas synthetic homoribopolymers were either inactive or only slightly active. Using a nitrocellulose membrane filter assay to measure complex formation between the replicase preparation and various RNA species, all natural RNA species tested, except that of the comovirus radish mosaic virus, appeared to be unable to compete with (32)P-labeled CPMV RNA in binding to replicase. We propose that CPMV replicase is actually template specific but does not display this property in a polymerase assay, since labile complexes between heterologous templates and replicase become stabilized by the formation of phosphodiester bonds. From homoribopolymer competition binding experiments we conclude that the polyadenylic acid on the CPMV genome might be a part of the replicase binding site.

Journal Article↗
PubMed1982

Antibodies Against the Genome-Linked Protein VPg of Cowpea Mosaic Virus Recognize a 60,000-Dalton Precursor Polypeptide.

We have prepared a rabbit antiserum specifically directed against the genome-linked protein (VPg) of cowpea mosaic virus by injecting an hydrolysate of purified virion RNA. Using this antiserum as a probe in combination with "Western" (protein) blots of subcellular fractions of cowpea mosaic virus-infected cowpea (Vigna unguiculata) cells, we have detected a bottom component RNA-encoded, 60,000-dalton polypeptide which is membrane bound and presumably represents the immediate precursor of VPg.

Journal Article↗
PubMed2005

Promoters, transcripts, and regulatory proteins of Mungbean yellow mosaic geminivirus.

Geminiviruses package circular single-stranded DNA and replicate in the nucleus via a double-stranded intermediate. This intermediate also serves as a template for bidirectional transcription by polymerase II. Here, we map promoters and transcripts and characterize regulatory proteins of Mungbean yellow mosaic virus-Vigna (MYMV), a bipartite geminivirus in the genus Begomovirus. The following new features, which might also apply to other begomoviruses, were revealed in MYMV. The leftward and rightward promoters on DNA-B share the transcription activator AC2-responsive region, which does not overlap the common region that is nearly identical in the two DNA components. The transcription unit for BC1 (movement protein) includes a conserved, leader-based intron. Besides negative-feedback regulation of its own leftward promoter on DNA-A, the replication protein AC1, in cooperation with AC2, synergistically transactivates the rightward promoter, which drives a dicistronic transcription unit for the coat protein AV1. AC2 and the replication enhancer AC3 are expressed from one dicistronic transcript driven by a strong promoter mapped within the upstream AC1 gene. Early and constitutive expression of AC2 is consistent with its essential dual function as an activator of viral transcription and a suppressor of silencing.

Base Sequence↗
PubMed2005

Suppression of RNA silencing by a geminivirus nuclear protein, AC2, correlates with transactivation of host genes.

Bipartite geminiviruses encode a small protein, AC2, that functions as a transactivator of viral transcription and a suppressor of RNA silencing. A relationship between these two functions had not been investigated before. We characterized both of these functions for AC2 from Mungbean yellow mosaic virus-Vigna (MYMV). When transiently expressed in plant protoplasts, MYMV AC2 strongly transactivated the viral promoter; AC2 was detected in the nucleus, and a split nuclear localization signal (NLS) was mapped. In a model Nicotiana benthamiana plant, in which silencing can be triggered biolistically, AC2 reduced local silencing and prevented its systemic spread. Mutations in the AC2 NLS or Zn finger or deletion of its activator domain abolished both these effects, suggesting that suppression of silencing by AC2 requires transactivation of host suppressor(s). In line with this, in Arabidopsis protoplasts, MYMV AC2 or its homologue from African cassava mosaic geminivirus coactivated >30 components of the plant transcriptome, as detected with Affymetrix ATH1 GeneChips. Several corresponding promoters cloned from Arabidopsis were strongly induced by both AC2 proteins. These results suggest that silencing suppression and transcription activation by AC2 are functionally connected and that some of the AC2-inducible host genes discovered here may code for components of an endogenous network that controls silencing.

DNA, Viral↗
PubMed2003

Scanning electron microscopy of native biofilms on mung bean sprouts.

Native biofilms present on the adaxial surface of cotyledons of mung bean sprouts (Vigna radiata) were studied by use of scanning electron microscopy. Biofilms were abundant on the cotyledon surfaces and were comprised of rod-shaped bacteria, cocci-shaped bacteria, or yeasts, often with one type of microbe predominant. In contrast to our earlier study of biofilms on green sprouts (alfalfa, clover, broccoli, and sunflower), yeast and cocci were abundant on mung bean. Filamentous fungi were not observed. Sheet-like or fibrillar material (presumably composed of secreted microbial polysaccharides, proteins, lipids, and nucleic acids) fully or partially covered the biofilms. Biofilms up to 5 mm in length were observed, and some biofilms were comprised of more than just a monolayer of microbial cells. Native biofilms on sprout surfaces undoubtedly play an important role in the ecology of plant epiphytic microbes and may also afford protected sites for plant and human bacterial pathogens.

Bacteria↗
PubMed2002

Radioprotective effects of vitexina for breast cancer patients undergoing radiotherapy with cobalt-60.

Vitexina, a product containing the flavonoid vitexin as the main component, is derived from a plant, Vigna radiata (L.), that has been traditionally used in Vietnam for detoxification. This remedy is also used to treat the symptoms of conditions classified as "hot" in traditional medicine. The present study is a randomized, placebo-controlled comparative clinical trial for investigating the radioprotective effects of Vitexina for breast cancer patients undergoing radiotherapy with cobalt-60. No relevant weight loss, (even weight gain), occurred in 70% of patients in the Vitexina group, whereas 73% of the placebo group lost 1 to 2 kg of weight after 6 weeks of radiation therapy. The administration of Vitexina produced a significantly protective effect in peripheral blood cells in amount and in lymphocyte blast-transformation function. Condition of hot was observed in almost all cancer patients in this study by tongue examination. Hot condition did not change in the Vitexina group, but the incidence of hot and extreme hot cases were significantly increased in the placebo group after 6 weeks of radiation therapy. The results suggest that application of medicinal plants of the "clearing heat and detoxification" classification as an adjuvant would be a potential solution in integrative cancer therapy.

Apigenin↗
PubMed1981

Use of purified endopolygalacturonase for a topochemical study of elongating cell walls at the ultrastructural level.

Endopolygalacturonase, a fungal enzyme purified by Albersheim and his group which specifically degrades the galacturonosyl linkages of pectic polysaccharides, was used at the ultrastructural level on intact tissues from differentiated organs. Specimens were taken from the elongating zone of mung bean (Vigna radiata) hypocotyls. Incubation with the enzyme solution was performed en bloc, prior to embedding and to ultrastructural cytochemistry (PATAg test for polysaccharides), or by flotation of ultrathin frozen sections. From the morphological viewpoint, the images obtained are sharp and reproducible. They support the plywood model for the organization of expanding walls. The ordering of walls appears to be built up very early, at the very beginning of elongation, in the upper part of the hypocotyl (hook). The specificity of the enzyme allows a topochemical study of the wall. Data indicate an uneven distribution of polysaccharides of pectic type across a single wall and among the different cells. Outer and inner wall areas are highly resistant to extraction by endopolygalacturonase. In the middle lamella the insolubility of amorphous components probably indicates a local concentration of highly methyl-esterified carboxyl groups not susceptible to endopolygalacturonase attack; conversely, adjacent parts of the middle lamella are regularly extracted, indicating a high concentration of galacturonans. An intense extraction occurs in the bow-shaped zone, revealing the occurrence of a massive embedding of unesterified pectic polysaccharides around the fibrillar subunits responsible for the twisted patterns. In the inner and recent part of the wall, the ordering of fibrillar subunits seems progressive and possibly related to the peptic embedding. This incrusting material could play a role in the morphogenesis of the ordered wall by means of specific assembly and interconnections of wall subunits.

Cell Wall↗
PubMed1997

Expression and characterization of sucrose synthase from mung bean seedlings in Escherichia coli.

The cDNA fragment coding for mung bean (Vigna radiata Wilczek) sucrose synthase was introduced into the expression vector pET-20b resulting in the construction of plasmid pEB-01. After transformation of Escherichia coli strain BL21(DE3) cells by pEB-01 and induction with isopropyl thio-beta-galactoside, high level expression of the recombinant enzyme was obtained. The enzyme had a tetrameric form that conserved the activity of sucrose synthase. Although the Km and Vmax of the recombinant enzyme acting on either UDP-glucose or fructose were very close to those of the native enzyme isolated from mung bean seedlings, the Km for sucrose was higher by a factor of 10 for the recombinant enzyme. This suggests that the recombinant sucrose synthase has a tendency to synthesize sucrose, although the native enzyme catalyzes a freely reversible reaction.

Blotting, Western↗
PubMed2001

In vitro biosynthesis of homogalacturonan by a membrane-bound galacturonosyltransferase from epicotyls of azuki bean.

A membrane preparation of 7-d-old seedlings from azuki bean (Vigna angularis) contained galacturonosyltransferase (GalAT) capable of transferring galacturonic acid (GalA) from UDP-GalA into polygalacturonic acid (PGA) as an exogenous acceptor. The enzyme was maximally active at pH 6.8-7.8 and 25-35 degrees C in the presence of 5 mM Mn2+ and 0.5% (w/v) Triton X-100. Acid-soluble low-Mr (average Mr 10,000) PGA was a more efficient acceptor substrate than acid-insoluble polymer (Mr 70,000). The apparent Michaelis constants for UDP-GalA and low-Mr PGA were 0.14 mM and 0.02 mg/ml, respectively. Various pectins with different degrees of methyl-esterification (DE) were poor acceptors, and the enzyme activity tended to decrease with decreasing DE of the pectins. The transfer products from incubation of the enzyme with UDP-14C-GalA and the low-Mr PGA yielded 14C-GalA2 as the major product upon digestion with an endopolygalacturonase (EPGase), confirming the incorporation of GalA into PGA through contiguous alpha-1,4-linkages.

Carbohydrate Conformation↗
PubMed2004

The seed coat of Phaseolus vulgaris interferes with the development of the cowpea weevil [Callosobruchus maculatus (F.) (Coleoptera: Bruchidae)].

We have confirmed here that the seeds of the common bean (Phaseolus vulgaris, L.) do not support development of the bruchid Callosobruchus maculatus (F.), a pest of cowpea [Vigna unguiculata (L.) Walp] seeds. Analysis of the testa (seed coat) of the bean suggested that neither thickness nor the levels of compounds such as tannic acid, tannins, or HCN are important for the resistance. On the other hand, we have found that phaseolin (vicilin-like 7S storage globulin), detected in the testa by Western blotting and N-terminal amino acid sequencing, is detrimental to the development of C. maculatus. As for the case of other previously studied legume seeds (Canavalia ensiformis and Phaseolus lunatus) we suggest that the presence of vicilin-like proteins in the testa of P. vulgaris may have had a significant role in the evolutionary adaptation of bruchids to the seeds of leguminous plants.

Animals↗
PubMed2002

The leaves of green plants as well as a cyanobacterium, a red alga, and fungi contain insulin-like antigens.

We report the detection of insulin-like antigens in a large range of species utilizing a modified ELISA plate assay and Western blotting. We tested the leaves or aerial parts of species of Rhodophyta (red alga), Bryophyta (mosses), Psilophyta (whisk ferns), Lycopodophyta (club mosses), Sphenopsida (horsetails), gymnosperms, and angiosperms, including monocots and dicots. We also studied species of fungi and a cyanobacterium, Spirulina maxima. The wide distribution of insulin-like antigens, which in some cases present the same electrophoretic mobility as bovine insulin, together with results recently published by us on the amino acid sequence of an insulin isolated from the seed coat of jack bean (Canavalia ensiformis) and from the developing fruits of cowpea (Vigna unguiculata), suggests that pathways depending on this hormone have been conserved through evolution.

Animals↗
PubMed2002

Immune response induced in mice oral immunization with cowpea severe mosaic virus.

There is increasing interest in the immune response induced by plant viruses since these could be used as antigen-expressing systems in vaccination procedures. Cowpea severe mosaic virus (CPSMV), as a purified preparation (300 g of leaves, 2 weeks post-inoculation), or crude extract from cowpea (Vigna unguiculata) leaves infected with CPSMV both administered by gavage to Swiss mice induced a humoral immune response. Groups of 10 Swiss mice (2-month-old females) were immunized orally with 10 daily doses of either 50 microg viral capsid protein (boosters of 50 microg at days 21 and 35 after immunization) or 0.6 mg protein of the crude extract (boosters of 0.6 mg at days 21 and 35 after immunization). Anti-CPSMV antibodies were quantified by ELISA in pooled sera diluted at least 1:400 at days 7, 14, 21, 28, 35 and 42 after the 10th dose. IgG and IgA against CPSMV were produced systemically, but IgE was not detected. No synthesis of specific antibodies against the proteins of leaf extracts from V. unguiculata, infected or not with CPSMV, was detected. The use of CPSMV, a plant-infecting virus that apparently does not induce a pathogenic response in animals, induced a humoral and persistent (at least 6 months) immune response through the administration of low antigen doses by gavage. These results raise the possibility of using CPSMV either as a vector for the production of vaccines against animal pathogens or in quick and easy methods to produce specific antisera for viral diagnosis.

Administration, Oral↗
PubMed2004

Resistance of Glycine species and various cultivated legumes to the soybean aphid (Homoptera: Aphididae).

The soybean aphid, Aphis glycines Matsumura, is a new pest of soybean, Glycine max (L.) Merr., in North America. It has become widespread on soybean in North America since it was first identified in the Midwest in 2000. Species of Rhamnus L. (buckthorn) are the primary hosts of A. glycines, and soybean is known as a secondary host. There is limited information about the secondary host range of A. glycines. Aphid colonization on various legume hosts was compared in choice experiments. Aphid colonization occurred on species in the genus Glycine Wild. No colonization occurred on Lablab purpureus (L.) Sweet, Lens culinaris Medik, Phaseolus vulgaris L., Pisum sativum L., or species of Vicia L. and Vigna Savi. Colonization was limited or aphids were transient on species of Medicago L., Phaseolus L., and Trifolium L. There were significant differences in aphid colonization among Medicago truncatula accessions with numbers ranging from 7 to 97 aphids per plant. Six Glycine soja Sieb. & Zucc. accessions were as resistant as G. max accessions to A. glycines; these may represent novel sources of A. glycines resistance not found in G. max. Antibiosis was found to play a large role in the expression of resistance in three of the G. soja accessions. Results of this study indicated that G. max and G. soja were the best secondary hosts of A. glycines; however, its secondary host range may include other leguminous species. Therefore, A. glycines did not seem to have a highly restricted monophagous secondary host range.

Animals↗
PubMed2002

Susceptibility of the bruchid Callosobruchus maculatus (Coleoptera: Bruchidae) and its parasitoid Dinarmus basalis (Hymenoptera: Pteromalidae) to three essential oils.

The bruchid Callosobruchus maculatus (F.) causes major losses during the storage of seeds of Vigna unguiculata (Walp.) in West Africa. An endemic parasitoid, the pteromalid Dinarmus basalis (Rond.) reduces the increase in bruchid populations in stores and could be used for biological control. African farmers often introduce essential oils into granaries at harvest time. In Togo, essential oils were extracted from two Gramineae, Cymbopogon nardus (L.) and Cymbopogon schoenanthus (L.) and from a Lamiaceae, Ocimum basilicum (L.). The major components of these essential oils were citronellal in C. nardus, carene-2 and piperitone in C. schoenanthus and estragol in O. basilicum. Cymbopogon schoenanthus was the most toxic oil for C. maculatus adults. D. basalis adults were more susceptible to the three essential oils than the adults of their hosts C. maculatus. In the presence of cowpea seeds, the LC50s of the three essential oils were lower than in their absence, suggesting that the seeds may absorb a part of the volatiles. High doses of three essential oils slightly affected the survival of the fourth instar or the pupae of C. maculatus. This high survival was due to protection of larvae from volatiles by the surrounding seeds. The D. basalis were more affected by the oil volatiles than their hosts. Sub-lethal doses of essential oils reduced the duration of the adult life of both insect species and fecundity of the females. The differences in sensitivity of the host and its parasitoid could influence their population dynamics. The introduction of the essential oils into storage systems potentially could reduce density of parasitoid populations and increase seed losses.

Animals↗
PubMed2003

Variation in responses to susceptible and resistant cowpeas among West African populations of Callosobruchus maculatus (Coleoptera: Bruchidae).

The cowpea seed beetle, sometimes also known as the cowpea weevil, Callosobruchus maculatus (F.), is a major pest of stored cowpeas (Vigna unguiculata Walpers) in West Africa. Control methods have included development of 'resistant' varieties as an environmentally benign alternative to insecticides, but there is concern over their effectiveness because of population variation among the insects and the possibility of adaptation overcoming seed resistance. Populations of C. maculatus from Nigeria, Ghana, Benin, and Niger, were used to examine variation in response to resistant and susceptible cowpea varieties at two geographical scales. Among seven Nigerian populations, there were significant differences in development times, the pattern of adult emergence, adult weights, and female fecundity when reared under identical conditions. Development in the resistant variety was retarded, produced higher mortality and lower adult weights. Significant interactions between variety and population were evident in terms of their effects on adult weight and development time; development times in the resistant variety were longer and emergences occurred over a longer period in some populations than in others. Population responses to resistant seeds were therefore unpredictable, but there was no evidence to suggest adaptation to overcome seed resistance within three generations. On a larger geographical scale, variation in performance was much greater and therefore, even less predictable. Mortality in resistant seeds was also higher among populations collected from outside Nigeria and may be explained by significant adaptation among Nigerian populations to previous release of resistant varieties. The findings are discussed in relation to understanding the extent of intraspecific variation in C. maculatus and its implications for future pest management.

Animals↗