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Publikation

Fu, N.; Becker, T.; Brandt, W.; Kunert, M.; Burse, A.; Boland, W.; Involvement of CYP347W1 in neurotoxin 3‐nitropropionic acid based chemical defense in mustard leaf beetle Phaedon cochleariae Insect Sci. 29, 453–466, (2022) DOI: 10.1111/1744-7917.12944

Chrysomelina beetles store 3-nitropropionic acid in form of a pre-toxin, isoxazolin-5-one glucoside conjugated ester, to protect themselves against predators. Here we identified a cytochrome P450 monooxygenase, CYP347W1, to be involved in the production of the 3-nitropropionic acid moiety of the isoxazolin-5-one glucoside ester. Knocking down CYP347W1 led to a significant depletion in the concentration of the isoxazolin-5-one glucoside ester and an increase in the concentration of the isoxazolin-5-one glucoside in the larval hemolymph. Enzyme assays with the heterologously expressed CYP347W1 showed free β-alanine was not the direct substrate. Homology modeling indicated that β-alanine-CoA ester can fit into CYP347W1’s active site. Furthermore, we proved that Phaedon cochleariae eggs are not able to de novo synthesize 3-NPA, although both isoxazolin-5-one glucoside and its 3-NPA conjugated ester are present in the eggs. These results provide direct evidence for the involvement of CYP347W1 in the biosynthesis of a P. cochleariae chemical defense compound.
Preprints

Fu, N.; Yang, Z.-l.; Pauchet, Y.; Paetz, C.; Brandt, W.; Boland, W.; Burse, A.; A cytochrome P450 from juvenile mustard leaf beetles hydroxylates geraniol, a key step in iridoid biosynthesis bioRxiv (2019) DOI: 10.1101/634485

Juveniles of the leaf beetle Phaedon cochleariae synthesize iridoid via the mevalonate pathway to repel predators. The normal terpenoid biosynthesis is integrated into the dedicated defensive pathway by the ω-hydroxylation of geraniol to 8-hydroxygeraniol. Here we identify and characterize the geraniol 8-hydroxylase as a P450 monooxygenase using integrated transcriptomic and proteomic analyses. In the fat body, 73 individual cytochrome P450s were identified. The double stranded RNA (dsRNA)-mediated knock down of CYP6BH5 led to a significant reduction of 8-hydroxygeraniol-glucoside in the hemolymph and, later, of the chrysomelidial in the defensive secretion. Heterologously expressed CYP6BH5 converted geraniol to 8-hydroxygeraniol. In addition to geraniol, CYP6BH5 also catalyzes other monoterpenols, such as nerol and citronellol, into the corresponding α, ω-dihydroxy compounds.
Publikation

Fu, N.; Yang, Z.-l.; Pauchet, Y.; Paetz, C.; Brandt, W.; Boland, W.; Burse, A.; A cytochrome P450 from the mustard leaf beetles hydroxylates geraniol, a key step in iridoid biosynthesis Insect Biochem. Mol. Biol. 113, 103212, (2019) DOI: 10.1016/j.ibmb.2019.103212

Larvae of the leaf beetle Phaedon cochleariae synthesize the iridoid chysomelidial via the mevalonate pathway to repel predators. The normal terpenoid biosynthesis is integrated into the dedicated defensive pathway by the ω-hydroxylation of geraniol to (2E,6E)-2,6-dimethylocta-2,6-diene-1,8-diol (ω-OH-geraniol). Here we identify and characterize the P450 monooxygenase CYP6BH5 as the geraniol hydroxylase using integrated transcriptomics, proteomics and RNA interference (RNAi). In the fat body, 73 cytochrome P450s were identified, and CYP6BH5 was among those that were expressed specifically in fat body. Double stranded RNA mediated knockdown of CYP6BH5 led to a significant reduction of ω-hydroxygeraniol glucoside in the hemolymph and, later, of the chrysomelidial in the defensive secretion. Heterologously expressed CYP6BH5 converted geraniol to ω-OH-geraniol. In addition to geraniol, CYP6BH5 also catalyzes hydroxylation of other monoterpenols, such as nerol and citronellol to the corresponding α,ω-dihydroxy compounds.
Publikation

Wang, D.; Pentzold, S.; Kunert, M.; Groth, M.; Brandt, W.; Pasteels, J. M.; Boland, W.; Burse, A.; A subset of chemosensory genes differs between two populations of a specialized leaf beetle after host plant shift Ecol. Evol. 8, 8055-8075, (2018) DOI: 10.1002/ece3.4246

Due to its fundamental role in shaping host selection behavior, we have analyzed the chemosensory repertoire of Chrysomela lapponica. This specialized leaf beetle evolved distinct populations which shifted from the ancestral host plant, willow (Salix sp., Salicaceae), to birch (Betula rotundifolia, Betulaceae). We identified 114 chemosensory candidate genes in adult C. lapponica: 41 olfactory receptors (ORs), eight gustatory receptors, 17 ionotropic receptors, four sensory neuron membrane proteins, 32 odorant binding proteins (OBPs), and 12 chemosensory proteins (CSP) by RNA‐seq. Differential expression analyses in the antennae revealed significant upregulation of one minus‐C OBP (ClapOBP27) and one CSP (ClapCSP12) in the willow feeders. In contrast, one OR (ClapOR17), four minus‐C OBPs (ClapOBP02, 07, 13, 20), and one plus‐C OBP (ClapOBP32) were significantly upregulated in birch feeders. The differential expression pattern in the legs was more complex. To narrow down putative ligands acting as cues for host discrimination, the relative abundance and diversity of volatiles of the two host plant species were analyzed. In addition to salicylaldehyde (willow‐specific), both plant species differed mainly in their emission rate of terpenoids such as (E,E)‐α‐farnesene (high in willow) or 4,8‐dimethylnona‐1,3,7‐triene (high in birch). Qualitatively, the volatiles were similar between willow and birch leaves constituting an “olfactory bridge” for the beetles. Subsequent structural modeling of the three most differentially expressed OBPs and docking studies using 22 host volatiles indicated that ligands bind with varying affinity. We suggest that the evolution of particularly minus‐C OBPs and ORs in C. lapponica facilitated its host plant shift via chemosensation of the phytochemicals from birch as novel host plant.
Publikation

Rahfeld, P.; Haeger, W.; Kirsch, R.; Pauls, G.; Becker, T.; Schulze, E.; Wielsch, N.; Wang, D.; Groth, M.; Brandt, W.; Boland, W.; Burse, A.; Glandular β-glucosidases in juvenile Chrysomelina leaf beetles support the evolution of a host-plant-dependent chemical defense Insect Biochem. Mol. Biol. 58, 28-38, (2015) DOI: 10.1016/j.ibmb.2015.01.003

Plant-feeding insects are spread across the entire plant kingdom. Because they chew externally on leaves, leaf beetle of the subtribe Chrysomelina sensu stricto are constantly exposed to life-threatening predators and parasitoids. To counter these pressures, the juveniles repel their enemies by displaying glandular secretions that contain defensive compounds. These repellents can be produced either de novo (iridoids) or by using plant-derived precursors. The autonomous production of iridoids pre-dates the evolution of phytochemical-based defense strategies. Both strategies include hydrolysis of the secreted non-toxic glycosides in the defensive exudates. By combining in vitro as well as in vivo experiments, we show that iridoid de novo producing as well as sequestering species rely on secreted β-glucosidases to cleave the pre-toxins. Our phylogenetic analyses support a common origin of chrysomeline β-glucosidases. The kinetic parameters of these β-glucosidases demonstrated substrate selectivity which reflects the adaptation of Chrysomelina sensu stricto to the chemistry of their hosts during the course of evolution. However, the functional studies also showed that the broad substrate selectivity allows building a chemical defense, which is dependent on the host plant, but does not lead to an “evolutionary dead end”.
Publikation

Frick, S.; Nagel, R.; Schmidt, A.; Bodemann, R. R.; Rahfeld, P.; Pauls, G.; Brandt, W.; Gershenzon, J.; Boland, W.; Burse, A.; Metal ions control product specificity of isoprenyl diphosphate synthases in the insect terpenoid pathway Proc. Natl. Acad. Sci. U.S.A. 110, 4194-4199, (2013) DOI: 10.1073/pnas.1221489110

Isoprenyl diphosphate synthases (IDSs) produce the ubiquitous branched-chain diphosphates of different lengths that are precursors of all major classes of terpenes. Typically, individual short-chain IDSs (scIDSs) make the C10, C15, and C20 isoprenyl diphosphates separately. Here, we report that the product length synthesized by a single scIDS shifts depending on the divalent metal cofactor present. This previously undescribed mechanism of carbon chain-length determination was discovered for a scIDS from juvenile horseradish leaf beetles, Phaedon cochleariae. The recombinant enzyme P. cochleariae isoprenyl diphosphate synthase 1 (PcIDS1) yields 96% C10-geranyl diphosphate (GDP) and only 4% C15-farnesyl diphosphate (FDP) in the presence of Co2+ or Mn2+ as a cofactor, whereas it yields only 18% C10 GDP but 82% C15 FDP in the presence of Mg2+. In reaction with Co2+, PcIDS1 has a Km of 11.6 μM for dimethylallyl diphosphate as a cosubstrate and 24.3 μM for GDP. However, with Mg2+, PcIDS1 has a Km of 1.18 μM for GDP, suggesting that this substrate is favored by the enzyme under such conditions. RNAi targeting PcIDS1 revealed the participation of this enzyme in the de novo synthesis of defensive monoterpenoids in the beetle larvae. As an FDP synthase, PcIDS1 could be associated with the formation of sesquiterpenes, such as juvenile hormones. Detection of Co2+, Mn2+, or Mg2+ in the beetle larvae suggests flux control into C10 vs. C15 isoprenoids could be accomplished by these ions in vivo. The dependence of product chain length of scIDSs on metal cofactor identity introduces an additional regulation for these branch point enzymes of terpene metabolism.
Publikation

Burse, A.; Frick, S.; Schmidt, A.; Buechler, R.; Kunert, M.; Gershenzon, J.; Brandt, W.; Boland, W.; Implication of HMGR in homeostasis of sequestered and de novo produced precursors of the iridoid biosynthesis in leaf beetle larvae Insect Biochem. Mol. Biol. 38, 76-88, (2008) DOI: 10.1016/j.ibmb.2007.09.006

Insects employ iridoids to deter predatory attacks. Larvae of some Chrysomelina species are capable to produce those cyclopentanoid monoterpenes de novo. The iridoid biosynthesis proceeds via the mevalonate pathway to geranyl diphospate (GDP) subsequently converted into 8-hydroxygeraniol-8-O-β-d-glucoside followed by the transformation into the defensive compounds. We tested whether the glucoside, its aglycon or geraniol has an impact on the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key regulatory enzyme of the mevalonate pathway and also the iridoid biosynthesis. To address the inhibition site of the enzyme, initially a complete cDNA encoding full length HMGR was cloned from Phaedon cochleariae. Its catalytic portion was then heterologously expressed in Escherichia coli. Purification and characterization of the recombinant protein revealed attenuated activity in enzyme assays by 8-hydroxygeraniol whereas no effect has been observed by addition of the glucoside or geraniol. Thus, the catalytic domain is the target for the inhibitor. Homology modeling of the catalytic domain and docking experiments demonstrated binding of 8-hydroxygeraniol to the active site and indicated a competitive inhibition mechanism. Iridoid producing larvae are potentially able to sequester glucosidically bound 8-hydroxygeraniol whose cleavage of the sugar moiety results in 8-hydroxygeraniol. Therefore, HMGR may represent a regulator in maintenance of homeostasis between de novo produced and sequestered intermediates of iridoid metabolism. Furthermore, we demonstrated that HMGR activity is not only diminished in iridoid producers but most likely prevalent within the Chrysomelina subtribe and also within the insecta.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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