A Comparative Genomic Investigation of Niche Adaptation in Fungi”
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The Pennsylvania State University The Graduate School Huck Institute of the Life Sciences A COMPARATIVE GENOMIC INVESTIGATION OF NICHE ADAPTATION IN FUNGI A Dissertation in Integrative Biosciences by Venkatesh Moktali © 2013 Venkatesh Moktali Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2013 ! The dissertation of Venkatesh Moktali was reviewed and approved* by the following: Seogchan Kang Professor of Plant Pathology and Environmental Microbiology Dissertation Advisor Chair of Committee David M. Geiser Professor of Plant Pathology and Environmental Microbiology Kateryna Makova Professor of Biology Anton Nekrutenko Associate Professor of Biochemistry and Molecular Biology Yu Zhang Associate Professor of Statistics Peter Hudson Department Head, Huck Institute of the Life Sciences *Signatures are on file in the Graduate School ! ! Abstract The Kingdom Fungi has a diverse array of members adapted to very disparate and the most hostile surroundings on earth: such as living plant and/or animal tissues, soil, aquatic environments, other microorganisms, dead animals, and exudates of plants, animals and even nuclear reactors. The ability of fungi to survive in these various niches is supported by the presence of key enzymes/proteins that can metabolize extraneous harmful factors. Characterization of the evolution of these key proteins gives us a glimpse at the molecular mechanisms underpinning adaptations in these organisms. Cytochrome P450 proteins (CYPs) are among the most diversified protein families, they are involved in a number of processed that are critical to fungi. I evaluated the evolution of Cytochrome P450 proteins (CYPs) in order to understand niche adaptation in fungi. Towards this goal, a previously developed database the fungal cytochrome P450 database (FCPD) was improved and several features were added in order to allow for systematic comparative genomic and phylogenomic analysis of CYPs from numerous fungal genomes. Specifically, an in-house platform was developed to standardize and improve the protein sequence clustering procedure, more than 100 fungal and non-fungal genomes were added, putative functional classification of CYPs into three broad categories was added, the CYP clan/family classification was extended to 117 CYP clans and 292 families. With these new features the FCPD 1.2 (http://p450.riceblast.snu.ac.kr/) was published with systematic classification of 22,940 CYPs from 213 species. Using the CYP data from the FCPD 1.2 I carried out detailed phylogenomic analysis of 6108 CYPs belonging to 51 species from the subphylum Pezizomycotina. The analysis revealed CYPomes that were specific to the niches occupied by the different fungi. There was preferential ! iii ! ! presence of CYP families among pathogenic species. I introduced a putative functional diversification ratio to identify divergence of “CYPomes”, the ratio suggests that the non- pathogenic fungi tended to have comparatively more CYP families/clans compared to the pathogenic fungi. I also identified the CYP clans ancestral to Pezizomycotina fungi, the results confirm previously estimated ancestral CYP clans CYP51, CYP61 and add CYP52 and CYP58 to the set of clans present in the last common ancestor of Pezizomycotina fungi. Putative metabolic classification of CYPs in the group suggests increase in CYPs involved in secondary metabolism among fungal plant pathogens. I also carried out gene family evolution analysis with the CYP data, the analyses suggested high number of gains and losses among serious plant pathogens such as Magnaporthe oryzae and Fusarium spp. Calcium is a ubiquitous ion that plays a major role in myriad processes in the fungal cell. I developed the fungal calcium-signaling database (FCSD: http://fcsd.ifungi.org/) in order to understand the roles of genes interacting directly or indirectly with calcium. The computational pipeline standardized in the FCPD was used to cluster the calcium signaling gene dataset. Comparative genomics analyses of calcium signaling genes indicated varying complexity of the calcium-signaling pathway across fungal phyla. I also carried out detailed gene family analysis of different calcium signaling genes, niche-specific gene gains were observed through this analysis. I believe that the various features built into FCSD encourage community participation and research on understanding calcium signaling in fungi. ! iv ! ! Contents Pages List of Tables x List of Figures xii Acknowledgements xv 1 Introduction 1 1.1 History of Fungal Research…………………………………………………...............1 1.2 Diversity in Kingdom Fungi…………………………………………………………..2 1.3 Variation in Fungal genome content…………………………………………………..3 1.4 Emergence of Comparative Genomics and “Phylogenomics”………………………..7 1.5 Investigating evolution via comparative analysis of protein families………...............9 2 Pilot Project: Optimization of Cytochrome P450 sequence clustering procedure 10 2.1 Background…………………………………………………………………………..11 2.1.1 Cytochrome P450 proteins…………………………………………………11 2.1.2 Cytochrome P450 nomenclature system…………………………...............14 2.1.3 FCPD Pipeline……………………………………………………………..15 2.1.4 Fungal cytochrome P450 database: drawbacks …………………...............16 2.2 Results and Discussion………………………………………………………………17 2.2.1 Genus Aspergillus …………………………………………………………17 2.2.2 P450 Analysis Platform……………………………………………………21 2.2.3 Species-specific CYPs……………………………………………………..26 2.2.4 CYPs involved in Secondary Metabolism Clusters………………………..29 2.2.5 N. fischeri cluster…………………………………………………………..31 2.2.6 Putative secretory CYPs…………………………………………...............35 2.3 Materials & Methods………………………………………………………...............38 2.3.1 Data………………………………………………………………...............38 ! v ! ! 2.3.2 Analyses Tools……………………………………………………………..38 2.4 Conclusion …………………………………………………………………………..39 3 Systematic and searchable classification of cytochrome P450 proteins encoded by fungal and oomycete genomes 40 3.1 Background…………………………………………………………………………..42 3.2 Results and Discussion………………………………………………………………44 3.2.1 Identification of CYPs and optimization of clustering parameters...............44 3.2.2 CYP clustering in fungal cytochrome P450 database (FCPD) 1.2...............46 3.2.3 Wide variation of the CYPome……………………………………………47 3.2.4 Phyletic distribution of CYP families and clans in fungi………………….48 3.2.5 Functional annotation and classification of CYP clusters ………...............50 3.2.6 Detailed analysis of specific clans…………………………………………51 3.2.6a Clans 51 and 61 ………………………………………………….52 3.2.6b Clans 65 and 68…………………………………………………...53 3.2.6c Clan 505…………………………………………………………..54 3.2.6d Clan 52 …………………………………………………………...54 3.2.6e Clan 53 and Clan 504 …………………………………………….55 3.2.6f Clan 533 …………………………………………………………..56 3.2.7 CYPs in Mucoromycotina, Blastocladiomycota and Oomycota ………….57 3.2.8 CYPs with unusual phyletic profiles ………………………………………58 3.3 Conclusion…………………………………………………………………...............60 3.4 Materials and methods ………………………………………………………………62 3.4.1 Acquisition of sequence data and phylogenetic analyses …………………62 3.4.2 Clustering the CYPs using BLASTp and the optimized Tribe-MCL algorithm…………………………………………………………………………63 3.4.3 Clan Identification…………………………………………………………63 3.4.4 Classification of CYPs into putative functional categories………………..64 3.4.5 Online database architecture……………………………………………….64 3.5 Tables………………………………………………………………………...............65 3.7 Figures………………………………………………………………………………..77 ! vi ! ! 4 Phylogenomic investigation of Cytochrome P450 proteins from 51 Pezizomycotina species 85 4.1 Background…………………………………………………………………………..85 4.2 Results………………..………………………………………………………………87 4.2.1 CYP clan/family diversity in Pezizomycotina……………………………..87 4.2.2 Metabolic distribution of CYPs……………………………………………89 4.2.3 CYP clan gains and losses in Pezizomycotina………….………………….91 4.2.4 CYPs under selection pressure………………………….………………….93 4.2.5 CYP duplication in Pezizomycotina……..………………………...............94 4.2.6 Taxa and species-specific CYPs…………………………………...............95 4.2.7 Secretory CYPs of Pezizomycotina………………………………………..95 4.3 Discussion.………………………………………………….………………………..96 4.3.1 CYP functional diversity is correlated with pathogenic lifestyles…………96 4.3.2 CYP clan losses and gains are correlated with fungal lifestyles…...............97 4.3.3 Preferential gains/losses of CYPs in specific metabolic categories..………98 4.3.4 CYP clan evolution…………..…………………………………………...100 4.3.5 Species and Class-specific CYPs indicate niche specific diversification...101 4.3.6 Reasons to find species-specific CYPs…………….……………………..103 4.4 Conclusion………………………………………………….………………………104 4.5 Materials and methods……………………………………………………………...105 4.5.1 Description of the computational pipeline…………………………..……105 4.5.2 Comparison of Pezizomycotina with characterized CYPs……….............105 4.5.3 Calculating dN/dS ratio……………………………………………..……105 4.5.4 Phylogenetic tree construction……………………………………………106 4.5.5 CYP clan gain and loss analysis………………………………………….106 4.5.6 Finding secretory CYPs…………………………………………………..106 4.5 Supplementary Figures……………….……………….……………………………107 4.6 Supplementary Tables………………………………….…………………...............108 ! vii ! ! 5 Fungal Calcium Signaling Database (FCSD): A community resource for calcium signaling in fungi 119 5.1 Background………………………………………………….……………………...120 5.2 Construction and content…………………………………………………...............121 5.2.1 Computational pipeline utilized in FCPD………………………………...121 5.2.2 Searching and accessing sequence data in FCSD………………...............122 5.2.3 BLAST utility………………………………………………….…………123