Genome analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared t

RIME: Repeat Identification

We present an algorithm for detecting long similar fragments occurring at least twice in a set of biological sequences. The problem becomes computationally challenging when the frequency of a repeat is allowed to increase and when a non-negligible number of insertions, deletions and substitutions are allowed. We introduce in this paper an algorithm, Rime1 1 Rime is also a reference to Coleridge's poem "The Rime of an Ancient Mariner" which contains many repetitions as a poetic device. (for Repeat Identification: long, Multiple, and with Edits) that performs this task, and manages instances whose size and combination of parameters cannot be handled by other currently existing methods. This is achieved by using a filter as a preprocessing step, and by then exploiting the information gathered by the filter in the following actual repeat inference step. To the best of our knowledge, Rime is the first algorithm that can accurately deal with very long repeats (up to a few thousands), occurring possibly several times, and with a rate of differences (substitutions and indels) allowed among copies of a same repeat of 10-15% or even more

Analysis of the transcriptional program governing meiosis and gametogenesis in yeast and mammals

During meiosis a competent diploid cell replicates its DNA once and then undergoes two consecutive divisions followed by haploid gamete differentiation. Important aspects of meiotic development that distinguish it from mitotic growth include a highly increased rate of recombination, formation of the synaptonemal complex that aligns the homologous chromosomes, as well as separation of the homologues and sister chromatids during meiosis I and II without an intervening S-phase. Budding yeast is an excellent model organism to study meiosis and gametogenesis and accordingly, to date it belongs to the best studied eukaryotic systems in this context. Knowledge coming from these studies has provided important insights into meiotic development in higher eukaryotes. This was possible because sporulation in yeast and spermatogenesis in higher eukaryotes are analogous developmental pathways that involve conserved genes. For budding yeast a huge amount of data from numerous genome-scale studies on gene expression and deletion phenotypes of meiotic development and sporulation are available. In contrast, mammalian gametogenesis has not been studied on a large-scale until recently. It was unclear if an expression profiling study using germ cells and testicular somatic control cells that underwent lengthy purification procedures would yield interpretable results. We have therefore carried out a pioneering expression profiling study of male germ cells from Rattus norvegicus using Affymetrix U34A and B GeneChips. This work resulted in the first comprehensive large-scale expression profiling analysis of mammalian male germ cells undergoing mitotic growth, meiosis and gametogenesis. We have identified 1268 differentially expressed genes in germ cells at different developmental stages, which were organized into four distinct expression clusters that reflect somatic, mitotic, meiotic and post-meiotic cell types. This included 293 yet uncharacterized transcripts whose expression pattern suggests that they are involved in spermatogenesis and fertility. A group of 121 transcripts were only expressed in meiotic (spermatocytes) and postmeiotic germ cells (round spermatids) but not in dividing germ cells (spermatogonia), Sertoli cells or two somatic control tissues (brain and skeletal muscle). Functional analysis reveals that most of the known genes in this group fulfill essential functions during meiosis, spermiogenesis (the process of sperm maturation) and fertility. Therefore it is highly possible that some of the �30 uncharacterized transcripts in this group also contribute to these processes. A web-accessible database (called reXbase, which was later on integrated into GermOnline) has been developed for our expression profiling study of mammalian male meiosis, which summarizes annotation information and shows a graphical display of expression profiles of every gene covered in our study. In the budding yeast Saccharomyces cerevisiae entry into meiosis and subsequent progression through sporulation and gametogenesis are driven by a highly regulated transcriptional program activated by signal pathways responding to nutritional and cell-type cues. Abf1p, which is a general transcription factor, has previously been demonstrated to participate in the induction of numerous mitotic as well as early and middle meiotic genes. In the current study we have addressed the question how Abf1p transcriptionally coordinates mitotic growth and meiotic development on a genome-wide level. Because ABF1 is an essential gene we used the temperature-sensitive allele abf1-1. A phenotypical analysis of mutant cells revealed that ABF1 plays an important role in cell separation during mitosis, meiotic development, and spore formation. In order to identify genes whose expression depends on Abf1p in growing and sporulating cells we have performed expression profiling experiments using Affymetrix S98 GeneChips comparing wild-type and abf1-1 mutant cells at both permissive and restrictive temperature. We have identified 504 genes whose normal expression depends on functional ABF1. By combining the expression profiling data with data from genome-wide DNA binding assays (ChIPCHIP) and in silico predictions of potential Abf1p-binding sites in the yeast genome, we were able to define direct target genes. Expression of these genes decreases in the absence of functional ABF1 and whose promotors are bound by Abf1p and/or contain a predicted binding site. Among 352 such bona fide direct target genes we found many involved in ribosome biogenesis, translation, vegetative growth and meiotic developement and therefore could account for the observed growth and sporulation defects of abf1-1 mutant cells. Furthermore, the fact that two members of the septin family (CDC3 and CDC10 ) were found to be direct target genes suggests a novel role for Abf1p in cytokinesis. This was further substantiated by the observation that chitin localization and septin ring formation are perturbed in abf1-1 mutant cells

Virulence in smut fungi: Insights from evolutionary comparative genomics

Fungi and plants show a long history of co-evolution since about 400 million years. This lead to the development of diverse types of interactions which include for example parasitism, in which fungi reduce the fitness of their host. Parasitic fungi can establish biotrophic interactions, which require living plant tissues for successful colonization. To establish biotrophy, fungi secret effectors, which are proteins that prevent or mediate plant immune responses. They can also contribute to virulence by changing the host physiology towards the needs of the pathogen. Effectors and their plant targets evolve in a molecular arms race, where both pathogen and plants evolve new effectors and plant interactors, respectively. In this process, single nucleotide polymorphisms and species-specific orphan genes can play an important role. Smut fungi (order: Ustilaginales) are biotrophic pathogens, which parasitize mostly sweet grasses, including wheat, oat, barley, maize, sugar cane and Sorghum grass. The genomes of five related species with different host plants or colonization strategies (Ustilago hordei, Ustilago maydis, Sporisorium scitamineum, Sporisorium reilianum f. sp. zeae and S. reilianum f. sp. sorghi) were sequenced. Furthermore, methods allowing geneitc manipulations were developed, which makes this group of smut fungi an interesting model system for studying virulence and/or host specificity. The aim of the present work was to investigated to which extent positively selected or species-specific effectors contribute to virulence of the respective species. To detect positive selection, families of homologous proteins were built. Positive selection was then inferred by applying a non-homogenous branch model of sequence evolution. Most genes under positive selection were found in both formae speciales of S. reilianum. A role in virulence could be shown for sr10529 in S. reilianum f. sp. zeae. This gene is orthologous to pit2 of U. maydis, where it encodes an inhibitor of cysteine proteases. To get insights in differences in the inhibition of maize cysteine proteases by Pit2-orthologues, a yeast-2-hybrid assay was conducted In contrast to the expectaion that Pit2-orthologues of maize pathogens can better interact with maize cysteine proteases compared to Pit2 of the Sorghum pathogen, no host/pathogen-specific interaction could be observed. Besides this, a contribution to virulence could be demonstrated for three gene clusters containing positively selected genes inS. reilianum f. sp. zeae. Besides positively selected genes, species-specific orphan genes were bioinformatically identified. Most candidates could be detected in Pseudocyma flocculosa. Deletion of the orphan gene um02193 in U. maydis did not reveal a contribution to virulence for this protein

Functional genome annotation and transcriptome analysis of Pseudozyma hubeiensis BOT-O, an oleaginous yeast that utilizes glucose and xylose at equal rates

Pseudozyma hubeiensis is a basidiomycete yeast that has the highly desirable traits for lignocellulose valorisation of being equally efficient at utilization of glucose and xylose, and capable of their co-utilization. The species has previously mainly been studied for its capacity to produce secreted biosurfactants in the form of mannosylerythritol lipids, but it is also an oleaginous species capable of accumulating high levels of triacylglycerol storage lipids during nutrient starvation. In this study, we aimed to further characterize the oleaginous nature of P. hubeiensis by evaluating metabolism and gene expression responses during storage lipid formation conditions with glucose or xylose as a carbon source. The genome of the recently isolated P. hubeiensis BOT-O strain was sequenced using MinION long-read sequencing and resulted in the most contiguous P. hubeiensis assembly to date with 18.95 Mb in 31 contigs. Using transcriptome data as experimental support, we generated the first mRNA-supported P. hubeiensis genome annotation and identified 6540 genes. 80% of the predicted genes were assigned functional annotations based on protein homology to other yeasts. Based on the annotation, key metabolic pathways in BOT-O were reconstructed, including pathways for storage lipids, mannosylerythritol lipids and xylose assimilation. BOT-O was confirmed to consume glucose and xylose at equal rates, but during mixed glucose-xylose cultivation glucose was found to be taken up faster. Differential expression analysis revealed that only a total of 122 genes were significantly differentially expressed at a cut-off of |log2 fold change| ≥ 2 when comparing cultivation on xylose with glucose, during exponential growth and during nitrogen-starvation. Of these 122 genes, a core-set of 24 genes was identified that were differentially expressed at all time points. Nitrogen-starvation resulted in a larger transcriptional effect, with a total of 1179 genes with significant expression changes at the designated fold change cut-off compared with exponential growth on either glucose or xylose

Life and Death of Selfish Genes: Comparative Genomics Reveals the Dynamic Evolution of Cytoplasmic Incompatibility.

Cytoplasmic incompatibility is a selfish reproductive manipulation induced by the endosymbiont Wolbachia in arthropods. In males Wolbachia modifies sperm, leading to embryonic mortality in crosses with Wolbachia-free females. In females, Wolbachia rescues the cross and allows development to proceed normally. This provides a reproductive advantage to infected females, allowing the maternally transmitted symbiont to spread rapidly through host populations. We identified homologs of the genes underlying this phenotype, cifA and cifB, in 52 of 71 new and published Wolbachia genome sequences. They are strongly associated with cytoplasmic incompatibility. There are up to seven copies of the genes in each genome, and phylogenetic analysis shows that Wolbachia frequently acquires new copies due to pervasive horizontal transfer between strains. In many cases, the genes have subsequently acquired loss-of-function mutations to become pseudogenes. As predicted by theory, this tends to occur first in cifB, whose sole function is to modify sperm, and then in cifA, which is required to rescue the cross in females. Although cif genes recombine, recombination is largely restricted to closely related homologs. This is predicted under a model of coevolution between sperm modification and embryonic rescue, where recombination between distantly related pairs of genes would create a self-incompatible strain. Together, these patterns of gene gain, loss, and recombination support evolutionary models of cytoplasmic incompatibility.Wellcome Trust grant number WT094664MA - Wellcome Trust grant number WT202888/Z/16/Z - ERC grant 28166