Breeding a fungal gene into wheat

Every year, infection of wheat by the fungus Fusarium graminearum results in losses of ∼28 million metric tons of wheat grain (1), valued at $5.6 billion. The fungus reduces yields but also contaminates harvests with trichothecene toxins such as deoxynivalenol (DON; also called vomitoxin because of its effects on mammals) that render grain too poisonous to use. The disease is becoming more prevalent because of increasing cultivation of maize (also a host for the fungus) and reduced tillage (ploughing) agriculture, which promotes fungal survival on last season's plant debris. On page 844 of this issue, Wang et al. (2) reveal the molecular identity of the Fusarium head blight 7 (Fhb7) gene, which encodes a glutathione S-transferase that detoxifies DON. This gene was acquired through a “natural” fungus-to-plant gene transfer in a wild wheat relative. This naturally occurring genetically modified (GM) wheat strain is therefore exempt from regulation and can be grown directly by farmer

Symbiont-specific responses to environmental cues in a threesome lichen symbiosis

Photosymbiodemes are a special case of lichen symbiosis where one lichenized fungus engages in symbiosis with two different photosynthetic partners, a cyanobacterium and a green alga, to develop two distinctly looking photomorphs. We compared gene expression of thallus sectors of the photosymbiodeme-forming lichen Peltigera britannica containing cyanobacterial photobionts with thallus sectors with both green algal and cyanobacterial photobionts and investigated differential gene expression at different temperatures representing mild and putatively stressful conditions. First, we quantified photobiont-mediated differences in fungal gene expression. Second, because of known ecological differences between photomorphs, we investigated symbiont-specific responses in gene expression to temperature increases. Photobiont-mediated differences in fungal gene expression could be identified, with upregulation of distinct biological processes in the different morphs, showing that interaction with specific symbiosis partners profoundly impacts fungal gene expression. Furthermore, high temperatures expectedly led to an upregulation of genes involved in heat shock responses in all organisms in whole transcriptome data and to an increased expression of genes involved in photosynthesis in both photobiont types at 15 and 25 degrees C. The fungus and the cyanobacteria exhibited thermal stress responses already at 15 degrees C, the green algae mainly at 25 degrees C, demonstrating symbiont-specific responses to environmental cues and symbiont-specific ecological optima

The importance of subclasses of chitin synthase enzymes with myosin-like domains for the fitness of fungi

Acknowledgements TG and CF are funded by FEDER funds through the Operational Programme Competitiveness Factors – COMPETE and national funds by FCT – Foundation for Science and Technology under the strategic project UID/NEU/04539/2013. C.F. is a recipient of a postdoctoral fellowship from FCT-Fundação para a Ciência e Tecnologia (SFRH/BPD/63733/2009). NG is funded by The Wellcome Trust (080088, 086827, 075470, 099215 & 097377), the FungiBrain Marie Curie Network and the Medical Research Council (UK).Peer reviewedPostprin

Bacterial Transposons containing Markers for Fungal Gene Disruption

We have constructed 2 tn5-containing plasmids, pLH1 and pLH3, specialized towards mutagenesis of genes from fungi which are auxotrophic for arginine or sensitive to hygromycin (such as the filamentous fungi Aspergillus nidulans and Magnaporthe grisea.) These plasmids are also a useful means of integrating additional marker genes in the plasmid backbone

Fungal ecological strategies reflected in gene transcription - a case study of two litter decomposers.

Microbial communities interplay with their environment through their functional traits that can be a response or an effect on the environment. Here, we explore how a functional trait-the decomposition of organic matter, can be addressed based on genetic markers and how the expression of these markers reflect ecological strategies of two fungal litter decomposer Gymnopus androsaceus and Chalara longipes. We sequenced the genomes of these two fungi, as well as their transcriptomes at different steps of Pinus sylvestris needles decomposition in microcosms. Our results highlighted that if the gene content of the two species could indicate similar potential decomposition abilities, the expression levels of specific gene families belonging to the glycoside hydrolase category reflected contrasting ecological strategies. Actually, C. longipes, the weaker decomposer in this experiment, turned out to have a high content of genes involved in cell wall polysaccharides decomposition but low expression levels, reflecting a versatile ecology compare to the more competitive G. androsaceus with high expression levels of keystone functional genes. Thus, we established that sequential expression of genes coding for different components of the decomposer machinery indicated adaptation to chemical changes in the substrate as decomposition progressed

FunGeneClusterS:Predicting fungal gene clusters from genome and transcriptome data

Introduction: Secondary metabolites of fungi are receiving an increasing amount of interest due to their prolific bioactivities and the fact that fungal biosynthesis of secondary metabolites often occurs from co-regulated and co-located gene clusters. This makes the gene clusters attractive for synthetic biology and industrial biotechnology applications. We have previously published a method for accurate prediction of clusters from genome and transcriptome data, which could also suggest cross-chemistry, however, this method was limited both in the number of parameters which could be adjusted as well as in user-friendliness. Furthermore, sensitivity to the transcriptome data required manual curation of the predictions. In the present work, we have aimed at improving these features. Results: FunGeneClusterS is an improved implementation of our previous method with a graphical user interface for off- and on-line use. The new method adds options to adjust the size of the gene cluster(s) being sought as well as an option for the algorithm to be flexible with genes in the cluster which may not seem to be co-regulated with the remainder of the cluster. We have benchmarked the method using data from the well-studied Aspergillus nidulans and found that the method is an improvement over the previous one. In particular, it makes it possible to predict clusters with more than 10 genes more accurately, and allows identification of co-regulated gene clusters irrespective of the function of the genes. It also greatly reduces the need for manual curation of the prediction results. We furthermore applied the method to transcriptome data from A. niger. Using the identified best set of parameters, we were able to identify clusters for 31 out of 76 previously predicted secondary metabolite synthases/synthetases. Furthermore, we identified additional putative secondary metabolite gene clusters. In total, we predicted 432 co-transcribed gene clusters in A. niger (spanning 1.323 genes, 12% of the genome). Some of these had functions related to primary metabolism, e.g. we have identified a cluster for biosynthesis of biotin, as well as several for degradation of aromatic compounds. The data identifies that suggests that larger parts of the fungal genome than previously anticipated operates as gene clusters. This includes both primary and secondary metabolism as well as other cellular maintenance functions. Conclusion: We have developed FunGeneClusterS in a graphical implementation and made the method capable of adjustments to different datasets and target clusters. The method is versatile in that it can predict co-regulated clusters not limited to secondary metabolism. Our analysis of data has shown not only the validity of the method, but also strongly suggests that large parts of fungal primary metabolism and cellular functions are both co-regulated and co-located

Jaarverslag 2009 KNPV Werkgroep Graanziekten

Jaarverslag 2009 van de KNPV Werkgroep Graanziekten. De excursie naar Rothamsted Research in Harpenden (UK) op 28 mei 2009 wordt beschreven

Paxilline negative mutants of Penicillium paxilli generated by heterologous and homologous plasmid integration : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Genetics at Massey University, Palmerston North, New Zealand

Using a monoclonal antibody-based ELISA, 600 pAN7-1 plasmid-tagged mutants of Penicillium paxilli were screened for paxilline accumulation and one paxilline negative mutant, YI-20, was identified (Itoh, unpublished data). A molecular analysis of this mutant showed that pAN7-1 was inserted at a single site but was present as 4-6 copies arranged in a head-to tail tandem repeat. Rescue of flanking sequences and analysis of the corresponding genomic region revealed that YI-20 has an extensive deletion at the site of pAN7-1 integration. Probing of a CHEF gel with the same sequences showed that associated with the deletion is a rearrangement of chromosome Va. Targeted gene disruption of wild-type sequences adjacent to the site where pAN7-1 inserted, resulted in the generation of two additional paxilline-negative mutants; both were single crossovers with deletions extending outside the region mapped. Neither of these new mutants had a rearrangement of chromosome Va, suggesting that deletion of genes on this chromosome is responsible for the paxilline-negative phenotype

Further studies of dothistromin toxin genes in the fungal forest pathogen Dothistroma septosporum : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand

The fungal pathogen Dothistroma septosporum is the main causal agent of Dothistroma (red-hand) needle blight, which is a devastating foliar disease of a wide range of pine species. Dothistromin is a difuranoanthraquinone toxin produced by D. septosporum and is considered as a possible virulence factor for the disease. Based on the similarity of chemical structure between dothistromin and aflatoxin (AF) /sterigmatocystin (ST) precursors, nine putative dothistromin biosynthetic genes have been identified, which are homologous to their corresponding genes in the AF/ST gene clusters. However, in contrast to all 25 AF biosynthetic genes tightly clustered in one region (70-Kb) of the genome, the dothistromin gene clusters are located on a 1.3-Mb chromosome and separated into three mini-clusters along with non-dothistromin genes. The dotC gene, located in the mini-cluster 1, is predicted to encode a major facilitator superfamily (MFS) membrane transporter involved in secretion of dothistromin. In this work, by constructing DotC-eGFP fusion protein containing mutants, the subcellular localization of the DotC protein was determined to be mainly targeted to the plasma membrane. The biological function of the dotC gene was characterized by targeted gene disruption. The dotC gene disrupted mutants showed a significant reduction of dothistromin production in both the medium and mycelium. In addition, the exponential growth of dotC null mutants was inhibited when exogenous dothistromin was presented and these mutants also displayed more sensitivity than the wild type strain to exogenous dothistromin. The results indicated that the DotC protein is a membrane associated protein and might have a role in dothistromin production and be involved in secretion of exogenously supplied dothistromin toxin. Two novel dothistromin biosynthetic genes, norA/B and verB (partial sequence), were identified by using degenerate PCR and D. septosporum genomic library screening. The putative NorA/B and VerB are postulated to encode a dehydrogenase and a desaturase, respectively and are similar to AF/ST genes. These findings further confirmed that the dothistromin shares biosynthetic pathway steps with AF/ST

Optimizing Dothistroma septosporum infection of Pinus radiata and the development of red-band disease : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand

The filamentous fungus Dothistroma septosporum infects pine species throughout the world causing red-band disease, one of the most serious diseases of conifer species. In NZ, a clonally derived asexual strain of D. septosporum was identified in 1964, and has spread throughout the country. There are conflicting accounts on the environmental conditions required for infection, which has lead to difficulties in optimizing a laboratory-based system for infection. The pathogen is spread naturally through rain-splashed inoculum of conidiospores from mature stromata that have erupted through the pine needle tissue. Diseased needles become necrotic, often with a red band due to the mycotoxin dothistromin produced by the hyphae. Dothistromin has the chemical structure of a difuranoanthraquinone and shows similarity to the aflatoxin precursor, versicolorin B produced by Aspergillus parasiticus. The role of dothistromin in pathogenicity has not yet been determined, although experiments have shown injecting toxin into pine needles results in the characteristic red band lesion. In this study it was found that fluctuating temperature (16°C/24°C), a 12 h diurnal cycle (white and ultraviolet light), high relative humidity and continuous moisture are conditions conducive to development of red-band disease on inoculated pine trees in an artificial environment. A higher rate of infection was obtained using pine seedlings as opposed to pine cuttings, and using a spore suspension containing a yeast extract. A dothistromin minus mutant was able to infect pine needles, indicating that dothistromin is not a pathogenicity factor, though it may be a virulence factor. The use of GFP-expressing isolates allowed the initial infection process to be monitored with both wild type and mutant isolates. Additionally, a PCR-based diagnostic procedure to confirm infection was developed. The production of aflatoxin by Aspergillus species is regulated by nutritional parameters and extracellular pH, which affect both growth and aflatoxin gene expression. D. septosporum similarly has enhanced growth at acidic pH, but it does not appear that pH has a strong influence on physiological processes as toxin biosynthesis and gene expression do not appear to be pH regulated. Different carbon and nitrogen sources also affect the morphology of D. septosporum