Plenary Session Abstracts

Microtubules in polar growth of Ustilago maydis

Gero Steinberg

Polar growth of yeasts and filamentous fungi depends on cytoskeleton-based transport of vesicles and protein complexes towards the expanding cell pole. While solid evidence exists for a central function of F-actin in fungal growth, the importance of the tubulin cytoskeleton is by far less understood. In vivo observation of microtubules in growing cells of Ustilago maydis revealed that the dynamic ends ("plus"-ends) of these tubulin polymers are growing towards the cell poles, while cytoplasmic microtubule organizing centers focus the microtubule minus-ends at the neck region. This microtubule orientation suggests that minus-end directed dynein motors support transport towards the neck, whereas plus-end directed kinesin motors might be required for transport towards the poles of the cell. This concept is supported by the recent identification of the transport machinery for microtubule-based traffic of early endosomes that depends on both a Kif1A-like kinesin and cytoplasmic dynein. Interestingly, a balance of the activity of these motors determines the cell cycle-dependent formation of endosome clusters at both cell poles, where the endosomes apparently support polar growth, bipolar budding and septation. However, it emerges that, in addition to their function in membrane transport, both motors influence the organization of their transport "tracks" by modifying dynamic parameters of microtubules in U. maydis. Understanding this dual function of motors will be a fascinating future challenge, and might be key to the understanding of the role of microtubules in polar fungal growth.

Genetics analysis of the regulation of cytoplasmic dynein in Neurospora.

M. Plamann, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110-2499.

        Cytoplasmic dynein is the most complex of the cytoplasmic microtubule-associated motor proteins, and it has been shown to be required for retrograde vesicle transport and the movement and positioning of nuclei in fungi. Analysis of cytoplasmic dynein from mammals reveals that at least 16 different gene products are required for formation of the dynein motor, the dynein activator complex dynactin, and the LIS1 complex, required for dynein function. Examination of the recently completed genome sequence of Neurospora crassa reveals a high degree of conservation for nearly all these gene products. We have developed a simple genetic system for the isolation of mutants defective in cytoplasmic dynein function. We have shown previously that most of the genes defined through this screen encode subunits of cytoplasmic dynein or dynactin. We have now cloned all but one of the genes defined by our screen. Interestingly, we have not identified mutations in four genes encoding known dynein/dynactin subunits, although genomic sequence data suggests that apparent orthologs are present in N. crassa. In addition, we have also defined two genes that have not previously been implicated as required for dynein/dynactin function.

Septins in Aspergillus nidulans.

Michelle Momany. Department of Plant Biology, University of Georgia, Athens, GA. USA

Septins form scaffolds that organize division sites and areas of new growth in fungi and animals. Many proteins critical for cytokinesis and cell cycle regulation depend upon septins for proper localization. Five septins (AspA-AspE) have been identified in A. nidulans. AspB localizes to forming septa and conidiophore layers. It also localizes to emerging branches where it may play a role in cell cycle regulation of subapical compartments. Deletion of AspB is lethal and deletion of other septins results in branched conidiophores.

Nuclear migration in Aspergillus nidulans: Motors, microtubules and more

Reinhard Fischer, Max-Planck-Institute for terrestrial Microbiology and Philipps-University Marburg, Karl-von-Frisch-Str., D-35043 Marburg, Germany.

Nuclei migrate through growing hyphae of A. nidulans with a similar speed as hyphae elongate. However, nuclear behavior is much more complex than just a simple tipward travel. They oscillate on their way around a certain position, move backwards for short time periods, enter newly formed branches, devide while moving and populate the reproductive structures such as asexual and sexual spores. The movement requires the microtubule-dependent motor dynein and an intact microtubule cytoskeleton. We asked whether kinesins may play a role in the migration process and isolated three novel genes. The corresponding proteins displayed similarity to conventional kinesin and S. cerevisiae Kip2 (KipA) and Kip3 (KipB). Deletion analyses revealed that only conventional kinesin had an effect on nuclear migration. KipA-mutation affected polarized growth, likely through the positioning of the Spitzenkörper and KipB appeared to be involved in mitosis and meiosis. Likewise, the latter protein localised to cytoplasmic and to mitotic microtubules.

              In addition to motor proteins and the microtubule tracks, nuclear positioning in A. nidulans requires two proteins, ApsA and ApsB. In vivo localisation studies revealed that ApsA forms a protein gradient along the cortex. This gradient depends on the actin cytoskeleton to be maintained. In contrast, ApsB localised to the nucleus and appeared to be associated to the spindle pole body. Simultaneous observation of nuclear movement, microtubule dynamics and ApsB behavior suggested that ApsB might be invovled in the movement of nuclei along microtubule filaments. We observed migration of the nucleus along one microtubule filament towards the plus and the minus end of one microtubule, suggesting the involvement of motors with different polarity. We believe that this movement represents one possibility how nuclei can be translocated within a hyphal cell.

Fungal mitosis, closed or open?

Colin De Souza and Stephen Osmani. Department of Molecular Genetics, the Ohio State University, Columbus, OH43210, USA

The nuclear envelope is broken down at mitosis in higher eukaryotes leading to an "open" mitosis but in fungi the mitotic apparatus is enclosed within a nuclear envelope and so remains "closed". Recent work on the NIMA kinase of Aspergillus nidulans suggests that the closed mitosis of fungi may be more open than previously recognized.

We have determined the location of NIMA in real time through the cell cycle. At G2 it is excluded from the nucleus and at early mitosis it locates transiently to the nuclear pore complex (NPC) before locating to the nucleus. The C-terminal regulatory domain of NIMA is sufficient to target GFP to the NPC and this domain acts in a dominant negative fashion, delaying entry into mitosis. The data support a mitotic role for NIMA at the NPC.

We have previously isolated sonA as a NPC protein that could specifically suppress nimA1. A second extragenic suppressor of nimA1 has now been cloned (sonB) and identified as a NPC protein with homology to human Nup98/Nup96 proteins. Not only do mutations of sonA and sonB suppress the nimA1 mutation, these two NPC proteins physically interact as revealed by co-immunoprecipitation experiments. Moreover, the interaction between SONA and SONB is reduced by the sonB1 mutation which is within a domain of SONB responsible for binding to SONA. Thus, both genetic and biochemical data indicate that NIMA and two interacting NPC proteins play a role in mitotic regulation.

The dynamic localization of SONA and SONB further implicates these proteins in mitotic regulation, as both are precipitously lost from the NPC at mitotic onset and then locate back to the NPC at mitotic exit. These studies reveal for the first time that the protein makeup of the NPC radically changes during a closed fungal mitosis. We propose that these changes open the NPC to allow protein diffusion either in or out of nuclei during mitosis. Proteins which have an affinity for nuclear components concentrate within nuclei, for example tubulin and NIMA. Other nuclear proteins, with no affinity for nuclear components, escape from nuclei during mitosis into the cytoplasm. As daughter nuclei are produced, and SONA and SONB relocate to the NPC, normal nuclear transport is reestablished. This then plays a role in relocating proteins by active transport. Therefore, perhaps the "closed" mitosis of fungi is more open then previously realized.

Distinct endophyte genome evolution associated with differing degrees of antagonism or mutualism

Christopher L. Schardl, Department of Plant Pathology, University of Kentucky, Lexington, KY

Epichloë (anamorph = Neotyphodium) species are systemic and constitutive fungal symbionts (endophytes) of cool-season grasses (subfamily Poöideae). This single genus includes pathogenic and mutualistic symbionts. The more mutualistic endophytes transmit by infection of floral meristems and, ultimately, embryos (vertical transmission); the more pathogenic species transmit horizontally via ascomata that surround and abort host inflorescences (choke disease). In mixed transmission, only some inflorescences are choked, whereas others produce endophyte-bearing seeds. Vertical- and mixed-transmission Epichloë/Neotyphodium species are typically host specific, whereas more pathogenic species can have broader host ranges. Our recent phylogenetic analyses of endophyte and host species indicate a tendency toward co-phylogeny. However, our results also suggest that major host shifts often lead to loss of sexual expression, resulting in symbiota in which the endophyte (much like an organelle) is disseminated only via its host maternal lineage. Should a second Epichloë species superinfect such a symbiotum, the resident endophyte and newcomer may hybridize. Two thirds of the asexual endophytes characterized to date are interspecific hybrids, and repeated hybridizations are evident in some endophyte pedigrees. This, and the observation that hybrids tend to dominate symbiont populations in many host species, strongly suggest a fitness advantage conferred by hybridization. Possible bases for this advantage are (1) as a counter to Muller’s ratchet, and (2) pyramiding of genes that enhance host and, therefore, endophyte fitness. Examples of such fitness-enhancing genes include those that direct the biosynthesis of anti-herbivore alkaloids, and genes yet to be identified for drought tolerance, nematode resistance, and improved nutrient acquisition.

Of fungi and plants: specific developmental and metabolic processes required for their interactions

Marc-Henri Lebrun, CNRS - Bayer Cropscience, Lyon, France

Fungi have developed various strategies to attack plants. In most cases, however, early stage of the infection is marked by the differentiation of specialized structures, such as appressoria, that mediate the penetration of the fungus into host tissues. This process requires specific developmental programs and metabolic pathways that have been highlighted in the rice blast fungus Magnaporthe grisea. Analysis of a non-pathogenic mutant, defective for a B-zip transcription factor that is specifically expressed during infection, suggests the existence of infection specific regulatory networks. Infection-specific regulatory networks, as shown for ACE1, also control avirulence gene expression. Here, at least two different signals are involved in the control of ACE1 expression. The tetraspanin-encoding gene PLS1, that is essential for penetration, also displays an appressorium-specific expression. Control of PLS1 expression occurs at the post-transcriptional level and involves its 5'UTR sequence. Genome wide transcriptome analysis of mutants defective for host penetration that are involved in signaling pathways such as PLS1, or defective for infection-specific expression, should reveal the networks involved in the infection process. Although most appressorium signaling/regulatory pathways were identified in M. grisea, recent experiments suggest that these pathways are involved in the infection process of other fungal species.

Signal transduction in Ustilago maydis: for mating and more

Regine Kahmann, Max Planck Institute for terrestrial Microbiology, Marburg, Germany

Ustilago maydis is a dimorphic fungus that switches from a yeast-like haploid stage to a filamentous dikaryon after mating of sexually compatible strains. In nature it is the dikaryon that is able to differentiate infection structures and cause disease.

To understand the complex signaling processes involved in cell fusion and pathogenicity molecular tools for functional genome analysis were applied. These include REMI, the use of regulated promoters in differential screens as well as the exploitation of DNA micro arrays based on the annotation of the U. maydis genomic sequence. I will discuss these strategies and highlight their impact for the identification of pathogenicity determinants in this system. Special emphasis will be given to signaling molecules derived from either the fungus or the plant.

New strategies for C. albicans virulence gene discovery

Aaron P. Mitchell, Department of Microbiology, Columbia University, New York, NY

Candida albicans is the most frequently encountered fungal pathogen of humans. Although it is a benign inhabitant of mucosal surfaces in most individuals, it is a significant cause of infection when host or environmental factors are permissive. Much effort has focused on the definition of genes that govern C. albicans survival in the host and pathogenicity. Most known C. albicans survival and pathogenicity genes have been identified through their expression patterns, through properties of their gene products, or through phenotypes that arise from overexpression or heterologous expression in Saccharomyces cerevisiae. Ultimately, gene function is deduced from disruption mutations that cause loss-of-function defects. However, it has been largely impossible to identify genes through an initial screen of gene disruption mutants because C. albicans is an asexual diploid, so that creation of homozygous mutants requires involved genetic manipulations.

We report the results of an insertional mutagenesis of C. albicans for virulence gene discovery. We have described a gene disruption cassette, UAU1, that permits selection for homozygous mutants after insertion in a single allele. UAU1 was incorporated into a bacterial Tn7 transposon to permit in vitro mutagenesis of a genomic library. Insertions in C. albicans ORF sequences were then transformed into C. albicans to create a set of orf::Tn7/orf::Tn7 insertion mutants. We have thus far disrupted 217 ORFs, and have evidence that another 36 ORFs may be essential for viability. This collection is far from comprehensive – there are ~7110 ORFs in the C. albicans "haploid complement" – but screening of these mutants has permitted identification of new genes required for host cell damage, pH-dependent filamentation, azole drug resistance, biofilm formation, and other unique C. albicans biological properties.

Melanin biosynthesis and virulence of the human-pathogenic fungus Aspergillus fumigatus

Axel A. Brakhage1, Burghard Liebmann1, Kim Langfelder1, and Bernhard Jahn2

1 Institut für Mikrobiologie, Universität Hannover, Schneiderberg 50, D-30167 Hannover,

Germany; 2 Institut für Labordiagnostik und Hygiene, Dr.-Horst-Schmidt-Kliniken, Wiesbaden, Germany

Aspergillus fumigatus is an important opportunistic human-pathogenic fungus that causes invasive aspergillosis (IPA) in immunocompromised patients. Since conidia are the infectious agent in IPA, we focused on the elucidation of conidial factors contributing to pathogenicity ANGEBEN{ ADDIN REFMAN \11\05\19\01\00\00\00\00\01\00\00\13E:\5Cber\5CWinRM8\5Cbjinf\03\00\03412 412 /id Tsai, Chang, et al. 1998\00 \00 . Previously, we isolated a mutant of A. fumigatus which lacked the ability to form the grey-green pigment characteristic of wild-type conidia. Conidia of this mutant are white. Cloning of the gene defective in the mutant led to the identification of a gene designated pksP (=alb1) for polyketide synthase involved in pigment biosynthesis. Conidia of a pksP mutant strain showed reduced virulence in a mouse infection model and were 10-20-fold more sensitive against reactive oxygen species (ROS) compared with wild-type conidia. Wild-type conidia were able to scavenge ROS, presumably thereby detoxifying ROS. The pksP gene was found to be part of a cluster which is involved in the biosynthesis of 1,8-dihydroxynaphthalene (DHN)-melanin present in conidia. The analysis of an PpksP-egfp gene fusion in A. fumigatus showed that the pksP-egfp gene fusion was expressed in vivo in outgrowing hyphae isolated from the lungs of infected immunocompromised mice. Furthermore, our data suggest that the presence of a functional pksP gene in A. fumigatus conidia is associated with an inhibition of phagolysosome fusion in human monocyte derived macrophages (MDM). In summary, these findings provide a conceptual frame to understand the virulence of A. fumigatus.

Fatal Attraction: Heterokaryon incompatibility in Neurospora.

Glass, N. L., Sarkar, S., Xiang, Q. Iyer, G., Kaneko, I., Pandey, A. and S. Brown. 1Plant and Microbial Biology Department, University of California, Berkeley, CA 94563

Filamentous fungi grow by tip extension, branching and hyphal fusion (anastomosis) to form a hyphal network that makes up a fungal individual. In addition to "self" hyphal fusion, filamentous fungi are capable of undergoing hyphal fusion between individuals to make heterokaryons. Recognition of nonself in such heterokaryons is mediated by genetic differences at het (for heterokaryon incompatibility) loci. Heterokaryons or partial diploids that contain alternative het alleles show severe growth inhibition, suppression of conidiation and hyphal compartmentation and death, a phenomenon reminiscent of programmed cell death in other organisms. In Neurospora crassa, nonself recognition mediated by the het-c locus occurs by the formation of a HET-C heterocomplex, which is associated with the plasma membrane of dead hyphal compartments. Targeting of HET-C to the plasma membrane is not essential for HET-C heterocomplex formation or aggregation. Genetic analysis to identify mutations that suppress heterokaryon incompatibility revealed that a MAPK signal transduction pathway and a putative transcription factor are required for heterokaryon incompatibility. Some of the suppressor mutations also affect heterokaryon incompatibility mediated by other het loci. These data suggest that common cellular machinery is involved in mediating heterokaryon incompatibility by allelic differences at a number of het loci; hypotheses for how this death pathway may be activated can now be tested.

Establishment and maturation of hyphal growth in the filamentous ascomycete Ashbya gossypii

Hans-Peter Schmitz1, Philipp Knechtle1, Andreas Kaufmann1,, Philippe Laissue1, Hanspeter Helfer1, Kamila Wojnowska1, Michael Köhli1, Jürgen Wendland1,2 , Yasmina Bauer1, Fred Dietrich1,3, Sophie Brachat1, Tom Gaffney4 and Peter Philippsen1. Division of Molecular Microbiology, Biozentrum, University of Basel, Switzerland1; Friedrich-Schiller University, Jena, Germany2; Duke University N.C., USA3; Syngenta Research Triangle Park, N.C., USA4

A. gossypii was originally isolated as cotton pathogen. It gained attention as a model organism for the analysis of filamentous growth due to a few unique features. Linear transforming DNA is exclusively integrated by homologous recombination allowing efficient PCR-based gene targeting. Circular transforming DNA carrying a replication origin of the yeast S. cerevisiae can autonomously replicate in A. gossypii allowing homologous and heterologous complementation studies. The completely sequenced genome consists of only 9MB coding for 4720 proteins and each nucleus carries only one copy of this genome. 95% of the A. gossypii genes are homologues of S. cerevisiae genes, very often with conserved gene order (synteny).

Despite the apparent evolutionary relation to S. cerevisiae the hyphal growth of A. gossypii is typical for filamentous fungi. Spores develop into germlings which grow by sustained tip extensions, lateral branching and, at a later stage, by tip branching. The growth speed of hyphal tips can increase from 0.01mm /hour (germling) up to 0,25mm /hour in mature mycelium.

We identified in the Ashbya genome homologues to all S. cerevisiae genes known to be important for polar growth control. We also found an additional copy of a formin gene, a RHO gene, and a GEF gene. Several of these genes were deleted and/or fused to the GFP coding sequence. Analyses of growth dynamics of theses strains using video microscopy allowed important conclusions about polar growth control in A.gossypii. Key results of these studies will be presented.

PKC regulates the stability of WC-1 in response to light

Guiseppe Macino, Dipt di Biotecnologie Cellulari ed Ematologia, Univ La Sapienza, Roma, ITALY

Previous pharmachological studies have shown that Neurospora Protein Kinase C (PKC) is involved in the regulation of the light responsive genes. We have studied the function of PKC in the light response by investigating its biochemical and functional interaction with the blue light photoreceptor WC-1. We demonstrate that WC-1 and PKC interact in a light regulated manner in vivo, and that immunopurified PKC phosphorylates WC-1 zinc-finger region in vitro. In addition, we show that the mutated PKCs induce changes in WC-1 protein levels. A dominant negative PKC induces a dramatic increase in WC-1 protein levels, which is the result of slower WC-1 degradation rate. Consistently, in the presence of a constitutively active PKC we see decreased levels of WC-1. We show that the altered photoreceptor levels induced by PKC cause an altered light induced transcription of the al-2 mRNA. We see opposite effects in the presence of the dominant negative PKC. In addition we tested PKC effects on FRQ protein levels and degradation rate, and we see that that PKC induces a change in FRQ levels, affecting the robustness of the circadian rhythm. Together our data indicate that PKC is a novel component of the Neurospora light signal transduction pathway physically interacting with and phosphorylating WC-1.

Listening to Silenced Genes

Robert L. Metzenberg and Patrick K.-T. Shiu

Filamentous fungi occupy a unique position in the hierarchy of cellular organization. Their nuclei are self-contained like those of other microorganisms; they do not ordinarily undergo irreversible differentiation of function. However, their undivided cytoplasm is shared by thousands to many millions of nuclei, and from this point of view, fungi are thoroughly macroscopic, ranging in size from centimeters to kilometers. This suits them well in their niche, but it also leaves them as extraordinary targets of opportunity for the spread of viruses and retrotransposons.

It is not surprising, then, that fungi, here exemplified by Neurospora crassa, have evolved an array of mechanisms for combatting the proliferation of these renegade elements. Two of these, Repeat-Induced Point-mutation (RIP) and quelling, have been elegantly explored by Selker and his coworkers and by Macino and Cogoni and their group. Despite the extreme dissimilarity of these processes, they have one feature in common: sequences present in two or more copies in a cell which should contain only one copy are recognized as aberrant, and their effects are nullified. RIP occurs in a window of time in which pre-karyogamic nuclei reside in ascogenous hyphae. It introduces numerous GC to AT transitions into any DNA present in more than one copy in a nucleus, which in general, inactivates the gene or transposon irreversibly. Quelling, by contrast, operates during the vegetative phase of life. It acts against aberrant RNA molecules by fragmenting them, but not directly against the genes that gave rise to them. Both RIP and quelling presumably judge a sequence as illegitimate by the fact that it must have transposed at least once.

We have described another mechanism that could silence potentially damaging genetic elements. This process, Meiotic Silencing by Unpaired DNA (MSUD), operates primarily, but perhaps not exclusively, in the brief period following karyogamy. During pachytene, legitimate DNA sequences of a chromatid will be diploid, each copy being paired with those of a homolog. Any sequence that does not have a pairing partner at a corresponding position of its homolog is taken as "aberrant" and is silenced by a mechanism that involves double-stranded RNA. Furthermore, any sequence homologous to the unpaired DNA is silenced by MSUD, even if it is itself properly paired with a homolog. Thus, unlike RIP and quelling, MSUD has the properties necessary to silence a virgin transposon, but cannot act against a quiescent transposon that has established itself in canonical positions of both parents, as can RIP and quelling.

Investigating the genetics of appressorium-mediated plant infection by Magnaporthe grisea. Nicholas J. Talbot, Sara L. Tucker, Martin Egan, Karen Tasker, Martin J. Gilbert, and Darren M. Soanes. School of Biological Sciences, University of Exeter, Exeter, EX4 4QG, UK

Magnaporthe grisea infects its host by elaborating a specialised infection structure known as an appressorium. This cell forms in response to the hard, hydrophobic rice leaf surface and brings about infection by generation of hydrostatic pressure. M. grisea appressoria are melanin-pigmented cells with a thickened cell wall that allows turgor to develop within the cell due to accumulation of glycerol and subsequent influx of water. Mechanical rupture of the plant leaf cuticle occurs and a narrow penetration peg enters the leaf epidermis, providing the route for fungal colonization of plant leaf tissue. We are investigating mechanisms required for appressoria to form and the genetic control of appressorium formation via the PMK1 MAP kinase pathway. Subsequent to appressorium maturation, we are studying the origin of appressorial glycerol, its biosynthetic pathway during appressorium formation and genetic regulation of turgor generation. In particular the role of trehalose metabolism, lipid metabolism and glycogen metabolism during turgor generation are being investigated. The availability of substantial EST sets and the recent first draft of the M. grisea genome sequence is allowing us to adopt a more holistic approach to investigation of appressorium differentiation and function. We are therefore developing bioinformatic tools to compare M. grisea and related pathogenic fungi with free-living saprotrophic relatives.

Neurospora biology, from the genome up

Bruce Birren and the sequencing group at the Whitehead Inst./MIT Center for Genome Research, and the Neurospora Community Analysis Project.

A collaborative effort between the Genome Sequencing and Analysis Program at the Whitehead Genome Center and members of the Neurospora research community has produced and analyzed a high-quality draft sequence of the Neurospora crassa genome. The complexity of the filamentous fungi relative to the previously sequenced yeasts is highlighted by the number and kinds of genes we find. Neurospora contains ~10,000 protein-coding genes. This is more than twice the number found in the fission yeast S. pombe and only about 25% fewer than in the fruit fly D. melanogaster. Despite the decades of genetic analysis of this organism, the predicted gene set suggests significant biological processes remain to be explored in Neurospora. These include the potential for red light photobiology, secondary metabolism, and important differences in Ca2+ signaling as compared to plants and animals. The genome sequence has also permitted a global analysis of the process of Repeat Induced Point Mutation (RIP). Sequence analysis indicates that RIP has greatly slowed the creation of new genes through duplication and as a consequence Neurospora contains an unusually low proportion of closely related genes.

Neurospora represents the first of numerous fungal genomes that are being sequenced at the Whitehead Genome Center. The Fungal Genome Initiative will sequence a comprehensive collection of species spanning the entire fungal kingdom. These data will allow all fungal researchers to apply the power of comparative genomics to further our understanding of the remarkable diversity of these organisms.

Comparative genomics of plant pathogenic fungi

B. Gillian Turgeon and Members of the former Torrey Mesa Research Institute Fungal Group. Dept. of Plant Pathology

Cornell University. Ithaca, NY, 14853

Access to complete genomes of saprobic and pathogenic euascomycetes has allowed us to compare inventories of certain gene families involved in both primary and secondary metabolism among fungi that differ in pathogenic lifestyle. We will describe our comparative structural and functional analyses, as well as our comprehensive phylogenomic treatment of all polyketide synthases (PKS), non-ribosomal peptide synthetases (NPS), ABC transporters, histidine kinases (HK), monofunctional catalases and kinesins from complete genomic sequence of five taxonomically diverse euascomycete fungi [the saprobe Neurospora crassa, the maize pathogens Cochliobolus heterostrophus (Bipolaris maydis) and Gibberella moniliformis (Fusarium verticillioides), the general cereal pathogen G. zeae (F. graminearum), and the cosmopolitan dicot pathogen Botryotinia fuckeliana (Botrytis cinerea)], and three earlier diverging ascomycetes [the hemiascomycete yeast saprobe Saccharomyces cerevisiae, the hemiascomycete plant pest, Eremothecium (Ashbya) gossypii, and the archaeascomycete yeast saprobe Schizosaccharomyces pombe]. Some of these data (e.g., on PKSs) have been used to challenge the hypothesis that small molecule products of these genes are abundant only in phytopathogenic fungi and that presence of genes for secondary metabolism reflects a history of horizontal gene transfer, much as pathogenicity islands have been transferred among pathogenic bacteria. The phylogeny of PKSs shows no bias in favor of plant pathogenic fungi, nor any need for horizontal gene transfer. Furthermore, PKSs are far more abundant than known polyketides and there are few orthologs among taxa, even between closely related species, indicating that most polyketides have yet to be characterized and that there is an enormous untapped potential for small molecule production. In contrast, data on HKs show that the filamentous fungi encode an extensive family of two-component signaling proteins that fall into eleven classes. Many of these groups contain HKs that are highly conserved among filamentous ascomycetes. Other groups are more divergent, containing gene families that have expanded within species with few clear orthologs between species. These groupings suggest that some HKs are necessary for basic functions shared by most or all ascomycetes (ie. osmosensing) while others may have evolved to adapt to specific aspects of the pathogen’s lifestyle. In contrast, structural genes such as kinesin-like motor proteins have a near one-to-one correspondence of orthologs across ascomycete genomes.

Molecular adaptation in Phytophthora-plant interactions.

Sophien Kamoun, Jorunn Bos, Nicolas Champouret, Luis da Cunha, Shujing Dong, Elodie Gaulin, Edgar Huitema, Diane Kinney, Zhenyu Liu, Miaoying Tian, Trudy Torto. Department of Plant Pathology, The Ohio State University - OARDC, Wooster, OH.

Parasitic and pathogenic lifestyles have evolved repeatedly in eukaryotes. Several pathogenic eukaryotes represent deep phylogenetic lineages suggesting that they feature unique molecular processes for infecting their hosts. One such group is formed by the oomycetes, arguably the most devastating pathogens of dicot plants. Extensive structural genomic resources are available for the oomycete Phytophthora and the challenge in the post-genome era is to link sequences to phenotypes using computational tools for data mining and robust high throughput functional assays. We applied this strategy to the potato and tomato late blight pathogen Phytophthora infestans. Data mining for the identification of genes up-regulated during infection, encoding extracellular proteins, and undergoing diversifying selection, were combined with a variety of functional assays to identify P. infestans effector genes that trigger cellular and molecular responses in plant cells. The discovered effectors include: (1) The crinkler (CRN) family of general defense response elicitors; (2) Several classes of "orphan" avirulence genes that match previously unknown resistance genes; and (3) A family of extracellular protease inhibitors (EPI) that target host proteases. This research is allowing us to establish functional connections between Phytophthora effector genes and plant processes and to ask pertinent questions about the co-evolution of Phytophthora effectors with host factors.

The Magnaporthe grisea genome project

Ralph A. Dean and members of the International Rice Blast Consortium

Fungal Genomics Laboratory, Center for Integrated Fungal Genomics, NC State University, Raleigh NC 27695, USA.

Until recently opportunities to dissect the molecular pathways governing fungal pathogenesis have been limited, in large part by lack of basic knowledge of fungal genomes. At last this is beginning to change. Rice blast disease caused by Magnaporthe grisea is one of the most devastating threats to food security worldwide. The fungus is amenable to classical and molecular genetic manipulation and is a compelling experimental system for elucidating signaling pathways of pathogenesis, including infection-related morphogenesis, and host species and cultivar specificity. In 1998, an international consortium (IRBGP) was established to sequence the rice blast genome. Initially, a BAC physical map of strain 70-15 was used a framework to create a draft sequence (~5X coverage) of chromosome 7 using the “BAC by BAC” approach coupled with a comprehensive EST program. Most recently, in collaboration with the Whitehead Institute Center for Genome Research, a shotgun approach has been undertaken to sequence and assemble the entire genome. Sequenced BAC clones, known Magnaporthe genes and ESTs were used to validate the current 6X assembly. The current status of the genome project, including annotation, comparative and functional analyses pertaining to pathogenesis, will be presented. Sequence data and other information are publicly available at the consortium website and at the Whitehead Institute