Gene Discovery for Enzymes Involved in Limonene Modification or Utilization by the Mountain Pine Beetle-Associated Pathogen Grosmannia clavigera

Abstract

bTo 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.

Volatile Organic Compounds Emitted by Fungal Associates of Conifer Bark Beetles and their Potential in Bark Beetle Control

“…Grosmannia clavigera tolerates the high levels of monoterpenes in freshly attacked bark by employing specific ATP-binding cassette transporters (ABC) that export monoterpenes from the fungal cell (Wang et al 2013). This species also can utilize monoterpenes as a carbon source, making it exceptionally well-adapted for survival in resinous bark and wood tissue (Wang et al 2014) . …”

“…Many plant pathogenic fungi are reported to transform terpene olefins to oxygenated metabolites in vitro , such as the well-known horticultural pathogens Botrytis cinerea Persoon, Aspergillus niger Tieghem, and Penicillium digitatum (Persoon) Sacc. Among the ophiostomatoid fungi, G. clavigera transforms the host-derived monoterpene limonene to the volatile oxygenated metabolites, carvone, p -mentha-2,8-dienol, perillyl alcohol, and isopiperitenol in vitro (Wang et al 2014). This fungus utilizes limonene as a carbon source (DiGuistini et al 2011; Wang et al 2013) for growth.…”

Fungal Frequency and Mite Load Trends Interact with a Declining Mountain Pine Beetle Population

“…Another, Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield can influence MPB success by obtaining its needed carbon from limonene [22], a component of the oleoresin that is toxic to the beetle [23][24][25]. By metabolizing limonene, G. clavigera converts the beetle's deterrent into a nutritional fungal mass beneficial to the insect [22]. The third associate, Ophiostoma montium (Rumbold) von Arx, may be a more recent associate [16] or it may be more closely associated with mites that are phoretic on the MPB.…”

“…This species has been associated with cooler areas within the distribution of the beetle [21]. Another, Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield can influence MPB success by obtaining its needed carbon from limonene [22], a component of the oleoresin that is toxic to the beetle [23][24][25]. By metabolizing limonene, G. clavigera converts the beetle's deterrent into a nutritional fungal mass beneficial to the insect [22].…”

Catechol dioxygenases catalyzing the first step in Norway spruce phenolic degradation are key virulence factors in the bark beetle-vectored fungus Endoconidiophora polonica

“…It also has been reported that Grosmannia clavigera, a blue-stain fungus associated with the mountain pine beetle, could metabolize the oleoresin monoterpene defenses of pine trees (Pinus spp.) by oxidative conversion (Wang et al, 2014).…”