Ph.D. Candidate Exam Slides

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Bongsoo's Ph.D. Candidate Exam 8/27/2010

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<ul><li><p>Phylogenomic Analysis Demonstrates a Pattern of Rare and Ancient Horizontal Gene Transfer between Plants and Fungi(Richards et al. 2009 Plant Cell)</p></li><li><p>OutlineIntroductionResults MethodsDiscussion</p></li><li><p>What is Horizontal Gene Transfer?vs Vertical Gene Transfer (reproduction)HGT provides opportunities for rapid genome innovation.Abundant in prokaryotes (e.g. transfer of antibiotic resistance genes among bacteria, metabolic capability of certain substrates as nutrient sources, etc.)http://tolweb.org/tree/</p></li><li><p>Examples of HGTs in EukaryotesPlant Pathogen, Fusarium oxysporum (Li-Jun et al. Nature, 2010)</p><p>Pea Aphid, Acyrthosiphon pisum (Nancy A.M. Science, 2010)</p><p>Carotenoids biosynthesis genestransferred from fungi to an ancestor of aphids Entire Lineage-specific Chromosomes transferred to another strain of F. oxysporum, making the recipient pathogenic to Tomato.</p></li><li><p>Table 2. Nine putative HGT candidate genesResultsTable 1. Identification of Putative HGTs between Plants and Fungi for Phylogenetic AnalysesFungi &gt; PlantPlant &gt; Fungi</p><p>HGTPutative Functional Protein AnnotationDirection of Transfer1aL-Fucose permease, sugar transporterFungi &gt; Plant1bZinc binding alcohol dehydrogenasePlant &gt; Fungi1cMajor facilitator superfamily, membrane transporterFungi &gt; Plant2Phospholipase/carboxylesterase family proteinFungi &gt; Plant3aiucA/iucC family protein, siderophore biosynthesisFungi &gt; Plant3bUnknown/conserved hypothetical proteinFungi &gt; Plant4aDUF239 domain proteinPlant &gt; Fungi4bPhosphate-responsive 1 family proteinPlant &gt; Fungi4cUnknown/conserved hypothetical protein with similarity to zinc finger (C2H2-type) proteinPlant &gt; Fungi</p><p>Arabidopsis thaliana (Arabidopsis)Populus trichocarpa (Western poplar)Oryza sativa (Rice)Sorghum bicolor(Sorghum, edible grain)Selaginella moellendorffii (spikemosses)Physomitrella patens (Moss)Total number of proteins31,91345,55526,77735,89922,28535,938Top hit versus fungi1,2061,4261,0451,043802776Top hit versus fungi excluding TEs1,1601,331852965794764</p></li><li><p>BLAST, OrthoMCL pipelinePhylogenomic analysesPlants(Query)Fungi, Algae, Protists, Animals, Prokaryotes(Target)5,586 protein sequencesExcluding TEs(Putative functional proteome)1,689 groups Initial ScreeningRiceDB3,177 Orthology Groups4,866 sequence groups, Ready4,866 sequence groupsFour evidencesPutative 9 HGT candidates38GenBank NRGenBank EST, TBestDB(Improve sampling)3521OrthoMCL(e-value 1e-20; inflation value 1.5)14MethodsMrBayes, PHYML, RAxML, SH TestTarget DBBLASTp (e-value 10-20)</p></li><li><p>FungiPlantsStrong bootstrapvaluesAscomycete fungiSelaginellaWeak bootstrapvalues12EvidencesAlternative Topology Test(CONSEL)SH (Shimodaria-Hasegawa)P-value</p></li><li><p>1BayesianPHYMLRAxMLSH testL-Fucose permease, sugar transporter</p></li><li><p>FungiPlantsStrong bootstrap valuesA single fungal speciesPlants specific Gene family34EvidencesStrong bootstrap valuesProkaryotesHGT Based on a Prokaryote Tagged-Chain Transfer HypothesisWeak bootstrap values</p></li><li><p>3BayesianPHYMLRAxMLSH testDUF(Domain of Unknown Function)iucA/iucC family protein, siderophore biosynthesis</p></li><li><p>3A : iucA/iucC family protein, siderophore biosynthesisSiderophores are small, high-affinity iron chelating compoundsNon-pathogenic Bacteria, iron-poor environmentFungi, obtaining siderophore biosynthesissurvival in iron-poor environmentLycophyte (Selaginella moellendorffii)</p></li><li><p>44BayesianPHYMLRAxMLSH test</p></li><li><p>Putative functional assignmentsDiscussion</p><p>HGTPutative Functional Protein AnnotationDirection of Transfer1aL-Fucose permease, sugar transporterFungi &gt; Plant1bZinc binding alcohol dehydrogenasePlant &gt; Fungi1cMajor facilitator superfamily, membrane transporterFungi &gt; Plant2Phospholipase/carboxylesterase family proteinFungi &gt; Plant3aiucA/iucC family protein, siderophore biosynthesisFungi &gt; Plant3bUnknown/conserved hypothetical proteinFungi &gt; Plant4aDUF239 domain protein (Domain of Unknown function)Plant &gt; Fungi4bPhosphate-responsive 1 family proteinPlant &gt; Fungi4cUnknown/conserved hypothetical protein with similarity to zinc finger (C2H2-type) proteinPlant &gt; Fungi</p></li><li><p>AngiospermLycophyteBryophyteChytridiomycotaZygomyceteBasidiomyceteAscomyceteFig 8.Taphrinomycotina</p></li><li><p>My perspectivesGenome SamplingTransposable ElementsApplying this methods to detect HGTs in Fungal and Oomycete pathogens</p><p>This research, however, is a pioneer project to invest the eukaryote HGT by using currently available resources and methods.</p></li><li><p>Thank You</p></li><li><p>Supplementary Slides </p></li><li><p>History of HGTFirst described in Japan in 1959, 1960Transfer of antibiotic resistance between different species of bacteria; bacillary dysenteryTsutomu Watanabe, Bacteriology Review (1963)</p></li><li><p>Known mechanisms of HGTTransformation, introduction, uptake and expression of foreign genetic materialTransduction, transmission by virusBacterial conjugation, cell-to-cell contact</p></li><li><p>1BayesianPHYMLRAxMLSH test</p></li><li><p>1</p></li><li><p>1</p></li><li><p>22</p></li><li><p>3</p></li><li><p>3</p></li><li><p>44</p></li><li><p>4</p></li><li><p>Gene appears unique to genomeCould not confirm vertical inheritancePhylogeny suggests vertical inheritanceTaxon distribution of gene family suggests vertical inheritanceTransposable element1AL-fucose permease, sugar transporterPhpa(173818)1BZinc binding alcohol dehydrogenaseBDEG_06896Fig 7. Strong vertical inheritance</p></li><li><p>Table 1. Nine putative HGT candidate genes</p><p>HGTPutative Functional Protein AnnotationDirection of TransferGenBank Accession No.Phylogeny construction 1aL-Fucose permease, sugar transporterFungi &gt; PlantEDQ8358198 sequences and 341 length62 sequences and 349 length1bZinc binding alcohol dehydrogenasePlant &gt; FungiEDQ61140*95 sequences and 207 length1cMajor facilitator superfamily, membrane transporterFungi &gt; PlantEAU93280*40 sequences and 354 length2Phospholipase/carboxylesterase family proteinFungi &gt; PlantXP_389330*122 sequences and 158 length 62 sequences and 349 length3aiucA/iucC family protein, siderophore biosynthesisFungi &gt; PlantEDR08700*44 sequences and 218 length 15 sequences and 262 length3bUnknown/conserved hypothetical proteinFungi &gt; PlantEDQ6864255 sequences and 174 length 34 sequences and 247 length4aDUF239 domain proteinPlant &gt; FungiEDR0274787 sequences and 210 length4bPhosphate-responsive 1 family proteinPlant &gt; FungiABK92591*93 sequences and 198 length4cUnknown/conserved hypothetical protein with similarity to zinc finger (C2H2-type) proteinPlant &gt; FungiEDN2358413 sequences and 222 lengthGenBank accession numbers of transferred genes are given, and the asterisks indicate accession numbers of the closest BLAST hit in GenBank to the HGT, rather than that of the recipient gene</p></li><li><p>Challenging QuestionsHow long ago HGT processes occurred?DNA move back and forth between donors and recipients?How environmental factors (light, water, temperature, pH) affect the gene transfers?Any new mechanisms of transfer?How transferred genes can be fixed in a population successfully?</p></li><li><p>Good afternoon everyone. First, I would like to thank you for agreeing to serve on my candidacy exam committee.The topic I chose to present concerns Horizontal Gene Transfers between kingdoms of fungi and plants. </p><p>Here is the outline of my presentation. I will begin my presentation by briefly outlining what Horizontal Gene Transfer is, how it occurs, and why it matters. And then, I will summarize and discuss key findings in the paper I chosen, which is a Plant Cell paper published by Richards et al. in 2009. The main goal of this paper is to test whether Horizontal Gene Transfers have occurred between plants and fungi by using phylogenomic analysis of the proteomes of diverse plants, fungi, and other organisms. To explain what the Horizontal Gene Transfer is, I first need to talk about the Vertical Gene Transfer . The Vertical Gene Transfer refers to the passage of genes from one generation to the next through sexual or asexual production. However, Horizontal Gene Transfer doesnt require such reproduction process and involves the movement of genetic materials across species barriers. Although most genes transferred through HGT probably fail to function, probably because the recipient organism degrade the genes, dies because of mutations caused by the introduction, or lacks the ability to turn on the genes, in some cases the introduced genes can survive and be fixed in the recipient genome, especially when the introduced genes help the recipient organism survive under harmful conditions, such as the presence of antibiotics, or allows the recipient to utilize certain substrates as nutrients.Basically, HGT is a mechanism that provides opportunities for rapid genome innovation. It appears that HGT is a common mechanism of genome evolution in prokaryotes, but the roles and frequencies of HGT in eukaryotes have not yet been extensively studied. However, the rapid increase in genome sequencing has opened up opportunities for studying HGT in the evolution of eukaryotes.I would like to introduce two examples of HGTs in Eukaryotes. First example is plant pathogenic fungus, Fusarium oxysporum. Certain strains of the asexual species F. oxysporum have Lineage-specific (LS) chromosomes. These chromosomes cover about one-quarter of the genome, and many genes in the LS chromosomes are highly related to pathogenecity. The presence of these chromosomes allow the strains carrying them pathogenic to a particular host plant. Horizontal transfers of some of these chromosomes from a tomato pathogenic strain to a nonpathogenic strain have been experimentally demonstrated in the laboratory, and the recipient strains became pathogenic to tomato. Second example is the cartenoids biosynthe by pea aphid Acyrthosiphon pisum. The cartenoids biosynthetic genes were found in aphids. Usually, animals obtain carotenoids from food. However, Nancy Morgan at University of Arizona discovered the presence of a cartenoid gene set in insect (animal kingdom). Phylogenetic analyses imply the transfer of these genes from a fungus to an ancestor of aphid species, allowing aphids to synthesize cartenoides.Table 1 presents the six plant genome which were used in this study. This is total number of proteins. Second row, if the protein of plant is highly similar to fungi. They use BLASTp method here. They exclude the Transposable Element in this study. (5586)</p><p>Table 2 presents the nine candidate genes of potential HGT discovered by Richards et al.. There are five fungi-to-plant transfers, and four plant-to-fungi transfers. To find these nine candidates, they developed two pipelines which are a HGT detection pipeline based on BLAST search and gene clustering through OrthoMCL, and Phylogenomic analyses methods.</p><p>Angiospermae (Flowering plants)</p><p>Arabidopsis, 20-25cm tall, 3mm in diameter, Arabidopsis genome, 135 million bp, 5 chromosomes, 33410 protein-coding transcripts, one of the smallest genomes among plants, 2000 Arabidopsis Genome Initiative. T-DNA insertions, ATMT. Light microscopy analysis. Large number of ecotypes( genetically distinct geographic variety population, which is adapted to specific environmental conditions) 1986, T-DNA mediated transformation was first published. </p><p>Populus trichocarpa is known as black cottonwood or california popular, 2006 Full genome sequence, 30-50m, diameter of over 2m. Dioecious; maile and femail catkins are borne on separate trees. Model species for trees. Genome : 403 Mb, 45778 protein-coding transcripts, 19 chromosomes, Mitochondrial(803,000bp, 52genes), Chloroplast(157,000bp, 101genes)</p><p>Oryza sativa, Asian Rice. Genome 372Mb, 12Chromosomes. Easily genetically modified. (Temperate, Japonica, Tropical japonica, aromatic, indica), China 11,500 BP, Model organism cereal biology(JGI)When I compare the number of transcript, the data is not cosistent. (51258, protein-coding transcript)</p><p>Sorghum bicolor, 697Mbp, 2n=20 chromosome, 36,338 protein-coding transcripts(JGI)</p><p>Selaginella moellendorfii, spikemosses are among the few surviving members of the lycophytes, an ancient group of plants whose orgin can be trace back 400million years ago. Only three families of lycophytes survive today, including the Selaginellaceae. 212.5Mbp, 27 chromosomes</p><p>Physcomitrella patens, species of moss, basal lineage of land plants, having diverged before the acquision of well developed vasculate, 480Mb, 27 Chromosome, 39727 Loci, 39796 protein-coding transcripts(JGI)</p><p>*In their HGT detection pipeline, the proteins encoded by fully sequenced genomes of six plant species were used as query sequences to compared with proteins encoded by fungi, algae, protists, animals, and prokaryotes. Each plant protein sequences were compared with the proteins in the target sequence database using BLASTp. The best similarity hits from each species extracted e-value cutoff 10-20. They identified the query proteins that picked up fungal proteins as the best hit. The sequences that had a close similarity with transposons (Repbase) were excluded because they wanted to study the effect of HGT on the putative functional proteomes of fungi and plants. As a result, 5586 proteins were identified through this analysis. These proteins were clustered OrthoMCL, resulting in 943 clusters and 746 singletons. In total, 1,689 sequence groups were generated.The main concern of using BLASTp to identify candidates for HGT is that BLAST was not designed to accurately determine the evolutionary relationship between two sequences. So using BLASTp alone to identify candidates for HGT can potentially miss some candidate genes for HGT, as the the closest BLAST hit is often not the nearest neighbor on a phylogenetic tree. To test for this possibility, they also repeated this clustering step using the entire predicted proteome of rice, producing 3177 cluster groups. The combined set of 4,866 sequence groups were subjected to Phylogenomic analyses. </p><p>In here, they added highly homologus sequences from Target Database using BLASTp again. And then, they drew 4866 phylogenetic trees to identify potential HGT like events. The basic topology criteria is like this. Gene or gene family phylogenies demonstrating a plant gene branching within a cluster of sequences from fungal taxa (or vice versa) were identfied. After this analysis, only 38 phylogenies were remained.</p><p>(HGT Detection pipeline is ended here, in the middle of right box) </p><p>35 trees were derived from the proteins identified through the BLAST-based survey, and two of them were from the rice orthology cluster, and one candidate phylogeny was discovered by both methods. Before conducting more detailed analyses, they added more protein sequence data from GenBank, TBestDB database (EST database) and manually adjusted sequence alignments for more robust analysis. Adding additional sequences generated the better quality phylogeny judging from multiple statistical methods. The final testing of HGT was based whether the resulting phylogeny is supported by one of the four evidences, which I will summarize in the following slides. At the end, they discovered 9 candidate genes. *The first evidence is quiet same with previous criteria. A gene phylogeny demonstrating a plant gene sequence branching within a cluster of sequences from fungal taxa (or vice versa), However, the bootstrap values need to be...</p></li></ul>

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