Mycotoxins are a component of Fusarium graminearum stress-response system
- UR1264 - MycSA, Institut National de la Recherche Agronomique, Centre de Bordeaux-Aquitaine, Villenave d'Ornon, France
Secondary Metabolites in the Context of Phytopathogenic Fungal Life
Fusarium graminearum is a phytopathogenic Ascomycota that can cause Fusarium head blight in wheat and other cereals worldwide, leading to important yield losses as well as reduced grain quality. The analysis of the F. graminearum genome sequence revealed the presence of 20 non-ribosomal peptide synthases, 15 polyketide synthases, and 17 terpenoid synthases, all potentially involved in the production of a panel of secondary metabolites, including yet unknown ones, such as the mycotoxins deoxynivalenol (DON) and other type B trichothecenes (Cuomo et al., 2007). The toxicity of DON to humans and animals upon ingestion has been extensively illustrated (see Bonnet et al., 2012; Awad et al., 2013; Pinton and Oswald, 2014 for reviews), and the presence of this mycotoxin in cereal-derived food and feeds represents a serious threat for public health (reviewed in Marin et al., 2013; Sirot et al., 2013; Wu et al., 2014).
Secondary metabolites are a structurally diverse family of compounds that are often qualified as unessential for short-term development but important for long-term survival (see Roze et al., 2011a for a review). Secondary metabolism is tightly linked to primary metabolism, starting with the fact that primary metabolites “feed” secondary metabolite biosynthetic pathways (reviewed in Audenaert et al., 2014; Sheridan et al., 2015). For example, the biosynthesis of type B trichothecenes by F. graminearum derives from the isoprenoid pathway, an essential metabolic pathway involved in various cellular processes. Remarkably, the genes involved in the biosynthesis of isoprenoids are positively regulated by the transcription factor Tri6, which also regulates the genes involved in the trichothecenes biosynthetic pathway (Seong et al., 2009). Moreover, Menke et al. (2013) proposed that type B trichothecenes are produced in specific cellular vesicles, so-called “toxisomes,” and found pieces of evidence that the HMG-CoA reductase of the isoprenoid pathway also localizes to toxisomes when the production of toxins is induced (Menke et al., 2013). Whether these toxisomes are neo-formed for toxin biosynthesis or derive from vesicles hosting elements of the primary metabolism remains to be investigated. Nonetheless, the production of mycotoxins by F. graminearum could be the result of a tight coordination with primary metabolism.
Fungi are known to produce diverse families of secondary metabolites, biological functions of which are not yet fully understood. In the context of host-pathogen interactions, DON was shown to be a virulence factor for F. graminearum infecting wheat by promoting the spreading of the pathogen (Jansen et al., 2005). During infection and colonization, F. graminearum is particularly exposed to plant metabolites that can be constitutive components of the host or molecules produced as a consequence of the presence of the pathogen. Some of these metabolites can trigger fungal stress-response pathways. There are accumulated evidences showing that the production of fungal secondary metabolites, DON in particular, could be an element of the general stress response in F. graminearum.
Secondary Metabolites are Parts of Stress Response Pathways
Stresses caused by variations of the environment can be biotic (the surrounding microbiome) or abiotic (e.g., heat, pH, light, etc.), and both types can lead to either adaptation and survival, or cell death. Here, cell death does not necessary mean a failure to implement an effective stress response but can be the result of the response pathway itself when a destructive response is implemented. The effects of various stresses that phytopathogenic fungi are likely to encounter in the context of host invasion have been the subjects of numerous works. In particular, oxidative stress, as the result of the early defensive “oxidative burst” triggered in the host plant upon infection, has been intensively examined.
Oxidative stress has been extensively studied regarding its interaction with secondary metabolism in fungi. Secondary metabolites can counteract or, conversely, enhance the deleterious effects of oxidative stress, and fungi may use reactive oxygen species (ROS) as signals that initiate/modulate biosynthesis (reviewed in Hong et al., 2013a; Montibus et al., 2015; Sheridan et al., 2015). Response to oxidative stress and fungal secondary metabolism are indeed intertwined. Many of the regulators involved in such response described to date belong to the basic leucine zipper (bZIP) family of transcription factors as illustrated below.
In Aspergillus nidulans, the Yap-like bZIP factor NapA, was shown to be involved in tolerance to oxidative stress (Asano et al., 2007). Yin et al. (2013) showed that treatment with the pro-oxidant tert-butyl hydroperoxide (tBOOH) is associated with increased accumulation of the mycotoxin sterigmatocystin, an effect counteracted by over-expressing NapA (Yin et al., 2013). A similar trend was observed in the ochratoxin producer Aspergillus ochraceus and the NapA orthologue AoYap1 (Reverberi et al., 2012). Not only is ochratoxin production no longer stimulated by tBOOH in a mutant disrupted for AoYap1 but toxin accumulation is also enhanced in untreated conditions (Reverberi et al., 2012).
On the mechanistic side, mobility shift assays provided experimental evidence that antioxidant and aflatoxin biosynthetic genes in Aspergillus parasiticus have binding sites for the same bZIP transcription factor AtfB that is activated upon oxidative stress via MAPK signaling (Hong et al., 2013a,b). In addition, chromatin immunoprecipitation assays showed that the binding of AtfB on the promoters of aflatoxin genes occurs only when A. parasiticus is grown in toxin-inducing medium (Roze et al., 2011b), which supports the concept that toxin biosynthesis and oxidative stress are tightly linked. Considering the interconnectivity between oxidative stress response and mycotoxin production illustrated above, mycotoxin production as a way to cope with endogenous oxidative stress has been previously proposed in Aspergillus species (see Reverberi et al., 2010 for a review). In the gray mold fungus Botrytis cinerea, the BcAtf1 factor was also shown to positively regulate the production of secondary metabolites (Temme et al., 2012).
In F. graminearum, previous results indicate that oxidative stress with H2O2 could be a pre-requisite for the biosynthesis of the mycotoxin DON, which may suggest that DON production and endogenous oxidative stress could be connected (Ponts et al., 2006, 2007; Montibus et al., 2013). In this case, stress response and the regulation of DON synthesis are mediated by the transcription factor FgAP1 that activates the transcription of antioxidant enzymes (Montibus et al., 2013). Possible sources of endogenous oxidative stress in fungi include NAPDH oxidases (Nox) that generate superoxide anions. However, gene deletion experiments showed that the characterized NADPH oxidases NoxA and NoxB do not seem to play a role in the production of trichothecenes (Wang et al., 2014). Other sources of ROS such as mitochondrial respiration or monoamine oxidase could be involved. Similarly, the regulator of transcription FgSKN7 seem to be involved in both oxidative and cell wall stress response, as well as DON biosynthesis (Jiang et al., 2015). All together, it seems likely that the production of DON and its acetylated derivatives is part of an adaptive response to oxidative stress (reviewed in Audenaert et al., 2014; Montibus et al., 2015). In this context, the ROS produced by the host plant as an early defense mechanism in response to infection are used by F. graminearum to its own benefit. Recent data may bring clues as of the mechanism of activation of DON biosynthesis upon oxidative stress. A glycogen synthase kinase GSK3 was shown to be essential for both virulence and DON production F. graminearum, and to be up regulated upon oxidative stress by H2O2 (Qin et al., 2015). These considerations strongly suggest that mycotoxin biosynthesis and response to oxidative stress are intertwined.
The pH of the environment was shown to be particularly critical for the initiation of trichothecene B biosynthesis in F. graminearum. An acidic pH is a prerequisite for DON production (Merhej et al., 2010). The biosynthesis of type B trichothecenes has been shown to be negatively controlled by the transcription regulator FgPac1, homologous to the member of the pH regulator system PacC in A. nidulans (Merhej et al., 2011). It is noteworthy that oxidative stress and acidification have been described to occur together in various organisms including fungi (see examples in Jia et al., 2014; Pérez-Sampietro and Herrero, 2014). The relationship between these two stresses is however not clear.
Different response pathways can be triggered to counteract stresses that modify the organization and stability of the fungal cell wall. For example, the cell wall integrity pathway is responsive to changes in osmotic pressure and oxidative stress (see Hayes et al., 2014 for a review). Typically, the activation of the cell wall integrity pathway leads to the activation of cell wall biogenesis genes. In F. graminearum, the cell wall integrity pathway involves the MAP kinase FgMgv1 (Hou et al., 2002), itself activated by the MAP kinase FgMkk1 (Yun et al., 2014). When the pathway is altered by gene deletion, the production of DON is drastically reduced (Hou et al., 2002; Yun et al., 2014). Once more, the production of DON seems linked to a stress response pathway. It is noteworthy that the production of the secondary metabolite aurofusarin, a pigment, is also diminished in the absence of FgMgv1 (Hou et al., 2002). Such observation provides another example of the association between the activation of secondary metabolites pathways and stress response pathways. Remarkably, the MAP kinase FgOS-2 of the high osmolarity glycerol (HOG) response pathway is also activated by FgMkk1 (Yun et al., 2014), which illustrates the coupling between cell wall stress response and osmotic stress response. The HOG response pathway was initially described in the budding yeast as required for osmoadaptation (reviewed by Hohmann et al., 2007). In F. graminearum, FgHOG1 of the osmoregulation MAP kinase pathway, mediated by the FgOS-2 kinase, is involved in hyperosmotic as well as cell membrane stress responses. FgHOG1 also plays a role in ROS-mediated signaling. Its deletion causes a drastic reduction of DON accumulation, also caused by hyperosmotic conditions, as well as other developmental defects (Van Thuat et al., 2012; Zheng et al., 2012). Similarly, gene deletion experiments targeting FgOS-2 showed that DON production is strongly reduced in planta (Van Thuat et al., 2012).
A Secondary Metabolism Regulatory Pathway Part of Environmental Stress Response
Regulators of fungal secondary metabolite biosynthesis that play a role in regulating other aspects of fungal life, including response to stress, have been described. The example of the F. graminearum, transcription factor Tri6 has been evoked above (Seong et al., 2009). In Aspergilli, LaeA has been described as a secondary metabolism-specific regulator involved in switching from inactive heterochromatin to the transcriptionally permissive euchromatic state. In F. graminearum, FgLae1 is a regulator of secondary metabolism that activates the production of DON (Kim et al., 2013). The deletion of FgLae1 has pleiotropic effects in F. graminearum, also affecting sexual development for example (Kim et al., 2013). Typically LaeA is a member of the Velvet complex, involved in the response to light stress. In the absence of light, LaeA is available in the nucleus and chromatin at secondary metabolite gene clusters is active (and DON is produced for example). On the contrary, when light is applied, chromatin inactivates and secondary metabolite biosynthesis is shut down. Such a “secondary metabolism switch” could be conveniently associated with general stress response pathways to extend them to the production of secondary metabolites.
Previous observations made about F. graminearum's response to oxidative stress, i.e., defensive plant-produced H2O2 may serve as a signal to produce DON (Ponts et al., 2006, 2007; Montibus et al., 2013), also fit with this theory. Other examples of such hijacking have been proposed, for example the use of the plant carbon and nitrogen metabolisms by F. graminearum for its own development and secondary metabolism (Audenaert et al., 2014). Polyamines such as agmatine or putrescine are, indeed, excellent activators of DON biosynthesis (Gardiner et al., 2010). From the evolutionary point of view, the general components of oxidative, osmotic, and cell wall stress pathways appear well conserved among fungi (Nikolaou et al., 2009). However, specific sensors and regulators involved in those pathways are diverse and seem to have fairly recently rapidly evolved to adapt to fungal specific life traits (Nikolaou et al., 2009). In a general manner, plant pathogens are more sensitive to oxidative stress than human pathogens for example (Nikolaou et al., 2009). Along the same line, stress response pathways in fungal plant pathogens, especially oxidative, osmotic and cell wall stress, are typically involved in tolerance to fungicides targeting the fungal cell wall (e.g., caspofungin, nikkomycin Z, tunicamycin, fluconazole; see Hayes et al., 2014 for a review). Such aspects must definitely be considered and addressed by strategies aiming at controlling mycotoxin occurrence in cereals.
The hypothesis of a coupling between stress response and mycotoxin production is reinforced by recent evidence that proteins involved in secondary metabolite pathways, including the aflatoxin one in A. parasiticus, co-localize with stress response proteins to the endosome/transport vesicles/vacuoles fraction of a fungal cell extract (Linz et al., 2012). In F. graminearum, previous work showed that the endoplasmic reticulum stress response and oxidative stress are tightly linked (Malhotra et al., 2008). Remarkably, recent work found that vesicular sequestration of enzymes of the DON-producing pathway co-localize with the endoplasmic reticulum (Menke et al., 2013; Boenisch et al., 2015). When all elements are taken into consideration, F. graminearum secondary metabolism and stress response pathways are indisputably very closely interconnected. Although further investigation is required, an attractive hypothesis is that secondary metabolism pathways could be part of the fungus' stress-response system. Under this scenario, more than a coupling of pathways, the production of DON and other secondary metabolites would be integral part of the fungus' arsenal to cope and adapt to its always-changing environment, including in the context of host-pathogen exchanges.
Conflict of Interest Statement
The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Asano, Y., Hagiwara, D., Yamashino, T., and Mizuno, T. (2007). Characterization of the bZip-type transcription factor NapA with reference to oxidative stress response in Aspergillus nidulans. Biosci. Biotechnol. Biochem. 71, 1800–1803. doi: 10.1271/bbb.70133
Awad, W., Ghareeb, K., Böhm, J., and Zentek, J. (2013). The toxicological impacts of the Fusarium mycotoxin, deoxynivalenol, in poultry flocks with special reference to immunotoxicity. Toxins 5, 912–925. doi: 10.3390/toxins5050912
Cuomo, C. A., Güldener, U., Xu, J.-R., Trail, F., Turgeon, B. G., Di Pietro, A., et al. (2007). The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization. Science 317, 1400–1402. doi: 10.1126/science.1143708
Gardiner, D. M., Kazan, K., Praud, S., Torney, F. J., Rusu, A., and Manners, J. M. (2010). Early activation of wheat polyamine biosynthesis during Fusarium head blight implicates putrescine as an inducer of trichothecene mycotoxin production. BMC Plant Biol. 10:289. doi: 10.1186/1471-2229-10-289
Hayes, B. M. E., Anderson, M. A., Traven, A., van der Weerden, N. L., and Bleackley, M. R. (2014). Activation of stress signalling pathways enhances tolerance of fungi to chemical fungicides and antifungal proteins. Cell. Mol. Life Sci. 71, 2651–2666. doi: 10.1007/s00018-014-1573-8
Hong, S.-Y., Roze, L. V., Wee, J., and Linz, J. E. (2013b). Evidence that a transcription factor regulatory network coordinates oxidative stress response and secondary metabolism in aspergilli. Microbiologyopen 2, 144–160. doi: 10.1002/mbo3.63
Hou, Z., Xue, C., Peng, Y., Katan, T., Kistler, H. C., and Xu, J.-R. (2002). A mitogen-activated protein kinase gene (MGV1) in Fusarium graminearum is required for female fertility, heterokaryon formation, and plant infection. Mol. Plant Microbe Interact. 15, 1119–1127. doi: 10.1094/MPMI.2002.15.11.1119
Jansen, C., von Wettstein, D., Schäfer, W., Kogel, K.-H., Felk, A., and Maier, F. J. (2005). Infection patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted Fusarium graminearum. Proc. Natl. Acad. Sci. U.S.A. 102, 16892–16897. doi: 10.1073/pnas.0508467102
Jia, C., Yu, Q., Xu, N., Zhang, B., Dong, Y., Ding, X., et al. (2014). Role of TFP1 in vacuolar acidification, oxidative stress and filamentous development in Candida albicans. Fungal Genet. Biol. 71, 58–67. doi: 10.1016/j.fgb.2014.08.012
Jiang, C., Zhang, S., Zhang, Q., Tao, Y., Wang, C., and Xu, J.-R. (2015). FgSKN7 and FgATF1 have overlapping functions in ascosporogenesis, pathogenesis and stress responses in Fusarium graminearum. Environ. Microbiol. 17, 1245–1260. doi: 10.1111/1462-2920.12561
Kim, H.-K., Lee, S., Jo, S.-M., McCormick, S. P., Butchko, R. A. E., Proctor, R. H., et al. (2013). Functional roles of FgLaeA in controlling secondary metabolism, sexual development, and virulence in Fusarium graminearum. PLoS ONE 8:e68441. doi: 10.1371/journal.pone.0068441
Linz, J. E., Chanda, A., Hong, S.-Y., Whitten, D. A., Wilkerson, C., and Roze, L. V. (2012). Proteomic and biochemical evidence support a role for transport vesicles and endosomes in stress response and secondary metabolism in Aspergillus parasiticus. J. Proteome Res. 11, 767–775. doi: 10.1021/pr2006389
Malhotra, J. D., Miao, H., Zhang, K., Wolfson, A., Pennathur, S., Pipe, S. W., et al. (2008). Antioxidants reduce endoplasmic reticulum stress and improve protein secretion. Proc. Natl. Acad. Sci. U.S.A. 105, 18525–18530. doi: 10.1073/pnas.0809677105
Menke, J., Weber, J., Broz, K., and Kistler, H. C. (2013). Cellular development associated with induced mycotoxin synthesis in the filamentous fungus Fusarium graminearum. PLoS ONE 8:e63077. doi: 10.1371/journal.pone.0063077
Merhej, J., Boutigny, A. L., Pinson-Gadais, L., Richard-Forget, F., and Barreau, C. (2010). Acidic pH as a determinant of TRI gene expression and trichothecene B biosynthesis in Fusarium graminearum. Food Addit. Contam. A Chem. Anal. Control Expo. Risk Assess. 27, 710–717. doi: 10.1080/19440040903514531
Merhej, J., Richard-Forget, F., and Barreau, C. (2011). The pH regulatory factor Pac1 regulates Tri gene expression and trichothecene production in Fusarium graminearum. Fungal Genet. Biol. 48, 275–284. doi: 10.1016/j.fgb.2010.11.008
Montibus, M., Ducos, C., Bonnin-Verdal, M.-N., Bormann, J., Ponts, N., Richard-Forget, F., et al. (2013). The bZIP transcription factor Fgap1 mediates oxidative stress response and trichothecene biosynthesis but not virulence in Fusarium graminearum. PLoS ONE 8:e83377. doi: 10.1371/journal.pone.0083377
Montibus, M., Pinson-Gadais, L., Richard-Forget, F., Barreau, C., and Ponts, N. (2015). Coupling of transcriptional response to oxidative stress and secondary metabolism regulation in filamentous fungi. Crit. Rev. Microbiol. 41, 295–308. doi: 10.3109/1040841X.2013.829416
Nikolaou, E., Agrafioti, I., Stumpf, M., Quinn, J., Stansfield, I., and Brown, A. J. P. (2009). Phylogenetic diversity of stress signalling pathways in fungi. BMC Evol. Biol. 9:44. doi: 10.1186/1471-2148-9-44
Pérez-Sampietro, M., and Herrero, E. (2014). The PacC-family protein Rim101 prevents selenite toxicity in Saccharomyces cerevisiae by controlling vacuolar acidification. Fungal Genet. Biol. 71, 76–85. doi: 10.1016/j.fgb.2014.09.001
Ponts, N., Pinson-Gadais, L., Barreau, C., Richard-Forget, F., and Ouellet, T. (2007). Exogenous H(2)O(2) and catalase treatments interfere with Tri genes expression in liquid cultures of Fusarium graminearum. FEBS Lett. 581, 443–447. doi: 10.1016/j.febslet.2007.01.003
Ponts, N., Pinson-Gadais, L., Verdal-Bonnin, M.-N., Barreau, C., and Richard-Forget, F. (2006). Accumulation of deoxynivalenol and its 15-acetylated form is significantly modulated by oxidative stress in liquid cultures of Fusarium graminearum. FEMS Microbiol. Lett. 258, 102–107. doi: 10.1111/j.1574-6968.2006.00200.x
Qin, J., Wang, G., Jiang, C., Xu, J.-R., and Wang, C. (2015). Fgk3 glycogen synthase kinase is important for development, pathogenesis, and stress responses in Fusarium graminearum. Sci. Rep. 5:8504. doi: 10.1038/srep08504
Reverberi, M., Gazzetti, K., Punelli, F., Scarpari, M., Zjalic, S., Ricelli, A., et al. (2012). Aoyap1 regulates OTA synthesis by controlling cell redox balance in Aspergillus ochraceus. Appl. Microbiol. Biotechnol. 95, 1293–1304. doi: 10.1007/s00253-012-3985-4
Reverberi, M., Ricelli, A., Zjalic, S., Fabbri, A. A., and Fanelli, C. (2010). Natural functions of mycotoxins and control of their biosynthesis in fungi. Appl. Microbiol. Biotechnol. 87, 899–911. doi: 10.1007/s00253-010-2657-5
Roze, L. V., Chanda, A., and Linz, J. E. (2011a). Compartmentalization and molecular traffic in secondary metabolism: a new understanding of established cellular processes. Fungal Genet. Biol. 48, 35–48. doi: 10.1016/j.fgb.2010.05.006
Roze, L. V., Chanda, A., Wee, J., Awad, D., and Linz, J. E. (2011b). Stress-related transcription factor AtfB integrates secondary metabolism with oxidative stress response in aspergilli. J. Biol. Chem. 286, 35137–35148. doi: 10.1074/jbc.M111.253468
Seong, K.-Y., Pasquali, M., Zhou, X., Song, J., Hilburn, K., McCormick, S., et al. (2009). Global gene regulation by Fusarium transcription factors Tri6 and Tri10 reveals adaptations for toxin biosynthesis. Mol. Microbiol. 72, 354–367. doi: 10.1111/j.1365-2958.2009.06649.x
Sirot, V., Fremy, J.-M., and Leblanc, J.-C. (2013). Dietary exposure to mycotoxins and health risk assessment in the second French total diet study. Food Chem. Toxicol. Int. J. Publ. Br. Ind. Biol. Res. Assoc. 52, 1–11. doi: 10.1016/j.fct.2012.10.036
Temme, N., Oeser, B., Massaroli, M., Heller, J., Simon, A., Collado, I. G., et al. (2012). BcAtf1, a global regulator, controls various differentiation processes and phytotoxin production in Botrytis cinerea. Mol. Plant Pathol. 13, 704–718. doi: 10.1111/j.1364-3703.2011.00778.x
Van Thuat, N., Schäfer, W., and Bormann, J. (2012). The stress-activated protein kinase FgOS-2 is a key regulator in the life cycle of the cereal pathogen Fusarium graminearum. Mol. Plant Microbe Interact. 25, 1142–1156. doi: 10.1094/MPMI-02-12-0047-R
Wang, L., Mogg, C., Walkowiak, S., Joshi, M., and Subramaniam, R. (2014). Characterization of NADPH oxidase genes NoxA and NoxB in Fusarium graminearum. Can. J. Plant Pathol. 36, 12–21. doi: 10.1080/07060661.2013.868370
Yin, W.-B., Reinke, A. W., Szilágyi, M., Emri, T., Chiang, Y.-M., Keating, A. E., et al. (2013). bZIP transcription factors affecting secondary metabolism, sexual development and stress responses in Aspergillus nidulans. Microbiology 159, 77–88. doi: 10.1099/mic.0.063370-0
Yun, Y., Liu, Z., Zhang, J., Shim, W.-B., Chen, Y., and Ma, Z. (2014). The MAPKK FgMkk1 of Fusarium graminearum regulates vegetative differentiation, multiple stress response, and virulence via the cell wall integrity and high-osmolarity glycerol signaling pathways. Environ. Microbiol. 16, 2023–2037. doi: 10.1111/1462-2920.12334
Keywords: mycotoxins, adaptation, stress-response system, DON, Fusarium graminearum
Citation: Ponts N (2015) Mycotoxins are a component of Fusarium graminearum stress-response system. Front. Microbiol. 6:1234. doi: 10.3389/fmicb.2015.01234
Received: 08 January 2015; Accepted: 23 October 2015;
Published: 04 November 2015.
Edited by:Jonathan Palmer, US Forest Service, USA
Copyright © 2015 Ponts. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Nadia Ponts, firstname.lastname@example.org