Sugar Transporters Enable a Leaf Beetle to Accumulate Plant Defense Compounds

ARTICLE https://doi.org/10.1038/s41467-021-22982-8 OPEN Sugar transporters enable a leaf beetle to accumulate plant defense compounds Zhi-Ling Yang 1, Hussam Hassan Nour-Eldin 2, Sabine Hänniger 3, Michael Reichelt 4, ✉ Christoph Crocoll 2, Fabian Seitz1, Heiko Vogel 3 & Franziska Beran 1 Many herbivorous insects selectively accumulate plant toxins for defense against predators; however, little is known about the transport processes that enable insects to absorb and store 1234567890():,; defense compounds in the body. Here, we investigate how a specialist herbivore, the horseradish flea beetle, accumulates glucosinolate defense compounds from Brassicaceae in the hemolymph. Using phylogenetic analyses of coleopteran major facilitator superfamily transporters, we identify a clade of glucosinolate-specific transporters (PaGTRs) belonging to the sugar porter family. PaGTRs are predominantly expressed in the excretory system, the Malpighian tubules. Silencing of PaGTRs leads to elevated glucosinolate excretion, significantly reducing the levels of sequestered glucosinolates in beetles. This suggests that PaGTRs reabsorb glucosinolates from the Malpighian tubule lumen to prevent their loss by excretion. Ramsay assays corroborated the selective retention of glucosinolates by Mal- pighian tubules of P. armoraciae in situ. Thus, the selective accumulation of plant defense compounds in herbivorous insects can depend on the ability to prevent excretion. 1 Research Group Sequestration and Detoxification in Insects, Max Planck Institute for Chemical Ecology, Jena, Germany. 2 Department of Plant and Environmental Sciences, Faculty of Science, DynaMo Center, University of Copenhagen, Frederiksberg, Denmark. 3 Department of Entomology, Max Planck Institute for Chemical Ecology, Jena, Germany. 4 Department of Biochemistry, Max Planck Institute for Chemical Ecology, Jena, Germany. ✉ email: [email protected] NATURE COMMUNICATIONS | (2021) 12:2658 | https://doi.org/10.1038/s41467-021-22982-8 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22982-8 hysiological and chemosensory adaptations of herbivorous in vitro. Our results suggest that transporter-mediated reab- insects to plant defense compounds have played an sorption of glucosinolates in the Malpighian tubules enables P – important role in the evolution of insect plant interactions P. armoraciae to sequester high amounts of glucosinolates in the and insect host range1–4. Among the most remarkable insect hemolymph. adaptations is the ability to selectively accumulate (sequester) plant defense compounds for protection against generalist Results 5–7 predators . Indeed, it is increasingly recognized that predator Selection of candidate transporters. To elucidate the molecular pressure has promoted insect adaptations to plant defenses and basis of GTR in P. armoraciae, we generated a gut- and Mal- 7–10 driven the evolution of specialized host plant associations . pighian tubule-specific transcriptome. In this transcriptome, we Sequestration evolved in all major clades of herbivorous insects predicted a total of 1401 putative membrane transporters using and is particularly widespread in the megadiverse phytophagous the transporter automatic annotation pipeline (TransAAP)35.Of 11 Coleoptera and Lepidoptera ; however, the mechanisms by these, 353 are putative members of the MFS (Transporter Clas- which sequestering insects can accumulate ingested plant defense sification (TC)# 2.A.1; Supplementary Data 1), which are known compounds in their body remain poorly understood. to transport a broad spectrum of substrates including sugars and Glucosinolates are hydrophilic defense compounds produced amino acids across membranes36,37. β by plants of the order Brassicales that, together with the plant - We selected MFS transporters as our candidates and thioglucosidase enzyme myrosinase, constitute an activated two- additionally assumed that gene duplications have played a role 12–14 component defense system . When tissue damage brings both in the evolution of GTR activity in P. armoraciae. To detect components together, glucosinolates are rapidly hydrolyzed to species-specific expansions of MFS transporters, we annotated 15 unstable aglucones that give rise to different products , of which MFS transporters in the publicly available genomes of three non- 16–19 isothiocyanates are the most detrimental to small herbivores . sequestering beetle species (Supplementary Data 2). By phyloge- Some insects can prevent glucosinolate hydrolysis and accumu- netic analysis, we identified the largest P. armoraciae-specific 20–25 late ingested glucosinolates in the body . A defensive function clade comprising 21 putative sugar porters (TC# 2.A.1.1) for of glucosinolate sequestration has been demonstrated in specialist further study (Supplementary Data 3). Members of this clade fl aphids and ea beetles, which convergently evolved insect myr- share between 31 and 93% amino acid sequence identity and are osinases enabling glucosinolate activation in response to predator most similar to insect trehalose transporters. attack21,23,26. The turnip sawfly, Athalia rosae, sequesters glucosinolates but does not possess its own myrosinase activity, making Functional characterization of candidate transporters.We a role in defense less likely27,28. Instead, a rapid absorption of expressed the candidate transporters in High Five insect cells and ingested glucosinolates across the gut epithelium was proposed to screened them for glucoside import activity. In addition to glu- constitute a detoxification mechanism as it spatially separates cosinolates, we tested different types of nonhost glucosides as glucosinolates from the co-ingested plant myrosinase in the gut substrates, namely iridoid, cyanogenic, and phenolic glucosides lumen28,29. Due to their physicochemical properties, transport of (Fig. 1a). Insect cells expressing 15 different transporters accu- glucosinolates and other plant glucosides such as cyanogenic and mulated significantly higher levels of at least one tested glucoside phenolic glucosides is proposed to be mediated by membrane compared to control cells. Of these, 13 transporters were specific carriers6,23,29. For example, Strauss et al.30 demonstrated the role for glucosinolates (Fig. 1b, c), hereafter referred to as P. armor- of an ATP-binding cassette (ABC) transporter in the sequestra- aciae GTR (PaGTR) 1 to 13. Most PaGTRs showed broad and tion of the phenolic glucoside salicin in the defense glands of the overlapping substrate spectra; for example, ten different PaGTRs poplar leaf beetle, Chrysomela populi. More recently, Kowalski used 2-propenyl glucosinolate, the major glucosinolate in the host et al.31 identified an ABC transporter in the dogbane leaf beetle, plant of P. armoraciae, as a substrate. The other two transporters Chrysochus auratus, with high activity towards a plant cardeno- mediated iridoid glucoside uptake into insect cells, whereas lide, which adult beetles sequester in defense glands. Apart from phenolic and cyanogenic glucosides were not used as substrates these examples, no other membrane transporters involved in by any of the recombinant transporters (Fig. 1c and Supple- sequestration have been identified to date. mentary Fig. 1). We previously investigated the sequestration of glucosinolates in the flea beetle Phyllotreta armoraciae25,26. This species is fi PaGTR monophagous on horseradish in nature, but feeds on various Tissue-speci c expression of s. To determine the locali- brassicaceous plants in the laboratory32–34 and sequesters glu- zation of PaGTRsinP. armoraciae, we compared the transcript cosinolates mainly in the hemolymph25. Phyllotreta armoraciae levels in foregut, midgut, hindgut, Malpighian tubules, and other prefers to sequester aliphatic glucosinolates over indolic glucosi- tissues by quantitative PCR. All except two PaGTRs were speci- fi nolates; however, the sequestration pattern depends both on the cally expressed in the Malpighian tubules (Fig. 1d and Supple- levels and composition of glucosinolates in the food plant. mentary Fig. 2). Along the tubule, PaGTRs were predominantly – Interestingly, the beetles also selectively excrete sequestered glu- expressed in the proximal region close to the midgut hindgut cosinolates to balance the accumulation of new glucosinolates and junction (Fig. 1e and Supplementary Fig. 2d). This Malpighian maintain stable glucosinolate levels ~35 nmol/mg beetle fresh tubule region was previously shown to reabsorb metabolites to 38–40 weight25. Based on these findings, we hypothesize that glucosi- prevent their loss by excretion . nolate transporters (GTRs) are localized in the gut and Mal- pighian tubule epithelial membranes in P. armoraciae. Function of PaGTRs in vivo. The Malpighian tubules are bathed Here, we use a comparative phylogenetic approach to identify in hemolymph, the major storage site for sequestered glucosi- candidate GTR genes in the major facilitator superfamily (MFS). nolates in P. armoraciae. We analyzed the glucosinolate levels We focus on a P. armoraciae-specific clade comprising 21 puta- in the hemolymph and detected total concentrations of up to tive sugar porters, which are predominantly expressed in the 106 mM (Supplementary Table 1). Thus, the question arises what Malpighian tubules, and demonstrate glucosinolate-specific role PaGTR-mediated glucosiolate transport in Malpighian import activity for 13 transporters. We investigate the role of tubules plays in the storage

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