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PubMed2004

Protein proteinase inhibitor genes in combat against insects, pests, and pathogens: natural and engineered phytoprotection.

The continual need to increase food production necessitates the development and application of novel biotechnologies to enable the provision of improved crop varieties in a timely and cost-effective way. A milestone in this field was the introduction of Bacillus thuringiensis (Bt) entomotoxic proteins into plants. Despite the success of this technology, there is need for development of alternative strategies of phytoprotection. Biotechnology offers sustainable solutions to the problem of pests, pathogens, and plant parasitic nematodes in the form of other insecticidal protein genes. A variety of genes, besides (Bt) toxins that are now available for genetic engineering for pest resistance are genes for vegetative insecticidal proteins, proteinase inhibitors, alpha-amylase inhibitors, and plant lectins. This review presents a comprehensive summary of research efforts that focus on the potential use and advantages of using proteinase inhibitor genes to engineer insect- and pest-resistance. Crop protection by means of PI genes is an important component of Integrated Pest Management programmes.

Animals↗
PubMed2006

Molecular analysis of poplar defense against herbivory: comparison of wound- and insect elicitor-induced gene expression.

In order to characterize defense responses of hybrid poplar (Populus trichocarpax P. deltoides), we profiled leaf transcript patterns elicited by wounding and by regurgitant from forest tent caterpillar (FTC; Malacosoma disstria), a Lepidopteran defoliator of poplars. Macroarrays were used to compare transcript profiles. Both FTC-regurgitant (FTC-R) and mechanical wounding with pliers elicited expression of a variety of genes, and for these genes our analysis indicated that these treatments induced qualitatively similar responses. Similarly, a comparison of responses of directly treated and systemically induced leaves indicated extensive overlap in the sets of induced genes. FTC-R was found to contain the insect-derived elicitor volicitin. The simulated herbivory treatments resulted in the induction of genes involved in poplar defense and secondary metabolism. We also identified wound-responsive genes with roles in primary metabolism, including a putative invertase, lipase, and acyl-activating enzyme; some of these genes may have roles in defense signaling. In addition, we found three unknown genes containing a ZIM motif which may represent novel transcription factors.

Animals↗
PubMed1998

Characterization of the P25 silk gene and associated insertion elements in Galleria mellonella.

Insect silk genes attract attention by their precise territorial and developmental regulations and extremely high expression rates. Our present investigations demonstrated that the P25 silk gene of Galleria mellonella is down-regulated by ecdysteroid hormones. The gene was identified within 5217 nucleotides (nt) of two genomic clones. In contrast to other silk genes, Galleria P25 lacks the canonical TATA box. Transcription is initiated within a region of three nucleotides that lie at the end of a capsite initiator sequence ACAGT and about 90 nt downstream from a CAAT box. A stretch of 32 nt with a core sequence CTTTT was detected in the 5' region of Galleria P25 as well as in the presumptive regulatory regions of all other silk genes that are expressed in the posterior silk gland. However, consensus sequences reported for the regulatory regions of Bombyx silk genes are not obvious in Galleria P25. The coding sequence of this gene included 654 nt, is interrupted by 4 introns, and ends in position +3369; a potential polyadenylation signal starts at +4382. The gene contains 3 copies of a short interspersed nuclear element (SINE), which are located in the upstream region (-833 to -579) and in the first (+542 to +840) and second (+2259 to +2556) introns. The repeat, which was named Gm1, occurs in some other Galleria genes and exhibits homology to Bm1 SINE of the silkworm and to a similar element of a spider. Another insertion of at least 150 nt and with loosely defined borders is present in the 3' untranslated region (UTR) of Galleria P25. It includes a box (+3453 to +3552) of 99 nt that is tentatively called Lep1 because it was disclosed also in some other Lepidoptera. Lep1 seems to represent the core region of insertion elements that occur in the genomes of lepidopteran insects in various species specific and region specific modifications.

Amino Acid Sequence↗
PubMed1998

Cloning of a shrimp (Metapanaeus ensis) cDNA encoding a nuclear receptor superfamily member: an insect homologue of E75 gene.

Degenerate primers were derived from the amino acid sequence in the DNA binding domain of the Drosophila ecdysone receptor (DmEcR). Several partial cDNAs were amplified from the shrimp epidermis by reverse transcription polymerase chain reaction (RT-PCR). One of these fragments shows the highest amino acid sequence homology to the insect ecdysone inducible gene E75. This partial cDNA was used as a probe to screen the swimming leg cDNA library of the shrimp, Metapenaeus ensis. A 3.6 kb cDNA clone was obtained. The longest open reading frame of this cDNA consists of 606 amino acids and its deduced amino acid sequence has all five domains typical of a nuclear receptor. The putative polyadenylation signal is located at about 400 bp 3' to the stop signal. The deduced amino acid sequence of this cDNA shows the highest identity to that of the E75A reported in Manduca sexta, Galleria melonella, Drosophila melanogaster, and Choristoneura fumiferana. Based on the amino acid sequence comparison, the shrimp nuclear receptor is considered the insect homologue of E75A. Northern blot analysis shows that the shrimp E75 is expressed in the epidermis, eyestalk and the nerve cord of the pre-molt shrimp. Moreover, E75 transcripts can be detected in the epidermal tissues of early pre-molt shrimp by in situ hybridization. To determine whether the shrimp could also express other E75s like the insects, 5' end RACE and RT-PCR were performed on epidermal cDNA of a single shrimp. Subcloning and DNA sequence determination of the PCR products confirmed the presence of two other forms of E75 (tentatively called E75C and E75D) in shrimp. By RT-PCR, different levels of E75 expression can be detected in the epidermis, nerve cord and the eyestalk of early pre-molt shrimp. In addition to the different levels of expression of the shrimp E75s in the epidermis, the pattern of their expression is also different during the molting cycle. This is the first report on the cloning of a shrimp nuclear receptor superfamily member.

Amino Acid Sequence↗
PubMed1996

Characterization of a strawberry gene for auxin-binding protein, and its expression in insect cells.

A gene encoding an auxin-binding protein (ABP1) was isolated from strawberry by screening a genomic library with an ABP1 cDNA from maize. It resembles ABP1 genes from other sources both in structure (four introns) and in the high level of homology of the deduced amino acid sequence of the mature protein encoded in exons 2-5. Exon 1, encoding mainly the non-conserved signal peptide, was identified by a reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Northern analysis indicated that ABP1 transcript levels were low during fruit development, but transcripts were detected by RT PCR at all stages of receptacle swelling (auxin-dependent) and ripening (inhibited by auxin), consistent with a role for ABP1 in auxin perception. Southern blot analysis indicated a small ABP1 gene family in octoploid cultivated strawberry, and four genes were identified by comparison of genomic and cDNA sequences. RT PCR was used to amplify the complete coding region for cloning as cDNA, and a recombinant baculovirus was constructed for the expression of strawberry ABP1 in insect cells. The coding region contains three consensus glycosylation sites, and multiple bands representing a range of glycoforms of the protein were detected on western blots of insect cell extracts. Only a single band was observed in extracts of tunicamycin-treated cells, and glycosylated protein yielded a unique N-terminal amino acid sequence, allowing determination of the signal peptide cleavage site.

Amino Acid Sequence↗
PubMed1995

Analysis of a vitellogenin gene of the mosquito, Aedes aegypti and comparisons to vitellogenins from other organisms.

A genomic clone of the Aedes aegypti vitellogenin A1 gene was sequenced including 2015 bp of 5' untranscribed sequence, 6369 bp of open reading frame interrupted by two introns, and a short 3' untranslated region. Primer extension was used to identify the transcription initiation site. The amino termini of the large and small subunits were located by N-terminal sequencing of vitellin purified from eggs. The length of the signal sequence and the position of the cleavage site between the two subunits were also determined. Three sequential imperfect repeats were found near the beginning of the small subunit. The sequence of the coding region appears to be polymorphic. Comparison of the signal sequences of seven insect vitellogenin genes revealed several conserved leucines, and a conserved position of an intron. However, the signal sequences are not conserved between these genes and the yolk protein genes of Cyclorraphid Dipteran insects. The cleavage sites between the small and large subunits in the vitellogenins of the mosquito, A. aegypti, sawfly, Athalia rosae, boll weevil, Anthonomus grandis, and silkworm, Bombyx mori are flanked by sequences rich in serine. Pairwise dot matrix analysis at the protein level showed that the mosquito, boll weevil and silkworm vitellogenins are significantly related with approx. 50% similarity. One region of the three insect vitellogenin genes, near the N-terminal of the large subunit, showed the highest levels of similarity, from 57.5 to 64.4%. The position of cysteines in insect vitellogenins is conserved, particularly in the C-terminus of the large subunit. Dot matrix comparison of the mosquito vitellogenin with that of Xenopus laevis and Caenorhabditis elegans showed much lower, but still significant degrees of relationship. Pairwise comparisons of the mosquito vitellogenin and the Drosophila melanogaster yolk proteins did not show significant similarities. Potential regulatory regions in the mosquito VgA1 gene were identified by comparison to regulatory elements known from other organisms, especially D. melanogaster, which could provide useful information for further functional analysis.

Aedes↗
PubMed2001

Location, nucleotide sequence, and regulation of the p51 late gene of the hz-1 insect virus: identification of a putative late regulatory element.

An Hz-1 insect virus (Hz-1V) late gene encoding, a predicted polypeptide of 51 kDa was isolated from a cDNA library and mapped to the HindIII-J region (40-44.6 map units) of the viral genome. The p51 gene was characterized by DNA sequence, Northern blot, and primer extension analyses. The 1,152 bp open reading frame (ORF) is transcribed as a 1.8 kb RNA between 8 and 18 h post-infection (hpi) with maximum expression at 12 hpi. Homology was not detected between the nucleotide sequence upstream of the p51 ORF and the baculovirus conserved late promoter element NTAAG. Primer extension analysis detected one major late transcription initiation site at -205 nucleotides relative to the start of the p51 ORF and seven minor late initiation sites at positions upstream of this primary site. Comparison of the upstream regulatory regions of the p51 gene and the Hz- 1V p34 late gene revealed a region of significant homology comprised of the 9 bp sequence TTATAGTAT. The primary p51 transcription initiation site and all p34 transcription initiation sites were mapped to different nucleotides within this nonanucleotide sequence. This 9 bp motif was not observed in the ORFs of these genes, and no significant homology was detected between this motif and the 5' regulatory regions of any other characterized genes. The results of our study suggest that this conserved sequence may serve an important role in the regulation of Hz-1V late genes.

Amino Acid Sequence↗
PubMed2000

Insect population control using a dominant, repressible, lethal genetic system.

A major modification to the sterile insect technique is described, in which transgenic insects homozygous for a dominant, repressible, female-specific lethal gene system are used. We demonstrate two methods that give the required genetic characteristics in an otherwise wild-type genetic background. The first system uses a sex-specific promoter or enhancer to drive the expression of a repressible transcription factor, which in turn controls the expression of a toxic gene product. The second system uses non-sex-specific expression of the repressible transcription factor to regulate a selectively lethal gene product. Both methods work efficiently in Drosophila melanogaster, and we expect these principles to be widely applicable to more economically important organisms.

Animals↗
PubMed2006

Rhythmic expression of the cycle gene in a hematophagous insect vector.

BACKGROUND: A large number of organisms have internal circadian clocks that enable them to adapt to the cyclic changes of the external environment. In the model organism Drosophila melanogaster, feedback loops of transcription and translation are believed to be crucial for the maintenance of the central pacemaker. In this mechanism the cycle (or bmal1) gene, which is constitutively expressed, plays a critical role activating the expression of genes that will later inhibit their own activity, thereby closing the loop. Unlike Drosophila, the molecular clock of insect vectors is poorly understood, despite the importance of circadian behavior in the dynamic of disease transmission. RESULTS: Here we describe the sequence, genomic organization and circadian expression of cycle in the crepuscular/nocturnal hematophagous sandfly Lutzomyia longipalpis, the main vector of visceral leishmaniasis in the Americas. Deduced amino acid sequence revealed that sandfly cycle has a C-terminal transactivation domain highly conserved among eukaryotes but absent in D. melanogaster. Moreover, an alternative form of the transcript was also identified. Interestingly, while cycle expression in Drosophila and other Diptera is constitutive, in sandflies it is rhythmic in males and female heads but constitutive in the female body. Blood-feeding, which causes down-regulation of period and timeless in this species, does not affect cycle expression. CONCLUSION: Sequence and expression analysis of cycle in L. longipalpis show interesting differences compared to Drosophila suggesting that hematophagous vector species might present interesting new models to study the molecular control of insect circadian clocks.

Amino Acid Sequence↗
PubMed2001

The mitochondrial cytochrome oxidase II gene in Bacillus stick insects: ancestry of hybrids, androgenesis, and phylogenetic relationships.

Sequencing of a cytochrome oxidase II (COII) gene fragment in Bacillus taxa provided evidence that the bisexual B. rossius is the maternal ancestor of the hybridogenetic B. rossius-grandii strains and revealed the same ancestry for both parthenogenetic hybrids: the diploid B. whitei (B. rossius/grandii grandii) and the triploid B. lynceorum (B. rossius/grandii grandii/atticus). Present data clearly demonstrate that all Bacillus unisexuals arose through asymmetrical hybridization events and realized a paraphyletic derivation from the B. rossius redtenbacheri subspecies. The invention of B. rossius mitochondrial DNA haplotypes in specimens with B. grandii grandii nuclear genomes revealed the occurrence of androgenesis in nature. Natural androgens represent a peculiar escape from hybridity and can help maintain the hybridogenetic system through the production of the fathering taxon via hybrid females. Results from the COII gene support the phyletic relationships among taxa suggested by previous taxonomical approaches, but also indicate a departure of B. grandii subspecies from the established taxonomy. Assuming the existence of a molecular clock, the evaluated substitution rate brings the splitting between B. rossius and B. grandii/B. atticus back to 22.79 +/- 2.65 myr before present, while the origin of hybrids appears to be much more recent (1.06 +/- 0.53 myr).

Androgens↗
PubMed1996

Immediate-early baculovirus vectors for foreign gene expression in transformed or infected insect cells.

Baculovirus expression vectors are used routinely for foreign gene expression and are under intense development as improved biological pesticides. Conventional baculovirus expression vectors are recombinant viruses that can express a foreign gene in insect cells under the control of the polyhedrin promoter, which provides high-level transcription during the very late phase of infection. For some applications, including foreign glycoprotein production and insect pest control, it might be advantageous to have baculovirus vectors that could express foreign gene products in uninfected cells or earlier after infection. To fulfill this need, we have constructed a new set of plasmids that can be used to clone and express foreign genes under the control of a baculovirus ie1 promoter, which is active in uninfected insect cells and throughout infection. We used a subset of these new plasmids to isolate recombinant baculoviruses containing various foreign genes and compared expression of these genes by the resulting immediate-early baculovirus vectors and by conventional baculovirus vectors. As expected, the immediate-early vectors began to express each foreign gene earlier in infection but, by 36-48 h postinfection, the conventional vectors had produced more of each foreign protein. Conventional baculovirus vectors also produced more enzymatic activity from two different procaryotic genes than the immediate-early baculovirus vectors. However, immediate-early vectors produced as much or more enzymatic activity from two different eucaryotic genes encoding secretory pathway proteins than the conventional vectors, even at 48 h postinfection. Hence, this report describes a new set of plasmids that can be used to clone and express foreign genes under the control of the baculovirus ie1 promoter and suggests that immediate-early baculovirus vectors might be as useful as conventional baculovirus expression vectors for producing biologically active eucaryotic secretory pathway proteins.

Animals↗
PubMed2006

Evolutionary consequences of dispersal ability in cactus-feeding insects.

Although gene flow is an important determinant of evolutionary change, the role of ecological factors such as specialization in determining migration and gene flow has rarely been explored empirically. To examine the consequences of dispersal ability and habitat patchiness on gene flow, migration rates were compared in three cactophagous longhorn beetles using coalescent analyses of mtDNA sequences. Analyses of covariance were used to identify the roles of dispersal ability and habitat distribution in determining migration patterns. Dispersal ability was a highly significant predictor of gene flow (p< 0.001), and was more important than any other factor. These findings predict that dispersal ability may be an import factor shaping both microevolutionary and macroevolutionary patterns; this prediction is borne out by comparisons of species diversity in cactus-feeding groups.

Analysis of Variance↗
PubMed1991

Characterization of a candidate gene for GP72, an insect stage-specific antigen of Trypanosoma cruzi.

The Trypanosoma cruzi insect stage-specific antigen GP72 was purified from epimastigotes and the amino acid sequences of peptide fragments determined. Oligonucleotides derived from these data were used to amplify and clone a cDNA sequence, which was used to isolate a full-length gene. All the sequenced peptides were encoded within the gene. The characteristics of the encoded 62,600-Da protein, including a potential amino-terminal signal sequence, a hydrophobic carboxy-terminus, and a large number of potential O-glycosylation sites, are consistent with the properties of GP72. No sequence homologies were found in searches of DNA and protein data banks. GP72 is encoded by a single pair of non-telomeric allelic genes.

Amino Acid Sequence↗
PubMed1995

Gut-specific transcriptional regulatory elements of the carboxypeptidase gene are conserved between black flies and Drosophila.

Millions of people die every year in the tropical world from diseases transmitted by hematophagous insects. Failure of conventional containment measures emphasizes the need for additional approaches, such as transformation of vector insects with genes that restrict vectorial capacity. The availability of an efficient promoter to drive foreign genes in transgenic insects is a necessary tool to test the feasibility of such approach. Here we characterize the putative promoter region of a black fly midgut carboxypeptidase gene and show that these sequences correctly direct the expression of a beta-glucuronidase reporter in Drosophila melanogaster. By histochemical staining and mRNA analysis, we found that the gene is expressed strongly and gut-specifically in the transgenic Drosophila. This gut-specific black fly carboxypeptidase promoter provides a valuable tool for the study of disease vectors.

Amino Acid Sequence↗
PubMed2002

JNK signaling pathway is required for efficient wound healing in Drosophila.

Efficient wound healing including clotting and subsequent reepithelization is essential for animals ranging from insects to mammals to recover from epithelial injury. It is likely that genes involved in wound healing are conserved through the phylogeny and therefore, Drosophila may be an useful in vivo model system to identify genes necessary during this process. Furthermore, epithelial movement during specific developmental processes, such as dorsal closure, ressembles of those seen in mammalian wound healing. As puckered (puc) gene is a target of the JUN N-terminal kinase signaling pathway during dorsal closure, we investigated puc gene expression during wound healing in Drosophila. We showed that puc gene expression is induced at the edge of the wound in epithelial cells and Jun kinase is phosphorylated in wounded epidermal tissues, suggesting that the JUN N-terminal kinase signaling pathway is activated by a signal produced by an epidermal wound. In the absence of the Drosophila c-Fos homologue, puc gene expression is no longer induced. Finally, impaired epithelial repair in JUN N-terminal kinase deficient flies demonstrates that the JUN N-terminal kinase signaling is required to initiate the cell shape change at the onset of the epithelial wound healing. We conclude that the embryonic JUN N-terminal kinase gene cassette is induced at the edge of the wound. In addition, Drosophila appears as a good in vivo model to study morphogenetic processes requiring epithelial regeneration such as wound healing in vertebrates.

Animals↗
PubMed2005

Regulation of an insect cytochrome P450 monooxygenase gene (CYP6B1) by aryl hydrocarbon and xanthotoxin response cascades.

Many organisms respond to toxic compounds in their environment by inducing regulatory networks controlling the expression and activity of cytochrome P450 monooxygenase (P450s) detoxificative enzymes. In particular, black swallowtail (Papilio polyxenes) caterpillars respond to xanthotoxin, a toxic phytochemical in their hostplants, by activating transcription of the CYP6B1 promoter via several regions located within 150 nt of the transcription initiation site. One such element is the xenobiotic response element to xanthotoxin (XRE-Xan) that lies upstream of consensus XRE-AhR (xenobiotic response element to the aryl hydrocarbon receptor) and OCT-1 (octamer-1 binding site) element known to be utilized in mammalian aryl hydrocarbon response cascades. Two-plasmid transfections conducted in Sf9 cells have indicated that XRE-Xan, XRE-AhR and a number of other proximal elements, but not OCT-1, are critical for basal as well as xanthotoxin- and benzo[alpha]pyrene-induced transcription of the CYP6B1 promoter. Four-plasmid transfections with vectors co-expressing the spineless (Ss) and tango (Tgo) proteins, the Drosophila melanogaster homologues of mammalian AhR and ARNT, have indicated that these proteins enhance basal expression of the CYP6B1 promoter but not the magnitude of its xanthotoxin and benzo[alpha]pyrene induction. Based on these results, we propose that these Drosophila transcription factors modulate basal expression of this promoter in a ligand-independent manner and attenuate its subsequent responses to planar aryl hydrocarbons (benzo[alpha]pyrene) and allelochemicals (xanthotoxin).

Animals↗
PubMed2004

A conserved transcript pattern in response to a specialist and a generalist herbivore.

Transcript patterns elicited in response to attack reveal, at the molecular level, how plants respond to aggressors. These patterns are fashioned both by inflicted physical damage as well as by biological components displayed or released by the attacker. Different types of attacking organisms might therefore be expected to elicit different transcription programs in the host. Using a large-scale DNA microarray, we characterized gene expression in damaged as well as in distal Arabidopsis thaliana leaves in response to the specialist insect, Pieris rapae. More than 100 insect-responsive genes potentially involved in defense were identified, including genes involved in pathogenesis, indole glucosinolate metabolism, detoxification and cell survival, and signal transduction. Of these 114 genes, 111 were induced in Pieris feeding, and only three were repressed. Expression patterns in distal leaves were markedly similar to those of local leaves. Analysis of wild-type and jasmonate mutant plants, coupled with jasmonate treatment, showed that between 67 and 84% of Pieris-regulated gene expression was controlled, totally or in part, by the jasmonate pathway. This was correlated with increased larval performance on the coronatine insensitive1 glabrous1 (coi1-1 gl1) mutant. Independent mutations in COI1 and GL1 led to a faster larval weight gain, but the gl1 mutation had relatively little effect on the expression of the insect-responsive genes examined. Finally, we compared transcript patterns in Arabidopis in response to larvae of the specialist P. rapae and to a generalist insect, Spodoptera littoralis. Surprisingly, given the complex nature of insect salivary components and reported differences between species, almost identical transcript profiles were observed. This study also provides a robustly characterized gene set for the further investigation of plant-insect interaction.

Animals↗