Low genetic diversity among isolates of the nematode-trapping fungus Duddingtonia flagrans: evidence for recent worldwide dispersion from a single common ancestor

The genetic variation of Duddingtonia flagrans, which has become a promising biocontrol agent of animal parasitic nematodes, was investigated in a worldwide collection of 22 isolates. We analysed the sequence variation in four nuclear genes, tubA (beta-tubulin), CMD1 (calmodulin), EF1alpha (translation elongation factor 1alpha), and PII (extracellular serine protease). 1428 aligned base pairs (bp) were analysed from the four genes, including 709 bp of introns. In addition, the variations in three anonymous genomic regions comprising 1155 bp were examined. Three single nucleotide polymorphisms (SNPs) were detected in the seven loci, none of them in the protein encoding genes. The genetic variation was significantly higher in the nematode-trapping fungus Arthrobotrys oligospora, the closest evolutionary relative to D. flagrans. Analysis of 12 isolates of A. oligospora revealed 30 SNPs in tub A, CMD1, EF1alpha and PII. The genetic variation in the isolates of D. flagrans was further examined using AFLP analysis. Five primer combinations were used to detect 159 bands, of which 94 (59.1%) were polymorphic. A neighbour-joining tree based on the AFLP data showed no clear association between genotype and geographical origin. Furthermore, the AFLP data suggest that D. flagrans is mainly clonal and no recombination could be detected, not even within the same country. The low genetic variation in D. flagrans suggests that this fungus has recently diverged from a single progenitor. Based on estimations of mutation rates, it was calculated that this most recent common ancestor lived about 16000-23000 years ago

galaxieEST: addressing EST identity through automated phylogenetic analysis

BACKGROUND: Research involving expressed sequence tags (ESTs) is intricately coupled to the existence of large, well-annotated sequence repositories. Comparatively complete and satisfactory annotated public sequence libraries are, however, available only for a limited range of organisms, rendering the absence of sequences and gene structure information a tangible problem for those working with taxa lacking an EST or genome sequencing project. Paralogous genes belonging to the same gene family but distinguished by derived characteristics are particularly prone to misidentification and erroneous annotation; high but incomplete levels of sequence similarity are typically difficult to interpret and have formed the basis of many unsubstantiated assumptions of orthology. In these cases, a phylogenetic study of the query sequence together with the most similar sequences in the database may be of great value to the identification process. In order to facilitate this laborious procedure, a project to employ automated phylogenetic analysis in the identification of ESTs was initiated. RESULTS: galaxieEST is an open source Perl-CGI script package designed to complement traditional similarity-based identification of EST sequences through employment of automated phylogenetic analysis. It uses a series of BLAST runs as a sieve to retrieve nucleotide and protein sequences for inclusion in neighbour joining and parsimony analyses; the output includes the BLAST output, the results of the phylogenetic analyses, and the corresponding multiple alignments. galaxieEST is available as an on-line web service for identification of fungal ESTs and for download / local installation for use with any organism group at . CONCLUSIONS: By addressing sequence relatedness in addition to similarity, galaxieEST provides an integrative view on EST origin and identity, which may prove particularly useful in cases where similarity searches return one or more pertinent, but not full, matches and additional information on the query EST is needed

Divergence in gene expression related to variation in host specificity of an ectomycorrhizal fungus

Ectomycorrhizae are formed by mutualistic interactions between fungi and the roots of woody plants. During symbiosis the two organisms exchange carbon and nutrients in a specific tissue that is formed at the contact between a compatible fungus and plant. There is considerable variation in the degree of host specificity among species and strains of ectomycorrhizal fungi. In this study, we have for the first time shown that this variation is associated with quantitative differences in gene expression, and with divergence in nucleotide sequences of symbiosis-regulated genes. Gene expression and sequence evolution were compared in different strains of the ectomycorrhizal fungus Paxillus involutus; the strains included Nau, which is not compatible with birch and poplar, and the two compatible strains Maj and ATCC200175. On a genomic level, Nau and Maj were very similar. The sequence identity was 98.9% in the 16 loci analysed, and only three out of 1075 genes analysed by microarray-based hybridizations had signals indicating differences in gene copy numbers. In contrast, 66 out of the 1075 genes were differentially expressed in Maj compared to Nau after contact with birch roots. Thirty-seven of these symbiosis-regulated genes were also differentially expressed in the ATCC strain. Comparative analysis of DNA sequences of the symbiosis-regulated genes in different strains showed that two of them have evolved at an enhanced rate in Nau. The sequence divergence can be explained by a decreased selection pressure, which in turn is determined by lower functional constraints on these proteins in Nau as compared to the compatible strains

Evolutionary genomics of symbiotic fungi

Ectomycorrhizae is a mutualistic association between roots of woody plants and a diverse range of soil fungi. The fungi exchange soil derived mineral nutrients for photosynthetic sugars from the host plant. The mycorrhizal symbioses are commonly found in all forest ecosystems and have a major ecological and economical importance. I have used comparative genomics, DNA microarrays and computational approaches to gain insights into the evolution of the ectomycorrhizal symbiosis in two fungi Laccaria bicolor (Basidiomycetes; Agaricales) and Paxillus involutus (Basidiomycetes; Boletales). L. bicolor is the first symbiotic fungus to have its genome sequence determined. The genome assembly contains 65 million base pairs with about ~20,000 predicted protein-encoding genes. Here, I report the analysis of L. bicolor genome and its comparison with the genomes of four other basidiomycetes including the saprotrophic species Coprinopsis cinerea and Phanerochaete chrysosporium, the human pathogen Cryptococcus neoformans and the plant pathogen Ustilago maydis. The compared genomes cover about 550 million years of evolution. A total of 58,030 protein sequences from these five basidiomycetes were clustered into 7352 protein families. The evolution of protein families were analysed for accelerated rates of gain and loss along specific branches of a phylogenetic tree using a stochastic birth and death model. Analysis of the genome sequence of L. bicolor in comparison to other analysed basidiomycetes revealed large genome size, large number of protein families, larger size of protein families, many lineage specific and expanded families, and large number of recent duplicates. The evolution of two large and expanded protein families in L. bicolor having significant homology to protein kinases and Ras GTPases superfamilies were analysed in more detail. The analyses showed these families to contain many paralogs that have arisen through recent duplication events. The comparative analyses of gene families showed that the evolution of symbiosis in L. bicolor has been associated with the expansion of large multigene families. The functions of many of these families are unknown but many of them are differentially expressed during symbiosis. In the second part of my thesis, I have analysed duplicated and rapidly evolving genes that could be associated with symbiotic adaptations in the ectomycorrhizal fungus P. involutus. Strains of P. involutus forming ectomycorrhiza showing various degree of host-specificity were analysed by comparative genomic hybridizations using a cDNA microarray representing 1076 putative unique genes. Approximately 17% of the genes investigated on the array were detected as rapidly and presumably non-neutrally evolving within Paxillus. Among these genes, there were several hydrophobins. Hydrophobins are small, secreted hydrophobic cell surface proteins having several roles in growth and development of fungi. The evolutionary mechanisms responsible for generating sequence and expression divergence among members of the hydrophobin multigene family in P. involutus were examined in more detail

Autoimmune regulator is acetylated by transcription coactivator CBP/p300

The Autoimmune Regulator (AIRE) is a regulator of transcription in the thymic medulla, where it controls the expression of a large set of peripheral-tissue specific genes. AIRE interacts with the transcriptional coactivator and acetyltransferase CBP and synergistically cooperates with it in transcriptional activation. Here, we aimed to study a possible role of AIRE acetylation in the modulation of its activity. We found that AIRE is acetylated in tissue culture cells and this acetylation is enhanced by overexpression of CBP and the CBP paralog p300. The acetylated lysines were located within nuclear localization signal and SAND domain. AIRE with mutations that mimicked acetylated K243 and K253 in the SAND domain had reduced transactivation activity and accumulated into fewer and larger nuclear bodies, whereas mutations that mimicked the unacetylated lysines were functionally similar to wild-type AIRE. Analogously to CBP, p300 localized to AIRE-containing nuclear bodies, however, the overexpression of p300 did not enhance the transcriptional activation of AIRE-regulated genes. Further studies showed that overexpression of p300 stabilized the AIRE protein. Interestingly, gene expression profiling revealed that AIRE, with mutations mimicking K243/K253 acetylation in SAND, was able to activate gene expression, although the affected genes were different and the activation level was lower from those regulated by wild-type AIRE. Our results suggest that the AIRE acetylation can influence the selection of AIRE activated genes

Paralysis of nematodes: shifts in the transcriptome of the nematode-trapping fungus Monacrosporium haptotylum during infection of Caenorhabditis elegans

The transcriptional response in the parasitic fungus Monacrosporium haptotylum and its nematode host Caenorhabditis elegans were analyzed during infection using cDNA microarrays. The array contained 2,684 fungal and 372 worm gene reporters. Dramatic shifts occurred in the transcriptome of M. haptotylum during the different stages of the infection. An initial transcriptional response was recorded after 1h of infection when the traps adhered to the cuticle, but before immobilization of the captured nematodes. Among the differentially expressed genes were two serine proteases (spr1 and spr2), and several homologues to genes known to be regulated in other pathogenic fungi. After 4 hours, when approximately 40 % of the nematodes were paralyzed, we identified an up-regulated cluster of 372 fungal genes which were not regulated during the other phases of the infection. This cohort contained a large proportion (79%) of genes that appear to be specific for M. haptotylum and closely related species. These genes were of two different classes; those translating into presumably functional peptides and those with no apparent protein coding potential (noncoding RNAs). Among the infection-induced C. elegans genes were those encoding antimicrobial peptides, protease inhibitors and lectins

Characterization of Microstructure and Properties of Additively Manufactured Materials under Room and Elevated Temperatures

The utilisation of additive manufacturing (AM) has brought about a significant transformation in the manufacturing process of materials and components, since it allows for the creation of complex geometries and customised designs. The primary objective of this study is to conduct a thorough analysis of the microstructure and characteristics of materials produced by additive manufacturing techniques, including the effects of varying temperatures ranging from ambient temperature to increased levels. Microstructural analysis encompasses several methods, including optical microscopy, scanning electron microscopy (SEM), and X-ray diffraction (XRD), which are employed to investigate the grain structure, porosity, and phase composition. Standardised testing procedures are employed to assess mechanical qualities, such as tensile strength, hardness, and fracture toughness. temperature analysis methods, such as differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), are utilised in order to examine the temperature stability and phase transitions. This study investigates the impact of various printing factors, including layer thickness, printing speed, and build orientation, on the resultant microstructure and characteristics. This study aims to address the disparity between theoretical understanding and actual implementation, therefore facilitating the wider use of additively made materials in businesses that need exceptional performance in many environments