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Hans Merzendorfer - One of the best experts on this subject based on the ideXlab platform.
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a chymotrypsin like serine protease interacts with the chitin synthase from the midgut of the tobacco Hornworm
The Journal of Experimental Biology, 2007Co-Authors: Gunnar Broehan, Lars Zimoch, Anton Wessels, Beyhan Ertas, Hans MerzendorferAbstract:The chitin portion of the peritrophic matrix in the midgut of the tobacco Hornworm, Manduca sexta, is produced by chitin synthase 2 (CHS2), a transmembrane family II glycosyltransferase, located at the apical tips of brush border microvilli. To look for proteins that potentially interact with CHS2, we performed yeast two-hybrid screening, identifying a novel chymotrypsin-like protease (CTLP1) that binds to the extracellular carboxyterminal domain of CHS2. The occurrence of this interaction in vivo is supported by co-localization and co-immunoprecipitation data. Based on our findings we propose that chitin synthesis is controlled by an intestinal proteolytic signalling cascade linking chitin synthase activity to the nutritional state of the larvae.
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immunolocalization of chitin synthase in the tobacco Hornworm
Cell and Tissue Research, 2002Co-Authors: Lars Zimoch, Hans MerzendorferAbstract:To start investigation of chitin synthesis and peritrophic membrane formation in the midgut of Manduca sexta, we have cloned a cDNA fragment encoding chitin synthase. Northern blots with a corresponding RNA probe revealed a single transcript of 4.7 kb, which was most prominent in poly(A) RNA isolated from the anterior and median midgut as well as from tracheal cells. In situ hybridization showed that the amount of chitin synthase transcripts in the cytoplasm of columnar cells decreased from the anterior to the posterior midgut. Moreover, in the anterior midgut they were localized in the apical region of columnar cells. Southern blots suggested more than one gene locus for chitin synthase in the Manduca genome. To analyze the distribution of chitin synthases on the protein level, we expressed a polymerase chain reaction (PCR) fragment of 119 amino acids in Escherichia coli and generated polyclonal antibodies to the purified recombinant protein. In immunoblots of crude extracts derived from the anterior midgut as well as from partially purified brush border membranes of columnar cells the affinity-purified anti-chitin synthase antiserum labeled a single protein with an apparent molecular mass of 150–200 kDa. Immunohistochemistry showed intense labeling in midgut brush border membranes. Immunofluorescence was restricted to the apical ends of microvilli. Apical membranes of salivary glands and tracheal cells were labeled as well, but not those of Malpighian tubules. This is the first time that chitin synthase expression has been visualized in insect tissues on the level of proteins.
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the plasma membrane h v atpase from tobacco Hornworm midgut
Journal of Bioenergetics and Biomembranes, 1999Co-Authors: Helmut Wieczorek, Gerhard Gruber, William R Harvey, Markus Huss, Hans MerzendorferAbstract:The midgut plasma membrane V-ATPase from larval Manduca sexta,the tobacco Hornworm, is the sole energizer of any epithelial ion transportin this tissue and is responsible for the alkalinization of the gut lumen upto a pH of more than 11. This mini-review deals with those topics of researchon this enzyme which may have contributed or are expected to contribute noveland general aspects to the field of V-ATPases. Topics dealt with includenovel subunits or the quaternary structure of the V1 complex, aswell as the regulation of the enzyme's function by reversible dissociation ofthe V1 from the V0 complexes and by genetic control onthe transcriptional and posttranscriptional level.
Ian T Baldwin - One of the best experts on this subject based on the ideXlab platform.
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herbivory induced volatiles function as defenses increasing fitness of the native plant nicotiana attenuata in nature
eLife, 2012Co-Authors: Meredith C Schuman, Kathleen Barthel, Ian T BaldwinAbstract:As the population of the world continues to increase beyond 7 billion, and agricultural pests continue to rapidly evolve resistance to pesticides, it is becoming ever more important to cultivate arable land in a way that is sustainable for both humans and the environment. A better understanding of the different mechanisms used by wild plants to deter herbivores will help to increase crop production without harming the environment. Plants use both direct and indirect methods to fend off herbivores. Direct defense methods include the production of chemicals that are toxic to herbivores or give them indigestion, and the growth of sticky prickles and spines that can injure or kill the herbivore. Indirect defense methods, on the other hand, generally rely on the plant attracting organisms that are either predators or parasites of the herbivore. Plants produce odors known as herbivory-induced plant volatiles (HIPVs) that are thought to offer indirect defense against herbivores by betraying their location to predators and parasites. However, HIPVs also influence other members of the ecological community, sometimes in ways that are detrimental to plants. Moreover, despite 30 years of research, no study has demonstrated that HIPVs increase the fitness of a plant, so it is unclear what they have evolved to do. Now, a 2-year field study by Schuman et al. has shown plants that emit green leaf volatiles (which are a type of HIPV) produce twice as many buds and flowers—a measure of fitness—as plants that have been genetically engineered not to emit green leaf volatiles. This study was conducted with Nicotiana attenuata, which is a wild tobacco plant that is often targeted by Manduca sexta, a type of moth that is also known as the tobacco Hornworm. Green leaf volatiles only increased plants' fitness when various species of Geocoris—a bug that preys on Manduca sexta—reduced the number of herbivores by a factor of two. This is the first evidence that HIPVs offer indirect defense against herbivores. Schuman et al. also studied the effects of molecules called protease inhibitors that are thought to function as direct defenses by making it difficult for herbivores to digest plants. They found that the ability to produce protease inhibitors did not increase the fitness of plants under herbivore attack; however, tobacco Hornworms that had been fed plants containing protease inhibitors were found to be more sluggish in response to attack, which suggests that protease inhibitors can enhance the indirect defenses of plants. The results suggest that employing both direct and indirect defenses—such as a combination of biological pesticides and genetic engineering to produce both HIPVs and protease inhibitors—is the best approach for defending agricultural plants against pests.
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silencing geranylgeranyl diphosphate synthase in nicotiana attenuata dramatically impairs resistance to tobacco Hornworm
Plant Physiology, 2008Co-Authors: Amir Reza Jassbi, Klaus Gase, Axel Schmidt, Christian Hettenhausen, Ian T BaldwinAbstract:In bioassays with artificial diets, the 17-hydroxygeranyllinalool diterpenoid glycosides (HGL-DTGs) of Nicotiana attenuata function as antifeedants for the plant's adapted herbivore, tobacco Hornworm (Manduca sexta). To determine whether HGL-DTGs have a defensive function in planta, we suppressed HGL-DTG production by silencing the source of the geranylgeranyl diphosphates (GGPPs) required for geranyllinalool biosynthesis, a key intermediate. We used virus-induced gene silencing to suppress transcript levels of GGPP synthase gene (Naggpps) and farnesyl diphosphate (FPP) synthase gene (Nafpps), northern blotting and real-time polymerase chain reaction to quantify transcript accumulations, and radio gas chromatography to analyze prenyltransferase specificity. Silencing Nafpps had no effect on the accumulation of HGL-DTGs but decreased leaf steroid content, demonstrating that DTG-synthesizing enzymes do not use GGPP derived from FPP and confirming FPP's role as a steroid precursor. Unlike plants silenced in the phytoene desaturase gene (Napds), which rapidly bleached, Naggpps-silenced plants had reduced HGL-DTG but not carotenoids or chlorophyll contents, demonstrating that Naggpps supplies substrates for GGPP biosynthesis for HGL-DTGs, but not for phytoene or phytol. Expression of Naggpps in Escherichia coli revealed that the recombinant protein catalyzes the GGPP synthesis from isopentenyl diphosphate and dimethylallyl diphosphate. When fed on silenced plants, Hornworm larvae gained up to 3 times more mass than those that fed on empty vector control plants or plants silenced in Nafpps, the trypsin protease inhibitor gene, or the putrescine N-methyltransferase gene. We conclude that HGL-DTGs or other minor undetected diterpenoids derived from GGPP function as direct defenses for N. attenuata and are more potent than nicotine or trypsin protease inhibitors against attack by Hornworm larvae.
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herbivore induced plant vaccination part i the orchestration of plant defenses in nature and their fitness consequences in the wild tobacco nicotiana attenuata
Plant Journal, 2004Co-Authors: Andre Kessler, Ian T BaldwinAbstract:A plant's responses to attack from particular pathogens and herbivores may result in resistance to subsequent attack from the same species, but may also affect different species. Such a cross-resistance, called immunization or vaccination, can benefit the plant, if the fitness consequences of attack from the initial attacker are less than those from subsequent attackers. Here, we report an example of naturally occurring vaccination of the native tobacco plant, Nicotiana attenuata, against Manduca Hornworms by prior attack from the mirid bug, Tupiocoris notatus (Dicyphus minimus), which results from the elicitation of two categories of induced plant responses. First, attack from both herbivore species causes the plants in nature to release predator-attracting volatile organic compounds (VOCs), and the attracted generalist predator, Geocoris pallens, preferentially attacks the less mobile Hornworm larvae. Second, attack from both mirids and Hornworms increases the accumulation of secondary metabolites and proteinase inhibitors (PIs) in the leaf tissue, which is correlated with the slow growth of Manduca larvae. Mirid damage does not significantly reduce the fitness of the plant in nature, whereas attack from the Hornworm reduces lifetime seed production. Consequently, plants that are attacked by mirids realize a significant fitness advantage in environments with both herbivores. The combination of growth-slowing direct defenses and predator-attracting indirect defenses results in greater Hornworm mortality on mirid-attacked plants and provides the mechanism of the vaccination phenomenon.
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manipulation of endogenous trypsin proteinase inhibitor production in nicotiana attenuata demonstrates their function as antiherbivore defenses
Plant Physiology, 2004Co-Authors: Jorge A Zavala, Klaus Gase, Aparna G Patankar, Ian T BaldwinAbstract:Evidence for the in planta defensive function of trypsin protease inhibitors (TPIs) comes from observations of enhanced herbivore resistance after heterologous TPI expression or the manipulation of signal cascades that activate numerous defense responses, including TPI production; no studies have altered the expression of an endogenous pi gene to examine defensive function. We isolated two genes with seven- and six-repeat TPI domains from Nicotiana attenuata from the potato (Solanum tuberosum) PI-II family. To determine whether endogenous TPIs in N. attenuata function defensively against the native herbivores, Hornworm (Manduca sexta) and mirids (Tupiocoris notatus), we expressed 175 bp of the seven-domain pi from N. attenuata in an antisense orientation in a TPI-producing genotype to reduce TPI expression and expressed the full-length seven-domain pi in a sense orientation under control of a constitutive promoter to restore TPI activity in a natural genotype from Arizona unable to produce TPIs. Constitutive and inducible TPI production in two antisense lines were diminished by 80% to 90% and 33% to 52%, respectively, and sense expression restored 67% of the activity found in the TPI-producing genotype after caterpillar attack in the TPI-deficient A genotype. Hornworm larvae fed on genotypes with low or no TPI activity grew faster, had higher survivorship, and produced heavier pupae than those that fed on genotypes with high TPI activity. T. notatus showed higher preference for genotypes with low or no TPI activity than for genotypes with high TPI levels. We conclude that endogenous TPIs are an effective defense against these native herbivores.
Lee A. Bulla - One of the best experts on this subject based on the ideXlab platform.
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cry1a toxins of bacillus thuringiensis bind specifically to a region adjacent to the membrane proximal extracellular domain of bt r1 in manduca sexta involvement of a cadherin in the entomopathogenicity of bacillus thuringiensis
Insect Biochemistry and Molecular Biology, 2002Co-Authors: J A Dorsch, Ratna K Vadlamudi, Mehmet Candas, Natalya B Griko, W S A Maaty, Eric G Midboe, Lee A. BullaAbstract:Abstract Many subspecies of the soil bacterium Bacillus thuringiensis produce various parasporal crystal proteins, also known as Cry toxins, that exhibit insecticidal activity upon binding to specific receptors in the midgut of susceptible insects. One such receptor, BT-R1 (210 kDa), is a cadherin located in the midgut epithelium of the tobacco Hornworm, Manduca sexta. It has a high binding affinity ( K d ∼ 1 nM) for the Cry1A toxins of B. thuringiensis. Truncation analysis of BT-R1 revealed that the only fragment capable of binding the Cry1A toxins of B. thuringiensis was a contiguous 169-amino acid sequence adjacent to the membrane-proximal extracellular domain. The purified toxin-binding fragment acted as an antagonist to Cry1Ab toxin by blocking the binding of toxin to the tobacco Hornworm midgut and inhibiting insecticidal action. Exogenous Cry1Ab toxin bound to intact COS-7 cells expressing BT-R1 cDNA, subsequently killing the cells. Recruitment of BT-R1 by B. thuringiensis indicates that the bacterium interacts with a specific cell adhesion molecule during its pathogenesis. Apparently, Cry toxins, like other bacterial toxins, attack epithelial barriers by targeting cell adhesion molecules within susceptible insect hosts.
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cloning and expression of a receptor for an insecticidal toxin of bacillus thuringiensis
Journal of Biological Chemistry, 1995Co-Authors: Ratna K Vadlamudi, Eric Weber, Lee A. BullaAbstract:Environmentally friendly toxins of Bacillus thuringiensis are effective in controlling agriculturally and biomedically harmful insects. However, little is known about the insect receptor molecules that bind these toxins and the mechanism of insecticidal activity. We report here for the first time the cloning and expression of a cDNA that encodes a receptor (BT-R1) of the tobacco Hornworm Manduca sexta for an insecticidal toxin of B. thuringiensis. The receptor is a 210-kDa membrane glycoprotein that specifically binds the cryIA(b) toxin of B. thuringiensis subsp. berliner and leads to death of the Hornworm. BT-R1 shares sequence similarity with the cadherin superfamily of proteins.
Lars Zimoch - One of the best experts on this subject based on the ideXlab platform.
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a chymotrypsin like serine protease interacts with the chitin synthase from the midgut of the tobacco Hornworm
The Journal of Experimental Biology, 2007Co-Authors: Gunnar Broehan, Lars Zimoch, Anton Wessels, Beyhan Ertas, Hans MerzendorferAbstract:The chitin portion of the peritrophic matrix in the midgut of the tobacco Hornworm, Manduca sexta, is produced by chitin synthase 2 (CHS2), a transmembrane family II glycosyltransferase, located at the apical tips of brush border microvilli. To look for proteins that potentially interact with CHS2, we performed yeast two-hybrid screening, identifying a novel chymotrypsin-like protease (CTLP1) that binds to the extracellular carboxyterminal domain of CHS2. The occurrence of this interaction in vivo is supported by co-localization and co-immunoprecipitation data. Based on our findings we propose that chitin synthesis is controlled by an intestinal proteolytic signalling cascade linking chitin synthase activity to the nutritional state of the larvae.
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immunolocalization of chitin synthase in the tobacco Hornworm
Cell and Tissue Research, 2002Co-Authors: Lars Zimoch, Hans MerzendorferAbstract:To start investigation of chitin synthesis and peritrophic membrane formation in the midgut of Manduca sexta, we have cloned a cDNA fragment encoding chitin synthase. Northern blots with a corresponding RNA probe revealed a single transcript of 4.7 kb, which was most prominent in poly(A) RNA isolated from the anterior and median midgut as well as from tracheal cells. In situ hybridization showed that the amount of chitin synthase transcripts in the cytoplasm of columnar cells decreased from the anterior to the posterior midgut. Moreover, in the anterior midgut they were localized in the apical region of columnar cells. Southern blots suggested more than one gene locus for chitin synthase in the Manduca genome. To analyze the distribution of chitin synthases on the protein level, we expressed a polymerase chain reaction (PCR) fragment of 119 amino acids in Escherichia coli and generated polyclonal antibodies to the purified recombinant protein. In immunoblots of crude extracts derived from the anterior midgut as well as from partially purified brush border membranes of columnar cells the affinity-purified anti-chitin synthase antiserum labeled a single protein with an apparent molecular mass of 150–200 kDa. Immunohistochemistry showed intense labeling in midgut brush border membranes. Immunofluorescence was restricted to the apical ends of microvilli. Apical membranes of salivary glands and tracheal cells were labeled as well, but not those of Malpighian tubules. This is the first time that chitin synthase expression has been visualized in insect tissues on the level of proteins.
John Kemper - One of the best experts on this subject based on the ideXlab platform.
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influence of plant allelochemicals on the tobacco Hornworm and its parasitoid cotesia congregata
Ecology, 1991Co-Authors: Pedro Barbosa, Paul S. Gross, John KemperAbstract:Experiments were conducted to determine if three plant defenses affected the parasitoid Cotesia congregate in a fashion that would influence herbivore-parasitoid interactions. We evaluated the developmental rate, size, and survival of the parasitoid Cotesia congregate, when reared from Manduca sexta fed on diets containing either nic- otine, rutin, or hordenine. These results were compared to the effects of the same three chemicals on unparasitized M. sexta. The influences of the three plant defenses differed. In general, however, the effects of these allelochemicals on the parasitoid paralleled those on the unparasitized Hornworm. The one major exception was that concentrations of nicotine that had little or no effect on the Hornworm caused significant mortality of par- asitoids. Neither rutin nor hordenine had large or consistent negative effects, although rutin had a pronounced effect on both Hornworm and parasitoid developmental times.