Thesis (Ph. D.)--Brigham Young University. Dept. of Integrative Biology, 2003. / Includes bibliographical references.

The basidiomycete cereal pathogens Ustilago hordei and U. maydis are closely related and possess genomes with a high degree of homology and synteny. I report on the disparity of the RNAi phenomenon between U. hordei and U. maydis. Using an RNAi expression vector I targeted both a GUS transgene and an endogenous mating-type gene and confirmed the presence of double-stranded (ds)RNA in transgenic cells of both species. However, down-regulation of the GUS gene and production of siRNAs were seen only in U. hordei. The biological effect was a reduction in GUS protein and activity, and reduced mating only in U. hordei. In support of this experimental evidence, homologs to Dicer and Argonaute were found in the U. hordei genome but not in the published U. maydis genome. Interestingly, preliminary U. hordei sequences reveal conservation and synteny in U. maydis in the regions spanning these loci, with the only noticeable difference being the lack of Dicer and Argonaute genes in U. maydis. U. maydis also appears to differ from U. hordei with respect to genes presumed to be involved in transcriptional gene silencing and also has far fewer transposons in its genome. Efforts to clone the avirulent gene UhAvr1 led to a locus containing a large number of small proteins predicted to be secreted. This locus appears to be heterochromatic and is orthologous to the largest cluster of secreted proteins in U. maydis. Other laboratories have reported that deletion of this cluster in U. maydis results in a dramatic reduction in virulence. Genetic evidence for an avirulence gene at this locus in U. hordei suggests that the locus may also be important for U. hordei. Differences between these two smut fungi at this locus and at others identified in this study point to key differences in gene regulation and genome evolution.

Thèse (Ph. D.)--Université Laval, 2008. / Bibliogr. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (Ph. D.)--University of Minnesota, 1927. / Biography. Bibliography: p. 52-55.

München, Techn. Univ., Diss., 2002.

The basidiomycete cereal pathogens Ustilago hordei and U. maydis are closely related and possess genomes with a high degree of homology and synteny. I report on the disparity of the RNAi phenomenon between U. hordei and U. maydis. Using an RNAi expression vector I targeted both a GUS transgene and an endogenous mating-type gene and confirmed the presence of double-stranded (ds)RNA in transgenic cells of both species. However, down-regulation of the GUS gene and production of siRNAs were seen only in U. hordei. The biological effect was a reduction in GUS protein and activity, and reduced mating only in U. hordei. In support of this experimental evidence, homologs to Dicer and Argonaute were found in the U. hordei genome but not in the published U. maydis genome. Interestingly, preliminary U. hordei sequences reveal conservation and synteny in U. maydis in the regions spanning these loci, with the only noticeable difference being the lack of Dicer and Argonaute genes in U. maydis. U. maydis also appears to differ from U. hordei with respect to genes presumed to be involved in transcriptional gene silencing and also has far fewer transposons in its genome. Efforts to clone the avirulent gene UhAvr1 led to a locus containing a large number of small proteins predicted to be secreted. This locus appears to be heterochromatic and is orthologous to the largest cluster of secreted proteins in U. maydis. Other laboratories have reported that deletion of this cluster in U. maydis results in a dramatic reduction in virulence. Genetic evidence for an avirulence gene at this locus in U. hordei suggests that the locus may also be important for U. hordei. Differences between these two smut fungi at this locus and at others identified in this study point to key differences in gene regulation and genome evolution. / Science, Faculty of / Botany, Department of / Graduate

The question investigated in this study was whether or not somatic recombination can occur in Ustilago hordei whilst it is in the parasitic phase on barley. The investigation was carried out in two parts. In the first and major part, barley seeds were inoculated artificially with mixtures of nutritionally deficient mutants of U. hordei. This was done in such a way that infection of the mature plants could only occur if at least one recombination event had taken place asexually, between at least two infective dikaryons, prior to teliospore formation. One smutted plant was found. Detailed analysis of the teliospores from this infection was carried out. The second part of the study was designed to demonstrate that more than one infective dikaryon can simultaneously occupy the host. Again, mixtures of nutritionally deficient mutants were used to inoculate barley seeds but this time so that the origin of teliospores from any infection could be traced back to the parental types in the infective dikaryon. It was concluded that somatic recombination can in fact occur whilst the fungus occupies the host tissue. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Genetic studies were carried out on the fungal parasite Ustilago hordei (Pers.) Lagerh. and on its host, Hordeum vulgave L. (cultivated barley). In these studies of the host-parasite relationship, special emphasis was placed on the genetic investigation of the pathogenicity. The thesis is divided into four parts. Part I deals with multial1 elism of genes for virulence (v-genes) in the parasite, and demonstrated that four different levels of virulence (obtained on the barley cultivar Trebi) are controlled by alternative alleles at a single genetic locus in the parasite. This is the first demonstrated example of a series of multiple alleles determining different levels of virulence. Part II concerns the identification and characterization of v-genes in U. hordei and of resistance genes (R-genes) in H. vulgave. Three v-genes (two of them new, one of them previously known) were identified. It was shown that the previously identified gene was expressed either as a dominant or a recessive, dependingoon the conditions under which it was tested, and that the newly-identified genes were both recessive. Cultures possessing the newly-discovered v-genes were used in identifying two new R-genes in the barley host. A study of interactions involving the newly discovered v- and R-genes led to the conclusion that these interactions have their basis in gene-for-gene relationships. Part III deals with the synthesis of a complex biotype of u. hordei possessing v-genes at two genetic loci. Disease reaction obtained with this complex biotype were compared both qualitatively and quantitatively with those obtained with the simpler, parental biotypes. In tests on certain cultivars the complex biotype produced either the same or higher levels of disease reaction. Because the new biotype has ah extended host range it is considered that under certain conditions it would be comparatively more fit than either of the parental biotypes from which it was derived. Part IV of the thesis concerns the effects of nutritional deficiency on the action of v-genes. Dikaryons which were homozygous for arg, ad or met were in all cases non-pathogenic; for those which were homozygous for pdx, pathogenicity was unaffected. For dikaryons which were heterozygous for one or more nutritional deficiences, pathogenicity was either unimpaired or reduced, depending on the combination (deficiency: virulence gene: host cultivar) which was tested. It was concluded that the specificity of pathogen biotypes was not determined by the availability or non-availability of specific nutritional factors. However, the effects were not entirely non-specific, since changes in levels of virulence were shown only in certain tests. / Science, Faculty of / Botany, Department of / Graduate

Microtubules in the fungal pathogen Ustilago maydis have important roles, which include polar budding, morphogenesis and nuclear migration. They also serve as tracks for molecular motors, responsible for intracellular transport of organelles and membrane trafficking. Moreover, microtubules are indispensable during both interphase and cell division, and they play a crucial role in long-distance microtubule-based transport, which occurs in neurons or fungal hypha. Therefore, in order to carry out their functions correctly they need to be well organised and stabilised, which is achieved mainly by various microtubule-associated proteins. In this thesis, different aspects of microtubule (MT) cytoskeleton organisation in U. maydis were investigated, using bioinformatics and experimental approaches. In the first part of the thesis I studied the microtubule-associated protein (MAP) repertoire in U. maydis, which has never been done before in a comprehensive way. For this purpose, searches across five eukaryotic model organisms were conducted to identify all of their known MAPs, to query the U. maydis database. In addition, all of the proteins were checked for their domain architecture, to help decide if an orthologue had been found. As a result, 66 potential MAP orthologues were identified. The second part of this thesis focused on identifying novel factors involved in the organisation of the microtubule cytoskeleton using a specially designed genetic screen. This work involved five microtubule-organisation defect (MOD) mutants, generated by UV-mutagenesis, which were characterised by inability to produce long hyphae as well as by short, fragmented microtubules. To find which genes were responsible for this phenotype, the genomes of all mutants were sequenced and compared with a wild-type genome, and mutations in many genes were found. The analysis revealed potential candidate genes responsible for the specific phenotype of the mutants. However, most probably, UV-generated point mutations in more than one gene played a part in the defective microtubule array. In the final part of this thesis, the function of two beta-tubulin isotypes in U. maydis was analysed. Using conditional mutants, I demonstrated that there are subtle functional differences between the two beta tubulins.

Peroxisomes are ubiquitous organelles found in almost all eukaryotes. They are sensitive to changes in cellular homeostasis and involved in various metabolic processes. Deficiencies in peroxisome function cause severe neurological problems. Here I report, investigation of peroxisome motility and its relation to peroxisomal functions in the fungal model system Ustilago maydis. Peroxisomes are mostly motile in Ustilago maydis. Motile peroxisomes show different motility patterns: short-range pulse type movements and long range bidirectional motility. Motility behaviour is not static as oscillating peroxisomes may start long-range motility. Here, I present evidence that long-range bidirectional peroxisome motility is an energy driven process and is essential for homogeneous distribution of peroxisomes. Similar to early endosomes and endoplasmic reticulum, microtubule motors kinesin-3 and dynein are responsible for long-range peroxisome transport. In addition to using the same molecular motors for transport, early endosomes, endoplasmic reticulum and peroxisomes have the same transport velocity. Interestingly, motile peroxisomes and endoplasmic reticulum tubules co-localize with early endosomes. Functional investigation of early endosome mutants, Δrab5a and Yup1ts has revealed a novel transport mechanism where endoplasmic reticulum and peroxisomes hitch hike on early endosomes. Additionally, I report functional characterization of an AAA-ATPase, um05592, which has high homology to human protein NP_055873. Altogether these results reveal molecular mechanism of peroxisome transport in Ustilago maydis. Similarities in transport machinery illustrate Ustilago maydis as a model system to study peroxisome function in mammalian cells.