Frontalin pheromone biosynthesis in the mountain , ponderosae, and the role of isoprenyl diphosphate synthases

Christopher I. Keelinga, Christine C. Chiua, Tidiane Awb, Maria Lia, Hannah Hendersona, Claus Tittigerb, Hong-Biao Wengb, Gary J. Blomquistb, and Joerg Bohlmanna,1

aMichael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1A4; and bDepartment of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89557

Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved October 7, 2013 (received for review September 3, 2013) The mountain pine beetle (Dendroctonus ponderosae Hopkins) is cochleariae) (12), and a unique GPPS from the pine engraver the most destructive pest of western North American pine forests. beetle (Ips pini) that also produces myrcene (13, 14). To assess Adult males produce frontalin, an eight-carbon antiaggregation their importance in pheromone biosynthesis, we identified and pheromone, via the mevalonate pathway, as part of several pher- functionally characterized two IDSs in MPB. Based on three omones that initiate and modulate the mass attack of host different lines of evidence, gene-expression patterns of the IDSs, trees. Frontalin acts as a pheromone, attractant, or kairomone in enzyme activity of recombinant proteins, and the frontalin con- most Dendroctonus species, other , and even elephants. tent of dsRNA-treated , we show that frontalin originates 6-Methylhept-6-en-2-one, a frontalin precursor, is hypothesized to from GGPP rather than the shorter isoprenyl diphosphates. originate from 10-carbon geranyl diphosphate (GPP), 15-carbon far- nesyl diphosphate (FPP), or 20-carbon geranylgeranyl diphosphate Results (GGPP) via a dioxygenase- or cytochrome P450-mediated carbon– Cloning and Functional Characterization of IDSs. We identified two carbon bond cleavage. To investigate the role of isoprenyl diphos- putative full-length MPB IDS cDNA clones, DPO044_J12 and phate synthases in pheromone biosynthesis, we characterized a bi- DPO061_E16. The complete cDNA sequence of DPO044_J12 is functional GPP/FPP synthase and a GGPP synthase in the mountain 1,601 bp long, includes 82 bp and 229 bp of 5′ and 3′ UTRs, pine beetle. The ratio of GPP to FPP produced by the GPP/FPP syn- respectively, and encodes a predicted 429-aa protein with a mass thase was highly dependent on the ratio of the substrates isopen- of 49.4 kDa and a pI of 9.00. The complete cDNA sequence of tenyl diphosphate and dimethylallyl diphosphate used in the assay. DPO061_E16 is 1,033 bp long, includes 73 bp and 66 bp of 5′ and Transcript levels in various tissues and life stages suggested that 3′ UTRs, respectively, and encodes a predicted 297-aa protein GGPP rather than GPP or FPP is used as a precursor to frontalin. with a mass of 34.3 kDa and a pI of 5.97. Both proteins had the Reduction of transcript levels by RNA interference of the isoprenyl two aspartate-rich motifs (Fig. S1) characteristic of IDSs (15). diphosphate synthases identified GGPP synthase as having the Heterologous expression and in vitro assays with IPP and DMAPP largest effect on frontalin production, suggesting that frontalin is as substrates produced both GPP and FPP for DPO044_J12 and derived from a 20-carbon isoprenoid precursor rather than from the 10- or 15-carbon precursors. only GGPP for DPO061_E16 (Fig. 2). We thus designated the clones as DponGPPS/FPPS and DponGGPPS, respectively. When | midgut | semiochemistry assayed with a range of IPP:DMAPP substrate ratios between 5:1 and 1:5, DponGGPPS produced GGPP with only a trace of GPP he mountain pine beetle (Dendroctonus ponderosae Hopkins; Significance TMPB) is the most destructive pest of wester