Interactions between Co-Habitating fungi Elicit Synthesis of Taxol from an Endophytic Fungus in Host Taxus Plants

Abstract

Within a plant, there can exist an ecosystem of pathogens and endophytes, the latter described as bacterial and fungal inhabitants that thrive without causing disease to the host. Interactions between microbial inhabitants represent a novel area of study for natural products research. Here we analyzed the interactions between the fungal endophytes of Taxus (yew) trees. Fungal endophytes of Taxus have been proposed to produce the terpenoid secondary metabolite, Taxol, an anti-cancer drug. It is widely reported that plant extracts stimulate endophytic fungal Taxol production, but the underlying mechanism is not understood. Here, Taxus bark extracts stimulated fungal Taxol production 30-fold compared to a 10-fold induction with wood extracts. However, candidate plant-derived defense compounds (i.e., salicylic acid, benzoic acid) were found to act only as modest elicitors of fungal Taxol production from the endophytic fungus Paraconiothyrium SSM001, consistent with previous studies. We hypothesized the Taxus plant extracts may contain elicitors derived from other microbes inhabiting these tissues. We investigated the effects of co-culturing SSM001 with other fungi observed to inhabit Taxus bark, but not wood. Surprisingly, co-culture of SSM001 with a bark fungus (Alternaria) caused a ∼threefold increase in Taxol production. When SSM001 was pyramided with both the Alternaria endophyte along with another fungus (Phomopsis) observed to inhabit Taxus, there was an ∼eightfold increase in fungal Taxol production from SSM001. These results suggest that resident fungi within a host plant interact with one another to stimulate Taxol biosynthesis, either directly or through their metabolites. More generally, our results suggest that endophyte secondary metabolism should be studied in the context of its native ecosystem.

Keywords: Paraconiothyrium; Taxol; Taxus; elicitor; endophyte; fungus.

Figure 1

Effect of plant extracts or candidate plant compounds, based on the habitat of Paraconiothyrium SSM001, on fungal Taxol production at 3 weeks following inoculation in fungal liquid culture. (A) Trypan blue-stained SSM001 from 12-h-old cultured Taxus x media wood sections showing SSM001 localization to the wood vascular cells (wood medullary rays). (B) Effect of added Taxus and Pinus tissues. (C) Effect of added Taxus and Pinus alcohol extracts. (D) Effect of added taxane-producing Taxus suspension culture cells to test whether Taxus contributes precursors for fungal Taxol biosynthesis. (E) Effect of the antimicrobial phytoalexin, resveratrol. (F) Effect of plant defense hormones. (G) Effect of benzoic acid, a derivative related to salicylic acid. (H) Effect of strigolactone, a plant hormone that stimulates fungal germination and penetration, and which is localized to vascular cells. (I) Effect of indole acetic acid (IAA), a plant growth hormone important in vascular tissue biogenesis.

Figure 2

Co-habitating fungi present in Taxus x media outer bark stimulate Taxol production from Paraconiothyrium SSM001 fungi. (A) DNA fingerprinting (fungal 18S tRFLP) of Taxus wood (excluding bark). The x-axis reflects the fragment size following HaeIII digestion. The dendrogram shows one peak (blue) corresponding to SSM001 along with other very minor peaks. (B) Control tRFLP of pure cultured Paraconiothyrium SSM001 showing a peak at 490 nucleotides. (C) DNA fingerprinting (fungal 18S tRFLP) of Taxus outer bark. The dendrogram shows five novel peaks (blue) other than SSM001. (D) SSM001 spores. (E) Non-SSM001 fungal spores in the outer bark in a transverse section of a Taxus branch. (F) Close-up of spores from Taxus outer bark, stained with Trypan blue, showing the diversity of fungi. (G) Isolation and culturing of pure Alternaria fungus from the outer bark of T. x media. (H) Effect of co-culturing of pure Alternaria fungus with SSM001 in liquid culture.

Figure 3

Co-habitating fungi present in Taxus x media needles stimulate Taxol production from Paraconiothyrium SSM001 fungi. (A) tRFLP analysis of DNA pooled from Taxus needles showing several fungal peaks, none of which corresponded to SSM001. (B) Detection of different fungal spores from T. x media needles. (C) Effect of co-culturing of pure Phomopsis fungus isolated from Taxus needles with SSM001 in liquid culture.

Figure 4

Expression ratio of total fungal community in Taxus needles in comparison to Taxus bark and wood. (A) Expression ratio of total fungal community in Taxus needles and bark in comparison to Taxus wood. (B) Effect of co-culturing of Alternaria and Phomopsis fungi isolated from outer bark and needles, respectively on SSM001 Taxol production in liquid culture.