Abstract
Deoxynivalenol (DON) and Zearalenone (ZEA) are among the most predominant mycotoxins produced by Fusarium head blight pathogens. DON is a potent protein inhibitor while ZEA is a potent estrogenic metabolite. The toxins are known to inhibit growth and development of fungi with the exception of the mycoparasitic fungus Clonostachys rosea IK726, which is effective in controlling mycotoxin-producing Fusarium species. This thesis reports on aspects regarding (I) detoxification of ZEA in connection to biocontrol activities of the IK726, (II) mechanisms conferring resistance to DON and ZEA and (III) evolution of ABC transporters in mycoparasitic fungi focused on C. rosea and Trichoderma spp.
We showed that expression of zhd101 depended on concentrations of ZEA and that the gene was not induced by other agents, suggesting specificity of the enzyme towards ZEA and its derivates. To investigate effects of ZEA detoxification on mycoparasitism of C. rosea IK726, the Δzhd101 mutants impaired in detoxifying ZEA were generated through Agrobacterium-mediated transformation. In vivo interaction of sand seedling test between the Δzhd101 mutants and the non-producing ZEA ΔPKS4 strains showed a significant decrease in disease severity in comparison with the wild-type strain, whereas the C. rosea IK726 wild type provided complete protection against both F. graminearum strains. This suggested that ZEA detoxification is important for successful mycoparasitism of IK726. Furthermore, we generated DON- and ZEA-induced cDNA libraries to investigate whether additional mechanisms contributing tolerance to DON and ZEA in the strain IK726. Analysis of the DON-induced library showed no transcripts encoding enzyme associated with known DON detoxifying mechanisms. Instead, the majority of up-regulated transcripts encoded enzymes involved in metabolic processes, e.g., Cytochome P450 55A3, Cytochrome C oxidase and ATP synthase. This provided evidence that metabolic adjustment is a major component contributing to tolerance to DON in C. rosea. On the other hand, substantial upregulation of transcripts encoding ABC transporter species was observed in the ZEA-induced library together with transcripts encoding the ZEA-detoxifying ZHD101. The use of bioinformatic tools to predict full-length sequence from those that identified in the ZEA-induced library unveiled 2 putative ABC proteins, which belong to subfamily G of fungal ABC transporter as identified by phylogenetic analysis. This is the first time we showed the involvement of ABC proteins in providing resistance to ZEA in C. rosea. Lastly, we used bioinformatic approaches to identify a total of 85 putative ABC transporters from the draft C. rosea genome. Evolutionary analysis of gene birth and death identified expansion of ABC transporter families in C. rosea as in Trichoderma virens and T. harzianum, but not in T. reesei and T. atroviridae. The expansion was profoundly observed in the subfamily G of C. rosea ABC transporters and the subfamily C of Trichoderma spp, repectively. The subfamilies C and G are responsible for xenobiotic transport in many organisms, suggesting that the expansion of ABC transporters might be reflected in resistance to broad xenobiotics in C. rosea, while the contraction of subfamily C in T. reesei and T. atroviridae may be reflected in distinct lifestyles in these latter species.
We showed that expression of zhd101 depended on concentrations of ZEA and that the gene was not induced by other agents, suggesting specificity of the enzyme towards ZEA and its derivates. To investigate effects of ZEA detoxification on mycoparasitism of C. rosea IK726, the Δzhd101 mutants impaired in detoxifying ZEA were generated through Agrobacterium-mediated transformation. In vivo interaction of sand seedling test between the Δzhd101 mutants and the non-producing ZEA ΔPKS4 strains showed a significant decrease in disease severity in comparison with the wild-type strain, whereas the C. rosea IK726 wild type provided complete protection against both F. graminearum strains. This suggested that ZEA detoxification is important for successful mycoparasitism of IK726. Furthermore, we generated DON- and ZEA-induced cDNA libraries to investigate whether additional mechanisms contributing tolerance to DON and ZEA in the strain IK726. Analysis of the DON-induced library showed no transcripts encoding enzyme associated with known DON detoxifying mechanisms. Instead, the majority of up-regulated transcripts encoded enzymes involved in metabolic processes, e.g., Cytochome P450 55A3, Cytochrome C oxidase and ATP synthase. This provided evidence that metabolic adjustment is a major component contributing to tolerance to DON in C. rosea. On the other hand, substantial upregulation of transcripts encoding ABC transporter species was observed in the ZEA-induced library together with transcripts encoding the ZEA-detoxifying ZHD101. The use of bioinformatic tools to predict full-length sequence from those that identified in the ZEA-induced library unveiled 2 putative ABC proteins, which belong to subfamily G of fungal ABC transporter as identified by phylogenetic analysis. This is the first time we showed the involvement of ABC proteins in providing resistance to ZEA in C. rosea. Lastly, we used bioinformatic approaches to identify a total of 85 putative ABC transporters from the draft C. rosea genome. Evolutionary analysis of gene birth and death identified expansion of ABC transporter families in C. rosea as in Trichoderma virens and T. harzianum, but not in T. reesei and T. atroviridae. The expansion was profoundly observed in the subfamily G of C. rosea ABC transporters and the subfamily C of Trichoderma spp, repectively. The subfamilies C and G are responsible for xenobiotic transport in many organisms, suggesting that the expansion of ABC transporters might be reflected in resistance to broad xenobiotics in C. rosea, while the contraction of subfamily C in T. reesei and T. atroviridae may be reflected in distinct lifestyles in these latter species.
| Original language | English |
|---|
| Publisher | Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen |
|---|---|
| Number of pages | 103 |
| Publication status | Published - 2013 |