The goal of this study was to discover novel partners for perlecan, a major heparan sulfate proteoglycan of basement membranes, and to examine new interactions through which perlecan may influence cell behavior. We employed the yeast two-hybrid system and used perlecan domain V as bait to screen a human keratinocyte cDNA library. Among the strongest interacting clones, we isolated a ϳ1.6-kb cDNA insert that encoded extracellular matrix protein 1 (ECM1), a secreted glycoprotein involved in bone formation and angiogenesis. The sequencing of the clone revealed the existence of a novel splice variant that we name ECM1c. The interaction was validated by co-immunoprecipitation studies, using both cell-free systems and mammalian cells, and the specific binding site within each molecule was identified employing various deletion mutants. The C terminus of ECM1 interacted specifically with the epidermal growth factor-like modules flanking the LG2 subdomain of perlecan domain V. Perlecan and ECM1 were also co-expressed by a variety of normal and transformed cells, and immunohistochemical studies showed a partial expression overlap, particularly around dermal blood vessels and adnexal epithelia. ECM1 has been shown to regulate endochondral bone formation, stimulate the proliferation of endothelial cells, and induce angiogenesis. Similarly, perlecan plays an important role in chondrogenesis and skeletal development, as well as harboring pro-and anti-angiogenic activities. Thus, a physiological interaction could also occur in vivo during development and in pathological events, including tissue remodeling and tumor progression.
The two-spotted spider mite Tetranychus urticae is an extremely polyphagous crop pest. Alongside an unparalleled detoxification potential for plant secondary metabolites, it has recently been shown that spider mites can attenuate or even suppress plant defenses. Salivary constituents, notably effectors, have been proposed to play an important role in manipulating plant defenses and might determine the outcome of plant-mite interactions.Here, the proteomic composition of saliva from T. urticae lines adapted to various host plants-bean, maize, soy, and tomato-was analyzed using a custom-developed feeding assay coupled with nano-LC tandem mass spectrometry. About 90 putative T. urticae salivary proteins were identified. Many are of unknown function, and in numerous cases belonging to multimembered gene families. RNAseq expression analysis revealed that many genes coding for these salivary proteins were highly expressed in the proterosoma, the mite body region that includes the salivary glands. A subset of genes encoding putative salivary proteins was selected for whole-mount in situ hybridization, and were found to be expressed in the anterior and dorsal podocephalic glands. Strikingly, host plant dependent expression was evident for putative salivary proteins, and was further studied in detail by micro-array based genome-wide expression profiling. This meta-analysis revealed for the first time the salivary protein repertoire of a phytophagous chelicerate. The availability of this salivary proteome will assist in unraveling the molecular interface between phytophagous mites and their host plants, and may ultimately facilitate the development of mite-resistant crops. Furthermore, the technique used in this study is a time-and resourceefficient method to examine the salivary protein composition of other small arthropods for which saliva or salivary glands cannot be isolated easily. The family of spider mites (Chelicerata: Acari: Tetranychidae) comprises well over 1000 species, including several that are important pests on crops, and about 0.9 billion euro is being spent annually for their control worldwide (1, 2). These minute herbivores-about 0.5 mm in size-use their stylets to pierce leaf mesophyll cells and to inject saliva, after which they suck out the cytoplasm. This results in cell death visible as chlorotic spots sometimes accompanied by necrosis, and ultimately in leaf abscission (3,4). Among the spider mites, the From the ‡Laboratory
Pesticide resistance arises rapidly in arthropod herbivores, as can host plant adaptation, and both are significant problems in agriculture. These traits have been challenging to study as both are often polygenic and many arthropods are genetically intractable. Here, we examined the genetic architecture of pesticide resistance and host plant adaptation in the two-spotted spider mite, Tetranychus urticae, a global agricultural pest. We show that the short generation time and high fecundity of T. urticae can be readily exploited in experimental evolution designs for high-resolution mapping of quantitative traits. As revealed by selection with spirodiclofen, an acetyl-CoA decarboxylase inhibitor, in populations from a cross between a spirodiclofen resistant and a susceptible strain, and which also differed in performance on tomato, we found that a limited number of loci could explain quantitative resistance to this compound. These were resolved to narrow genomic intervals, suggesting specific candidate genes, including acetyl-CoA decarboxylase itself, clustered and copy variable cytochrome P450 genes, and NADPH cytochrome P450 reductase, which encodes a redox partner for cytochrome P450s. For performance on tomato, candidate genomic regions for response to selection were distinct from those responding to the synthetic compound and were consistent with a more polygenic architecture. In accomplishing this work, we exploited the continuous nature of allele frequency changes across experimental populations to resolve the existing fragmented T. urticae draft genome to pseudochromosomes. This improved assembly was indispensable for our analyses, as it will be for future research with this model herbivore that is exceptionally amenable to genetic studies. .
Arthropod herbivores cause dramatic crop losses, and frequent pesticide use has led to widespread resistance in numerous species. One such species, the two-spotted spider mite, Tetranychus urticae, is an extreme generalist herbivore and a major worldwide crop pest with a history of rapidly developing resistance to acaricides. Mitochondrial Electron Transport Inhibitors of complex I (METI-Is) have been used extensively in the last 25 years to control T. urticae around the globe, and widespread resistance to each has been documented. METI-I resistance mechanisms in T. urticae are likely complex, as increased metabolism by cytochrome P450 monooxygenases as well as a target-site mutation have been linked with resistance. To identify loci underlying resistance to the METI-I acaricides fenpyroximate, pyridaben and tebufenpyrad without prior hypotheses, we crossed a highly METI-I-resistant strain of T. urticae to a susceptible one, propagated many replicated populations over multiple generations with and without selection by each compound, and performed bulked segregant analysis genetic mapping. Our results showed that while the known H92R target-site mutation was associated with resistance to each compound, a genomic region that included cytochrome P450-reductase (CPR) was associated with resistance to pyridaben and tebufenpyrad. Within CPR, a single nonsynonymous variant distinguished the resistant strain from the sensitive one. Furthermore, a genomic region linked with tebufenpyrad resistance harbored a noncanonical member of the nuclear hormone receptor 96 (NHR96) gene family. This NHR96 gene does not encode a DNA-binding domain (DBD), an uncommon feature in arthropods, and belongs to an expanded family of 47 NHR96 proteins lacking DBDs in T. urticae. Our findings suggest that although cross-resistance to METI-Is involves known detoxification pathways, structural differences in METI-I acaricides have also resulted in resistance mechanisms that are compound-specific.
Carotenoids underlie many of the vibrant yellow, orange, and red colors in animals, and are involved in processes ranging from vision to protection from stresses. Most animals acquire carotenoids from their diets because de novo synthesis of carotenoids is primarily limited to plants and some bacteria and fungi. Recently, sequencing projects in aphids and adelgids, spider mites, and gall midges identified genes with homology to fungal sequences encoding de novo carotenoid biosynthetic proteins like phytoene desaturase. The finding of horizontal gene transfers of carotenoid biosynthetic genes to three arthropod lineages was unprecedented; however, the relevance of the transfers for the arthropods that acquired them has remained largely speculative, which is especially true for spider mites that feed on plant cell contents, a known source of carotenoids. Pigmentation in spider mites results solely from carotenoids. Using a combination of genetic approaches, we show that mutations in a single horizontally transferred phytoene desaturase result in complete albinism in the two-spotted spider mite, , as well as in the citrus red mite, Further, we show that phytoene desaturase activity is essential for photoperiodic induction of diapause in an overwintering strain of , consistent with a role for this enzyme in provisioning provitamin A carotenoids required for light perception. Carotenoid biosynthetic genes of fungal origin have therefore enabled some mites to forgo dietary carotenoids, with endogenous synthesis underlying their intense pigmentation and ability to enter diapause, a key to the global distribution of major spider mite pests of agriculture.
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