43 research outputs found
Comparative genomics of a plant-pathogenic fungus, pyrenophora tritici-repentis, reveals transduplication and the impact of repeat elements on pathogenicity and population divergence
Pyrenophora tritici-repentis is a necrotrophic fungus causal to the disease tan spot of wheat, whose contribution to crop loss has increased significantly during the last few decades. Pathogenicity by this fungus is attributed to the production of host-selective toxins (HST), which are recognized by their host in a genotype-specific manner. To better understand the mechanisms that have led to the increase in disease incidence related to this pathogen, we sequenced the genomes of three P. tritici-repentis isolates. A pathogenic isolate that produces two known HSTs was used to assemble a reference nuclear genome of approximately 40 Mb composed of 11 chromosomes that encode 12,141 predicted genes. Comparison of the reference genome with those of a pathogenic isolate that produces a third HST, and a nonpathogenic isolate, showed the nonpathogen genome to be more diverged than those of the two pathogens. Examination of gene-coding regions has provided candidate pathogen-specific proteins and revealed gene families that may play a role in a necrotrophic lifestyle. Analysis of transposable elements suggests that their presence in the genome of pathogenic isolates contributes to the creation of novel genes, effector diversification, possible horizontal gene transfer events, identified copy number variation, and the first example of transduplication by DNA transposable elements in fungi.Overall, comparative analysis of these genomes provides evidence that pathogenicity in this species arose through an influx of transposable elements, which created a genetically flexible landscape that can easily respond to environmental changes
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Identification of New Pathogenicity Genes in Magnaporthe Oryzae through the Construction of an Agrobacterium Tumefacines-Mediated Insertion Mutant Library
An understanding of plant pathogen-host interactions is essential to design efficient strategies to control disease in crops. Magnaporthe oryzae, an ascomyceteous fungus and causal agent of rice blast disease, is a model organism to study host-microbe interactions. The overall aim of this dissertation project was to identify genes involved in pathogenicity through the construction and characterization of a random insertional mutagenesis library. In order to saturate the genome with DNA inserts, a collection of >54,000 insertion lines of the M. oryzae strain 70-15 was generated via two transformation methods, PEG/CaCl2 (polyethylene glycol)-mediated protoplast transformation and Agrobacterium tumefaciens-mediated transformation. The first part of this dissertation describes the optimization of both transformation approaches, compares their efficiency and provides a description of the high-throughput processing and phenotypic analysis of the insertion lines. An in vitro appressorium assay of 12,000 T-DNA insertion strains allowed the identification of 135 lines that were classified as morphologically or functionally different than wild-type. Rice infection assays demonstrated that 112 of these strains exhibited defects in pathogenicity.The second part of this dissertation project analyzed the T-DNA integration patterns in a subset of pathogenicity mutants. This section aimed to identify the disrupted genes via recovery of M. oryzae sequences adjacent to the sites of T-DNA insertion. Genomic mapping of 61 T-DNA insertions in pathogenicity mutants via rescuing M. oryzae chromosomal T-DNA flanking sequences using inverse PCR resulted in the identification of 22 conserved hypothetical genes with predicted function, 11 predicted open reading frames without a GenBank significant match, two unannotated regions of the genome assembly and seven intergenic regions. The final part of this dissertation describes the characterization of a M. oryzae pathogenicity mutant that contains a T-DNA insertion in the upstream region of two divergently transcribed genes that encode the vacuolar type-ATPase subunit c`` and the general transcription factor TFIIA subunit γ. Genetic complementation demonstrated the insertion of the T-DNA in the promoter region of the general transcription factor TFIIA subunit γ is responsible for observed defects in conidiation, appressorium morphogenesis, and appressorium function. This is the first report relating the function of TFIIA subunit γ to pathogenicity
Reprogramming of telomerase activity and rebuilding of telomere length in cloned cattle
Nuclear reprogramming requires the removal of epigenetic modifications imposed on the chromatin during cellular differentiation and division. The mammalian oocyte can reverse these alterations to a state of totipotency, allowing the production of viable cloned offspring from somatic cell nuclei. To determine whether nuclear reprogramming is complete in cloned animals, we assessed the telomerase activity and telomere length status in cloned embryos, fetuses, and newborn offspring derived from somatic cell nuclear transfer. In this report, we show that telomerase activity was significantly (P < 0.05) diminished in bovine fibroblast donor cells compared with embryonic stem-like cells, and surprisingly was 16-fold higher in fetal fibroblasts compared with adult fibroblasts (P < 0.05). Cell passaging and culture periods under serum starvation conditions significantly decreased telomerase activity by approximately 30–50% compared with nontreated early passage cells (P < 0.05). Telomere shortening was observed during in vitro culture of bovine fetal fibroblasts and in very late passages of embryonic stem-like cells. Reprogramming of telomerase activity was apparent by the blastocyst stage of postcloning embryonic development, and telomere lengths were longer (15–23 kb) in cloned fetuses and offspring than the relatively short mean terminal restriction fragment lengths (14–18 kb) observed in adult donor cells. Overall, telomere lengths of cloned fetuses and newborn calves (≈20 kb) were not significantly different from those of age-matched control animals (P > 0.05). These results demonstrate that cloned embryos inherit genomic modifications acquired during the donor nuclei's in vivo and in vitro period but are subsequently reversed during development of the cloned animal