Gene discovery for enzymes involved in limonene modification or utilization by the mountain pine beetle-associated pathogen Grosmannia clavigera

Abstract

To successfully colonize and eventually kill pine trees, Grosmannia clavigera (Gs cryptic species), the main fungal pathogen associated with the mountain pine beetle (Dendroctonus ponderosae), has developed multiple mechanisms to overcome host tree chemical defenses, of which terpenoids are a major component. In addition to a monoterpene efflux system mediated by a recently discovered ABC transporter, Gs has genes that are highly induced by monoterpenes and that encode enzymes that modify or utilize monoterpenes [especially (+)-limonene]. We showed that pine-inhabiting Ophiostomale fungi are tolerant to monoterpenes, but only a few, including Gs, are known to utilize monoterpenes as a carbon source. Gas chromatography-mass spectrometry (GC-MS) revealed that Gs can modify (+)-limonene through various oxygenation pathways, producing carvone, p-mentha-2,8-dienol, perillyl alcohol, and isopiperitenol. It can also degrade (+)-limonene through the C-1-oxygenated pathway, producing limonene-1,2-diol as the most abundant intermediate. Transcriptome sequencing (RNA-seq) data indicated that Gs may utilize limonene 1,2-diol through beta-oxidation and then valine and tricarboxylic acid (TCA) metabolic pathways. The data also suggested that at least two gene clusters, located in genome contigs 108 and 161, were highly induced by monoterpenes and may be involved in monoterpene degradation processes. Further, gene knockouts indicated that limonene degradation required two distinct Baeyer-Villiger monooxygenases (BVMOs), an epoxide hydrolase and an enoyl coenzyme A (enoyl-CoA) hydratase. Our work provides information on enzyme-mediated limonene utilization or modification and a more comprehensive understanding of the interaction between an economically important fungal pathogen and its host's defense chemicals.

FIG 1

Differentially expressed Gs genes on MEA with limonene (MEA plus LIM) versus MEA and on YNB with a monoterpene mixture (YNB plus MT) versus YNB with mannose as a carbon source. (A) Significantly upregulated genes in MEA plus LIM and YNB plus MT (P value < 0.05); (B) significantly downregulated genes in MEA plus LIM and YNB plus MT (P value < 0.05); (C) most important GO terms in YNB plus MT (inner circle) compared with the total GO terms in the Gs genome (outer circle); (D) most important GO terms in MEA plus LIM (inner circle) compared with the total gene ontology (GO) terms in the Gs genome (outer circle).

FIG 2

Upregulated genes with mRNA abundance fold changes greater than 100 under at least one growth condition. Gray bars indicate growth in MEA with limonene (MEA plus LIM) versus MEA, while diagonal bars indicate growth in YNB with a monoterpene mixture (YNB plus MT) versus YNB with mannose as a carbon source.

FIG 3

Expressed clusters and genes potentially involved in monoterpene degradation. The heatmap was generated by the MultiExperimental Viewer (MeV). Relative abundances of each gene (rows) in each growth condition (columns) are shown as log-transformed fold change (FC) relative to the condition's control. Red versus green shows low versus high FC.

FIG 4

qRT-PCR validates the mRNA abundance of selected genes on YNB plus MT (diagonal bars) and MEA plus LIM (gray bars). Growth and treatment conditions were the same as for transcriptome analyses. mRNA abundance was normalized using the β-tubulin gene, a housekeeping gene; error bars show standard deviations based on three technical replicates.

FIG 5

Maximum likelihood phylogenetic tree of Baeyer-Villiger monooxygenases from 12 species. Amino acid sequences were retrieved from GenBank and aligned using multiple-sequence comparisons by log expectation (MUSCLE); poorly aligned positions and divergent regions were removed using Gblocks, and the maximum likelihood tree was generated by PhyLM (www.phylogeny.fr). The two Gs BVMOS are highlighted by asterisks. The typical bacterial cyclohexanone monooxygenase group is highlighted in light gray.