Supporting Information Beran et al. 10.1073/pnas.1523468113 SI Materials and Methods using the BLAST2GO PRO software suite v2.6.1 (https://www. Plants and Insects. Seeds of Brassica juncea cv. “Bau Sin” or blast2go.com/) as described in ref. 27. Brassica rapa cv. “Yo Tsai Ching” were purchased from Known- P. striolata You Seed Company. Field-collected Phyllotreta striolata adults Protein Extraction from . To establish the presence of were provided from the Entomology Unit of AVRDC–The farnesyl diphosphate synthase (FPPS) and sesquiterpene syn- World Vegetable Center in Shanhua, Taiwan. The import au- thase activity in P. striolata, crude protein extracts were pre- thorization for Brassica seeds and P. striolata adults to Germany pared separately from male and female adults with three biological was obtained under Directive 2008/61/EC. Beetles were reared replicates for each sex. Beetles were cold anesthetized by placing − on potted 3- to 5-wk-old B. juncea or B. rapa plants in a controlled them for 2 min at 20 °C. For each sample, 40 adults were – μ environment chamber (24 °C, 65% relative humidity, 14-h/10-h pooled (30 40 mg fresh weight) and homogenized in 350 L light/dark period). of extraction buffer [25 mM 3-(N-morpholino)-2-hydroxy- In September and October 2012, Phyllotreta armoraciae and propanesulfonic acid (MOPSO) (pH 7.2), 10% (vol/vol) glycerol] Psylliodes chrysocephala adults were collected from host plants supplemented with protease inhibitors (cOmplete EDTA-free; (Armoraciae rusticana and Brassica rapa subsp. rapa plants, re- Roche) using a 2-mL potter tissue grinder. After an initial cen- × spectively) in Laasdorf, Thuringia, Germany. Laboratory rearings trifugation step at 4 °C for 10 min at 16,000 g, the supernatant × of P. armoraciae and P. chrysocephala were established on potted was ultracentrifuged at 4 °C for 45 min at 100,000 g. The protein B. juncea and B. rapa plants, respectively, in a controlled envi- concentration in each sample was determined using the Bradford ronment chamber (conditions as described above). protein assay (Bio-Rad) according to the manufacturer’s instructions. Foliage of Abies nordmanniana for volatile collections was trans-IDS provided by the botanical garden in Jena, Germany. Identification of Putative Genes from Flea Beetles. Several putative trans-IDS genes were identified in transcriptomes of RNASeq and de Novo Transcriptome Assembly. The preparation of P. striolata (nine transcripts), P. armoraciae (eight transcripts), and the P. striolata transcriptome was described previously (27). P. chrysocephala (eight transcripts) based on homology to known RNASeq was performed with adult P. armoraciae and Psylliodes insect trans-IDS enzymes. Total RNA isolation and cDNA syn- chrysocephala flea beetles. Total RNA was extracted from whole thesis was carried out as described by Beran et al. (27). Because adults as well as from adults dissected into gut tissue and most transcripts in the database were truncated, we performed remaining body tissues using the InnuPREP RNA Mini Kit rapid amplification of cDNA ends–PCR (RACE-PCR) using (Analytik Jena). An additional sample was prepared from the Advantage 2 Polymerase mix according to the manufacturer’s P. chrysocephala beetles treated with a mixture of Gram-positive protocols (Clontech) to obtain the complete ORFs (primer se- and Gram-negative bacteria and fungi for 48 h to induce immune quences are listed in Dataset S1). RACE-PCR products were responses. RNA integrity was verified using an Agilent 2100 cloned into the TOPO TA 2.1 vector (Invitrogen) and sequenced. Bioanalyzer with the RNA 6000 Nano Kit (Agilent Technolo- The manually curated sequences have been deposited in the Gen- gies), and RNA quantity was determined using a Nanodrop ND- Bank database under accession numbers KT959237–KT959261. 1000 spectrophotometer. RNASeq was performed on the HiSeq 2500 Sequencing System from Illumina (www.illumina.com/) Cloning and Expression of Putative trans-IDS Genes from P. striolata. using paired end (2 × 100 bp) read technology with RNA frag- The putative trans-IDSs from P. striolata were amplified from mented to an average of 150 nt. Library construction and se- cDNAs by PCR (Phusion High-Fidelity DNA Polymerase; Fin- quencing was performed by the Max Planck Genome Center zymes), using gene-specific primers (primer sequences listed in ′ (Cologne, Germany) (mpgc.mpipz.mpg.de/home/). Illumina se- Dataset S1). The primers were designed to amplify a 5 -trun- quencing resulted in a total of ∼25 million reads for each of the cated sequence (Fig. S2) including the native stop codon, except three P. armoraciae and four P. chrysocephala tissue and treat- for PsTPS2, which was amplified full-length. Eukaryotic signal ment samples, respectively. Quality control including filtering of peptides may affect protein expression in E. coli and the activity high-quality reads based on the score value given in “fastq” files, of the recombinant enzymes as previously shown for IDSs (10). and trimming of read length was done using the CLC Genomics The PCR products were ligated into the expression vector Workbench software, version 7.0.1 (www.clcbio.com). After pET100/D-TOPO or pET200/D-TOPO (Invitrogen). Both vec- these initial filtering steps, 70 million total sequence reads were tors encode an N-terminal His-tag and only differ in the selec- used for each species for a de novo transcriptome assembly using tion marker (ampicillin and kanamycin, respectively). Full-length the CLC Genomics Workbench software by comparing an as- and 5′-truncated PsTPS1 including the native stop codon were sembly with standard settings and two additional CLC-based additionally cloned into the pET101/D-TOPO expression vector assemblies as described in more detail previously (27). Scaffolding for heterologous expression without His-tag. Constructs were was selected and conflicts among individual bases were resolved in checked by sequencing and transformed in Escherichia coli strain all assemblies by voting for the base with the highest frequency. BL21(DE3)pLysS (Life Technologies) for heterologous gene ex- Contigs shorter than 250 bp were removed from the final analysis. pression. A 20-mL preculture was inoculated from a single colony and incubated at 18 °C and 220 rpm using a Certomat IS orbital Annotation. Annotation of the consensus transcriptome used shaker (Sartorius) overnight. The Overnight Express Auto- BLAST searches conducted on a local server using the National induction System 1 (Novagen) was used for the 200-mL main Center for Biotechnology Information blastall program. Ho- culture. To start the protein production, the main culture was mology searches (BLASTx and BLASTn) of unique sequences inoculated with the preculture to an OD600 of 0.05 and then and functional annotation by gene ontology terms (GO) terms cultivated 3 d at 18 °C and 220 rpm on a Certomat IS orbital (geneontology.org/), InterPro terms (InterProScan, EBI), en- shaker (Sartorius). Afterward, the cells were harvested by centri- zyme classification codes (EC), and metabolic pathways (KEGG, fugation at 4 °C for 10 min at 4,000 × g. The pellets were re- Kyoto Encyclopedia of Genes and Genomes) were determined suspended in 5 mL of extraction buffer [50 mM Tris·HCl, 100 mM Beran et al. www.pnas.org/cgi/content/short/1523468113 1of13 NaCl, 10% (vol/vol) glycerol, 10 mM MgCl2, 2 mM DTT, 20 mM Tris·HCl, 10% (vol/vol) glycerol, 1 mM DTT (pH 7) with 10 mM imidazol, 10% (vol/vol) glycerol (pH 7.4)] supplemented with MgCl2]and10μM(E,E)-FPP (Sigma), (Z,Z)-FPP (Echelon), or 0.3 mg/mL lysozyme (AppliChem), 2.5 U/mL benzonase (Novagen), (Z,E)-FPP as substrate. (Z,E)-FPP was synthesized according to and proteinase inhibitors (Protease Inhibitor Mix HP; SERVA) method of Cane et al. (44) and was kindly provided by Nathalie and incubated at 4 °C for 30 min on a laboratory shaker. After- Gatto and Wilhelm Boland, Max Planck Institute for Chemical ward, cells were disrupted by sonication using a sonotrode (40% Ecology, Jena, Germany. Heterologously expressed TPS enzymes amplitude, 5-s pulse, 10-s pause, 100-s overall pulse duration). The (50 μL) were incubated with geranyl diphosphate [(E)-GPP] lysate was centrifuged at 4 °C for 30 min at 15,000 × g to obtain (Sigma), all available FPP substrates, as well as geranylgeranyl the soluble fraction. The heterologously expressed protein was diphosphate [(E,E,E)-GGPP] (Sigma) as described above. Assays purified via the His-tag using 1 mL of HisPur Ni-NTA Resin (Life were performed in 1-mL glass vials, and enzyme products were Technologies) according to the manufacturer’s instructions. After collected using solid-phase microextraction (SPME). After pene- washing of the column with extraction buffer, protein was eluted trating the PTFE-lined silicone septum of the cap with a SPME with 1 mL of elution buffer [50 mM Tris·HCl, 100 mM NaCl, fiber holder, a SPME fiber coated with 100-μm polydimethylsiloxane 10% (vol/vol) glycerol, 10 mM MgCl2, 2 mM DTT, 400 mM (Supelco) was exposed to the headspace in the vial for 2 h at 30 °C. imidazol (pH 7.5)]. For enzymatic assays, the buffer was ex- Afterward, the SPME fiber was directly inserted into the inlet of changedto25mMMOPSO,10%(vol/vol)glycerol,1mM a gas chromatograph. For collection and analysis of (E,E,E)- – DTT, 10 mM MgCl2 (pH 7.2) using PD-10 Desalting Columns GGPP derived diterpene products, assays were overlaid with (GE Healthcare Life Sciences). 100 μL of hexane and incubated for 2 h at 30 °C. After mixing for PsTPS5 could not be expressed successfully in E. coli, neither 60 s, 1 μL of the organic phase was removed and directly injected when it was cloned into the high-copy expression vector pET100/ into a gas chromatograph. D-TOPO nor cloned into the low-copy expression vector pASK- For analyzing the influence of different divalent metal cofac- IBA37plus (IBA GmbH).