Dissertations / Theses on the topic 'Fungal Mating Type Genes'

This work is concerned with the molecular analysis of ascomycete mating type genes of various Sordaria species. Work previously published has reported the cloning and characterisation of mating type genes from several Neurospora species. In heterothallic species the genotype at the mating type locus (mtA or mta) determines the mating type. Homothallic species, which proceed through the sexual cycle without the need to mate, have no obvious mating types but molecular analysis has been used to demonstrate the presence of mating type genes in species with this life cycle. Neurospora species and Sordaria species both belong to the Sordariaceae and are closely related. Several λ clones containing putative Sordaria mating type genes from heterothallic and homothallic species had been isolated previously using N. crassa mtA and mta probes. In this study the mtA-1 gene of the heterothallic species S. sclerogenia was subcloned from a λ clone and sequenced. The equivalent gene from S. equina (a homothallic species containing only the mtA sequence) was also subcloned and sequenced. A λ clone for the species S. fimicola was found to hybridise with both the mtA and mta probes. S. fimicola is a homothallic species containing mtA and mta in the same nucleus. On using the lambda clone it was found that the mtA and mta genes are linked in this species. All the Sordaria mtA-1 genes contained putative DNA binding domains, α domains. The mta-1 gene sequenced from s. fimicola contained a putative HMG box. The S. equina mtA-1 gene was expressed in a sterile N. crassa mta mutant and was found to restore mating type function to the mutant. The mtA-1 gene did not however confer homothallic behaviour on the recipient mutant. S. equina and S. sclerogenia contain a 59bp common region following on from the mtA-1 gene which is conserved in both these species and in Neurospora species. A variable region continues on from the common region in S. equina and S. sclerogenia and in Neuropora species.

Orientador: Gonçalo Amarante Guimarães Pereira
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O ciclo sexual de basidiomicetos é controlado pelo sistema mating type. Este é formado por dois loci multigênicos não ligados A e B, o locus A codifica duas proteínas homeodomínio HD1 e HD2, capazes de heterodimerização, enquanto o locus B apresenta genes para receptores de feromônio e feromônios. Em fungos heterotálicos, o desenvolvimento sexual depende da especificidade entre os quatro alelos, sistema este chamado tetrapolar, e é ativado apenas por interações específicas entre alelos parentais necessariamente diferentes, assegurando que hifas geneticamente iguais sejam incompatíveis. Em contrapartida, a condição na qual hifas geneticamente iguais são compatíveis é denominada homotalismo. Fungos basidiomicetos são tipicamente heterotálicos, no entanto, apesar de pertencer a este filo, o fitopatógeno Moniliophthora perniciosa, causador da doença Vassoura de Bruxa no cacaueiro, é classificado como homotálico primário. Curiosamente, apesar desta classificação, M. perniciosa contém um sistema genético tetrapolar, sendo o primeiro fungo descrito com essa característica. Neste trabalho, foi realizada a caracterização dos loci mating type em M. perniciosa e verificamos o perfil transcricional destes genes com o objetivo de entender os mecanismos moleculares que atuam no seu comportamento homotálico. Primeiramente, foram identificados no genoma um locus A e um locus B, além de genes atuantes no processamento e sinalização em resposta aos feromônios. O estudo do perfil transcricional destes genes revelou que um receptor tem um perfil de expressão condizente com a fase do ciclo de vida do fungo na qual ocorre o processo de dicariotização. A análise funcional dos receptores foi realizada em um sistema expressão heteróloga, promissor para o estudo de GPCRs (G coupled proteins receptors), porém não permitiu confirmar a presença de alelos compatíveis de receptores e precursores de feromônios no genoma de M. perniciosa como uma possível explicação ao comportamento homotálico. Tendo em vista o locus A, este é formado por um par MpHD1 e MpHD2, o que difere de outros basidiomicetos devido a inserção de uma sequência (11,958kb) interrompendo seus promotores. A hipótese neste cenário é que o transposon encontrado no locus A poderia ter permitido um crossover desigual que trariam genes compatíveis para o mesmo alelo, sendo responsável pelo homotalismo na espécie. Contrariando essa hipótese, os dados obtidos neste projeto indicam que uma possível transição prévia ao homotalismo resultou em uma pressão seletiva relaxada sobre os loci mating type, cuja consequência foi a degeneração nos genes destes loci. Neste contexto, os genes do mating type poderiam não estar mais envolvidos na dicariotização. Este trabalho, portanto, fornece importantes dados para o entendimento da biologia sexual deste fungo, o que futuramente poderá ser correlacionado a sua fitopatogenicidade
Abstract: The basidiomycetes¿ sexual cycle is controlled by the mating type system. The structure of this system comprises two unlinked multigenic loci, A and B. The A locus codes for homeodomain proteins, HD1 e HD2 which form a heterodimer, and B locus presents pheromone receptors and pheromones. In outcrossing (heterothallic) fungi, sexual development depends on the compatibility of four genes in two different allelic versions in a so-called tetrapolar system, and is strictly activated by specific interactions between different parental alleles, ensuring that genetically identical hyphae are incompatible. The phytopathogen Moniliophthora perniciosa causes Witches¿ broom disease in cacao plants, and it is a typical basidiomycete fungi. However, it completes its sexual development through the crossing of genetically identical hyphae, and is the first described homothallic fungi with a complete tetrapolar genetic system. Here we show the characterization of the mating type loci of M. perniciosa and the transcriptional profile of these genes, to uncover the mechanisms underpinning its homothallic behavior. First, we identified an A locus, a B locus and a set of genes that participates in pheromone processing and signalization. Considering the transcriptional profile of these genes, one receptor shows an expression profile consistent with an involvement in dikaryotization. The functional evaluation of the receptors was performed in a heterologous expression system, a promising tool for GPCR (G coupled proteins receptors) proteins study. This system did not allow the confirmation if M. perniciosa contains compatible alleles for receptors and pheromones, one possible explanation for homothallism. Considering A locus, it codes for a pair MpHD1 and MpHD2, which has a sequence insertion (11,958kb) interrupting their promoters, differing from others basidiomycetes. The hypothesis in this scenario is that the insertion of a transposon could have allowed an unequal crossover that brought together compatible genes in the same allele, causing the homothallism in this species. Interestingly, in an opposite direction, our data indicates that a previous transition for homothallism could have resulted in a relaxed selective pressure on mating type loci, with consequences such as the presence of degenerated genes on these loci. In this context, the mating type genes could not necessarily play a role in dikaryotization process. This work provides valuable data for understanding the sexual biology of M. perniciosa, which hereafter could be correlated with its phytopathogenicity
Mestrado
Genetica de Microorganismos
Mestra em Genética e Biologia Molecular

A hierarchy of different chromosome conformations plays a role in many biological systems. These conformations contribute to the regulation of gene expression, cellular development, chromosome transmission, and defects can lead to human disease. The highest functional level of this hierarchy is the partitioning of the genome into compartments of active and inactive chromatin domains (1’s -10’s Mb). These compartments are further partitioned into Topologically Associating Domains (TADs) that spatially cluster co-regulated genes (100’s kb – 1’s Mb). The final level that has been observed is long range loops formed between regulatory elements and promoters (10’s kb – 100’s Mb). At all of these levels, mechanisms that establish these conformations remain poorly understood. To gain new insights into processes that determine chromosome folding I used the mating type switching system in budding yeast to study the chromosome conformation at length scales analogous to looping interaction. I specifically examined the role in chromosome conformation in the mating type switching system. Budding yeast cells can have two sexes: MATa and MATα. The mating types are determined by allele-specific expression of the MAT locus on chromosome III. The MATa allele encodes for transcription factors responsible for the MATa mating type and the MATα allele encodes transcription factors responsible for the MATα mating type. Yeast cells can switch their mating type by a process that repairs a break at MAT using one of two silent loci, HML or HMR, as a donor to convert the allele at the MAT locus. When MATa cells switch they prefer to use HML, which contains the MATα allele, located at the end of the left arm. MATα cells prefer to use HMR, which contains the MATa allele, located on the end of the right arm of chromosome III. The sequences of the HM loci are not important for donor preference. Instead the cell chooses the donor on the left arm in MATa cells and chooses the donor on the right arm in MATα cells. This lack of sequence specificity has led to the hypothesis that the conformation of the chromosome may play a role in donor preference. I found that the conformation of chromosome III is, indeed, different between the two mating types. In MATa cells the chromosomes displays a more crumpled conformation in which the left arm of the chromosome interacts with a large region of the right arm which includes the centromere and the MAT locus. In MATα cells, on the other hand, the left arm of the chromosomes displays a more extend conformation. I found that the Recombination Enhancer (RE), which enhances recombination along the left arm of the chromosome in MATa cells, is responsible for these mating type-specific conformations. Deleting the RE affects the conformation of the chromosomes in both MATa and MATα cells. The left portion of the RE, which is essential for donor preference during the switching reaction in MATa cells, does not contribute to the conformation in MATa. This region does have a minor effect on the conformation in MATα cells. However, I found that the right portion of the RE is responsible for the conformation of chromosome III in both mating types prior to initiation of switching. This work demonstrates that chromosome conformation is determined by specific cis regulatory elements that drive cell-type specific chromosome conformation.

Orientadores: Gonçalo Amarante Guimarães Pereira, Lyndel W. Meinhardt
Dissertção (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A doença vassoura-de-bruxa em Theobroma cacao (cacaueiro), causada pelo fungo Crinipellis perniciosa, é uma das doenças de maior impacto econômico nos países produtores de cacau, sendo o Brasil um destes países. Esse fungo infecta os tecidos meristemáticos do cacaueiro em duas fases: parasítica e saprofítica. Pesquisas com outros fitopatógenos têm demonstrado que a mudança da fase parasítica para a saprofítica é regulada por genes do sistema mating type tornando este estudo extremamente importante para inferir estratégias de combate à doença. O presente trabalho teve por finalidade identificar os genes mating type de C. perniciosa através da análise dos dados gerados pelo seu projeto genoma. Para a identificação dos genes do sistema mating type (Hd1, Hd2, Rc e Fe) foram feitas buscas no banco de dados do genoma de C. perniciosa comparando seqüências similares àquelas codificadas pelos fungos basidiomicetos Coprinus cinereus, Coprinus bilanatus, Schizophyllum commune e Ustilago maydis, identificadas e disponibilizadas em rede. Foram identificados seis genes do sistema mating type de C. perniciosa. Um gene que codifica a proteína regulatória Hd1, um gene que codifica a proteína regulatória Hd2 e quatro genes que codificam as proteínas receptoras de ferormônio Rc1, Rc2, Rc3 e Rc4. Não foram identificados genes que codificam ferormônios. Portanto, concluímos que a organização molecular do sistema mating type de C. perniciosa é tetrapolar, contendo o locus HD bialélico e o locus FRF multialélico
Abstract: The witch's broom disease in Theobroma cacao, caused by mushroom Crinipellis perniciosa, is one of the diseases with the biggest economic impact in cocoa producing countries, and Brazil is one of them. The mushroom infects the meristematics tissues of the cocoa tree in two phases: parasitic and saprofitic. Research with other phytopathogens have demonstrated that the change from the parasitic to the saprophytic phase is regulated by genes of the mating type system, making this study extremely important to infer fighting strategies to this disease. This work's proposal is to identify mating type genes of C. perniciosa through the analysis of data generated by its genome project. The genes identification of the (Hd1, Hd2, Rc and Fe) mating type system was made by database search in the C. perniciosa genome comparing similar sequences with the ones codified by the basidiomycetes mushrooms Coprinus cinereus, Coprinus bilanatus, Schizophyllum commune and Ustilago maydis, identified and available in the internet. Six genes were identified in C. perniciosa mating type system. One gene that codifies the regulatory protein Hd1, another that codifies the regulatory protein Hd2 and four genes that codify the pheromone receptor proteins Rc1, Rc2, Rc3 and Rc4. Genes that codify pheromones were not identified. Therefore, we concluded that the molecular organization of C. perniciosa mating type system is tetrapolar, containing the bialelic HD locus and the multialelic FRF locus
Mestrado
Bioquimica
Mestre em Biologia Funcional e Molecular

Mating-type and other ‘sex-related’ genes in the filamentous ascomcyete genera Aspergillus and Penicillium, were examined to investigate the potential sexual capacity of supposedly asexual species and also the possible evolutionary route and ancestry of mating strategy and mating-type genes. Two heterothallic and one homothallic sexual species were screened to determine the presence and genomic organisation of mating-type genes. An additional gene has previously been detected in Neosartorya fischeri, N. fumigata and Penicillium marneffei. This gene was also detected and sequenced in the heterothallic species, Emericella heterothallica and the homothallic species, Eurotium repens. The expression of this gene was investigated under conditions that cause expression of mating-type genes in these species. Mating-type and other ‘sex-related’ genes were investigated in asexual Aspergilli that have been genome sequenced. Expression of mating-type, α-factor pheromone precursor, pheromone receptor and two transcription factor encoding genes were also investigated. Gene expression varied between species, but no genes displayed mating type-dependent expression. Previous studies had developed a degenerate PCR diagnostic approach to identify putative MAT1-1-1 and MAT1-2-1 gene fragments. This degenerate PCR diagnostic was performed on Penicillium species in the subgenus Penicillium to determine the presence or absence of mating-type genes. Mating-type gene fragments or whole open reading frames were sequenced from four of these Penicillium species. RT-PCR analyses were also performed on these species, and MAT1-1-1 and MAT1-2-1 gene expression was confirmed in three of the four Penicillium species. The overall structure of the mating-type loci and idiomorphs of the Aspergillus and Penicillium species revealed certain common features. The ancestral mating strategy of the Eurotiomycetes has been suggested to be homothallism. Whilst this remains possible, alternative evolutionary scenarios are suggested from this investigation.

Reproductive isolation is an essential stage in speciation. In Ascomycetes, the ubiquitous distribution of many species suggests that sympatric speciation through assertive mating should be an important factor. The MAT locus and the pheromone/receptor system could both potentially contribute to the development of such sexual isolation. Alterations at the MAT loci could lead to distinct reproductive habits or a change in mating system, both of which can reduce gene-flow between species. However, if deliberate pre-mating sexual preferences exist, they are more likely to be determined by the pheromone/receptor system. This study of Fusarium reproductive genes, and comparisons with other Ascomycetes, has yielded numerous interesting findings regarding the evolution of these mate-recognition mechanisms and the implications thereof. The G. fujikuroi and F. graminearum species complexes have offered an interesting comparison between heterothallic and homothallic MAT locus evolution. The value of comparative sequence analysis has been demonstrated in the discovery of a previously unknown gene, MAT1-2-3, which may be specific to members of the Order Hypocreales. While all MAT genes share similar regulatory elements, this is the first report of evidence that a transition to homothallism can be accompanied by the recruitment of distinct elements that could facilitate alternate expression of MAT genes. The MAT genes are also highly divergent between Fusarium spp., largely due to relaxed selective constraint, particularly in homothallic species. However, inter-specific gene-flow could curb MAT gene divergence among homothallic species. Despite strong reproductive barriers in the G. fujikuroi complex, the F. sacchari MAT1-1 sequence appears to have been acquired through lateral transfer from a distant relative. Analytical analysis of the MAT locus novelties reported here, including the new MAT gene, will be necessary to determine their biological significance. To investigate the extent of pheromone diversity in the Ascomycetes, and to gain clues as to its biological importance, pheromone peptides from seventy ascomycete species were compared. A number of reproductively incompatible species, such as those in the G. fujikuroi complex, share identical pheromones; which implies that another mechanism must be responsible for the observed reproductive barriers. However, on the whole, pheromones are highly divergent among species. Both adaptive and non-adaptive evolution could have contributed to this pattern. In fact the structure of the á-class pheromone precursor gene, which consists of multiple repeats of the pheromone module, could facilitate rapid diversification through “birth-and-death” evolution. Within species, selection maintains pheromone peptides, implying that much of the inter-specific variation is functionally relevant. This further suggests that pheromone evolution could contribute to the generation of reproductive isolation between species. The most general trend in the findings of this study is that ascomycete reproductive genes are highly divergent. This is in agreement with findings in other Kingdoms. A number of evolutionary forces are probably involved but weaker selective constraint, resulting from the fact that reproduction is not essential in these fungi, appears to be a common factor. This reproductive gene variability could be directly linked to speciation and, therefore, the great diversity in Ascomycetes. Additional information on the appendices is available on a CD, stored at the Merensky Library on Level 3
Dissertation (MSc)--University of Pretoria, 2011.
Genetics
Unrestricted

Sporisorium scitamineum é um fungo basidiomiceto causador do carvão da cana-de-açúcar, uma doença com impacto negativo no cultivo da cana-de-açúcar, e com ocorrência em todos os países produtores. A manifestação da doença na cultura da cana depende da formação de uma hifa dicariótica a partir da anastomose de duas hifas haplóides compatíveis com relação ao tipo de reação sexual (mating-type). O controle do cruzamento sexuado (mating) é realizado pela expressão de um conjunto de genes presentes em dois loci, a e b. O locus a codifica um lipopeptídeo com função de feromônio e um receptor de feromônio, responsáveis pelo reconhecimento de células compatíveis e fusão de hifas, enquanto o locus b codifica fatores de transcrição que controlam a expressão de genes responsáveis pela manutenção das hifas dicarióticas durante o processo de infecção e crescimento do fungo dentro da planta. Apesar de desempenharem função essencial no processo de infecção e manutenção da doença em cana-de-açúcar, o conhecimento a respeito da organização genômica ou da função dos demais genes presentes nos loci a e b em S. scitamineum e em outros fungos causadores de carvão é ainda incipiente. Desta forma, o objetivo geral do presente trabalho foi isolar as regiões genômicas relacionadas aos genes de cruzamento em S. scitamineum e analisar comparativamente com regiões similares já descritas e depositadas em bancos de dados públicos. Para o isolamento destas regiões, foi construída uma biblioteca genômica em BAC de uma linhagem haplóide de S. scitamineum, a Ssc39 (+), isolada de uma variedade de cana-de-açúcar com sintomas de alta susceptibilidade. Foram selecionados 11 clones por PCR. Os insertos foram sequenciados e utilizados para confirmação da montagem dos loci no sequenciamento do genoma do fungo. Apesar do fungo S. scitamineum apresentar sistema bipolar de reação sexual assim como o fungo U. hordei, as análises comparativas de ambos os locus indicaram que S. scitamineum apresenta maior similaridade com o fungo S. reilianum principalmente com o alelo a1, no qual apresenta sistema tetrapolar de reação sexual. A anotação e caracterização dos genes do tipo de reação sexual (mating type) possibilitaram a comparação e melhor entendimento sobre esses genes de grande importância na patogenicidade e no ciclo de vida do fungo.
Sporisorium scitamineum is a basidiomycete fungus causing the smut disease in sugarcane, with a negative impact on the cultivation of sugarcane, and occurring in all producing countries. The manifestation of the disease in sugarcane crop depends on the formation of a dikaryotic hyphae originated of the anastomosis of two haploid mating type compatible cells. The control of the sexual crossing (mating) is performed by expression of a set of genes present in two loci, a and b. The locus a encodes a lipopeptide with the function of pheromone and pheromone membrane receptor responsible for cell recognition and compatible hyphal fusion, whereas the locus b encodes transcription factors that control the expression of genes responsible for the maintenance of the dikaryotic hyphal growth in plant. Although they play an essential role in the maintenance of infection and disease in sugarcane process, knowledge about the genomic organization and function of other genes in these two loci of S. scitamineum and other smut fungi is still incipient. Thus, the overall goal of this work was to isolate genomic regions related to the mating type in S. scitamineum and to perform a comparative analyze with similar regions described and deposited in public databases. For the isolation of these regions, we constructed a genomic BAC library of a haploid strain of S. scitamineum, the Ssc39 (+), isolated from a variety of sugarcane with symptoms of high susceptibility. Eleven clones were selected by PCR. The inserts were sequenced and used to confirm the assembly of both loci in the genome sequencing of the fungus. Although S. scitamineum belongs to the class of bipolar system of sexual response as well as the fungus U. hordei , the comparative analysis of both loci indicated that S. scitamineum shows greater similarity to the S. reilianum mainly with A1 allele, which has a tetrapolar system sexual response. The annotation of the genes and characterization mating type genes enabled the comparison and better understanding of the importance of these genes in the life cycle of the fungus.

Dissertação para obtenção do Grau de Doutor em Biologia
This work concerns the investigation of the molecular mechanisms of sexual reproduction in fungi and their possible implication for fungal lifestyles (parasitic vs. saprobic) and for the emergence of asexual fungal lineages. The association between pathogenicity and sexuality is well-known in the basidiomycete plant parasite Ustilago maydis (subphylum Ustilaginomycotina), an economically important smut fungus. However, Ustilago species are phylogenetically interspersed with species of the genus Pseudozyma, which are considered saprobic and asexual. In this work, a study focused on genes involved in determining sexual identity (mating type or MAT genes), showed that Pseudozyma prolifica retains full sexual competence and pathogenicity, being therefore indistinguishable from U. maydis. For other Pseudozyma species, molecular analyses of PRF1, a gene that encodes a master regulator of sexual reproduction in U. maydis, showed no substantial evidence of loss of sexual reproduction. However, some clues were also found suggesting that some Pseudozyma species may be evolving towards a saprobic lifestyle. The earliest derived lineage of Basidiomycota (subphylum Pucciniomycotina) includes also important plant pathogens (rust and anther smut fungi) as well as lineages composed solely of saprobic organisms. Among the latter, the red yeasts of the order Sporidiobolales have the advantage of completing their life cycle in culture media, but have remained very little explored concerning the characterization of mating systems, the identification of MAT genes and the evolutionary relationships between sexual and asexual species. A comprehensive analysis of more than 200 strains belonging to 32 species of the Sporidiobolales indicated that asexuality seems to originate frequently from sexual lineages, but does not seem to persist long enough to form truly asexual species devoid of MAT genes. A more in-depth investigation of the red yeasts Rhodosporidium toruloides and Sporidiobolus salmonicolor allowed the identification for the first time in the Pucciniomycotina of the complete set of MAT genes. A detailed and multidisciplinary characterization of the mating system in the latter species yielded surprising results. A novel mating system that differs substantially from the two mating paradigms in basidiomycetes, the bipolar and tetrapolar systems, was brought to light. Given the basal phylogenetic position of the Pucciniomycotina within the Basidiomycota, this new system designated pseudo-bipolar, constitutes a significant contribution to the study of the evolution of MAT systems in fungi.
Fundação para a Ciência e a Tecnologia - PhD grant(SFRH/BD/29580/2006)

Gibberella zeae (anamorph Fusarium graminearum) is a homothallic ascomycete pathogen that is responsible for causing Fusarium head blight (FHB) of wheat and small grains. In addition to causing a reduction in yield, harvested grain is frequently contaminated with trichothecene mycotoxins that are harmful for human and animal health. Use of wheat varieties with resistance to FHB is an important strategy to lower its impact. In order to produce varieties with durable resistance, we must understand the origin and degree of genetic diversity present in the pathogen population. In my research, I focused my efforts on an investigation of the role of mating and sexual development in the generation of genotypic and phenotypic variability in G. zeae. The goal of one part of my work was to develop new genetic markers that can be used to monitor out-crossing and genetic diversity in the population. I also optimized gene deletion protocols for G. zeae so that I could produce mutant and control strains to address my research hypothesis that MAT genes play a direct role in pathogenicity. Application of novel repetitive RFLP probes to a group of G. zeae isolates originating from and near Kentucky revealed a surprisingly high degree of diversity in these local populations. Diversity between locations was greater than that within locations, suggesting the relative importance of local inoculum sources. The probes were also useful as genetic markers for segregation analysis. I crossed two genetically closely related, and commonly used, laboratory strains of G. zeae and found that this resulted in transgressive segregation for both aggressiveness and toxigenicity. I showed that the very high and very low levels of aggressiveness and toxigenicity in transgressive segregants are heritable. I also showed that selfing produced a higher degree of diversity in these traits among the progeny than was observed among conidial progeny. This suggests the presence of epigenetic factors that impact pathogenicity. Sexual behavior in G. zeae is under the control of MATing type genes. I deleted the complete MAT1 locus, and the MAT1-1-1, and MAT1-2-1 genes separately. Deletion of each of the targeted sequences produced the expected shifts in fertility phenotype. The mat1KO strains became asexual, while mat1-1-1KO and mat1-2-1KO strains shifted to obligate heterothallism. Deletion of the MAT1-1-1 and MAT1-2-1 genes had a negative effect on aggressiveness and mycotoxin production in planta, but deletion of the complete MAT1 locus had no effect. The set of mutant and ectopic control strains that I generated will be a useful asset that will be made available to the research community.

Doutoramento em Engenharia Agronómica - Instituto Superior de Agronomia
Considering the growing importance of black foot disease of grapevine, this study was aimed to deeply understand details on taxonomy, genetics, biology and pathological behaviour of its main causal agents, previously attributed mostly to Ilyonectria liriodendri and I. macrodidyma. A multi-gene analysis of a collection of Ilyonectria isolates, along with morphological characterisation, enabled the description of 12 species from I. radicicola and four from I. macrodidyma complexes. Among these, pathogenicity experiments revealed I. lusitanica, I. estremocensis and I. europaea as more virulent to grapevine than I. liriodendri and I. macrodidyma. The entire mating-type loci of I. liriodendri and of species from the I. macrodidyma complex were obtained. While the idiomorph structure of species from the latter matches that of other heterothallic Hypocreales, the organization of the mating-type loci in I. liriodendri seems unique, suggesting a potential pseudo-heterothallism. Soilborne inoculum is accepted to contribute significantly to initiate black foot disease in grapevine plants. qPCR amplification from DNA soil samples demonstrate that rotation can reduce the levels of Ilyonectria in nurseries, and that levels of infestation in vineyard soils are lower than in nursery or mother-plant soils. Additionally, a protoplast transformation protocol is presented for the stable integration of the GFP gene in the genome of I. liriondendri, enabling future downstream functional genetic studies.

Le champignon filamenteux, Podospora anserina, possède deux types sexuels, mat+ et mat-, caractérisés chacun par une séquence spécifique. La séquence mat+ contient un seul gène FPR1; la séquence mat- contient trois gènes : FMR1, SMR1 et SMR2. La fonction moléculaire de SMR1 est inconnue, les autres gènes codent des facteurs de transcription qui contrôlent la fécondation (reconnaissance intercellulaire), et le passage d'un syncytium à un hyphe spécialisé binucléé contenant un noyau mat+ et un noyau mat- (reconnaissance internucléaire). Il n'y a pas eu d'analyse exhaustive des gènes impliqués dans la reconnaissance intercellulaire et le mécanisme de la reconnaissance internucléaire est encore inconnu. Afin de déterminer les cibles de FPR1 et FMR1, et les différents mécanismes impliqués, nous avons utilisé une approche microarray. Le profil transcriptomique des souches mat+ et mat- compétentes pour la fécondation a permis d'identifier 157 gènes cibles, et l'analyse transcriptomique des souches mutantes fpr1- et fmr1- a révélé que ces cibles peuvent être soit réprimées, soit activées par FMR1 ou FPR1, ou être sous le contrôle de ces deux facteurs. Ces expériences ont aussi détecté l'existence de 10 gènes activés ou réprimés au même niveau dans mat+ et mat-. La délétion de 32 gènes choisis parmi ces 167 gènes cibles n'a permis de mettre en évidence que deux gènes impliqués dans la fécondation. Les comparaisons des gènes cibles des facteurs de transcription MAT de Gibberella moniliformis et Sordaria macrospora avec ceux de P. anserina révèlent un nombre significatif de gènes cibles communs entre ces espèces, mais ces gènes ont des profils transcriptomiques différents, soulevant la question du rôle de ces gènes cibles. La recherche des gènes cibles de FPR1, FMR1 et SMR2 impliqués dans la reconnaissance internuléaire a été effectuée en comparant le transcriptome des périthèces issus de deux croisements, l'un n'exprimant que les gènes spécifiques mat+, l'autre que les gènes spécifiques mat-. Les résultats ont été interprétés selon le modèle d'identité nucléaire et le modèle de ségrégation aléatoire. Le premier modèle a conduit à l'identification de 27 gènes cibles, tandis que 154 gènes cibles ont été identifiés en appliquant le deuxième modèle. Au total 46 souches mutantes ont été construites. Cependant aucune délétion n'a affecté le développement sexué. En parallèle de ces expériences transcriptomiques, nous avons invalidé tous les gènes à HMG-box de P. anserina. Les résultats montrent que ces derniers ont un rôle très important dans le développement sexué, particulièrement Pa_1_13940 qui code un régulateur des gènes des types sexuels, le premier identifié chez les Pezizomycotina.

The filamentous fungus Neurospora crassa has two mating-types, A and a, responsible for mating and vegetative heterokaryon incompatibility. The mt locus is composed of dissimilar DNA sequences termed idiomorphs. In a individuals, mt a-1 is the gene responsible for regulation of all sexual and vegetative functions of the mating-type locus. In A strains, mt A-1 is responsible for fertilisation and heterokaryon incompatibility functions. Two additional genes of the A idiomorph, mt A-2 and mt A-3, were isolated and characterised in this study. The mt A-2 and mt A-3 genes code for proteins which have characteristics of transcription factors. Isolation of mt A-2 and mt A-3-specific mutants indicated that mutations at these genes lead to arrest of the sexual development at about 3 days after fertilisation, but only if both mt A-2 and mt A-3 are affected. Six different mating-type mutants defective at mt A-1, mt A-2, mt A-3 and mt a-1 were used in transcriptional analyses of genes preferentially expressed during early sexual development (sdv genes). The results obtained with these analyses suggested the involvement of all four mating-type proteins in regulation of some of these genes. Regulation of different genes by N. crassa mating-type seems to depend on combinatorial interactions between the different mating-type proteins.

碩士
國立臺灣大學
植物病理與微生物學研究所
94
Phytophthora parasitica is an important plant pathogen with a broad host range capable of infecting more than 72 plant genera. P. parasitica is heterothallic with A1 and A2 mating types have been isolated in Taiwan. The sexual reproduction of Phytophthora plays an important role not only in genetics exchange, but also as a survival structure. In spite of numerous efforts made to determine the mating type of Phytophthora not much is known at the molecular level. Retroposon-base DNA fingerprint of 98130/A1 and 98130/A2 isolates of P. parasitica were analyzed.The hybridization pattern of 98130/A1 was almost the same as 98130/A2 with the exception of few bands, indicating that these two strain were isogenic. In AFLP experiment, a total 32 AFLP primer sets were analyzed for their ability to distinguish isolates of opposite mating type. Although the AFLP patterns were similar in most cases, 8 bands were found only in one mating type but not the other. Sequence analysis showed three of to containe homologous sequences of Rumex acetosa Y chromosome specific tandem repeat however, further study is needed to confirm its genetic link to mating type. In cDNA-AFLP experiment, a total of 40 combination primer sets were analyzed for their ability to distinguish between 98130/A1 and 98130/A2 in stable environmental conditions. Although the cDNA-AFLP patterns were similar in most cases but there did exit 44 differential transcription derivate fragments. The transcription derivate fragment A15T13-2 revealed by cDNA-AFLP expressed higher in 98130/A2 than 98130/A1. The validity of this result was comfirmed by northern hybridization and real-time PCR. The performance of northern hybridization with increased number of A1 and A2 isolates showed similar result, indicating that the phenomenon of higher expression of A15T13-2 in A2 isolates of P. parasitica is universal. A15T13-2 showed high similarity with Danio rerio aquaporin3 by sequence analysis. These differential transcription derivate fragments need further study to confirm its linkage with either mating type determination or mating process.

碩士
國立臺灣大學
植物病理與微生物學研究所
93
Ganoderma lucidum is a well-known and important medicinal mushroom demonstrated with anti-cancer and immunomodulatory activities. Although classic study on the mating and fruiting in G. lucidum have been carried out in recent decades and accumulated tremendous valuable knowledge, in-depth exploration of the biologically interesting phenomenon via molecular biology approaches was unavailable and deserved further study. In order to understand the genes in G. lucidum which regulated the mating and fruiting, first we BLAST the Expressed Sequence Tag (EST) from G. lucidum cDNA library, and accessed 4 mating type genes in A mating type locus, and 20 pheromone receptor genes and 19 pheromone precursor genes which controlled mating in B mating locus. A 15 kbp A mating type locus was assembled by linking contigs in whole genomic DNA library of G. lucidum. Full-length open reading frames of genes, a1, a2, encoded homeodomain protein HD1 and HD2, respectively, were cloned by Rapid Amplification of cDNA Ends (RACE). a1 gene is 1428 bp in full-length, having 48% similarity to HD1 of Pleurotus djamor, and also with Nuclear Localization Signal (NLS)：RKRRR, while a2 gene is 1680 bp in full length, having 39% similarity to conserved HD2 of P. djamor, and also with NLS：RRSRCRKE. By polymerase chain reaction using specific primers derived from mating genes a1, a2, b1, b2, PR3, PR5, PR7, PR8, PR15 and PR19, A1 mating type was demonstrated possessing a1, a2 genes; A2 mating type b1, b2 genes; B1 PR3, PR5, PR15 and PR19 genes; B2 PR3, PR7 and PR8 genes, respectively. The mating genes in A1B1, A1B2, A2B1, A2B2 of G. lucidum were further verified by dual mating in vitro on YMSA plates based on cytology, and ontology of clamp connection and pseudoclamp connection formation. And also, two binary vectors pCGl-a1 and pCGl-a2, having the a1 or a2 gene insertions, driven by glyceraldehyde - 3 - phosphate dehydrogenase (gpd) promoter and the downstream hygromycin resistance gene, were constructed, and will be transformed into compatible mating type to prove the function of these genes in mating. In addition, a Fosmid library constructed from monokaryotic strain BCRC 37180 (A2B2) of G. lucidum screened by labeled b1 and PR7 gene probes indicated 8 clones showing positive signals against A locus genes, and 5 clones to B locus genes. These specific Fosmid clones will be both terminal ends sequenced and assembled with the EST and genome databases to constructed a complete A, B, mating type locus genetic map. We also constructed a subtracted cDNA library of G. lucidum, genes which differentially expressed during fruiting body development will be cloned and their function proved by gene disruption.

Vegetative incompatibility is a ubiquitous phenomenon in many filamentous fungi. Neurospora crassa vegetative incompatibility involves the prevention of vigorous heterokaryons, mediated by genetic dissimilarity at eleven heterokaryon incompatibility (het) loci. During the vegetative phase, the mating-type (mat) locus functions as a het locus, while in the sexual phase, the mat locus confers mating identity and post-fertilization functions for completion of the sexual cycle, tol is an unlinked recessive mutation that suppresses mating-type vegetative incompatibility. TOL encodes a 1044 amino acid polypeptide that contains putative proteinprotein interaction domains. TOL contains a region called the SET domain that has similarity to HET-6 in N. crassa and HET-E in Podospora anserina (SET = HET-SIX, HET-E and TOL). Searches of the N. crassa genome yielded a large number of TOL-like sequences, which also share the SET domain and may indicate a novel motif region. BLASTP searches with TOL have identified similarity to Mms21p, a DNA repair protein in Saccharomyces cerevisiae. To identify additional proteins in the mating-type vegetative incompatibility pathway, TOL was used as bait in a yeast two-hybrid system. One of the putative pit genes (proteins that interact with TOL) was ncvipl, which has homology with vipl, an uncharacterized gene in Schizosaccharomyces pombe. RIP (repeat-induced point) mutational analyses of the ncvipl was conducted and progeny examined as to their effects on mating-type associated vegetative incompatibility. At the termination of this study, the influence of ncvipl remained unclear. Strains were constructed that will provide the tools for future analyses. A novel mutation was discovered that caused a partial suppression of mating-type vegetative incompatibility (Sup [mvi]) phenotype. This mutation appears to be linked to or in mat a, localizing the gene causing the Sup (mvi) phenotype to linkage group I. Partial diploid analysis and complementation assays failed to determine if the mutation was in mat a or not and demonstrated that the Sup (mvi) phenotype does not suppress mating-type vegetative incompatibility when the mating-type gene products are in the same nucleus. Further analysis is needed to determine the nature of the mutation and the full influence of the Sup (mvi) phenotype in a homoallelic condition.

The mating type genes of Neurospora crassa were shown to function abnormally when located at ectopic chromosomal positions. Crosses involving strains with ectopic mating type genes produce defective perithecia. Ascus number is reduced. The hypothesis that perithecial development requires physical proximity of opposite mating type homologs during meiosis was tested. The sterility of the crosses made between strains with both mating type regions relocated to the same ectopic position failed to support the hypothesis. To test the hypothesis that normal expression levels of the mating type genes require distant cw-acting sequences not present on ectopic fragments, autoradiograms of mRNA from wild type and ectopic-w^ strains were compared. Differences between expression levels in ectopic-/rtf and wild type cells were observed, but their significance cannot be assessed without additional studies. The hypothesis that nuclear identity is disrupted in ectopic-m/ strains was tested. Strains with disturbed nuclear identity (dual mating type) were crossed to wild type. The appearance of the reduced ascus number phenotype suggested that the affected function in ectopic-m/ strains is nuclear identity. The homothallic species N. terricola contains mt A- and mt a-like sequences. The genes were cloned and sequenced to determine whether or not they were functional. The genes specifying identity, mt A-l and mt a-l, are more than 95% similar at the amino acid level to the N. crassa homologs, but the putative MT A-2 polypeptide is truncated. N. terricola ml A-l and mt a-l genes induced mating and vegetative incompatibility in N. crassa mating type mutants. Expression in N. terricola of mt A-l and mt a-l was detected by reverse transcriptase PCR, upholding the hypothesis that the genes are functional. To determine the pattern of evolution of homothallism, a phylogeny of Neurospora was reconstructed from mt A-l DNA and amino acid sequences. Homothallic Neurospora species that carry both mating type genes are more closely related to heterothallic species than they are to the ^4-only homothallic species, suggesting that either heterothallic species are derived from a homothallic ancestor or that homothallism arose twice.

Sexual reproduction in fungi is governed by a specialized genomic region called the mating-type locus (MAT). The ascomycetes, the largest phylum of fungi, primarily possess a bipolar mating system while the basidiomycetes, the second largest group, are mostly tetrapolar. The human fungal pathogen and basidiomycetous yeast Cryptococcus neoformans has evolved a bipolar mating system that encodes homeodomain (HD) and pheromone/receptor (P/R) genes. The MAT locus of C. neoformans is unusually large, spans greater than 100 kb, and encodes more than 20 genes. To understand how the pathogenic Cryptococcus species complex evolved this unique bipolar mating system, we investigated the evolution of MAT in closely and distantly related species and discovered an extant sexual cycle in Cryptococcus amylolentus.

Phylogenetic analysis using a six-gene multi-locus sequencing (MLS) approach identified the most closely related species to the pathogenic Cryptococcus species complex that are currently known. The two non-pathogenic sibling species, Tsuchiyaea wingfieldii and Cryptococcus amylolentus, and the more distantly related species Filobasidiella depauperata define the Filobasidiella clade. We also resolved the phylogeny of the species located in the sister clade, Kwoniella. A comprehensive tree dendrogram revealed that the 15 Tremellales species examined suggests a common saprobic ancestor. Moreover, the pathogenic Cryptococcus species have a saprobic origin but later emerged as pathogens. We further characterized the mating-type locus for T. wingfieldii and C. amylolentus by cloning and sequencing two unlinked genomic loci encoding the HD and P/R genes. Interestingly, linked and likely divergently transcribed homologs for SXI1 and SXI2 are present in T. wingfieldii and C. amylolentus, while the P/R alleles contain many genes also found in the MAT locus of the pathogenic Cryptococcus species. Also, hypothetical genes present in C. neoformans MAT are also MAT-linked in both species and indicate a possible translocation event between chromosomes 4 and 5 of C. neoformans. Our analysis of MAT in the sibling species indicates that T. wingfieldii is likely tetrapolar, and the C. amylolentus sequence comparison of the dimorphic SXI1 and SXI2 region and the pheromone receptor, STE3, suggests that C. amylolentus is also tetrapolar. The examination of MAT in these sibling species confirms the model for MAT evolution previously proposed in which this structure in C. neoformans and C. gattii evolved from an ancestral tetrapolar mating system. Moreover, the organization of MAT in these sibling species mirrors key aspects of the proposed intermediates in the evolution of MAT in the pathogenic Cryptococcus species, and for sex chromosomes in plants, animals, and alga in general.

We discovered an extant sexual cycle for C. amylolentus, a species previously thought to be asexual. Matings between two strains of opposite mating-types produce dikaryotic hyphae with fused clamp connections and uni- and bi-nucleate basidiospores. Genotyping of basidiospores using markers linked and unlinked to MAT revealed that genetic exchange (recombination) occurs during the sexual cycle of C. amylolentus, and it is likely that either aneuploids are generated during sex or more than one meiosis event occurs within each basidium. This is in contrast to C. neoformans, where only one meiotic event per basidium has been observed. Uniparental mitochondrial inheritance has also been observed in C. amylolentus progeny; similar to the pathogenic Cryptococcus species, mtDNA is inherited from the C. amylolentus MATa parent. Analysis of sex in C. amylolentus has provided insight into the mechanisms that phylogenetically related fungi employ in orchestrating sexual reproduction.

We also extended our analysis to include the distantly related tetrapolar basidiomycete Tremella mesenterica. We completed comparisons of MAT-specific genes between five strains of T. mesenterica and identified the regions that define its mating-type system. The HD locus is limited to the SXI1- and SXI2-like genes while the P/R locus is defined by STE3, STE12, STE20, and the pheromone gene, tremerogen a-13. Interestingly, many of the genes associated with the MAT locus of the pathogenic Cryptococcus species flank the HD and P/R locus and are not incorporated in MAT in T. mesenterica. The MAT region includes transposons and C. neoformans hypothetical genes also present in T. wingfieldii and C. amylolentus. The mating-type system in T. mesenterica reflects an ancestral intermediate in the evolution of the MAT locus in the pathogenic Cryptococcus species. In conclusion, this study provides an in-depth analysis on the structure, function, and evolution of an unusual mating-type locus with broader implications for the transitions in modes of sexual reproduction in fungi that impact gene flow in populations.

Dissertation

The genus Arthroderma contains predominantly geophilic dermatophytes (naturally occuring in soil). Some species, especially those from Trichophyton terrestre complex, cause human and animal dermatomycosis. In the past, the species boundaries were determined mainly on the basis of biological species concept using in vitro mating experiments. But these nearly 70-years-old findings have not been tested by means of modern taxonomic methods. In total 194 species of the genus Arthroderma (including all available ex-type strains) originating predominantly in USA, Canada and Europe were studied in this thesis. They were mostly isolated from soil (n = 77), animals (n = 50), human clinical material (n = 41) and cave sediment (n = 9). The main goal of the thesis was to elucidate the species boundaries between species A. insingulare, A. lenticulare and A. quadrifidum, that were classified into the T. terrestre complex because of their seemingly identical asexual stage. Further, this work aimed to resolve the relationship between Arthroderma species using the multigene phylogeny and clarify which species are clinically relevant. A multigene phylogeny of the genus Arthroderma was based on the sequences of the ITS rDNA region, β-tubulin (TUB2) and translation elongation factor 1α (TEF1α) genes. The genus...

Dermatophytes (order Onygenales, Ascomycota) are microscopic filamentous keratinophilic fungi that can cause skin infections known as dermatophytosis. The most diverse but not very studied genus Arthroderma has been revised recently (Míková 2018) which was essential for further research. This genus comprises mostly species with a supposed reservoir in soil. Lack of information about their ecology and frequent isolation of some species from the hair of free- living mammals (mainly rodents) may testify a strong host association. Rodents could thus represent the hidden reservoir of this species. For this thesis, I have chosen three ecologically distinct rodent species: Mus musculus, Apodemus flavicollis, and Clethrionomys glareolus. I obtained the material by brushing the hair of asymptomatic individuals and used this material for cultivation on selective medium. I identified the isolates of dermatophytes (n = 30) using molecular methods. I used sequences of three highly variable loci (ITS, tubb a tef1α) to incorporate these isolates in the phylogenetic analysis based on the monography of the genus Arthroderma (Míková 2018). I characterized the phenotype of selected strains based on morphological and physiological data including the ability to utilize keratin and the production of siderophores. The...