Cytochrome P450s from the fall armyworm (Spodoptera frugiperda): responses to plant allelochemicals and pesticides.

Spodoptera frugiperda is a polyphagous lepidopteran pest that encounters a wide range of toxic plant metabolites in its diet. The ability of this insect to adapt to its chemical environment might be explained by the action of major detoxification enzymes such as cytochrome P450s (or CYP). Forty-two sequences coding for P450s were identified and most of the transcripts were found to be expressed in the midgut, Malpighian tubules and fat body of S. frugiperda larvae. Relatively few P450s were expressed in the established cell line Sf9. In order to gain information on how these genes respond to different chemical compounds, larvae and Sf9 cells were exposed to plant secondary metabolites (indole, indole-3-carbinol, quercetin, 2-tridecanone and xanthotoxin), insecticides (deltamethrin, fipronil, methoprene, methoxyfenozide) or model inducers (clofibrate and phenobarbital). Several genes were induced by plant chemicals such as P450s from the 6B, 321A and 9A subfamilies. Only a few genes responded to insecticides, belonging principally to the CYP9A family. There was little overlap between the response in vivo measured in the midgut and the response in vitro in Sf9 cells. In addition, regulatory elements were detected in the promoter region of these genes. In conclusion, several P450s were identified that could potentially be involved in the adaptation of S. frugiperda to its chemical environment.

Gene expression profiling of Spodoptera frugiperda hemocytes and fat body using cDNA microarray reveals polydnavirus-associated variations in lepidopteran host genes transcript levels.

BACKGROUND: Genomic approaches provide unique opportunities to study interactions of insects with their pathogens. We developed a cDNA microarray to analyze the gene transcription profile of the lepidopteran pest Spodoptera frugiperda in response to injection of the polydnavirus HdIV associated with the ichneumonid wasp Hyposoter didymator. Polydnaviruses are associated with parasitic ichneumonoid wasps and are required for their development within the lepidopteran host, in which they act as potent immunosuppressive pathogens. In this study, we analyzed transcriptional variations in the two main effectors of the insect immune response, the hemocytes and the fat body, after injection of filter-purified HdIV. RESULTS: Results show that 24 hours post-injection, about 4% of the 1750 arrayed host genes display changes in their transcript levels with a large proportion (76%) showing a decrease. As a comparison, in S. frugiperda fat body, after injection of the pathogenic JcDNV densovirus, 8 genes display significant changes in their transcript level. They differ from the 7 affected by HdIV and, as opposed to HdIV injection, are all up-regulated. Interestingly, several of the genes that are modulated by HdIV injection have been shown to be involved in lepidopteran innate immunity. Levels of transcripts related to calreticulin, prophenoloxidase-activating enzyme, immulectin-2 and a novel lepidopteran scavenger receptor are decreased in hemocytes of HdIV-injected caterpillars. This was confirmed by quantitative RT-PCR analysis but not observed after injection of heat-inactivated HdIV. Conversely, an increased level of transcripts was found for a galactose-binding lectin and, surprisingly, for the prophenoloxidase subunits. The results obtained suggest that HdIV injection affects transcript levels of genes encoding different components of the host immune response (non-self recognition, humoral and cellular responses). CONCLUSION: This analysis of the host-polydnavirus interactions by a microarray approach indicates that the presence of HdIV induces, directly or indirectly, variations in transcript levels of specific host genes, changes that could be responsible in part for the alterations observed in the parasitized host physiology. Development of such global approaches will allow a better understanding of the strategies employed by parasites to manipulate their host physiology, and will permit the identification of potential targets of the immunosuppressive polydnaviruses.

T-DNA activation tagging as a tool to isolate regulators of a metabolic pathway from a genetically non-tractable plant species.

T-DNA activation tagging is a method used to generate dominant mutations in plants or plant cells by the insertion of a T-DNA which carries constitutive enhancer elements that can cause transcriptional activation of flanking plant genes. We applied this approach to the species Catharanthus roseus (L.) G. Don (Madagascar periwinkle), in an attempt to isolate regulators of genes that are involved in the biosynthesis of secondary metabolites of the terpenoid indole alkaloid (TIA) class. Several TIAs have pharmaceutically interesting activities, including the anti-tumour agents vincristine and vinblastine. The use of suspension-cultured cells enabled us to screen in a relatively easy way hundreds of thousands of T-DNA-tagged cells for resistance to a toxic substrate of one of the TIA biosynthetic enzymes: tryptophan decarboxylase. This screening yielded several interesting tagged cell lines. Further characterisation of one of the tagged cell lines led to the isolation of Orca3, a gene encoding an AP2/ERF-domain transcription factor that acts as a master regulator of primary and secondary metabolism. The T-DNA activation tagging results described in detail in this paper illustrate the usefulness of this approach to isolate regulators of a complex metabolic pathway from a genetically non-tractable plant species.

Nuclear factors GT-1 and 3AF1 interact with multiple sequences within the promoter of the Tdc gene from Madagascar periwinkle: GT-1 is involved in UV light-induced expression.

Plant secondary metabolites of the terpenoid indole alkaloid (TIA) class comprise several compounds with pharmaceutical applications. A key step in the TIA biosynthetic pathway is catalysed by the enzyme tryptophan decarboxylase (TDC), which channels the primary metabolite tryptophan into TIA metabolism. In Catharanthus roseus (Madagascar periwinkle), the Tdc gene is expressed throughout plant development. Moreover, Tdc gene expression is induced by external stress signals, such as fungal elicitor and UV light. In a previous study of Tdc promoter architecture in transgenic tobacco it was shown that the -538 to -112 region is a quantitative determinant for the expression level in different plant organs. Within this sequence one particular region (-160 to -99) was identified as the major contributor to basal expression and another region (-99 to -37) was shown to be required for induction by fungal elicitor. Here, the in vitro binding of nuclear factors to the -572 to -37 region is described. In extracts from tobacco and C. roseus, two binding activities were detected that could be identified as the previously described nuclear factors GT-1 and 3AF1, based on their mobility and binding characteristics. Both factors appeared to interact with multiple regions in the Tdc promoter. Mutagenesis of GT-1 binding sites in the Tdc promoter did not affect the basal or elicitor-induced expression levels. However, induction of the Tdc promoter constructs by UV light was significantly lower, thereby demonstrating a functional role for GT-1 in the induction of Tdc expression by UV light.

Forskolin induces the reorganization of extracellular matrix fibronectin and cytoarchitecture in 3T3-F442A adipocytes: its effect on fibronectin gene expression.

We studied the effect of forskolin on fibronectin and actin gene expression in 3T3-F442A adipogenic cell line. The structural organizations of extracellular matrix fibronectin and actin cytoskeleton were investigated in parallel. Immunofluorescence experiments showed that preadipocytes treated for 48 h with 10 microM forskolin exhibited an intensified network of both actin and fibronectin when compared to control. A similar picture was obtained with adipocytes given long-term exposure to forskolin. As determined by Western analysis, fibronectin protein levels were increased by 50-75% over control, both in preadipocytes and adipocytes. A parallel increase of fibronectin mRNA content was observed in forskolin-treated cells. In contrast, forskolin treatment of preadipocytes and adipocytes did not elicit any change in the steady-state level of either actin mRNA or protein. Nuclear run-on experiments showed that forskolin increased the fibronectin gene transcription rate but left that of the actin gene in adipocytes unchanged. These findings suggest the reorganization of the actin network in forskolin-treated adipocytes to be a consequence of fibronectin-enhanced biosynthesis and reorganization.

The antiglucocorticoid RU38486 is a potent accelerator of adipose conversion of 3T3-F442A cells.

We examined the effects of RU38486, a potent glucocorticoid and progestin antagonist, upon several aspects of 3T3-F442A adipocyte differentiation. RU38486 accelerated the onset of differentiation, as monitored by cell morphological changes, accumulation of lipid droplets and widespread increases in the rate of expression of several enzyme adipose markers and specific mRNAs. RU38486, at a maximal concentration of 1 microM, dramatically hastened the emergence of both fatty-acid synthetase (FAS) and glycerol-3-phosphate dehydrogenase (G3PDH) enzyme activities (550% and 450% above control values 4 days after confluence, respectively). RU38486 induction of G3PDH-specific activity ran parallel to an increase in G3PDH mRNA content (2.4-fold the control content 4 days after confluence). Moreover, RU38486-treated cells exhibited enhancement of adenylate cyclase sensitivity to both isoproterenol and ACTH (160% and 350% above control activities 8 days after confluence, respectively). While the level of expression of lipogenic markers reached similar values at the mature stage, RU38486 enabled cells to acquire hypersensitivity in terms of ACTH-stimulated adenylate cyclase activity. Similarly, adipsin gene expression was highly potentiated by the drug at day 15 post-confluence (5-fold the control value). RU38486 responsiveness observed in differentiating 3T3-F442A cells is dependent upon their prior developmental activation; none of the studied markers could be induced by the drug in the undifferentiating 3T3-C2 cell subclone. Finally, this antiglucocorticoid appears to be a useful tool for studies on adipose conversion in vitro; it could permit a re-evaluation of the role of glucocorticoids in the understanding of adipocyte development.

Decreased biosynthesis of actin and cellular fibronectin during adipose conversion of 3T3-F442A cells. Reorganization of the cytoarchitecture and extracellular matrix fibronectin.

Differentiation of 3T3-F442A cells was accompanied by changes in cell morphology, decreased synthesis and assembly of actin and fibronectin. The network of microfilament stress fibers detected with NBD-phallacidin was altered during adipose conversion of 3T3-F442A cells. Parallel to this, the disappearance of fibrillar bundles of extracellular matrix fibronectin was observed by immunofluorescence staining. The pericellular fibronectin content, detected by immunoblotting, strongly diminished during the differentiation process. An altered rate of biosynthesis of both proteins was also measured by [35S]-methionine pulse-labeling and immunoprecipitation. A 4-5-fold decrease in cellular fibronectin synthesis was observed in adipocytes compared to control preadipocytes. Conversely, non-differentiating 3T3-C2 control cells did not reorganize either the cytoskeletal architecture or the extracellular matrix fibronectin in the resting state. These results suggest that the decreased rate of biosynthesis of cell-associated fibronectin is correlated with that of actin. Moreover, both events can essentially be ascribed to differentiation.

Growth and differentiation of 3T3-F442A preadipocytes in three-dimensional gels of native collagen.

Three-dimensional gels of native type I collagen have been used as a substrate for growth and differentiation in 3T3 adipocyte precursors. Such hydrated lattices can support a sustained cell growth leading to several 10-fold increases in cell number within 2 weeks. During this period, the cells condense the hydrated collagen lattice to a tissue-like structure one-fourth of the area of the initial gel. From Days 10 to 12, the cells progressively exhibit morphological characteristics of adipocytes and accumulate lipid droplets as evidenced by Oil Red O staining. Lipoprotein lipase activity appears very early; between Days 8 and 22 it sharply increases 15-fold and then remains stable at a very high level (about 30 nmol/min/10(6) cells). The emergence of glycerophosphate dehydrogenase activity is delayed; it becomes detectable at Day 15 and progressively increases up to 700 nmol/min/10(6) cells at Days 35-40. Thus, this adipose tissue equivalent appears to be a potential model for studying adipocyte function.