ECFG 9 KEYNOTE AND
Sunday April 6
Sex in fungi: revelations of genomic analysis
University of Oxford, Oxford, United Kingdom
Sexual reproduction is a familiar feature of most eukaryotic life cycles, playing a crucial role in maintaining genetic diversity and species survival. In fungi, just a few genes, the mating type genes, control the sexual identity of an individual and determine how it attracts and recognises a compatible mating partner, and how cell fusion then initiates the major changes in gene expression that lead to sexual development. It is not surprising that the mating type genes have been such an important focus of attention. The cloning of these genes from model species has identified their function and contributed extensively to our understanding of signal transduction pathways and protein interactions that we now know to be ubiquitous in nature. Significantly, the mating pathway appears to be highly conserved in ascomycete and basidiomycete fungi, but it is evident also that it has evolved to match different life styles. Indeed, fungi are sexually versatile, recombination may be achieved by meiotic and mitotic events, some species are capable of both outcrossing and selfing whereas others appear to be totally asexual. With ever increasing access to genomic sequence in more and more fungal species we can identify homologues of the mating type genes and the genes they are predicted to regulate and we can seek to understand how different mating behaviours have evolved. We can, moreover, look at the chromosomal context of the mating type genes and see how, in some species, the genes have been sequestered into extensive regions that can no longer recombine due to DNA rearrangements and insertions, events that resemble those that have led to the evolution of sex chromosomes in other eukaryotes. Genome sequence data is leading to an exciting resurgence of interest in mating type genes and fungal lifestyles.
Investigating the biology of plant infection by the rice blast fungus Magnaporthe grisea using functional genomics
Nicholas Talbot, Darren Soanes, Diane Saunders, Tom Richards, Martin Egan, Michael Kershaw, Elise Lambeth, Zaira Caracuel-Rios
University of Exeter, Devon, United Kingdom
Magnaporthe grisea is the causal agent of rice blast, one of the most devastating diseases of cultivated rice. The availability of genome sequences for M. grisea and its host, Oryza sativa, has allowed an unparalleled opportunity to explore this fungal-plant interaction and to study the evolution of fungal pathogenicity. We have utilized comparative and evolutionary genomics to define pathogenicity-associated gene functions in M. grisea and have also discovered instances of lateral gene transfer that may have contributed to the evolution of plant pathogenic micro-organisms. During plant infection, M. grisea elaborates a specialised infection structure known as an appressorium. This unicellular, dome-shaped structure generates cellular turgor, which is translated into mechanical force to allow rupture of the rice cuticle and entry into plant tissue. Development of a functional appressorium is linked to the control of cell division. Blocking completion of mitosis by generation of a temperature-sensitive mgnimA mutant, for instance, prevents appressorium morphogenesis. Furthermore, following mitosis, conidia always undergo cell collapse and programmed autophagic cell death. The absence of the autophagic cell machinery in M. grisea is sufficient to render the fungus non-pathogenic. Taken together, our results indicate that appressorium morphogenesis requires completion of mitosis and initiation of autophagic recycling of the contents of the fungal spore to the appressorium. Appressorium formation is also associated with an oxidative burst that requires NADPH oxidases that a virulence determinants of M. grisea. To study appressorium physiology and function in greater detail we have used proteomics to define the major changes in protein abundance associated with plant infection by M. grisea and metabolite fingerprinting by electrospray ionisation mass spectrometry and GC-ToF-MS to define major metabolic changes in both the fungus and its host during the onset of rice blast disease. Colonisation of host plant tissue by M. grisea also requires a P-type ATPase involved in Golgi function and exocytosis. This membrane-associated P-type ATPase, encoded by the MgAPT2 gene, is necessary for pathogenicity and for induction of host defences in an incompatible response and may be associated with protein delivery by the fungus during rice blast disease.
Molecular adaptation of Candida albicans to its human host
University of Aberdeen, Aberdeen, United Kingdom
The pathogen Candida albicans causes a range of infections in humans. Depending largely upon the immune status of its host, this fungus can thrive in diverse niches in its human host: on the skin, in the gastrointestinal and urogenital tracts, in the bloodstream and internal organs. This suggests that C. albicans must adapt effectively to the diverse microenvironments it occupies as well as controlling the expression of its virulence factors. This has been confirmed by a number of laboratories using a combination of molecular, cellular and genomic approaches. C. albicans regulates its metabolism, for example to assimilate available carbon sources, and activates specific stress responses in a niche-specific fashion during disease progression. Much of this regulation appears reminiscent of the signalling pathways that exist in the relatively benign model yeast Saccharomyces cerevisiae. However, significant differences have evolved between C. albicans and S. cerevisiae with regard to their metabolic and stress regulation. These will be discussed in the context of their probably significance in C. albicans pathogenicity.
The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis
INRA, Champenoux, France
Mycorrhizal symbioses -- the union of roots and soil fungi -- are universal in terrestrial ecosystems and may have been fundamental to land colonization by plants. Boreal, temperate, and montane forests all depend upon ectomycorrhizae. Identification of the primary factors that regulate symbiotic development and metabolic activity will therefore open the door to understanding the role of ectomycorrhizae in plant development and physiology, allowing the full ecological significance of this symbiosis to be explored.
Here, I will present the main features of the genome sequence from the ectomycorrhizal basidiomycete Laccaria bicolor and I will highlight gene sets involved in rhizosphere colonization and symbiosis. This 65-million-base genome assembly contains ~ 20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences. We detected unexpected genomic features most notably a battery of effector-type small secreted proteins (SSP) with unknown function, several of which are only expressed in symbiotic tissues. The most highly expressed SSP accumulates in the proliferating hyphae colonizing the host root. The ectomycorrhizae-specific SSP are likely to play a decisive role in the establishment of the symbiosis. The unexpected observation that the genome of L. bicolor lacks carbohydrate-active enzymes involved in degradation of plant cell walls, but maintains the ability to degrade non-plant cell walls, reveals the dual saprotrophic and biotrophic lifestyle of the mycorrhizal fungus which enables it to grow within both soil and living plant roots.
The predicted gene inventory of the L. bicolor genome, therefore, points to previously unknown mechanisms of symbiosis operating in biotrophic mycorrhizal fungi. The availability of this genome provides an unparalleled opportunity to develop a deeper understanding of the processes by which symbionts interact with plants within their ecosystem in order to perform vital functions in the carbon and nitrogen cycles that are fundamental to sustainable plant productivity.
Martin F et al. (2008) Nature, in press
Monday April 7
The fungal pH signal transduction pathway and its intimacies with endocytosis
Miguel A. Peñalva
Centro de Investigaciones Biológicas CSIC, Madrid, Spain
The fungal pH signal transduction pathway represents an example of how transduction of environmental signals to transcription factors can only understood by combining molecular and cell biology approaches. In this pathway six proteins (denoted PalA, PalB, PalC, PalF, PalH and PalI in Aspergillus nidulans) are required for the proteolytic activation of the transcription factor PacC/Rim101 in response to ambient alkaline pH. These proteins appear to be organized into two complexes: an ‘upstream’ complex at the plasma membrane involving the 7-TMD receptor PalH, the 3-TMD protein PalI and the PalF arrestin and a ‘downstream’ complex on endosomal membranes involving the Vps32-interacting protein PalA, the cysteine-protease PalB and PacC/Rim101. A key question is how these spatially segregated complexes communicate, i.e. how the alkaline-pH stimulated plasma membrane complex activates the downstream complex to initiate the proteolytic activation of PacC/Rim101. While recent findings in both S. cerevisiae and A. nidulans have led to major improvements in our understanding of this issue, this work has opened additional questions regarding the molecular basis of this communication. PalF strongly binds the cytosolic tail of PalH and is phosphorylated and ubiquitinated (a sorting signal for endocytic internalization) in an ambient pH-dependent manner. The fact that this dedicated arrestin-like protein is positively required for pH signal transduction strongly suggests that this communication involves endocytosis, as mammalian arrestins specifically bind to the agonist-activated conformation of 7-TMD receptors and promote their endocytosis, acting as endocytic adaptors. This hypothetical trafficking from the plasma membrane to endosomes could actually fill the gap between the PalH/PalF complex and the endosomal complex, resulting in the activation of the latter and leading to PacC signalling proteolysis. Thus the fungal pH signalling pathway would represent a novel paradigm of a positively-acting partnership between endocytosis and signalling. A likely candidate to liaise the plasma membrane and endosomal pH signalling complexes is PalC, which is recruited to cortical punctate structures in a PalH-dependent manner. PalC contains a Bro1-like domain through which it binds Vps32, and this binding is necessary for pH signalling.
Developmental pathways from the conidium in Neurospora crassa
University of Edinburgh, Edinburgh, United Kingdom
Three developmental pathways can be undergone during conidial germination in Neurospora crassa resulting in the formation of three specialized types of hyphae: (1) conidial germ tubes involved in colony establishment; (2) conidial anastomosis tubes (CATs) involved in conidial germling fusion, and which we discovered three years ago (Roca et al. 2005 Eukaryot. Cell 4, 911); and (3) conidial sex tubes (CSTs) involved in mating, and which we have only very recently discovered (Kuo & Read, subm). In my presentation I will describe these three different pathways of differentiation, focusing primarily on the signalling mechanisms involved in CATs and CSTs. My group has established the CAT system as a simple and experimentally amenable model in which to analyze self-signalling and self fusion in filamentous fungi. Screening deletion mutants compromised in signalling has greatly helped us to identify many of the signalling proteins involved in CAT induction and homing. Characterization of the cell-cell communication involved is also being characterized using optical tweezers to micromanipulate homing CATs. Together with pharmacological studies, these techniques are providing exciting new insights into the possible mechanistic basis of self-signalling in filamentous fungi. We have shown that conidial sex tubes are morphologically and genetically different from germ tubes and CATs, and under separate genetic control. Their distinctive features will be described. Besides being stimulated by sex pheromone, CST induction is also regulated by red light and is thus phytochrome regulated. Neurospora crassa possesses two phytochromes: PHY-1 and PHY-2, and they play contrasting and mating dependent roles in the photoregulation of CST induction.
The cytoskeleton in hyphal growth
University of Exeter, Exeter, United Kingdom
Much of the biological success of fungi is based on the ability of the fungal hypha to invade tissue or substrate. Such invasive growth requires the expansion of the cell at the tip of the hypha. Recent research indicates that motor-driven transport processes along the fibrous polymers of the cytoskeleton participate in tip growth. On average fungal cells contain ~10 kinesins, 1 dynein and 4 myosins, which deliver cargo along microtubules or F-actin towards the expanding tip. In addition, backward traffic mediates endocytosis and supports recycling of molecular motors themselves. Although we are beginning to understand the importance of molecular motors, their cargo still remains unknown. However, one class of motors comprise fusion proteins that contain an N-terminal myosin motor domain and a C-terminal chitin synthase region. This suggests that this motor takes its own chitin synthase domain to the growth region, where it participates in wall-synthesis. However, experimental proof for a role of the myosin domain in transport is missing. In this presentation I will give an overview of our current on the cellular role of all kinesins, dynein, and the myosin motors in Ustilago maydis.
Mitotic regulation of nuclear structure in Aspergillus nidulans
Stephen Osmani, Leena Ukil, Hui-Lin Liu, Colin De Souza
Ohio State University, Columbus, United States
The mitotic specific NIMA protein kinase of Aspergillus nidulans is essential for all structural aspects of mitosis. However, in Saccharomyces cerevisiae and Schizosaccharomyces pombe orthologous kinases are not essential for mitosis. This suggests there are aspects of mitosis that are regulated by NIMA in A. nidulans that do not occur in the yeasts. Recent studies lend support to this idea because the nuclear pore complex (NPC) undergoes a process of disassembly and reassembly during A. nidulans mitosis whereas NPCs remain intact during yeast mitoses. As described below, we have identified new NPC proteins and defined further similarities with higher eukaryotic mitosis.
Affinity purification and analysis using Mass Spectrometry have identified two new NPC proteins of A. nidulans previously thought to be specific to higher eukaryotes. The role of these NPC proteins to maintain the core structure of the NPC during mitosis has been defined using deletion analysis and live cell imaging of multiple endogenously tagged NPC proteins. The findings shed light on the molecular mechanisms of NPC disassembly-reassembly during mitosis in A. nidulans and higher eukaryotes.
Another mitotic change occurring to higher eukaryotic nuclei, but not to yeast nuclei, is the disassembly then reassembly of the entire structure of the nucleolus. From studies of A. nidulans NIMA copy number suppressors we identified a novel nucleolar associated protein which led to investigation of how the nucleolus is segregated during A. nidulans mitosis. Amazingly, we find the nucleolus is expelled into the cytoplasm during mitosis. This involves a double pinch of the nuclear envelope during mitosis which generates two daughter nuclei and a nuclear remnant containing the nucleolus. This cytoplasmic nucleolus then undergoes stepwise disassembly. The sequentially released proteins are imported into daughter nuclei to generate new nucleoli during exit from mitosis and entry into G1. How this process is regulated, and the significance of this unique pattern of nucleolar segregation, will be discussed.
Tuesday April 8
The clock in the cell: Neurospora as a window on clocks in 'higher' organisms
University of Groningen, Haren, Netherlands
The daily biological (circadian) clock rules our lives. It controls biology from the level of gene expression through behaviour. Most of what we know concerning clock genes and mechanisms comes from studies in free running conditions. Yet, circadian clocks were shaped through their systematic responses to highly predictable, daily environmental changes. This entrainment yields a distribution of chronotypes in natural populations. Although a general rule exists predicting that a short free running period leads to an early entrained phase and, conversely, a long one to late, clocks with equivalent periods can – in theory – yield different entrained states. We propose to use entrainment to reveal novel clock properties and mechanisms.
To understand entrainment within natural degrees of freedom, we have entrained the Neurospora crassa conidiation rhythm in circadian surface protocols using both light and temperature. These experiments call for 'all' photo- and thermo-periods in three different cycle lengths using a panel of clock mutants with different endogenous periods. Thus, a grid describing all combinations of endogenous and exogenous period and zeitgeber cycles is established. The data reveal rules of entrainment for Neurospora. The results are being used to screen potential clock mutants in cycles that are more revealing or challenging than symmetrical (12 h light/12 h dark) light cycles. Further, comparison of the Neurospora 'rules' with those for plants or animals, which use different sets of clock genes, should reveal characteristics that are fundamental to the clock versus those that are adaptive for Neurospora, in particular.
The role of G proteins and two component regulatory systems in Neurospora sexual reproduction
University of California, Riverside, California, United States
Differentiation of female reproductive structures and their subsequent fertilization by males, followed by meiosis and ascospore production, together comprise a complex, temporally-regulated process in Neurospora. Our laboratory has demonstrated a requirement for two distinct signaling pathways--heterotrimeric G proteins and two-component regulatory systems—in female fertility and ascospore development. All five heterotrimeric G protein subunits play roles in sexual reproduction, including protoperithecial differentiation, the pheromone response and ascospore production. The involvement of G protein coupled receptors and other regulatory proteins in female fertility will be presented.
We have shown that a two component system that includes the OS-1 histidine kinase and the response regulator RRG-1 regulates protoperithecial development, at least partially via the downstream OS-4/OS-5/OS-2 MAPK cascade. Results from analysis of a ∆rrg-1 knockout mutation as well as an rrg-1 allele mutated in the predicted site of phosphorylation suggest that blocking RRG-1 phosphorylation affects the timing of protoperithecial development, while the absence of RRG-1 protein completely blocks differentiation of female reproductive structures. Recent work, including analysis of the interactions between pathway proteins and the importance of various domains within RRG-1, will presented.