Genomic and transcriptomic analyses of Microbotryum lychnidis-dioicae provide insights into the biology of a fascinating fungal phytopathogen.

This study made use of the Silene latifolia/Microbotryum lychnidis-dioicae phytopathogen system as the focal system to establish the first reference genome for Microbotryum violaceum sensu lato. In silico analysis was performed on the genome assembly to identify various characteristics of the genome. Using RNA-Sequencing technologies on the Illumina platform, we collected transcriptomic data for both in vitro and in planta life stages of the fungus, providing the most comprehensive look at the gene expression and regulation of this fungus. Due to a lack of identifiable domains on the predicted genes, gene set enrichment analysis was done in context, by including gene sets like “secreted proteins”, “small secreted proteins” and “unique proteins”, to aid discovery of the features in the different datasets. To further research into Microbotryum species in general, we developed, for the first time, a robust and repeatable Agrobacterium-mediated transformation system. Using genomic and transcriptomic data, we were able to select native promoters that drive transcription in specific conditions, making it a highly versatile and controllable system

Annotation and comparative analysis of fungal genomes: a hitchhiker's guide to genomics

This thesis describes several genome-sequencing projects such as those from the fungi Laccaria bicolor S238N-H82, Glomus intraradices DAOM 197198, Melampsora laricis-populina 98AG31, Puccinia graminis, Pichia pastoris GS115 and Candida bombicola, as well as the one of the haptophyte Emiliania huxleyi CCMP1516. These species are important organisms in many aspects, for instance: L. bicolor and G. intraradices are symbiotic fungi growing associate with trees and present an important ecological niches for promoting tree growth; M. laricis- populina and P. graminis are two devastating fungi threating plants; the tiny yeast P. pastoris is the major protein production platform in the pharmaceutical industry; the biosurfactant production yeast C. bombicola is likely to provide a low ecotoxicity detergent and E. huxleyi places in a unique phylogeny position of chromalveolate and contributes to the global carbon cycle system. The completion of the genome sequence and the subsequent functional studies broaden our understanding of these complex biological systems and promote the species as possible model organisms. However, it is commonly observed that the genome sequencing projects are launched with lots of enthusiasm but often frustratingly difficult to finish. Part of the reason are the ever-increasing expectations regarding quality delivery (both with respect to data and analyses). The Introductory Chapter aims to provide an overview of how best to conduct a genome sequencing project. It explains the importance of understanding the basic biology and genetics of the target organism. It also discusses the latest developments in new (next) generation high throughput sequencing (HTS) technologies, how to handle the data and their applications. The emergence of the new HTS technologies brings the whole biology research into a new frontier. For instance, with the help of the new sequencing technologies, we were able to sequence the genome of our interest, namely Pichia pastoris. This tiny yeast, the analysis of which forms the bulk of this thesis, is an important heterologous production platform because its methanol assimilation properties makes it ideally suitable for large scale industrial production. The unique protein assembly pathway of P. pastoris also attracts much basic research interests. We used the new HTS method to sequence and assemble the GS115 genome into four chromosomes and made it publicly available to the research community (Chapter 2 and Chapter 3). The public release of the GS115 brought broader interests on the comparison of GS115 and its parental strains. By sequencing the parental strain of GS115 with different new sequencing platforms, we identified several point mutations in the coding genes that likely contribute to the higher protein translocation efficiency in GS115. The sequence divergence and copy number variation of rDNA between strains also explains the difference of protein production efficiency (Chapter 4). Before 2008, the Sanger sequencing method was the only technology to obtain high quality complete genomes of eukaryotes. Because of the high cost of the Sanger method, regarding the other genome projects discussed in this thesis, it was necessary to team up with many other partners and to rely on the U.S. Department of Energy Joint Genome Institute (DOE-JGI) and the Broad Institute to generate the genome sequence. The M. larici-populina srain 98AG31 and the Puccinia graminis f. sp. tritici strain CRL 75-36-700-3 are two devastating basidiomycete ‘rusts’ that infect poplar and wheat. Lineage-specific gene family expansions in these two rusts highlight the possible role in their obligate biotrophic life-style. Two large sets of effector-like small-secreted proteins with different pri- mary sequence structures were identified in each organism. The in planta-induced transcriptomic data showed upregulation of these lineage-specific genes and they are likely involved in the establishing of the rust-host interaction. An additional immunolocalization study on M. larici-populina confirmed the accumulation of some candidate effectors in the haustoria and infection hyphae, which is described in Chapter 5

Evolution of sex-biased expression in Caenorhabditis

Mating systems have a profound impact on genome structure evolution, both indirectly through their effects on population genetics and directly due to the genetic control of reproductive traits. Most extant Caenorhabditis species are gonochoristic (males and females), while the most studied species, C. elegans and C. briggsae, are androdioecious (self-fertile hermaphrodites and males). The latter two species display an overall reduced ability to mate, suggesting that the selective pressure on maintaining efficient mating was weakened as selfing arose. The genes underlying these traits were likely to have been expressed in a sex-biased fashion in the gonochoristic ancestor, and we hypothesized that as selfing emerged their regulation was modified or they were lost altogether. This hypothesis is especially interesting given that selfing species have consistently smaller genome sizes than their gonochoristic relatives. I sought to address whether a disproportionate loss of genes with sex-biased expression accompanies the loss of mating-related traits in Caenorhabditis hermaphrodites. I first examine sex-biased expression in a gonochoristic species, C. remanei, and identify genes with highly sex-biased expression. I find that these genes are more likely to be missing in selfing species than expected by chance. I then select some of these genes based on their phylogenetic conservation patterns in the genus, and characterize them more thoroughly to shed some light on their functions. Through this study I identify a novel male-associated candidate cis-regulatory element. Lastly, I broaden the scope of the study by determining transcriptome wide sex-biased expression patterns in four Caenorhabditis species. I confirm that C.elegans displays a decrease in the proportion of strong female-biased expression, as well as a modification of the expression of genes with male-biased expression both in males and in hermaphrodites, when compared to gonochoristic Caenorhabditis. Taken together, this study illustrates the transcriptomic consequences of a modification of the mating system, and begins to address its effect on genome structure

Assessing the evolutionary patterns of plastid genome reduction in a group of non-photosynthetic parasitic Angiosperms (Orobanchaceae)

Der Plastid, der als Schlüsselfunktion der autotrophen Lebensweise die Photosynthese ausführt, besitzt ein semi-autonomes genetisches System mit eigenem Genom (Plastom), welches für Proteine des Photosyntheseapparates sowie wenige Enzyme anderer metabolischer Prozesse und Untereinheiten grundlegender genetischer Prozesse kodiert. Aufgrund des Überganges zu einer unterschiedlich stark ausgeprägten heterotrophen Lebensweise weisen parasitische Pflanze enorm modifizierte Plastome auf. Diese sind durch eine extreme funktionelle und strukturelle Reduktion sowie stark erhöhte DNS-Substitutionsraten charakterisiert. Gegenstand dieser Dissertation ist es, evolutive Trends der Plastomreduktion bei verminderten Selektionsdrücken zu rekonstruieren. Zu diesem Zweck wurden die Plastome verschiedener zur Photosynthese fähiger und unfähiger Vertreter der Sommerwurzgewächse (Orobanchaceae) vollständig sequenziert und mittels moderner Methoden der vergleichenden Genomanalyse hinsichtlich folgender Aspekte analysiert: (i) strukturelle Änderungen, (ii) potentielle Funktionalität von Photosynthese-assoziierten Plastidengenen, (iii) Pseudogenisierung und Gendeletion, und (iv) Evolution und Auswirkung erhöhter plastidärer DNS-Substitutionsraten. Darüber hinaus behandelt ein Kapitel methodologische Aspekte der Sequenzierung von Plastidengenomen mittels Pyrosequenzierung gesamt-genomischer DNS-Extrakte. Die Analyse plastidärer DNS nah-verwandter Orobanchaceae erlaubt es erstmals, komplexe Muster der Genomreduktion in parasitischen Pflanzen aufzudecken. Unter anderem kann gezeigt werden, dass bereits der Übergang zu einer heterotrophen Lebensweise für strukturelle Änderungen des Plastoms ausschlaggebend ist und zu einem Verlust der Funktionalität bestimmter Gene und einer Erhöhung der Substitutionsraten führt. Innerhalb der Orobanchaceae schreitet die Plastomreduktion mit zunehmender Heterotrophie mit linienspezifischer Geschwindigkeit voran. Das Ausmaß von Pseudogenisierung und Deletion nicht-essentieller Genomabschnitte wird dabei maßgeblich durch die Distanz zu essentiellen genischen Elementen und von der plastidären Operonstruktur beeinflusst. Darüber hinaus weisen die zusammengetragenen Ergebnisse darauf hin, dass parasitische Pflanzen die Funktion einzelner Photosynthese-assoziierter Proteinkomplexe möglicherweise aufrechterhalten und eine erhöhte Rate an intrazellulärem DNS-Transfer aufweisen. Veränderungen der DNS-Substitutionsmuster bei zur Photosynthese fähigen heterotrophen Orobanchaceae implizieren eine Korrelation des Übertritts zur parasitischen Lebensweise mit der Verminderung der Selektion bestimmter plastidärer Genen. Im Vergleich zu photosynthetisch aktiven Pflanzen, weisen Vollparasiten differenzierte Muster bezüglich DNS-Substitutionen auf, einschließlich linienspezifischer Ratenerhöhung und –reduktion. In der vorliegenden Arbeit wird anhand der Analyse von simulierten und experimentell generierten 454-Datensätzen erstmals gezeigt, dass die erfolgreiche Plastomrekonstruktion signifikant von der verwendeten Sequenzdatenmenge bestimmt wird. Darüber hinaus wird eine Methode zur a priori Schätzung der optimalen Datenmenge unter Verwendung weniger Parameter erarbeitet.The prime function of the plastid organelle is to carry out photosynthesis thereby providing autotrophy to the plant kingdom. Plastids retain a semi-autonomous genetic system including a genome (plastome) encoding subunits for photosynthesis-related and unrelated processes as well as proteins for basic functions of the genetic apparatus. Due to the transition from an autotrophic to a semi- or holo-heterotrophic lifestyle, parasitic plants show major plastomic reconfigurations with extreme reductions of plastome size and coding capacity as well as extraordinarily elevated nucleotide substitution rates. Using the broomrape family (Orobanchaceae) as a model group, this dissertation thesis reconstructs molecular evolutionary patterns of reductive plastome evolution of the plastid chromosome under relaxed evolutionary constraints. Employing comparative-evolutionary analyses of completely sequenced plastid genomes from several hemi- and holoparasitic members of Orobanchaceae this work examines aspects concerning the (i) co-linearity and structural rearrangements of plastomes, (ii) potential functionality of genes involved in photosynthesis, (iii) pseudogenization and gene loss, and (iv) accelerated substitution rates in plastid genomes. In addition, one chapter evaluates methodological aspects of plastid genome sequencing employing whole-genome shotgun pyrosequencing. This work reveals that genetic and genomic changes concerning plastome structure, nucleotide substitution rates and selectional constraints occur in a complex and highly lineage specific manner, and it provides novel insights into factors influencing reductive evolution of plastome. Increasing host-dependency notably seeds excessive non-functionalization of plastid genes due to pseudogenization or deletion, and severely relaxes the structural maintenance of the plastid chromosome. Pseudogenization and segmental deletions of newly dispensable regions depend significantly on the operon-structure of the plastid chromosomes as well as on the distribution of essential genes in Orobanchaceae. There is evidence for maintained or alternative function of a photosynthesis-related complex as well as for putatively increased rates of intracellular gene transfer in parasitic plants. Analyses of nucleotide substitutions reveal significantly elevated rates in both housekeeping and photosynthesis genes already in photosynthetic heterotrophs indicating that relaxation of selective constraints relates to the transition to a parasitic lifestyle. Compared to hemiparasites and autotrophs, distinctive trends of rate and selectional changes exist among holoparasite lineages including both local accelerations and rate reductions. Above that, this thesis shows for the first time that the successful reconstruction of plastid chromosomes from whole-genome shotgun pyrosequencing strongly depends on the size of the assembled read pool. Using the results of simulated and empirical 454 datasets in combination with a resampling scheme for automated quality assessment, a method for a parameter-less a priori assessment of the optimal read pool size is established that should ease assembly efforts

Conservation of different mechanisms of Hox cluster regulation within chordates

[eng] In this thesis we have covered the importance of finding underlying conservation events to better understand the regulatory mechanisms of important development orchestrators like the Hox cluster. As an example of these non-evident conservation, we have shown two cases, as described below. The first case studied, after developing a software able to detect homologous long noncoding RNAs by means of microsynteny analyses, is the conservation of Hotairm1 in Chordata. For assessing the homology of this lncRNA, first we had to identify the lncRNA fraction within the B. lanceolatum transcriptome. With a reliable lincRNA dataset, we used our pipeline, LincOFinder, to identify orthologs between human and amphioxus through microsynteny. After the identification of Hotairm1 as one of the lincRNAs with conserved microsynteny, we used Xenopus as a proxy to analyse the homologies in the expression and the function. We had to proceed this way due to the difficulties associated with the inhibition of genes in B. lanceolatum, and the unavailability of expression patterns for Hotairm1 in the bibliography. After we successfully characterised Hotairm1 expression in amphioxus and Xenopus, we injected morpholino oligonucleotides to target and inhibit the splicing of Hotairm1 to promote an isoform imbalance. Through the phenotype obtained and the performing of qPCRs, we were able to deduct the mechanism of Hotairm1 and successfully relate this mechanism with the one described in human cells. With all the data obtained we were able to strongly suggest that the amphioxus Hotairm1 is homologous to the Xenopus and human Hotairm1, thus being conserved in most of the lineages within chordates. The second case studied was the conservation of the regulation of the Hox cluster mediated by Cdx. When analysing the B. floridae knockouts of Cdx and Pdx obtained using the TALEN technique, we found a severe phenotype of the developing larvae in Cdx-/- and a mild phenotype in Pdx-/-. The Cdx-/- phenotype consisted in the disruption of posterior gut development, as well as an underdevelopment of the postanal tail, coupled with a non-opening anus. When looking at changes in the expression of the Hox cluster in this Cdx-/- embryos, we found collinear misregulation of the expressed Hox genes, with the most anterior Hox cluster genes upregulated, and the most posterior ones downregulated. This is very similar to findings seen in triple morpholino knockdowns of the Cdx genes in Xenopus, indicating that in both, Xenopus and amphioxus, Cdx is regulating the Hox cluster through a homologous mechanism

Mammalian comparative genomics and epigenomics

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student submitted PDF version of thesis.Includes bibliographical references.The human genome sequence can be thought of as an instruction manual for our species, written and rewritten over more than a billion of years of evolution. Taking a complete inventory of our genome, dissecting its genes and their functional components, and elucidating how these genes are selectively used to establish and maintain cell types with markedly different behaviors, are key challenges of modern biology. In this thesis we present contributions to our understanding of the structure, function and evolution of the human genome. We rely on two complementary approaches. First, we study signatures of evolutionary processes that have acted on the genome using comparative sequence analysis. We generate high quality draft genome sequences of the chimpanzee, the dog and the opossum. These species share a last common ancestor with humans approximately 6 million, 80 million and 140 million years ago, respectively, and therefore provide distinct perspectives on our evolutionary history. We apply computational methods to explore the functional organization of the genome and to identify genes that contribute to shared and species-specific traits. Second, we study how the genome is bound by proteins and packaged into chromatin in distinct cell types. We develop new methods to map protein-DNA interactions and DNA methylation using single-molecule based sequencing technology. We apply these methods to identify new functional sequence elements based on characteristic chromatin signatures, and to explore the relationship between DNA sequence, chromatin and cellular state.by Tarjei Sigurd Mikkelsen.Ph.D

ESTABLISHMENT OF BIOTROPHY BY THE MAIZE ANTHRACNOSE PATHOGEN \u3cem\u3eCOLLETOTRICHUM GRAMINICOLA\u3c/em\u3e: USE OF BIOINFORMATICS AND TRANSCRIPTOMICS TO ADDRESS THE POTENTIAL ROLES OF SECRETION, STRESS RESPONSE, AND SECRETED PROTEINS

Colletotrichum graminicola is a hemibiotrophic pathogen of maize that causes anthracnose leaf and stalk rot diseases. The pathogen penetrates the host and initially establishes an intracellular biotrophic infection, in which the hyphae are separated from the living host cell by a membrane that is elaborated by the host, apparently in response to pathogen signals. A nonpathogenic mutant (MT) of C. graminicola was generated that germinates and penetrates the host normally, but is incapable of establishing a normal biotrophic infection. The mutated gene is Cpr1, conserved in eukaryotes and predicted to encode a component of the signal peptidase complex. How can we explain why the MT is normal in culture and during early stages of pathogenicity, but is deficient specifically in the ability to establish biotrophy? To address this, first I characterized the insertion in the 3’ UTR of the MT strain in detail, something that had not been done before. The wild-type (WT) transcript did not differ from predictions, but the MT produced several aberrant transcript species, including truncated and non-spliced transcripts, and the normal one. Aberrant splicing of MT cpr1 was observed both in RNAseq transcriptome data and reverse-transcription polymerase chain reaction (RT-PCR), under different growth conditions and in planta. I also conducted a bioinformatic analysis of other conserved components of the secretory pathway in the MT and WT in planta. One explanation for nonpathogenicity of the MT is that it cannot cope with an increase in secretory activity during infection, and fails to produce necessary pathogenicity factors. With the transcriptome data, I was able to identify effector proteins that were expressed in the WT but not in the MT. Another possible explanation for the MT phenotype is that the MT can’t adapt to stress imposed by the plant. I developed a growth assay to characterize the effect of chemical stressors in vitro. The MT was more sensitive to most stressors, when compared to the WT. The transcriptome data indicates that the genes involved in different stress pathways are expressed in planta in both WT and MT, although very few genes are differentially expressed across the different growth stages

“Deciphering the inter-kingdom RNA exchanges in the Alternaria-Tomato Pathosystem”

Fungal infections make up most plant crop losses in the agricultural industry. This contrasts with mammals, in which the major threat to our existence is viruses and bacteria with fungi making up only a small minority of the infections we suffer. Due to this there has been a large investment in the prevention of fungal infection, mainly using chemical fungicides. However, chemical fungicides have many disadvantages, most notably the runoff resulting in damage to wildlife and other forms of ecological impact, occurrence of resistant pathogen isolates and health risks for consumers and farmers using them. This has gradually led to public rejection of chemical fungicides as shown by the growth of the organic food industry. The growing technology of RNA silencing shows great potential in alleviating this problem. Host induced gene silencing (HIGS), naturally exists in plants as a means of protection from pathogens and has already become cited as a potential new resistance engineering method in crops. Either by spraying the plant directly with RNA molecules or inducing the RNA production in the plant using gene delivery vectors like Agrobacterium and viruses. The more we understand these interactions the more we can perfect this technology. Here the A. alternata-tomato pathosystem will be used to evaluate the role of sRNA in infection and defence of the plant and evaluate genes that may have value as a target for sRNA. In addition, this project will look at the potential of certain genes as targets for targeted RNA silencing to protect tomato from Alternaria alternata. Specifically, the Chitin synthase genes (CHS) and the effect silencing RNAs has on these genes has and whether they inhibit fungal infection. The methods used in this project focused on two areas, determining if CHS is a viable target for spray induced silencing through treating spores with small RNA solutions and determining any effect on germination rates. These experiments found limited effect on germination however did discover a significant effect on germination tube size and spore production, with tube size increasing significantly on treatment with sRNA and spore production occuring earlier and in larger quantities. sRNA produced by the plant during infection was also evaluated using sRNA sequencing, to identify novel miRNA. My conclusion for this project are that CHS is likely a poor candidate for sRNA silencing however with the number of miRNA identified to show upregulation during infection it can be concluded that natural miRNA play a role in infection and many of these miRNA could be the targets of future research to improve the defence response of the tomato plant or to develop new SIGS or HIGS systems