Cyclooxygenases and lipoxygenases are used by the fungus Podospora anserina to repel nematodes

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Abstract

Oxylipins are secondary messengers used universally in the living world for communication and defense. The paradigm is that they are produced enzymatically for the eicosanoids and non-enzymatically for the isoprostanoids. They are supposed to be degraded into volatile organic compounds (VOCs) and to participate in aroma production. Some such chemicals composed of eight carbons are also envisoned as alternatives to fossil fuels. In fungi, oxylipins have been mostly studied in Aspergilli and shown to be involved in signalling asexual versus sexual development, mycotoxin production and interaction with the host for pathogenic species. Through targeted gene deletions of genes encoding oxylipin-producing enzymes and chemical analysis of oxylipins and volatile organic compounds, we show that in the distantly-related ascomycete Podospora anserina, isoprostanoids are likely produced enzymatically. We show the disappearance in the mutants lacking lipoxygenases and cyclooxygenases of the production of 10-hydroxy-octadecadienoic acid and that of 1-octen-3-ol, a common volatile compound. Importantly, this was correlated with the inability of the mutants to repel nematodes as efficiently as the wild type. Overall, our data show that in this fungus, oxylipins are not involved in signalling development but may rather be used directly or as precursors in the production of odors against potential agressors.

Significance

We analyzse the role in inter-kingdom communication of lipoxygenase (lox) and cyclooxygenase (cox) genes in the model fungus Podospora anserina.

Through chemical analysis we define the oxylipins and volatile organic compounds (VOCs)produce by wild type and mutants for cox and lox genes,

We show that the COX and LOX genes are required for the production of some eight carbon VOCs.

We show that COX and LOX genes are involved in the production of chemicals repelling nematodes.

This role is very different from the ones previously evidenced in other fungi.

Introduction

The complete arsenal of molecules used by microorganisms to sense and adapt to their biotic and abiotic environment is far from being known. Oxylipins derived from the oxidation of polyunstaurated fatty acids (PUFAs) are used throughout the plant, animal and fungal kingdoms to signal defense mechanisms, but also development and reproduction [1]. In fungi, their roles are well studied in the genus Aspergillus mostly through the deletion of cyclooxygenase genes (Table 1) and in this genus oxylipins are at the crossroads of several biologically-significant mechanisms, such as reproduction and growth, pathogen interactions and secondary metabolite production [2, 3]. Few genes involved in oxylipin production have been analyzed in other fungi and developmental roles may or may not be found in the studied fungi (Table 1). For the model ascomycete Aspergillus nidulans, it has been shown that specific oxylipins, known as Precocious Sexual Inducers (psi factors), play a crucial role in the balance between sexual and asexual reproduction [4, 5]. Similar roles were observed in Aspergillus flavus [6, 7], Aspergillus fumigatus [8] and Fusarium verticillioides [9]. In Trichoderma atroviride, oxylipins have been hypothesized to play a role in wounding response [10], and in Ascocoryne sarcoides in the production of eight-carbon volatile organic compounds (VOCs) [11]. Many microorganisms, both prokaryotic and eukaryotic, generate VOCs [12, 13]. Fungal VOCs can have a variety of applications ranging from the control of bacteria and fungi [14] to clean biofuels [15]. Like oxylipins, in nature, VOCs are responsible for inter- and intra-organismic communication, leading to attraction, repulsion, as well as growth and differentiation enhancement [16]. The volatile emission profile is a consequence of specific metabolic activities of each microorganism. Fungi produce VOCs as mixtures of alcohols, ketones, esters, small alkenes, monoterpenes, sesquiterpenes, and derivatives originating from a variety of precursors [17]. They especially synthesize many VOCs with eight carbons that are responsible for the fungal odor [18, 19]. The exact pathways used by fungi to produce these eight-carbon VOCs are not well-known [20, 21] and fungi, like plants, may utilize PUFAs and/or oxylipins to produce volatile compounds, because PUFAs may first be oxidized and then cleaved to produce the short-chain volatiles [21].

Oxylipins are synthesized in two ways, directly chemically by reactive oxygen species (ROS) [22] and indirectly by enzymes belonging to the dioxygenase family like cyclooxygenases (COX) [22], lipoxygenases (LOX) [22] or the monooxygenase family like the recently-discovered Abm monooxygenase [23]. Eicosanoids are supposed to be produced mainly enzymatically, while isoprostanoids mainly non-enzymatically [[24], [25], [26], [27]]. Presently, the exact contribution of the enzymatic versus non-enzymatic oxidation of lipids in vivo is not well known, nor it is proven that oxylipins are indeed important precursors of VOCs in fungi [21, 28]. Here, we describe the role of two LOX and two COX by systematic targeted gene deletion in the production of oxylipins and VOCs, as well as in the general physiology of the model fungus Podospora anserina. This fungus inhabits herbivore dung and, thanks to its rapid and easy culture and manipulation in the laboratory, is frequently used to rapidly address the role of genes [29]. We provide evidence that (1) this fungus produces isoprostanoids by an enzymatic route, (2) oxygenases are necessary for the production of some of the eight-carbon VOCs, especially 1-octen-3-ol, and (3) oxylipins and/or VOCs are used by the fungus to repel nematodes.

Section snippets

Strains and growth conditions

The P. anserina strains (Table S1) used in this study derived from the “S” (uppercase S) wild-type strain [30] used for sequencing [31, 32]. Standard culture conditions, media and genetic methods for P. anserina have been described [29, 33]. The M2 medium had the following composition KH2PO4 0.25 g/l, K2HPO4 0.3 g/l, MgSO4/7H2O 0.25 g/l, Urea 0.5 g/l, Thiamine 0.05 mg/l, Biotine 0.25 μg/l, Citric Acid 2.5 mg/l, ZnSO4 2.5 mg/l, CuSO4 0.5 mg/l, MnSO4 125 μg/l, Boric Acid 25 μg/l, Sodium Molybdate

The P. anserina genome encodes four oxylipin-producing enzymes

Mining the genome of P. anserina for genes coding oxylipin-producing enzymes (Table 1) revealed that this fungus has two genes coding for LOX of the Ile-group [56], PaLox1 (=Pa_2_4370 according to the nomenclature of the P. anserina genome sequencing project [32]) and PaLox2 (Pa_6_8140), and two genes coding for COX, PaCox1 (Pa_1_4690) and PaCox2 (Pa_5_1240). The P. anserina genome does not contain any gene coding for LOX enzymes of the Val-group [56] nor any homologue of the Abm monooxygenase

Discussion

Oxylipins are natural products that may be formed enzymatically or non-enzymatically. COX and LOX are the two major classes of enzymes involved in their production. In P. anserina, inactivation of COX and LOX genes clearly has an effect on oxylipins production. Analysis of the patterns of oxylipins in the wild type and the mutants shows that the COX and LOX enzymes may have complex activities. Firstly, they are involved in the synthesis of 10-HODE, which appears to be a major eicosanoid in P.

Conclusion

In P. anserina, oxylipins are produced by four COX and LOX genes and are involved in providing a unique signature of VOCs. These peroxidized lipids and/or the VOCs they produce are used by the fungus to repel potential mycophagous animals. The worm C. elegans that we assayed here to show this effect is bacterivorous and does not consume P. anserina. Nevertheless, many nematodes and other small animals, such as mites, eat fungi and there is thus a clear advantage for P. anserina to produce

Conflict of interest statement

We have no conflict of interest.

Acknowledgements

We thank Dr. Batool Ossareh-Nazari for the gift of the strain N2 of C. elegans and for her help in assessing that the worm does not eat P. anserina and Sylvie Cangemi for expert technical assistance. This work was supported by intramural funding from Université Paris 7 and by Region Ile de France (grant P3AMB).

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