Progress 07/15/03 to 07/14/07
Outputs
1) We were awarded a patent: United States Patent US 7,183,098 B2 Date of Patent: February 27, 2007 Inventors: Schardl, Christopher, Heather Wilkinson, and Martin Spiering. Loline Alkaloid Gene Clusters of the Fungal Endophyte Neotyphodium uncinatum 2) I am a member of the Epichole festucae Annotation Consortium
Impacts
LOL is a novel secondary metabolite gene cluster associated with the production of loline alkaloids (saturated 1-aminopyrrolizidine alkaloids with an oxygen bridge) exclusively in closely related grass-endophyte species in the genera Epichloe and Neotyphodium. In this study we characterized the LOL cluster in E. festucae, including 10 individual lol genes. Furthermore we defined the boundaries of the cluster and the genomic DNA region flanking LOL in E. festucae. In addition to characterizing the LOL cluster in E. festucae, we sequenced LOL from two additional species, Neotyphodium coenophialum and Neotyphodium sp. PauTG-1. Together with two recently published LOL clusters from N. uncinatum, these data allow for a powerful phylogenetic comparison of five clusters from four closely related species. There is a high degree of microsynteny (conserved gene order and orientation) among the five LOL clusters, allowing us to predict potential transcriptional co-regulatory
binding motifs in lol promoter regions. The relatedness of LOL clusters is especially interesting in light of the history of interspecific hybridizations that generated the asexual, Neotyphodium lineages. In fact, three of the clusters appear to have been introduced to different Neotyphodium species by the same ancestral Epichloe species, for which present day isolates are no longer able to produce lolines. To address the evolutionary origins of the cluster we investigated the phylogenetic relationships of particular lol ORFs to their paralogous primary metabolism genes (and gene families) from endophytes, other fungi and even other kingdoms. Further investigation of two individual lol genes reveals that they evolved from primary metabolism genes within the fungal ancestors of endophytes, as opposed to introduction via horizontal gene transfer. Complementation studies in Neurospora crassa were conducted to explore the functional divergence of one lol gene (lolD) from its primary
metabolism paralog (ornithine decarboxylase). The results of this project support the hypothesis that the LOL secondary metabolite cluster was present in an early ancestor to the fungal endophytes and therefore, the cluster has likely been lost from extant lineages that lack it. Furthermore, phylogenetic analysis of two of the genes and functional characterization of one supports the hypothesis that some, if not all, lol genes are neofunctionalized paralogs of primary metabolism genes from an ancestor of the endophytes.
Publications
- Kutil, Brandi L, Charles J Greenwald, Gang Liu, Martin J Spiering, Christopher L Schardl, Heather H. Wilkinson. 2007. Comparison of loline alkaloid gene clusters across fungal endophytes: predicting both the co-regulatory sequence motifs and the evolutionary history of the trait. Fungal Genetics and Biology 44(10):1002-10.
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Progress 01/01/06 to 12/31/06
Outputs
LOL is a novel secondary metabolite gene cluster associated with the production of loline alkaloids (saturated 1-aminopyrrolizidine alkaloids with an oxygen bridge) exclusively in closely related grass-endophyte species in the genera Epichloe and Neotyphodium. In this study we characterized the LOL cluster in E. festucae, including 10 individual lol genes. Furthermore we defined the boundaries of the cluster and the genomic DNA region flanking LOL in E. festucae. In addition to characterizing the LOL cluster in E. festucae, we sequenced LOL from two additional species, Neotyphodium coenophialum and Neotyphodium sp. PauTG-1. Together with two recently published LOL clusters from N. uncinatum, these data allow for a powerful phylogenetic comparison of five clusters from four closely related species. There is a high degree of microsynteny (conserved gene order and orientation) among the five LOL clusters, allowing us to predict potential transcriptional co-regulatory
binding motifs in lol promoter regions. The relatedness of LOL clusters is especially interesting in light of the history of interspecific hybridizations that generated the asexual, Neotyphodium lineages. In fact, three of the clusters appear to have been introduced to different Neotyphodium species by the same ancestral Epichloe species, for which present day isolates are no longer able to produce lolines. To address the evolutionary origins of the cluster we investigated the phylogenetic relationships of particular lol ORFs to their paralogous primary metabolism genes (and gene families) from endophytes, other fungi and even other kingdoms. Further investigation of two individual lol genes reveals that they evolved from primary metabolism genes within the fungal ancestors of endophytes, as opposed to introduction via horizontal gene transfer. Complementation studies in Neurospora crassa were conducted to explore the functional divergence of one lol gene (lolD) from its primary
metabolism paralog (ornithine decarboxylase).
Impacts
The results of this project support the hypothesis that the LOL secondary metabolite cluster was present in an early ancestor to the fungal endophytes and therefore, the cluster has likely been lost from extant lineages that lack it. Furthermore, phylogenetic analysis of two of the genes and functional characterization of one supports the hypothesis that some, if not all, lol genes are neofunctionalized paralogs of primary metabolism genes from an ancestor of the endophytes.
Publications
- Kutil, Brandi L, Charles J Greenwald, Gang Liu, Martin J Spiering, Christopher L Schardl, Heather H. Wilkinson. 2007. Comparison of loline alkaloid gene clusters across fungal endophytes: predicting both the co-regulatory sequence motifs and the evolutionary history of the trait. Fungal Genetics and Biology, Accepted with revisions (Jan. 2007).
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Progress 01/01/05 to 12/31/05
Outputs
Progress has been made on both objectives. For Objective 1 we have completed characterizing the degree to which lolD and Ef-odc complement the Nc-odc mutant of Neurospora crassa. It turns out Ef-odc is sufficient to rescue the mutation, while lolD seems to have diverged enough in fuction that it does not. For Objective 2. Comparative analysis reveals conserved putative transcription factor binding sites associated with all the lol genes. Also gene family analysis of two lol genes support the hypothesis that the cluster arose, at least in part, via gene duplication and recruitment in an early ancestor to the endophytes. Furthermore, we have gained additional insights about these particular gene families. They are members of the most ancient of all gene families, those responsible for production of PLP-binding enzymes. We have extended our analysis of both lolD and lolC to include analysis of these gene families across a broad sample of taxa. Additional collaborative
efforts include studies of gene family diversification, and gene expression analysis in other fungi and plants.
Impacts
This work confirms the prediction that while lolD has evolved from the primary metabolism odc, there has been sufficient functional divergence since recruitment into the LOL cluster to render it unable to rescue an odc mutation. This result fits well with a model of evolution via gene duplication and neofunctionalization. Furthermore, there is clear support for the hypothesis that the genes in LOL are coregulated, based on shared transcription factor binding sites across and within LOL.
Publications
- Li, D., Bobrowicz, P., Wilkinson, H.H., and Ebbole, D.J. 2005. A mitogen-activated protein kinase pathway essential for mating and contributing to vegetative growth in Neurospora crassa. Genetics 170, 1091-1104.
- Zhang, J., Simmons, C., Yalpani, N., Crane, V., Wilkinson, H., and Kolomiets, M. 2005. Genomic Analysis of the 12-oxo-phytodienoic Acid Reductase Gene Family of Zea mays. Plant Molecular Biology 59, 323-343.
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Progress 01/01/04 to 12/31/04
Outputs
Sufficient progress has been made on both objectives. Specifically, for Objective 1 (Characterize lolD, a putative loline alkaloid biosynthesis gene, as a further test of the hypothesis that LOL governs loline alkaloid expression.) we have made the four constructs to complement the Neurospora mutant. Two transformations tried thus far have been successful. Objective 2. (Continue comparative mapping of LOL and the corresponding flanking regions in E. festucae, N. coenophialum and N. uncinatum.) We now have sequence for five LOL clusters from 4 endophyte species. Comparative analysis reveals conserved putative transcription factor binding sites associated with all the lol genes. Also gene family analysis of two lol genes support the hypothesis that the cluster arose, at least in part, via gene duplication and recruitment in an early ancestor to to the endophytes.
Impacts
This work helps us to understand how genes involved in both beneficial secondary metabolite production and in mycotoxin production work within the organism and how they have evolved. Understanding this helps us to predict better the roles of genes in other filamentous fungi. Strategies for management of agriculturally important fungi (mutualists and pests) may result through targeting genes for secondary metabolism.
Publications
- Li, D., P. Bobrowicz, H.H. Wilkinson and D.J. Ebbole. 2005. A MAP kinase pathway essential for mating and contributing to vegetative growth in Neurospora crassa. Genetics (accepted 12/06/04 pending revision)
- Spiering, M. J., C. D. Moon, H. H. Wilkinson and C. L. Schardl. 2005. Gene clusters for insecticidal loline alkaloids in the grass-endophytic fungus Neotyphodium uncinatum. Genetics (accepted 12/01/04).
- Xie, X., H. H. Wilkinson, A. Correa, Z. Lewis, D. Bell-Pedersen and D. J. Ebbole. 2004. Transcriptional response to glucose starvation and functional analysis of a glucose transporter of Neurospora crassa. Fungal Genetics & Biology 41:1104-1119
- Wilkinson, H. H., A. Ramaswamy, S. C. Sim and N. P. Keller. Increased conidiation associated with progression along the sterigmatocystin biosynthetic pathway. 2004 Mycologia 96:1190-1198
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Progress 07/15/03 to 12/31/03
Outputs
Steps have been taken to initiate both objectives. Specifically, for Objective 1 (Characterize lolD, a putative loline alkaloid biosynthesis gene, as a further test of the hypothesis that LOL governs loline alkaloid expression.) we have a) secured the appropriate Neurospora crassa mutant, b) cloned the odc gene out of Epichloe festucae and c) made 2 of the 4 constructs necessary for the transformation. For Objective 2 (Continue comparative mapping of LOL and the corresponding flanking regions in E. festucae, N. coenophialum and N. uncinatum.) we have constructed a BIBAC vector appropriate for use in fungi so that when we construct the Neotyphodium coenophialum library we should be able to use agrobacterium assisted transformation with the clones. We have cloned and sequenced much of the N. coenophialum LOL cluster.
Impacts
This work helps us to understand how genes involved in both beneficial secondary metabolite production and in mycotoxin production work within the organism and how they have evolved. Understanding this helps us to predict better the roles of genes in other filamentous fungi. Strategies for management of agriculturally important fungi (mutualists and pests) may result through targeting genes for secondary metabolism.
Publications
- Kutil, B.L., L. Gang, J. Vrebalov, and H. H. Wilkinson. 2004. Contig assembly and microsynteny analysis using a bacterial artificial chromosome library for Epichloe festucae, a mutualistic fungal endophyte of grasses Fungal Genetics and Biology. 41:23-32.
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