A specialized ABC efflux transporter GcABC‐G1 confers monoterpene resistance to Grosmannia clavigera, a bark beetle‐associated fungal pathogen of pine trees

Abstract

Summary Grosmannia clavigera is a bark beetle‐vectored pine pathogen in the mountain pine beetle epidemic in western North America. Grosmannia clavigera colonizes pines despite the trees' massive oleoresin terpenoid defences. We are using a functional genomics approach to identify G. clavigera's mechanisms of adaptation to pine defences. We annotated the ABC transporters in the G. clavigera genome and generated RNA‐seq transcriptomes from G. clavigera grown with a range of terpenes. We functionally characterized GcABC‐G1, a pleiotropic drug resistance (PDR) transporter that was highly induced by terpenes, using qRT‐PCR, gene knock‐out and heterologous expression in yeast. Deleting GcABC‐G1 increased G. clavigera's sensitivity to monoterpenes and delayed development of symptoms in inoculated young lodgepole pine trees. Heterologous expression of GcABC‐G1 in yeast increased tolerance to monoterpenes. G. clavigera but not the deletion mutant, can use (+)‐limonene as a carbon source. Phylogenetic analysis placed GcABC‐G1 outside the ascomycete PDR transporter clades. G. clavigera appears to have evolved two mechanisms to survive and grow when exposed to monoterpenes: GcABC‐G1 controls monoterpene levels within the fungal cells and G. clavigera uses monoterpenes as a carbon source. This work has implications for understanding adaptation to host defences in an important forest insect–fungal system, and potentially for metabolic engineering of terpenoid production in yeast.

Strong Induction of Minor Terpenes in Italian Cypress, Cupressus sempervirens, in Response to Infection by the Fungus Seiridium cardinale

“…This strategy had been observed in Picea abies, Abies grandis, and Pinus resinosa, where their main monoterpenes (pinenes), lowered proportions in infected trees in favor of minor monoterpenes such as sabinene and terpinolene (Klepzig et al 1995;Raffa and Berryman 1982a;Zhao et al 2010). Some tree terpenes (usually the main compounds) have low inhibiting effects (Kusumoto et al 2014) or can even enhance the growth of some fungal pathogens (Bridges 1987;Cakir et al 2004;Davis and Hofstetter 2011), because some pathogenic fungi have developed the ability to survive in the presence of the major compounds of their common hosts, detoxifying them or even exploiting them as carbon sources (Kusumoto et al 2014;Wang et al 2013). One plausible hypothesis accounting for our results is that a strong concentration and proportion increase of minor terpenes in infected cypresses would help to lower the success of S. cardinale infection or slow its growth considerably, thereby allowing the tree to react effectively, at least in resistant varieties.…”

A Common Fungal Associate of the Spruce Bark Beetle Metabolizes the Stilbene Defenses of Norway Spruce

“…For example, the virulence of pathogens on pea (Pisum sativum) plants depends on their ability to detoxify the pea phytoalexin pisatin (Van Etten et al, 2001), and the virulence of B. cinerea isolates on grape could be at least partially correlated with their ability to metabolize stilbenes (Sbaghi et al, 1996). Moreover, in G. clavigera, it was shown that the export of antifungal components of pine resin by an ATP-binding cassette transporter is essential for fungal growth in its host (Wang et al, 2013). In this study, we show that the virulence of C. polonica correlated with differential usage of the various pathways for stilbene biotransformation and the degradation of structurally similar compounds.…”

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

“…The glass plates were sealed with DuraSeal film (laboratory sealing film, catalog number 89031-573; VWR) and incubated with the lid down in a sealed glass container. For additional details see the work of Wang et al (18). For growth and utilization of single or mixtures of monoterpenes as a carbon source, a similar procedure was used, with a yeast nitrogen base medium (YNB) that contained no carbon source except the monoterpenes tested (18).…”

“…Gs can effectively detoxify monoterpenes and utilize monoterpenes (especially limonene) as a sole carbon source. To decrease the toxicity of monoterpenes that enter Gs cells, the fungus has evolved an active efflux pump system through an ABC transporter (18). A mutant lacking this transporter was more sensitive to monoterpene treatments on malt extract agar (MEA) and was unable to grow on a minimal medium in which monoterpenes were the sole carbon source.…”

“…While these fungal lineages inhabit distinct ecological niches, they are closely related phylogenetically and may have evolved by adapting to the specific chemistry of their host trees. Because G. clavigera Gs is one of the most pathogenic fungi carried by MPB in the current epidemic in western North Amer-ican, diverse genomic resources have been developed for this fungus (16)(17)(18). Gs can effectively detoxify monoterpenes and utilize monoterpenes (especially limonene) as a sole carbon source.…”