Welcome to IMC 2018 International Mycological Congress
Dual roles of fungal phytochrome: a light receptor and a temperature sensor
- Z. Yu
- A. Ali
- C. Streng
- R. Fischer
AbstractTo survive in the ever-changing environment, fungi have evolved to sense internal and external signals and adapt to various stresses. Light as one of the most important environmental signals regulates morphogenetic and physiological processes (1). To sense the light, fungi have been equipped with different photoreceptors during the evolution. Aspergillus nidulans is able to sense red and blue light with the red light sensor phytochrome (FphA) and the blue light senor white collar-1 (LreA), respectively. Phytochrome consists of a photosensory, a histidine kinase and a response-regulator domain and uses the SakA/HogA MAP kinase pathway to transmit light signal (2). The phosphotransfer protein YpdA interacted with the response regulator domain of FphA. The conserved histidine (H770) in the histidine kinase domain and aspartate (D1181) in the response regulator (RR) domain are essential for the activity of FphA. When H770 was mutated to glutamic acid (E), the light-regulated genes ccgA and conJ were already induced in the dark. Thus the negatively charged amino acid glutamate mimicked phosphorylation of histidine. FphA wild type and mutant forms were further purified from E. coli. The H770E mutated form migrated faster in size exclusion chromatography (SEC) than the wild-type form, which implying the negative charges cause the conformational change of FphA.
Intriguingly, phytochrome is more than a light receptor. In vitro, the spectra properties of recombinant FphA were changed with temperature. The dark reversion of FphA was slightly increased when the temperature was increased. In vivo, higher temperature caused the transient activation of SakA/HogA pathway and ccgA and ccgB were induced. In fphA-deletion strain, SakA phosphorylation and the gene induction in higher temperature were reduced in comparison to wildtype strain. These results demonstrate phytochrome plays dual roles in sensing temperature and light.
Mycelial growth under light improves conidial stress tolerance and virulence of Metarhizium robertsii and up-regulates stress related genes
- D. Rangel
Light conditions during fungal growth are well known to cause several physiological adaptations in the produced conidia; thus, conidia of the insect-pathogenic fungi Metarhizium robertsii were produced on: 1) potato dextrose agar (PDA) medium in the dark; 2) PDA medium under white light; 3) PDA medium under blue light; and 4) PDA medium under red light. The conidial production, the speed of conidial germination, the virulence to the insect Tenebrio molitor, as well as gene expression, and tolerances to osmotic stress and to UV radiation were evaluated. Conidia produced under white light or blue light germinated faster and were the most tolerant to UV radiation and osmotic stress. White light improved conidial virulence as compared with conidia produced in the dark. Growth under blue light produced more conidia than the fungus grown in the dark. The small (Mrhsp30) and large (Mrhsp101) heat shock protein genes were highly up-regulated under white light condition, suggesting an active role of heat shock proteins in fungal exposition to the different visible spectrum components. The cytosolic catalase Mrcatc gene was not induced under all light conditions assayed. Conidia produced under red light germinated slower than conidia produced in the dark and were the least tolerant to osmotic stress and UV radiation. The virulence of conidia produced under red light was similar to conidia produced in the dark. In conclusion, white light produced conidia that germinated faster and killed the insects faster; in addition, blue light afforded the highest conidial production. Both white light and blue light afforded the highest tolerance to both stress conditions.
Shining light on a creature of the dark: extreme sensitivity to ultraviolet light in the fungal pathogen causing white-nose syndrome of bats
- D. Lindner
- J. Palmer
AbstractFungi have evolved complex light-sensing and regulatory systems that control core metabolic processes involved in many aspects of fungal biology, including the ability to repair DNA damage from ultra-violet (UV) light. Repair of UV-damaged DNA lesions in fungi is mediated by several different conserved mechanisms and these repair systems have typically been studied in fungi that have evolved in the presence of light. The fungal pathogen causing white-nose syndrome of bats (WNS), Pseudogymnoascus desctructans, affords a rare opportunity to study a fungus that has evolved for millions of years in the absence of light. WNS has decimated North American hibernating bats since its introduction to North America in 2006. In order to better understand this disease, we utilized a comparative genomics approach for P. destructans, comparing its genome to those of six closely related non-pathogenic Pseudogymnoascus species. A large reduction (~ 65%) in carbohydrate utilizing enzymes (CAZYmes), a reduction in the predicted secretome (~50%), an increase in unique gene models, and estimation of last common ancestor of 23.5 MYA indicate that P. destructans has a long evolutionary history with bats and likely evolved alongside bats in the absence of light. P. destructans has lost a key enzyme, UVE1, in the alternate excision repair (AER) pathway, which functions to repair DNA-lesions induced by ultra-violet (UV) light. Consistent with a non-functional AER pathway, P. destructans is extremely sensitive to UV-light as well as the DNA alkylating agent methyl methanesulfonate (MMS). A better understanding of light-sensing and regulatory systems may be gained by examining fungi that have evolved for long periods of time in dark environments, for example the digestive tracts of animals.
What makes a zombie ant tick: the daily rhythms in a fungal behavioral manipulator
- C. De Bekker
Adaptive manipulation of host behavior is an effective way for parasites to increase transmission rates. Manipulations resulting in summiting of insect hosts can be observed in Zygomycetes and Ascomycetes of the genera Entomophthora and Ophiocordyceps. Climbing, biting to fix the host at elevated positions, following death, and spore release all appear to display time-of-day synchronization. Biological clocks of the insect host and the fungal parasite, as well as environmental factors such as light and temperature, likely play an important role in these parasite-host interactions. To begin to test these hypotheses we use an integrative approach, combining ecology with behavioral analyses and next-generation sequencing, on a variety of Ophiocordyceps unilateralis sensu lato - carpenter ant interactions. Field data indicates that light influences the manipulated behavior of Ophiocordyceps-infected carpenter ants. This suggests that the fungal parasite, which is fully pulling the strings at this point, can sense light, and has a light entrainable biological clock. To demonstrate this, as well as identify candidate molecular clock components of Ophiocordyceps, we made use of bioinformatics and transcriptional profiling. We did this for the recently sequenced behavior-manipulating parasite Ophiocordyceps kimflemingiae (a named species of the O. unilateralis complex). Through a bioinformatics approach, we identified putative homologs of known clock genes. RNA-Seq was performed on 48 h time courses of O. kimflemingiae to determine daily rhythms and enrichment patterns in the transcriptome. Liquid media cultures were entrained under 24 h light-dark (LD) cycles and harvested at 4 h intervals under LD or continuous darkness. We identified a significant number of fungal transcription factors with peaked activity during the light phase (day time). In contrast, a significant number of secreted enzymes and small bioactive compounds, proteases, and toxins peaked during the dark phase or subjective night. These findings support a model whereby Ophiocordyceps species use their biological clock for phase-specific activity and light-dependent manipulation. This may be a general mechanism involved in parasite-host interactions across taxa.
Light-Sensing, optogenetics and photographic memory: developing biotechnological solutions and pushing the boundaries between science and art
- L. Larrondo
- V. Rojas
- V. Delgado
- P. Canessa
- C. Olivares-Yañez
AbstractThe filamentous fungus Neurospora crassa perceives and responds to blue light through a transcriptional heterodimer named White Collar Complex (WCC). One of its components, WC-1, possesses a LOV (Light Oxygen Voltage) domain capable of detecting blue wavelengths, which promotes a conformational change that leads to dimerization, resulting in strong transcriptional activation, in a light-intensity dependent manner. In order to design and improve optogenetic switches that can be utilized in other organisms as orthogonal controllers, we have been exploring the dynamics of light responses in this fungus. Thus, through the development of Neurospora-based optogenetic switches we have successfully implemented a blue-light responding transcriptional system in Saccharomyces cerevisiae. Therefore, in yeast, now we can efficiently induce gene expression over 1000-fold and control biotechnological relevant phenotypes such as flocculation by switching on/off the lights.
We have also adopted optogenetic approaches to further delve into Neurospora’s circadian and light-responses. In doing so, we were able to genetically program 2D-images in this organism. Thus, we can project a photograph on top of a Neurospora carrying a luciferase reporter under the control of a light responsive promoter and obtain back a bioluminescent pattern mimicking the original image. Thus, we have established a live canvas in which images are genetically processed and reconstituted with real-time dynamics. Such technology not only allows studying light-responses with great resolution, but also provides a powerful artistic substrate. Remarkably, since the live canvas circuit is integrated in the Neurospora circadian regulatory network, the fungus reproduces on subsequent days -in a circadian manner- the image that it had originally “seen”, creating an eidetic (photographic) memory effect. Such phenomenon, based on local discrete phase changes, not only will provide new insights on phase responses, but it also allows for the opportunity to ponder on concepts such as vision and memory. MIISSB and FONDECYT 1171151 and HHMI International Research Scholar grant.
Identification of genes involved in fruiting body induction in Coprinopsis cinerea
- Y. Sakamoto
- S. Sato
- T. Nakazawa
- K. Osakabe
- H. Muraguchi
Light and nutrient are crucial environmental factors influencing fungal sexual reproduction. Gene expressions influenced by such environmental factors in Coprinopsis cinerea were investigated. Blue light induces simultaneous hyphal knot formation in mycelia grown on low glucose (0.2%) media, but not in mycelia grown on high glucose (1%) media in C. cinerea. Many hyphal knots are visible in arc near the edge of the colony one day after a 15min of blue light stimulation. These suggest that blue light accelerates hyphal knot induction in nutrient limiting condition. Trascriptome (Super-SAGE) analysis revealed that gene expression after light exposure divided into at least two major stages. In the first stage, genes coding for fasciclin (fas1), cyclopropane-fatty-acyl-phospholipid synthases (cfs1 and cfs2), and putative lipid exporter (nod1) are highly expressed at 1h after light exposure in the mycelial region where the hyphal knot will be developed. It is reported that the cfs1 mutated strain was defected in fruiting body development. These genes were up-regulated by blue light, and not influenced by glucose condition and mating. These results suggest that although some of the genes are critical for induction of the hyphal knots, they are not sufficient for hyphal knot development. In the second gene expression stage, genes encoding galectins (cgl1-3), farnesyl cysteine-carboxyl methyltransferases, mating pheromone containing protein, nucleus protein (ich1) and laccase (lcc1) are specifically upregulated at 10-16 h after blue light exposure when the mycelia are cultivated on low glucose media. These might be involved in building architecture of hyphal knot or signal transduction for further fruiting body development. These results contribute to the understanding of the effect of environmental factors on sexual reproduction in basidiomycetous fungi.