Intron architecture in Fusarium

The genus Fusarium constitutes fungi with diverse biological behaviours. This study focused on four plant pathogenic species. These were F. verticillioides which infects maize, F. oxysporum which infects tomato, F. graminearum, a pathogen of wheat and F. circinatum, which is pathogenic to pine. The genomes of F. verticillioides, F. oxysporum, F. graminearum and F. circinatum have been sequenced. These genomes were annotated using different gene prediction software. To study the architecture and distribution of Spliceosomal introns in these a set of Housekeeping (HK) genes common to all eukaryotes were used. These analyses revealed discrepancies in the annotations of these genomes, which most commonly included intron position incongruences, misidentified introns and sequencing errors. Spliceosomal introns have four cis-elements which include the 5ꞌ and 3ꞌ splice sites, the branch site and the polypyrimidine tract. Analysis of the first three elements of Spliceosomal introns in the four Fusarium species and comparisons to those in other fungi showed significant differences in the consensus sequences of these elements. Two additional branch site motifs were also found for Fusarium, while the polypyrimidine tract of these species was found to be very diverse. The results also indicated that the first introns of the HK genes of the Fusarium species significantly longer, which is consistent with what have been found for genes of other eukaryotic. Also consistent with what is known for other eukaryotes, the analysed Fusarium genes had much lower intron densities than those observed in higher eukaryotes. An average of 2.53 introns per gene was observed in Fusarium and most of these introns were located closer to the 5ꞌ end of the HK genes. This average is low compared A. thaliana and Homo sapiens which have averages of 4.3 and 8.82 introns per gene, respectively. With the aid of EST and genome data, F. circinatum was shown to harbour putative alternative introns at a frequency of approximately 0.3%. Homologues of a number of these genes from F. verticillioides, F. oxysporum and F. graminearum also harboured alternative splicing signals. Certain alternatively spliced transcripts harbored premature stop codons. These transcripts are targeted by non-sense mediated mRNA decay (NMD) system. The high rate at which these transcripts included premature stop codons suggested that such a quality control system is indeed needed for these fungi. Overall, however, it remains to be investigated whether these alternative transcripts are functional as is the case with some of them in humans, plants and insects. As more fungal genomes are being sequenced the need for accurate gene prediction methods is soaring. The incorporation of the findings of the architecture and distribution of Spliceosomal introns in Fusarium into gene prediction methods will thus increase the accuracy of such methods for Fusarium species, especially those related to F. circinatum. The identification of genes that are potentially regulated through alternative intron splicing also provide valuable targets for future studies on important biological processes such as pathogenicity and virulence. Note: The disc provided contains a spreadsheet with all the data included in this dissertation.Dissertation (MSc)--University of Pretoria, 2012.Microbiology and Plant PathologyMScUnrestricte

First fungal genome sequence from Africa : a preliminary analysis

Some of the most significant breakthroughs in the biological sciences this century will emerge from the development of next generation sequencing technologies. The ease of availability of DNA sequence made possible through these new technologies has given researchers opportunities to study organisms in a manner that was not possible with Sanger sequencing. Scientists will, therefore, need to embrace genomics, as well as develop and nurture the human capacity to sequence genomes and utilise the ’tsunami‘ of data that emerge from genome sequencing. In response to these challenges, we sequenced the genome of Fusarium circinatum, a fungal pathogen of pine that causes pitch canker, a disease of great concern to the South African forestry industry. The sequencing work was conducted in South Africa, making F. circinatum the first eukaryotic organism for which the complete genome has been sequenced locally. Here we report on the process that was followed to sequence, assemble and perform a preliminary characterisation of the genome. Furthermore, details of the computer annotation and manual curation of this genome are presented. The F. circinatum genome was found to be nearly 44 million bases in size, which is similar to that of four other Fusarium genomes that have been sequenced elsewhere. The genome contains just over 15 000 open reading frames, which is less than that of the related species, Fusarium oxysporum, but more than that for Fusarium verticillioides. Amongst the various putative gene clusters identified in F. circinatum, those encoding the secondary metabolites fumosin and fusarin appeared to harbour evidence of gene translocation. It is anticipated that similar comparisons of other loci will provide insights into the genetic basis for pathogenicity of the pitch canker pathogen. Perhaps more importantly, this project has engaged a relatively large group of scientists including students in a significant genome project that is certain to provide a platform for growth in this important area of research in the future.We thank the National Research Foundation (NRF) of South Africa, members of the Tree Protection Co-operative Programme, the THRIP initiative of the Department of Trade and Industry and the Department of Science and Technology (DST)/NRF Centre of Excellence in Tree Health Biotechnology and the Oppenheimer Foundation for funding.http://www.sajs.co.zanf201

Ras2 is important for growth and pathogenicity in Fusarium circinatum

In this study, we investigated to possible role of Ras2 in Fusarium circinatum- a fungus that causes pine pitch canker disease on many different pine species and has a wide geographic distribution. This protein is encoded by the RAS2 gene and has been shown to control growth and pathogenicity in a number of fungi in a mitogen-activated protein kinase- and/or cyclic adenosyl monophosphate pathway-dependent manner. The aim was therefore to characterize the phenotypes of RAS2 gene knockout and complementation mutants of F. circinatum. These mutants were generated by transforming protoplasts of the fungus with suitable split-marker constructs. The mutant strains, together with the wild type strain, were used in growth studies as well as pathogenicity assays on Pinus patula seedlings. Results showed that the knockout mutant strain produced significantly smaller lesions compared to the complementation mutant and wild type strains. Growth studies also showed significantly smaller colonies and delayed conidial germination in the knockout mutant strain compared to the complement mutant and wild type strains. Interestingly, the knockout mutant strain produced more macroconidia than the wild type strain. Collectively, these results showed that Ras2 plays an important role in both growth and pathogenicity of F. circinatum. Future studies will seek to determine the pathway(s) through which Ras2 controls these traits in F. circinatum.Supplementary data 1. Gene knockout and transformation protocol for Fusarium circinatum using split-marker constructs.Supplementary data 2. Maximum likelihood phylogeny for the RAS2 gene from selected Fusarium and closely related fungal species. Bootstrap values above 60% are shown at the nodes, and the scale bar shows substitutions per site. The tree was rooted using Aspergillus nidulans and Alternaria burnsii as outgroup species. GenBank genome assembly numbers are shown in brackets.The Tree Protection Cooperative Programme (TPCP), the University of Pretoria, and the National Research Foundation (NRF) and Department of Science and Innovation (DSI) via their DSI-NRF Centre of Excellence in Plant Health Biotechnology (CPHB) and the DSI-NRF South African Research Chairs Initiative (SARChI) Chair in Fungal Genomics.http://www.elsevier.com/locate/yfgbi2022-02-24hj2021BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog