Three Sitophilus species (S. granarius L., S. oryzae L., and S. zeamais Mots.) are closely related based on DNA analysis of their endosymbionts. All are seed parasites of cereal crops and important economic pest species in stored grain. The Sitophilus species that currently exist, including these three species, are generally believed to be endemic to Asia's forested areas, suggesting that the first infestations of stored grain must have taken place near the forested mountains of southwestern Asia. Previous archaeological data and historical records suggest that the three species may have been diffused by the spread of Neolithic agriculture, but this hypothesis has only been established for granary weevils in European and southwestern Asian archaeological records. There was little archeological evidence for grain pests in East Asia before the discovery of maize weevil impressions in Jomon pottery in 2004 using the “impression replica” method. Our research on Jomon agriculture based on seed and insect impressions in pottery continued to seek additional evidence. In 2010, we discovered older weevil impressions in Jomon pottery dating to ca. 10 500 BP. These specimens are the oldest harmful insects in the world discovered at archaeological sites. Our results provide evidence of harmful insects living in the villages from the Earliest Jomon, when no cereals were cultivated. This suggests we must reconsider previous scenarios for the evolution and propagation of grain pest weevils, especially in eastern Asia. Although details of their biology or the foods they infested remain unclear, we hope future interdisciplinary collaborations among geneticists, entomologists, and archaeologists will provide the missing details.
PA1b (Pea Albumin 1, subunit b) is a peptide extract from pea seeds showing significant insecticidal activity against certain insects, such as cereal weevils (genus Sitophilus), the mosquitoes Culex pipiens and Aedes aegyptii, and certain species of aphids. PA1b has great potential for use on an industrial scale and for use in organic farming: it is extracted from a common plant; it is a peptide (and therefore suitable for transgenic applications); it can withstand many steps of extraction and purification without losing its activity; and it is present in a seed regularly consumed by humans and mammals without any known toxicity or allergenicity. The potential of this peptide to limit pest damage has stimulated research concerning its host range, its mechanism of action, its three-dimensional structure, the natural diversity of PA1b and its structure-function relationships.
PA1b; insect; bioinsecticide; Legumes
The weevils Sitophilus spp. are among the most important cosmopolitan pests of stored cereal grains. However, their biology and physiology are poorly understood, mainly because the insect developmental stages take place within cereal grains and because of the lack of gene specific molecular manipulation.
To gain access to the different insect developmental stages, weevil females were allowed to lay their eggs on starch pellets and hatched embryos were collected by dissolving starch with water. Embryos were transferred between two Glass Plates filled with packed Flour (GPF) to mimic compact texture of the cereal grain, and this system allowed us to recover specific developmental stages. To knockdown the gene expressed in the bacteria-bearing organ (the bacteriome), whole larvae were injected with dsRNA to target the wpgrp1 gene and they were then left to develop for a further 4 days period. Quantitative RT-PCR and Western blot analyses on the bacteriome of these animals revealed a down-regulation of the wpgrp1 expression, both at transcript and protein levels.
These results demonstrate that whole larval injection with dsRNA results in a high and systemic decrease of both mRNA and protein in the bacteriome tissue. This, along with the possibility of access to the insect developmental stages, opens up a new research avenue for exploring gene specific functions in the cereal weevils.
Commensal symbionts, thought to be intermediary amid obligate mutualists and facultative parasites, offer insight into forces driving the evolutionary transition into mutualism. Using macroarrays developed for a close relative, Escherichia coli, we utilized a heterologous array hybridization approach to infer the genomic compositions of a clade of bacteria that have recently established symbiotic associations: Sodalis glossinidius with the tsetse fly (Diptera, Glossina spp.) and Sitophilus oryzae primary endosymbiont (SOPE) with the rice weevil (Coleoptera, Sitophilus oryzae). Functional biologies within their hosts currently reflect different forms of symbiotic associations. Their hosts, members of distant insect taxa, occupy distinct ecological niches and have evolved to survive on restricted diets of blood for tsetse and cereal for the rice weevil. Comparison of genome contents between the two microbes indicates statistically significant differences in the retention of genes involved in carbon compound catabolism, energy metabolism, fatty acid metabolism, and transport. The greatest reductions have occurred in carbon catabolism, membrane proteins, and cell structure-related genes for Sodalis and in genes involved in cellular processes (i.e., adaptations towards cellular conditions) for SOPE. Modifications in metabolic pathways, in the form of functional losses complementing particularities in host physiology and ecology, may have occurred upon initial entry from a free-living to a symbiotic state. It is possible that these adaptations, streamlining genomes, act to make a free-living state no longer feasible for the harnessed microbe.
Studies were conducted to evaluate the effect of a protein-enriched pea (Pisum sativum var. Bonneville) flour extract against the rice weevil, Sitophilus oryzae in its repellency, toxicity, effect on fecundity, stability and sensory properties. Milled rice admixed with pea flour extract at 1% concentration significantly repelled S. oryzae. Mortality of S. oryzae was found to increase and fecundity was markedly suppressed, in rice treated with 1% pea flour extract. The toxicity and reproductive effects of the pea protein-enriched rice were found to be stable for a period of 5 months. The sensory characteristics of stored rice when eaten were not affected by the treatment with pea flour extract. This study indicates that the protein-enriched flour extract obtained from the Bonneville pea may be feasible to protect stored milled rice from insect attack.
repellency; toxicity; fecundity; stability; sensory properties
The modification of lipid A with 4-amino-4-deoxy-L-arabinose (Ara4N) allows gram-negative bacteria to resist the antimicrobial activity of cationic antimicrobial peptides and antibiotics such as polymyxin. ArnA is the first enzyme specific to the lipid A-Ara4N pathway. It contains two functionally and physically separable domains: a dehydrogenase domain (ArnA_DH) catalyzing the NAD+-dependent oxidative decarboxylation of UDP-Glucuronic acid (UDP-GlcA), and a transformylase domain that formylates UDP-Ara4N. Here, we describe the crystal structure of the full-length bifunctional ArnA with UDP-GlcA and ATP bound to the dehydrogenase domain. Binding of UDP-GlcA triggers a 17 Å conformational change in ArnA_DH that opens the NAD+ binding site while trapping UDP-GlcA. We propose an ordered mechanism of substrate binding and product release. Mutation of residues R619 and S433 demonstrates their importance in catalysis and suggests that R619 functions as a general acid in catalysis. The proposed mechanism for ArnA_DH has important implications for the design of selective inhibitors.
Endo-polygalacturonase, one of the group of enzymes known collectively as pectinases, is widely distributed in bacteria, plants and fungi. The enzyme has also been found in several weevil species and a few other insects, such as aphids, but not in Drosophila melanogaster, Anopheles gambiae, or Caenorhabditis elegans or, as far as is known, in any more primitive animal species. What, then, is the genetic origin of the polygalacturonases in weevils? Since some weevil species harbor symbiotic microorganisms, it has been suggested, reasonably, that the symbionts' genomes of both aphids and weevils, rather than the insects' genomes, could encode polygalacturonase. We report here the cloning of a cDNA that encodes endo-polygalacturonase in the rice weevil, Sitophilus oryzae (L.), and investigations based on the cloned cDNA. Our results, which include analysis of genes in antibiotic-treated rice weevils, indicate that the enzyme is, in fact, encoded by the insect genome. Given the apparent absence of the gene in much of the rest of the animal kingdom, it is therefore likely that the rice weevil polygalacturonase gene was incorporated into the weevil's genome by horizontal transfer, possibly from a fungus.
Two laboratory workers, who spent a considerable time each day handling grain infested by the grain weevil (Sitophilus granarius), developed allergic responses to this insect varying from rhinitis and pruritus to marked asthma. These findings suggested that weevil protein present in mill dust could result in sensitization in those exposed continuously. A pilot study was therefore undertaken on 75 volunteer millworkers to determine whether such sensitivity existed. A millworker was defined as anyone who worked in a flour mill or mill producing animal feed from mixed cereals, or who worked in grain-storing silos.
Skin testing with weevil, mixed flour extracts, and a control was carried out on all 75 volunteers; 57% had a positive response to the weevil extract and 68% a positive response to the mixed flour extract. In a control group of 100 workers from two engineering firms matched for age and sex, 34% were positive to the weevil extract and 17% to mixed flour.
From the initial 75 millworkers, 15 were selected for further study based on a positive skin response to the weevil and a history of a productive cough and chest tightness and wheezing when exposed to mill dust. The forced expiratory volume in one second (F.E.V.1·0) was measured after control inhalations and after weevil and mixed flour inhalations. Significant reductions of 20% and 15·4% were found in two subjects after inhalation of weevil extract. In one case wheezing and cough developed. The changes in F.E.V.1·0 were reversed after inhalation of a bronchodilator aerosol.
Twenty-five of the control subjects with positive skin responses to the grain weevil were given similar provocation inhalations but none showed any significant change in F.E.V.1·0.
This pilot study suggests that grain weevil sensitivity is an additional factor in millworkers' asthma.
To improve their nutrition, most plants associate with soil microorganisms, particularly fungi, to form mycorrhizae. A few lineages, including actinorhizal plants and legumes are also able to interact with nitrogen-fixing bacteria hosted intracellularly inside root nodules. Fossil and molecular data suggest that the molecular mechanisms involved in these root nodule symbioses (RNS) have been partially recycled from more ancient and widespread arbuscular mycorrhizal (AM) symbiosis. We used a comparative transcriptomics approach to identify genes involved in establishing these 3 endosymbioses and their functioning. We analysed global changes in gene expression in AM in the actinorhizal tree C. glauca. A comparison with genes induced in AM in Medicago truncatula and Oryza sativa revealed a common set of genes induced in AM. A comparison with genes induced in nitrogen-fixing nodules of C. glauca and M. truncatula also made it possible to define a common set of genes induced in these three endosymbioses. The existence of this core set of genes is in accordance with the proposed recycling of ancient AM genes for new functions related to nodulation in legumes and actinorhizal plants.
Using nitrogen-fixing Sinorhizobium species that interact with Medicago plants as a model system, we aimed at clarifying how sex has shaped the diversity of bacteria associated with the genus Medicago on the interspecific and intraspecific scales. To gain insights into the diversification of these symbionts, we inferred a topology that includes the different specificity groups which interact with Medicago species, based on sequences of the nodulation gene cluster. Furthermore, 126 bacterial isolates were obtained from two soil samples, using Medicago truncatula and Medicago laciniata as host plants, to study the differentiation between populations of Sinorhizobium medicae, Sinorhizobium meliloti bv. meliloti, and S. meliloti bv. medicaginis. The former two can be associated with M. truncatula (among other species of Medicago), whereas the last organism is the specific symbiont of M. laciniata. These bacteria were characterized using a multilocus sequence analysis of four loci, located on the chromosome and on the two megaplasmids of S. meliloti. The phylogenetic results reveal that several interspecific horizontal gene transfers occurred during the diversification of Medicago symbionts. Within S. meliloti, the analyses show that nod genes specific to different host plants have spread to different genetic backgrounds through homologous recombination, preventing further divergence of the different ecotypes. Thus, specialization to different host plant species does not prevent the occurrence of gene flow among host-specific biovars of S. meliloti, whereas reproductive isolation between S. meliloti bv. meliloti and S. medicae is maintained even though these bacteria can cooccur in sympatry on the same individual host plants.
Medicago truncatula Gaertn. (barrel medic) is cultivated as a pasture legume for its high protein content and ability to improve soils through nitrogen fixation. Toxic concentrations of the micronutrient Boron (B) in agricultural soils hamper the production of cereal and leguminous crops. In cereals, the genetic analysis of B tolerance has led to the development of molecular selection tools to introgress and maintain the B tolerance trait in breeding lines. There is a comparable need for selection tools in legumes that grow on these toxic soils, often in rotation with cereals.
Genetic variation for B tolerance in Medicago truncatula was utilised to generate two F2 populations from crosses between tolerant and intolerant parents. Phenotyping under B stress revealed a close correlation between B tolerance and biomass production and a segregation ratio explained by a single dominant locus. M. truncatula homologues of the Arabidopsis major intrinsic protein (MIP) gene AtNIP5;1 and the efflux-type transporter gene AtBOR1, both known for B transport, were identified and nearby molecular markers screened across F2 lines to verify linkage with the B-tolerant phenotype. Most (95%) of the phenotypic variation could be explained by the SSR markers h2_6e22a and h2_21b19a, which flank a cluster of five predicted MIP genes on chromosome 4. Three CAPS markers (MtBtol-1,-2,-3) were developed to dissect the region further. Expression analysis of the five predicted MIPs indicated that only MtNIP3 was expressed when leaf tissue and roots were assessed. MtNIP3 showed low and equal expression in the roots of tolerant and intolerant lines but a 4-fold higher expression level in the leaves of B-tolerant cultivars. The expression profile correlates closely with the B concentration measured in the leaves and roots of tolerant and intolerant plants. Whereas no significant difference in B concentration exists between roots of tolerant and intolerant plants, the B concentration in the leaves of tolerant plants is less than half that of intolerant plants, which further supports MtNIP3 as the best candidate for the tolerance trait-defining gene in Medicago truncatula.
The close linkage of the MtNIP3 locus to B toxicity tolerance provides a source of molecular selection tools to pasture breeding programs. The economical importance of the locus warrants further investigation of the individual members of the MIP gene cluster in other pasture and in grain legumes.
Medicago truncatula; Abiotic stress; Legume; Boron toxicity tolerance; Genetic analysis; MtNIP3; Gene cluster; Differential expression
In the present work, we examined the insecticidal effect of spinosad, against adults of the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae), the rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae), the confused flour beetle, Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) on wheat and the larger grain borer, Prostephanus truncatus (Horn) (Coleoptera: Bostrychidae) on maize. The dose rates used were 0.01, 0.1, 0.5 and 1 ppm. The bioassays were carried out at three temperatures, 20, 25 and 30°C and two relative humidity levels, 55 and 75%. Mortality of R. dominica and S. oryzae was high even at 0.01 ppm of spinosad, reaching 100% at 55% relative humidity and 30° after 21 days of exposure. Generally, mortality of R. dominica, increased with temperature while for S. oryzae mortality increased with temperature and with the decrease of relative humidity. Moreover, for S. oryzae, mortality was low at 20°C. In the case of T. confusum, mortality was low at doses between 0.01 and 0.5 ppm even after 21 days of exposure. At 1 ppm, mortality exceeded 90% only at 30°C and only after 21 days of exposure. Mortality of P. truncatus was low on maize treated with 0.01 ppm, but increasing the dose to 0.1 ppm resulted in > 87% mortality after 14 days of exposure. In several combinations tested, spinosad efficacy notably varied according to the temperature and humidity regimes. Of the species tested, R. dominica and P. truncatus were very susceptible to spinosad, followed by S. oryzae, while T. confusum was the least susceptible.
spinosad; wheat; maize; abiotic conditions; grain protectants; Rhyzopertha dominica; Sitophilus oryzae; Tribolium confusum; Prostephanus truncatus
Genome-wide identification and phylogenetic and syntenic comparison were performed for the genes responsible for phenylalanine ammonia lyase (PAL) and peroxidase A (POX A) enzymes in nine plant species representing very diverse groups like legumes (Glycine max and Medicago truncatula), fruits (Vitis vinifera), cereals (Sorghum bicolor, Zea mays, and Oryza sativa), trees (Populus trichocarpa), and model dicot (Arabidopsis thaliana) and monocot (Brachypodium distachyon) species. A total of 87 and 1045 genes in PAL and POX A gene families, respectively, have been identified in these species. The phylogenetic and syntenic comparison along with motif distributions shows a high degree of conservation of PAL genes, suggesting that these genes may predate monocot/eudicot divergence. The POX A family genes, present in clusters at the subtelomeric regions of chromosomes, might be evolving and expanding with higher rate than the PAL gene family. Our analysis showed that during the expansion of POX A gene family, many groups and subgroups have evolved, resulting in a high level of functional divergence among monocots and dicots. These results will act as a first step toward the understanding of monocot/eudicot evolution and functional characterization of these gene families in the future.
Gram-negative bacteria have evolved mechanisms to resist the bactericidal action of cationic antimicrobial peptides of the innate immune system and antibiotics such as polymyxin. The strategy involves the addition of the positively charged sugar 4-amino-4-deoxy-L-arabinose (Ara4N) to lipid A in their outer membrane. ArnA is a key enzyme in the Ara4N-lipid A modification pathway. It is a bifunctional enzyme catalyzing (1) the oxidative decarboxylation of UDP-glucuronic acid (UDP-GlcA) to the UDP-4′′-ketopentose [UDP-β-(L-threo-pentapyranosyl-4′′-ulose] and (2) the N-10-formyltetrahydrofolate-dependent formylation of UDP-Ara4N. Here we demonstrate that the transformylase activity of the Escherichia coli ArnA is contained in its 300 N-terminal residues. We designate it the ArnA transformylase domain and describe its crystal structure solved to 1.7 Å resolution. The enzyme adopts a bilobal structure with an N-terminal Rossmann fold domain containing the N-10-formyltetrahydrofolate binding site and a C-terminal subdomain resembling an OB fold. Sequence and structure conservation around the active site of ArnA transformylase and other N-10-formyltetrahydrofolate-utilizing enzymes suggests that the HxSLLPxxxG motif can be used to identify enzymes that belong to this family. Binding of an N-10-formyltetrahydrofolate analogue was modeled into the structure of ArnA based on its similarity with glycinamide ribonucleotide formyltransferase. We also propose a mechanism for the transformylation reaction catalyzed by ArnA involving residues N102, H104, and D140. Supporting this hypothesis, point mutation of any of these residues abolishes activity.
Insects thriving on nutritionally poor habitats have integrated mutualistic intracellular symbiotic bacteria (endosymbionts) in a bacteria-bearing tissue (the bacteriome) that isolates the endosymbionts and protects them against a host systemic immune response. Whilst the metabolic and physiological features of long-term insect associations have been investigated in detail over the past decades, cellular and immune regulations that determine the host response to endosymbionts and pathogens have attracted interest more recently.
To investigate bacteriome cellular specificities and weevil immune responses to bacteria, we have constructed and sequenced 7 cDNA libraries from Sitophilus oryzae whole larvae and bacteriomes. Bioinformatic analysis of 26,886 ESTs led to the generation of 8,941 weevil unigenes. Based on in silico analysis and on the examination of genes involved in the cellular pathways of potential interest to intracellular symbiosis (i.e. cell growth and apoptosis, autophagy, immunity), we have selected and analyzed 29 genes using qRT-PCR, taking into consideration bacteriome specificity and symbiosis impact on the host response to pathogens. We show that the bacteriome tissue accumulates transcripts from genes involved in cellular development and survival, such as the apoptotic inhibitors iap2 and iap3, and endosomal fusion and trafficking, such as Rab7, Hrs, and SNARE. As regards our investigation into immunity, we first strengthen the bacteriome immunomodulation previously reported in S. zeamais. We show that the sarcotoxin, the c-type lysozyme, and the wpgrp2 genes are downregulated in the S. oryzae bacteriome, when compared to aposymbiotic insects and insects challenged with E. coli. Secondly, transcript level comparison between symbiotic and aposymbiotic larvae provides evidence that the immune systemic response to pathogens is decreased in symbiotic insects, as shown by the relatively high expression of wpgrp2, wpgrp3, coleoptericin-B, diptericin, and sarcotoxin genes in aposymbiotic insects.
Library sequencing significantly increased the number of unigenes, allowing for improved functional and genetic investigations in the cereal weevil S. oryzae. Transcriptomic analyses support selective and local immune gene expression in the bacteriome tissue and uncover cellular pathways that are of potential interest to bacteriocyte survival and homeostasis. Bacterial challenge experiments have revealed that the systemic immune response would be less induced in a symbiotic insect, thus highlighting new perspectives on host immunity in long-term invertebrate co-evolutionary associations.
A cDNA clone encoding pectinmethylesterase of the rice weevil, Sitophilus oryzae (L.) has been isolated and sequenced. The cDNA clone was expressed in cultured insect cells and active pectinmethylesterase was purified from the culture medium, thus confirming that the cDNA encodes pectinmethylesterase. In situ hybridization indicated that the enzyme's transcript was present in the midgut. Weevils treated with tetracycline so that they lack genes of known symbiotic organisms still contained the pectinmethylesterase gene, indicating that the gene is encoded by the rice weevil genome. The rice weevil enzyme is most similar in sequence to bacterial pectinmethylesterases. Given this and the enzyme's apparently rather general absence from animal species, we suggest the possibility that this gene was transferred horizontally to an ancient weevil, possibly from a bacterial symbiont, and exists in Sitophilus species now as a result of that ancestral horizontal transfer.
pectinase; pectin esterase; polygalacturonase; gut; digestive enzyme; pectin; symbiont; Wolbachia; SOPE; gene transfer
Nitric oxide (NO) is a gaseous molecule that participates in numerous plant signalling pathways. It is involved in plant responses to pathogens and development processes such as seed germination, flowering and stomatal closure.Using a permeable NO-specific fluorescent probe and a bacterial reporter strain expressing the lacZ gene under the control of a NO-responsive promoter, we detected NO production in the first steps, during infection threads growth, of the Medicago truncatula–Sinorhizobium meliloti symbiotic interaction. Nitric oxide was also detected, by confocal microscopy, in nodule primordia.Depletion of NO caused by cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide), an NO scavenger, resulted in a significant delay in nodule appearance. The overexpression of a bacterial hmp gene, encoding a flavohaemoglobin able to scavenge NO, under the control of a nodule-specific promoter (pENOD20) in transgenic roots, led to the same phenotype. The NO scavenging resulting from these approaches provoked the downregulation of plant genes involved in nodule development, such as MtCRE1 and MtCCS52A. Furthermore, an Hmp-overexpressing S. meliloti mutant strain was found to be less competitive than the wild type in the nodulation process.Taken together, these results indicate that NO is required for an optimal establishment of the M. truncatula–S. meliloti symbiotic interaction.
flavohaemoglobin; MtCRE1; nitric oxide; nitrogen fixation; nodule initiation and organogenesis; Rhizobium legumes symbiosis
During our screening program for new agrochemicals from Chinese medicinal herbs, the essential oil of Illicium difengpi stem bark was found to possess strong insecticidal activities against the maize weevil, Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae) and red flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae). A total of 37 components of the essential oil of I. difengpi were identified. The main components of the essential oil were safrole (23.61%), linalool (12.93%), and germacrene D (5.35%). Bioactivities-directed chromatographic separation on repeated silica gel columns led to the isolation of two compounds: safrole and linalool. Safrole showed pronounced contact toxicity against both insect species and (LD50 = 8.54 for S. zeamais; 4.67 µg/adult for T. castaneum) and was more toxic than linalool (LD50 = 24.88 for S. zeamais; 8.12 µg/adult for T. castaneum). The essential oil acting against the two species of insects showed LD50 values of 13.83 and 6.33 µg/adult, respectively. Linalool also possessed strong fumigant toxicity against both insect species (LC50 = 10.02 for S. zeamais; 9.34 mg/L for T. castaneum) and was more toxic than safrole (LD50 = 32.96 and 38.25 mg/L), while the crude essential oil acting against the two species of insects showed LC50 values of 14.62 and 16.22 mg/L, respectively. These results suggest that the essential oil of I. difengpi stem bark and the two compounds may be used in grain storage to combat insect pests.
contact toxicity; fumigant; Sitophilus zeamais; Tribolium castaneum
The use of Bacillus thuringiensis as a biopesticide is a viable alternative for insect control since the insecticidal Cry proteins produced by these bacteria are highly specific; harmless to humans, vertebrates, and plants; and completely biodegradable. In addition to Cry proteins, B. thuringiensis produces a number of extracellular compounds, including S-layer proteins (SLP), that contribute to virulence. The S layer is an ordered structure representing a proteinaceous paracrystalline array which completely covers the surfaces of many pathogenic bacteria. In this work, we report the identification of an S-layer protein by the screening of B. thuringiensis strains for activity against the coleopteran pest Epilachna varivestis (Mexican bean beetle; Coleoptera: Coccinellidae). We screened two B. thuringiensis strain collections containing unidentified Cry proteins and also strains isolated from dead insects. Some of the B. thuringiensis strains assayed against E. varivestis showed moderate toxicity. However, a B. thuringiensis strain (GP1) that was isolated from a dead insect showed a remarkably high insecticidal activity. The parasporal crystal produced by the GP1 strain was purified and shown to have insecticidal activity against E. varivestis but not against the lepidopteran Manduca sexta or Spodoptera frugiperda or against the dipteran Aedes aegypti. The gene encoding this protein was cloned and sequenced. It corresponded to an S-layer protein highly similar to previously described SLP in Bacillus anthracis (EA1) and Bacillus licheniformis (OlpA). The phylogenetic relationships among SLP from different bacteria showed that these proteins from Bacillus cereus, Bacillus sphaericus, B. anthracis, B. licheniformis, and B. thuringiensis are arranged in the same main group, suggesting similar origins. This is the first report that demonstrates that an S-layer protein is directly involved in toxicity to a coleopteran pest.
Aphids are a major family of plant insect pests. Medicago truncatula and Acyrthosiphon pisum (pea aphid, PA) are model species with a suite of resources available to help dissect the mechanism underlying plant–aphid interactions. A previous study focused on monogenic and relatively strong resistance in M. truncatula to PA and other aphid species. In this study a moderate resistance to PA was characterized in detail in the M. truncatula line A17 and compared with the highly susceptible line A20 and the more resistant line Jester. The results show that PA resistance in A17 involves both antibiosis and tolerance, and that resistance is phloem based. Quantitative trait locus (QTL) analysis using a recombinant inbred line (RIL) population (n=114) from a cross between A17 and A20 revealed that one locus, which co-segregated with AIN (Acyrthosiphon-induced necrosis) on chromosome 3, is responsible for the reduction of aphid biomass (indicator of antibiosis) for both PA and bluegreen aphid (BGA, A. kondoi), albeit to a lesser degree for PA than BGA. Interestingly, two independent loci on chromosomes 5 and 3 were identified for the plant biomass reduction (indicator of plant tolerance) by PA and BGA, respectively, demonstrating that the plant’s tolerance response to these two closely related aphid species is distinct. Together with previously identified major resistant (R) genes, the QTLs identified in this study are powerful tools to understand fully the spectrum of plant defence against sap-sucking insects and provide opportunities for breeders to generate effective and sustainable strategies for aphid control.
Antibiosis; antixenosis; EPG; herbivory; hypersensitive response; necrosis; phloem; sap-sucking insect
Cowpea aphid (CPA; Aphis craccivora) is the most important insect pest of cowpea and also causes significant yield losses in other legume crops including alfalfa, beans, chickpea, lentils, lupins and peanuts. In many of these crops there is no natural genetic resistance to this sap-sucking insect or resistance genes have been overcome by newly emerged CPA biotypes.
In this study, we screened a subset of the Medicago truncatula core collection of the South Australian Research and Development Institute (SARDI) and identified strong resistance to CPA in a M. truncatula accession SA30199, compared to all other M. truncatula accessions tested. The biology of resistance to CPA in SA30199 plants was characterised compared to the highly susceptible accession Borung and showed that resistance occurred at the level of the phloem, required an intact plant and involved a combination of antixenosis and antibiosis. Quantitative trait loci (QTL) analysis using a F2 population (n = 150) from a cross between SA30199 and Borung revealed that resistance to CPA is controlled in part by a major quantitative trait locus (QTL) on chromosome 2, explaining 39% of the antibiosis resistance.
The identification of strong CPA resistance in M. truncatula allows for the identification of key regulators and genes important in this model legume to give effective CPA resistance that may have relevance for other legume crops. The identified locus will also facilitate marker assisted breeding of M. truncatula for increased resistance to CPA and potentially other closely related Medicago species such as alfalfa.
Antibiosis; Antixenosis; EPG; Herbivory; Sap-sucking insect; Phloem
Root extracts of Decalepis hamiltonii were tested for insecticidal activity against the stored products pests, Rhyzopertha domonica, Sitophilus oryzae, Stigobium pancieum, Tribolium castaneum and Callosobruchus chinensis, in residual and contact toxicity bioassays. Methanolic extract showed LC50 value of 0.14 mg/cm2 for all the test species in a filter paper residual bioassay. The extract was effective as a grain protectant for wheat and green gram. Reduction of F1 progeny was observed in treated grain stored for 3–4 months. The extract did not affect the germination of the treated grains. Our results indicate that methanolic extracts of D. hamiltonii has a potential to control stored product pests and could serve as a natural grain protectant.
Decalepis hamiltonii; Methanolic extract; Insecticidal activity; Insect pests; Grain protection
The grain weevil (Sitophilus granarius) caused symptoms of allergic rhinitis and asthma in two laboratory workers, one after exposure to weevil dust for only six months and the other after three years. The former worker also noticed sneezing when she was cooking with National Flour. Skin testing with extracts of the grain weevil faecal dust gave positive skin responses. The antibodies were successfully transferred by passive sensitization using the Prausnitz-Kustner technique.
One hundred patients attending an allergy clinic for seasonal hay fever symptoms were also tested. All reacted to skin testing to a high dilution of grass pollens, and 51 gave some response to extracts of Sitophilus granarius; in 18 the wheal size was at least 6 mm. in diameter. The significance of the positive responses in these 18 patients is at present unexplained.
One of the laboratory workers was so sensitive to weevil dust that she had to leave the laboratory; the other worker, by using an extractor fan and wearing a mask, could remain free of symptoms. Sitophilus granarius, like other insects, easily gives rise to inhalant allergic symptoms and may be a possible cause of baker's asthma which has not been previously considered.
Legumes (Leguminosae or Fabaceae) play a major role in agriculture. Transcriptomics studies in the model legume species, Medicago truncatula, are instrumental in helping to formulate hypotheses about the role of legume genes. With the rapid growth of publically available Affymetrix GeneChip Medicago Genome Array GeneChip data from a great range of tissues, cell types, growth conditions, and stress treatments, the legume research community desires an effective bioinformatics system to aid efforts to interpret the Medicago genome through functional genomics. We developed the Medicago truncatula Gene Expression Atlas (MtGEA) web server for this purpose.
The Medicago truncatula Gene Expression Atlas (MtGEA) web server is a centralized platform for analyzing the Medicago transcriptome. Currently, the web server hosts gene expression data from 156 Affymetrix GeneChip® Medicago genome arrays in 64 different experiments, covering a broad range of developmental and environmental conditions. The server enables flexible, multifaceted analyses of transcript data and provides a range of additional information about genes, including different types of annotation and links to the genome sequence, which help users formulate hypotheses about gene function. Transcript data can be accessed using Affymetrix probe identification number, DNA sequence, gene name, functional description in natural language, GO and KEGG annotation terms, and InterPro domain number. Transcripts can also be discovered through co-expression or differential expression analysis. Flexible tools to select a subset of experiments and to visualize and compare expression profiles of multiple genes have been implemented. Data can be downloaded, in part or full, in a tabular form compatible with common analytical and visualization software. The web server will be updated on a regular basis to incorporate new gene expression data and genome annotation, and is accessible at: http://bioinfo.noble.org/gene-atlas/.
The MtGEA web server has a well managed rich data set, and offers data retrieval and analysis tools provided in the web platform. It's proven to be a powerful resource for plant biologists to effectively and efficiently identify Medicago transcripts of interest from a multitude of aspects, formulate hypothesis about gene function, and overall interpret the Medicago genome from a systematic point of view.
Previous studies have identified two distinct O-methyltransferases (OMTs) implicated in isoflavonoid biosynthesis in Medicago species, a 7-OMT methylating the A-ring 7-hydroxyl of the isoflavone daidzein and a 4’-OMT methylating the B-ring 4’-hydroxyl of 2,7,4′-trihydroxyisoflavanone. Genes related to these OMTs from the model legume Medicago truncatula cluster as separate branches of the type I plant small molecule OMT family. To better understand the possible functions of these related OMTs in secondary metabolism in M. truncatula, seven of the OMTs were expressed in E. coli, purified, and their in vitro substrate preferences determined. Many of the enzymes display promiscuous activities, and some exhibit dual regio-specificity for the 4′ and 7-hydroxyl moieties of the isoflavonoid nucleus. Protein structure homology modeling was used to help rationalize these catalytic activities. Transcripts encoding the different OMT genes exhibited differential tissue-specific and infection- or elicitor-induced expression, but not always in parallel with changes in expression of confirmed genes of the isoflavonoid pathway. The results are discussed in relation to the potential in vivo functions of these OMTs based on our current understanding of the phytochemistry of M. truncatula, and the difficulties associated with gene annotation in plant secondary metabolism.
Isoflavonoid; O-methyltransferase; Secondary metabolism; Molecular modeling; Gene family