Reinhard Fischer

Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.

Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus.

Aspergillus fumigatus is exceptional among microorganisms in being both a primary and opportunistic pathogen as well as a major allergen. Its conidia production is prolific, and so human respiratory tract exposure is almost constant. A. fumigatus is isolated from human habitats and vegetable compost heaps. In immunocompromised individuals, the incidence of invasive infection can be as high as 50% and the mortality rate is often about 50% (ref. 2). The interaction of A. fumigatus and other airborne fungi with the immune system is increasingly linked to severe asthma and sinusitis. Although the burden of invasive disease caused by A. fumigatus is substantial, the basic biology of the organism is mostly obscure. Here we show the complete 29.4-megabase genome sequence of the clinical isolate Af293, which consists of eight chromosomes containing 9,926 predicted genes. Microarray analysis revealed temperature-dependent expression of distinct sets of genes, as well as 700 A. fumigatus genes not present or significantly diverged in the closely related sexual species Neosartorya fischeri, many of which may have roles in the pathogenicity phenotype. The Af293 genome sequence provides an unparalleled resource for the future understanding of this remarkable fungus.

The Aspergillus nidulans Phytochrome FphA Represses Sexual Development in Red Light

Phytochrome photoreceptors sense red and far-red light through photointerconversion between two stable conformations, a process mediated by a linear tetrapyrrole chromophore. Originally, phytochromes were thought to be confined to photosynthetic organisms including cyanobacteria, but they have been recently discovered in heterotrophic bacteria and fungi, where little is known about their functions. It was shown previously in the ascomycetous fungus Aspergillus nidulans that asexual sporulation is stimulated and sexual development repressed by red light. The effect was reminiscent of a phytochrome response, and indeed phytochrome-like proteins were detected in several fungal genomes. All fungal homologs are more similar to bacterial than plant phytochromes and have multifunctional domains where the phytochrome region and histidine kinase domain are combined in a single protein with a C-terminal response-regulator domain. Here, we show that the A. nidulans phytochrome FphA binds a biliverdin chromophore, acts as a red-light sensor, and represses sexual development under red-light conditions. FphA-GFP is cytoplasmic and excluded from the nuclei, suggesting that red-light photoperception occurs in the cytoplasm. This is the first phytochrome experimentally characterized outside the plant and bacterial kingdoms and the second type of fungal protein identified that functions in photoperception.

Functional and Physical Interaction of Blue- and Red-Light Sensors in Aspergillus nidulans

Light sensing is very important for organisms in all biological kingdoms to adapt to changing environmental conditions. It was discovered recently that plant-like phytochrome is involved in light sensing in the filamentous fungus Aspergillus nidulans[1]. Here, we show that phytochrome (FphA) is part of a protein complex containing LreA (WC-1) and LreB (WC-2) [2, 3], two central components of the Neurospora crassa blue-light-sensing system. We found that FphA represses sexual development and mycotoxin formation, whereas LreA and LreB stimulate both. Surprisingly, FphA interacted with LreB and with VeA, another regulator involved in light sensing and mycotoxin biosynthesis. LreB also interacted with LreA. All protein interactions occurred in the nucleus, despite cytoplasmic subfractions of the proteins. Whereas the FphA-VeA interaction was dependent on the presence of the linear tetrapyrrole in FphA, the interaction between FphA and LreB was chromophore independent. These results suggest that morphological and physiological differentiations in A. nidulans are mediated through a network consisting of FphA, LreA, LreB, and VeA acting in a large protein complex in the nucleus, sensing red and blue light.

Nuclear traffic in fungal hyphae: in vivo study of nuclear migration and positioning in Aspergillus nidulans.

Nuclear migration and nuclear positioning are fundamental processes in all eukaryotic cells. They are easily monitored during hyphal growth of filamentous fungi. We expressed the green fluorescent protein (GFP) as a fusion protein with the putative nuclear localization domain of the transcriptional activator stuA in nuclei of Aspergillus nidulans and visualized these organelles in living cells. Nuclear staining was observed in interphase nuclei but not during mitosis. Nuclear division, nuclear migration, septum formation and branching were analysed with time‐lapse video microscopy during hyphal extension. Hyphae elongated at 0.1–1.2 μm min−1 and nuclei moved with similar speeds towards the hyphal tip until they had reached a defined position. An individual regulation of nuclear mobility in a given hyphal compartment was observed. Some representative movies are available on the Internet (http://www.blackwell‐science.com/products/journals/molextra.htm). Nuclear positioning was also studied at the molecular level. The ApsA protein, which regulates nuclear migration, was localized at the cytoplasmic membrane in germlings and hyphae by immunofluorescence and GFP tagging. A model of nuclear migration, nuclear positioning and the role of ApsA is presented.

Polarized growth in fungi – interplay between the cytoskeleton, positional markers and membrane domains

One kind of the most extremely polarized cells in nature are the indefinitely growing hyphae of filamentous fungi. A continuous flow of secretion vesicles from the hyphal cell body to the growing hyphal tip is essential for cell wall and membrane extension. Because microtubules (MT) and actin, together with their corresponding motor proteins, are involved in the process, the arrangement of the cytoskeleton is a crucial step to establish and maintain polarity. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, actin‐mediated vesicle transportation is sufficient for polar cell extension, but in S. pombe, MTs are in addition required for the establishment of polarity. The MT cytoskeleton delivers the so‐called cell‐end marker proteins to the cell pole, which in turn polarize the actin cytoskeleton. Latest results suggest that this scenario may principally be conserved from S. pombe to filamentous fungi. In addition, in filamentous fungi, MTs could provide the tracks for long‐distance vesicle movement. In this review, we will compare the interaction of the MT and the actin cytoskeleton and their relation to the cortex between yeasts and filamentous fungi. In addition, we will discuss the role of sterol‐rich membrane domains in combination with cell‐end marker proteins for polarity establishment.

Fungi, Hidden in Soil or Up in the Air: Light Makes a Difference

Light is one of the most important environmental factors for orientation of almost all organisms on Earth. Whereas light sensing is of crucial importance in plants to optimize light-dependent energy conservation, in nonphotosynthetic organisms, the synchronization of biological clocks to the length of a day is an important function. Filamentous fungi may use the light signal as an indicator for the exposure of hyphae to air and adapt their physiology to this situation or induce morphogenetic pathways. Although a yes/no decision appears to be sufficient for the light-sensing function in fungi, most species apply a number of different, wavelength-specific receptors. The core of all receptor types is a chromophore, a low-molecular-weight organic molecule, such as flavin, retinal, or linear tetrapyrrols for blue-, green-, or red-light sensing, respectively. Whereas the blue-light response in fungi is one of the best-studied light responses, all other light-sensing mechanisms are less well studied or largely unknown. The discovery of phytochrome in bacteria and fungi in recent years not only advanced the scientific field significantly, but also had great impact on our view of the evolution of phytochrome-like photoreceptors.

Growth, differentiation and sexuality

Vegetative processes and growth 1 Fungal cell types K.J. Boyce and A. Andrianapoulous 2 Organelle inheritance in budding yeast and other fungi L.J. Garcia-Rodriguez, A.C. Gay, and L.A. Pon 3 Mitosis in filamentous fungi S. Harris 4 Apical wall biogenesis J.H. Sietsma and J.G.H. Wessels 5 The fungal cell wall J.P. Latge and R. Calderone 6 Septation and cytokinesis in fungi J. Wendland and A. Walther 7 Re-wiring the network: understanding the mechanism and function of anastomosis in filamentous ascomycete fungi N. L. Glass and A. Fleissner 8 Heterogenic incompatibility in fungi K. Esser 9 Programmed cell death in fungi B.C.-K. Lu 10 Senescence and longevity H.D. Osiewacz and A. Hamann Signals in growth and development 11Autoregulatory signals in mycelial fungi U. Ugalde 12 Pheromone action in the fungal groups Chytridiomycota, and Zygomycota, and in the Oomycota C. Schimek and J. Wostemeyer 13 Photomorphogenesis and gravitropism L. Corrochano and P. Galland Reproductive Processes 14 Asexual sporulation in mycelial fungi R. Fischer and U. Kues 15 Regulation of sexual development in filamentous ascomycetes (Mating types, pheromones) R. Debuchy and G. Turgeon 16 Fruiting body development in ascomycetes S. Poggeler, M. Nowrousian, and U. Kuck 17 Mating type genes of the basidiomycetes L.A. Casselton and M.P. Challen 18 Regulatory and structural networks, orchestrating mating, dimorphism, cell shape, and pathogenesis in Ustilago M. Feldbrugge, M. Bolker, G. Steinberg, J. Kamper, and R. Kahmann 19 The emergence of fruiting bodies in basidiomyctes H.A.B. Wosten and J.G.H. Wessels 20 Meiosis in mycelial fungi D. Zickler Biosystematic Index Subject Index

Reactions and Enzymes Involved in Methanogenesis from CO2 and H2

This chapter concentrates on the reactions and enzymes involved in methanogenesis from CO2 and H2. The coenzymes and electron carriers involved are described only as far as necessary for the understanding of their functions; they are dealt with extensively in other chapters. The bioenergetics of CO2 reduction to CH4 are only briefly discussed. For details the reader is referred to Chapter 8. We have tried to cover the complete literature dealing with the enzymology of methanogenesis from CO2 and H2. However, mainly work is cited which was performed with purified or at least partially purified preparations following the famous dictum of Efraim Racker: don’t waste clean thinking on dirty enzymes.

The Aspergillus nidulans Kinesin-3 UncA Motor Moves Vesicles along a Subpopulation of Microtubules

The extremely polarized growth form of filamentous fungi imposes a huge challenge on the cellular transport machinery, because proteins and lipids required for hyphal extension need to be continuously transported to the growing tip. Recently, it was shown that endocytosis is also important for hyphal growth. Here, we found that the Aspergillus nidulans kinesin-3 motor protein UncA transports vesicles and is required for fast hyphal extension. Most surprisingly, UncA-dependent vesicle movement occurred along a subpopulation of microtubules. Green fluorescent protein (GFP)-labeled UncA(rigor) decorated a single microtubule, which remained intact during mitosis, whereas other cytoplasmic microtubules were depolymerized. Mitotic spindles were not labeled with GFP-UncA(rigor) but reacted with a specific antibody against tyrosinated alpha-tubulin. Hence, UncA binds preferentially to detyrosinated microtubules. In contrast, kinesin-1 (conventional kinesin) and kinesin-7 (KipA) did not show a preference for certain microtubules. This is the first example for different microtubule subpopulations in filamentous fungi and the first example for the preference of a kinesin-3 motor for detyrosinated microtubules.