Phyllotreta Striolata Flea Beetles Use Host Plant Defense Compounds to Create Their Own Glucosinolate-Myrosinase System
Total Page：16
File Type：pdf, Size：1020Kb
Phyllotreta striolata flea beetles use host plant defense compounds to create their own glucosinolate-myrosinase system Franziska Berana,1, Yannick Paucheta, Grit Kunertb, Michael Reicheltb, Natalie Wielschc, Heiko Vogela, Andreas Reinecked,2, Aleš Svatošc, Inga Mewise, Daniela Schmidf, Srinivasan Ramasamyg, Christian Ulrichsh, Bill S. Hanssond, Jonathan Gershenzonb, and David G. Heckela Departments of aEntomology, bBiochemistry, cMass Spectrometry, fBioorganic Chemistry, and dEvolutionary Neuroethology, Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; hUrban Plant Ecophysiology, Humboldt-Universität zu Berlin, 14195 Berlin, Germany; eQuality, Leibniz Institute of Vegetable and Ornamental Crops Grossbeeren/Erfurt e.V., 14979 Grossbeeren, Germany; and gEntomology, AVRDC–The World Vegetable Center, Tainan 74151, Taiwan Edited by May R. Berenbaum, University of Illinois at Urbana–Champaign, Urbana, IL, and approved April 4, 2014 (received for review November 20, 2013) The ability of a specialized herbivore to overcome the chemical leading to the formation of isothiocyanates (ITCs), thiocyanates, defense of a particular plant taxon not only makes it accessible as nitriles, and epithionitriles (6). Due to their chemical properties, a food source but may also provide metabolites to be exploited for ITCs are the most reactive hydrolysis products of glucosinolates communication or chemical defense. Phyllotreta flea beetles are (6, 9). Although several generalist lepidopterans detoxify ITCs via adapted to crucifer plants (Brassicales) that are defended by the conjugation with glutathione (10), the specialists investigated so glucosinolate-myrosinase system, the so-called “mustard-oil bomb.” far circumvent ITC formation (7, 11, 12). Several adapted herbi- Tissue damage caused by insect feeding brings glucosinolates into vores sequester intact host plant glucosinolates (13–16), and two contact with the plant enzyme myrosinase, which hydrolyzes them aphid species, Brevicoryne brassicae and Lipaphis erysimi, not only to form toxic compounds, such as isothiocyanates. However, we sequester glucosinolates but also convert these glucosinolates to Phyllotreta striolata ECOLOGY previously observed that beetles themselves toxic ITCs by using their own myrosinase, encoded in the aphid produce volatile glucosinolate hydrolysis products. Here, we show – P. striolata genome (17 20). that adults selectively accumulate glucosinolates from Flea beetles of the genus Phyllotreta (Coleoptera: Chrys- their food plants to up to 1.75% of their body weight and express omelidae) are closely associated with glucosinolate-containing plants their own myrosinase. By combining proteomics and transcriptomics, in the Brassicaceae, Resedaceae, Tropaeolaceae, and Capparaceae a gene responsible for myrosinase activity in P. striolata was identi- on which they feed throughout their life cycle (21–23). In fact, the fied. The major substrates of the heterologously expressed myrosinase only confirmed exception is Phyllotreta vittula, which prefers Poaceae were aliphatic glucosinolates, which were hydrolyzed with at least fourfold higher efficiency than aromatic and indolic glucosinolates, but also accepts crucifers as food plants (24). As specialized herbi- and β-O-glucosides. The identified beetle myrosinase belongs to the vores, Phyllotreta flea beetles are expected to be adapted to the glycoside hydrolase family 1 and has up to 76% sequence similarity to glucosinolate-myrosinase system, but the mechanism for such tol- other β-glucosidases. Phylogenetic analyses suggest species-specific erance is not known for any crucifer-feeding Coleoptera to date. diversification of this gene family in insects and an independent evo- Intact glucosinolates stimulate feeding of Phyllotreta spp., and thus lution of the beetle myrosinase from other insect β-glucosidases. Significance convergent evolution | host plant specialization Associations of plants and herbivores are regarded as the result hytophagous insects are confronted with an arsenal of con- of coevolution, which has produced an astonishing diversity of Pstitutive and inducible chemical plant defenses (1). To avoid plant defenses and corresponding insect counteradaptations. We deleterious effects from plant toxins, herbivores require appro- focus on the leaf beetle Phyllotreta striolata, which is adapted to priate biochemical adaptations allowing them to deal with these the glucosinolate-myrosinase system present in its cruciferous metabolites, for example, by developing insensitive target sites or host plants. We show that P. striolata adults not only selectively by enzymatic detoxification. Many specialized insects are able to sequester intact glucosinolates from their host plants but also sequester plant defense compounds and exploit them for their express their own myrosinase, a member of the β-glucosidase own protection against natural enemies and/or as semiochemicals family capable of hydrolyzing glucosinolates to form toxic deg- in interspecific communication (2, 3). radation products. Our results reveal the convergent evolution of The glucosinolate-myrosinase system characteristic of plants in a glucosinolate-myrosinase system in P. striolata that enables this the order Brassicales is one of the best-studied activated plant herbivore to use glucosinolate hydrolysis products for its own defense systems (4–6), and several different strategies for avoiding purposes. glucosinolate toxicity have been described in specialized insect Author contributions: F.B., Y.P., M.R., A.R., A.S., I.M., S.R., C.U., B.S.H., J.G., and D.G.H. de- herbivores of the orders Lepidoptera, Hemiptera, and Hymenop- signed research; F.B., Y.P., M.R., N.W., H.V., and D.S. performed research; F.B., Y.P., G.K., tera (7). Glucosinolates are a structurally diverse group of β-thio- M.R., N.W., and H.V. analyzed data; and F.B. and D.G.H. wrote the paper. glucoside-N-hydroxysulfates that are classified according to their The authors declare no conflict of interest. precursor amino acid as aliphatic, aromatic, or indolic glucosinolates This article is a PNAS Direct Submission. (6, 8). Plant myrosinases, ascorbate-dependent β-thioglucosidases Data deposition: The sequences reported in this paper have been deposited in the Gen- (EC 3.2.1.147) belonging to glycoside hydrolase family 1 (GH1), Bank database (accession nos. KF377828–KF377855). are stored separately from the glucosinolates and only come into 1To whom correspondence should be addressed. E-mail: [email protected]. contact with their substrate when the spatial compartmentalization 2Present address: Behavioural Ecology and Evolutionary Genetics, Max Planck Institute for is destroyed, for example, by herbivore feeding (6). The profile of Ornithology, 82319 Seewiesen, Germany. hydrolysis products then depends on glucosinolate structure, cel- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lular conditions, and the presence of so-called “specifier proteins” 1073/pnas.1321781111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1321781111 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 play a role in host acceptance (22), but genetically increased myr- 1400 R= osinase activity in Brassica rapa plants enhanced resistance to Phyl- A 1200 lotreta cruciferae (25). However, ITCs are suggested to play a role in B. juncea interspecific communication of Phyllotreta spp. because they were 1000 O S shown to enhance or synergize the response to the male-produced 800 A. thaliana aggregation pheromone in several species (26–29). In previous studies with Phyllotreta striolata (Coleoptera: Chrys- 600 B. rapa omelidae), we detected several unexpected ITCs in the volatile 400 S. alba emission of adults feeding on different Brassica host plants (30). For 200 example, we identified alkenyl ITCs to be present in the headspace OH of adult P. striolata feeding on Brassica oleracea var. italica, although 0 this plant was shown to accumulate methylsulfinylalkyl and indolic 800 glucosinolates but no alkenyl glucosinolates (Fig. S1A). We thus B hypothesized that the beetles themselves are able to emit these B. juncea 600 unexpected ITCs. Because the putative emission of volatile ITCs B. rapa relative to plant content [%] from feeding adult P. striolata was independent of the glucosinolate profile of the current food plant (30) (Fig. S1B), we investigated 400 R S Glc how these metabolites could be produced in beetles. Accumulation of glucosinolates per mg beetle Here, we demonstrate that P. striolata adults selectively se- N 200 - quester glucosinolates during feeding on host plants, and probably OSO3 are able to retain these previously accumulated glucosinolates when 0 feeding on a plant with a distinct glucosinolate profile. Hence, by 012345678 using their own endogenous myrosinase, the adults may be able to Day release bioactive hydrolysis products independent of the current host plants’ glucosinolate profile. A corresponding gene was cloned Fig. 1. P. striolata adults sequester a variety of glucosinolates from their and heterologously expressed, and the enzyme was analyzed for its host plants. Values are given as the amount per insect expressed relative to plant glucosinolate content (set to 100%) after 1, 3, and 7 d of feeding on B. activity toward different glucosinolate structures. We further used ± phylogenetic analyses to trace the evolution of this myrosinase in juncea, B. rapa, A. thaliana, and S. alba. All values are given as mean SEM. (A)