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. 2021 Feb:129:103513.
doi: 10.1016/j.ibmb.2020.103513. Epub 2021 Jan 1.

RNA-Seq used to identify ipsdienone reductase (IDONER): A novel monoterpene carbon-carbon double bond reductase central to Ips confusus pheromone production

Katherine E Fisher  1 , Richard L Tillett  2 , Misha Fotoohi  3 , Cody Caldwell  4 , Juli Petereit  5 , Karen Schlauch  6 , Claus Tittiger  7 , Gary J Blomquist  8 , Marina MacLean  9
Affiliations

RNA-Seq used to identify ipsdienone reductase (IDONER): A novel monoterpene carbon-carbon double bond reductase central to Ips confusus pheromone production

Katherine E Fisher et al. Insect Biochem Mol Biol. 2021 Feb.

Abstract

The pinyon ips beetle, Ips confusus (LeConte) is a highly destructive pest in pine forests in western North America. When colonizing a new host tree, I. confusus beetles coordinate a mass attack to overcome the tree's defenses using aggregation pheromones. Ips confusus, as with other Ips spp. beetles, biosynthesize ipsdienol and ipsenol in a specific enantiomeric blend and ratio as aggregation pheromones. While several of the initial steps in the pheromone biosynthetic pathway have been well defined, the final steps were unknown. We used comparative RNA-Seq analysis between fed and unfed male I. confusus midgut tissue to identify candidate genes involved in pheromone biosynthesis. The 12,995 potentially unique transcripts showed a clear separation based on feeding state. Differential expression analysis identified gene groups that were tightly connected. This analysis identified all known pheromone biosynthetic genes and suggested a novel monoterpene double bond reductase, ipsdienone reductase (IDONER), with pheromone biosynthetic gene expression patterns. IDONER cDNA was cloned, expressed, and functionally characterized. The coding DNA sequence has an ORF of 1101 nt with a predicted translation product of 336 amino acids. The enzyme has a molecular weight of 36.7 kDa with conserved motifs of the medium chain dehydrogenases/reductase (MDR) superfamily in the leukotriene B4 dehydrogenases/reductases (LTB4R) family. Tagged recombinant protein was expressed and purified. Enzyme assays and GC/MS analysis showed IDONER catalyzed the reduction of ipsdienone to form ipsenone. This study shows that IDONER is a monoterpene double bond reductase involved in I. confusus pheromone biosynthesis.

Keywords: Bark beetle; Ips confusus; Ipsdienone; Ipsenone; Monoterpene carbon-carbon double bond reductase; Pheromone; Pinyon ips; Prostaglandin reductase; RNA-Seq.

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Conflict of interest statement

Declarations of conflicts of interest: none

Figures

Figure 1.
Figure 1.
Ips spp. pheromone biosynthetic pathway.
Figure 2.
Figure 2.
Verification of RNA-Seq data.
Figure 3.
Figure 3.
Clustering of significantly feeding status regulated genes with expression differences ≤ −5 or ≥ 5.
Figure 4.
Figure 4.
GC-MS analysis of ipsenone formed from ipsdienone and NADPH by recombinant IDONER.
Figure 5.
Figure 5.
Metabolism of ipsdienone to ipsenone by recombinant IDONER.
Figure 6.
Figure 6.
Monomeric structure of IDONER and VvCurA.
See this image and copyright information in PMC

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