Brian Shaw, Srijana Upadhyay, Soo Chan Lee

Filamentous fungi are ideal model systems in which to study the process of polarized growth, since their life cycle is dominated by growth of hyphae exclusively at the cell apex. The actin cytoskeleton plays an important role in growth of hyphae. Until now, there have been no tools to visualize actin or the actin binding protein fimbrin in live cells of a filamentous fungus. We investigated the roles of actin (ActA) and fimbrin (FimA) in hyphal growth in A. nidulans. We examined the localization of ActA::GFP and FimA::GFP in live cells, and each displayed a similar localization pattern. In actively growing hyphae, cortical ActA::GFP and FimA::GFP patches were highly mobile throughout the hypha and were concentrated near hyphal apices. A patch depleted zone occupied the apical 0.5 µm of growing hypha. Both FimA::GFP and Act::GFP also localize transiently to septa. Movement and later localization of both was compromised after cytochalasin treatment. Disruption of fimA resulted in delayed polarity establishment during conidium germination, abnormal hyphal growth and endocytosis defects in apolar cells. Endocytosis was severely impaired in apolar fimA disruption cells. To further probe the role of endocytosis in hyphal growth we examined the function of ArfB. A. nidulans ArfB is an ortholog of S. cerevisiae arf3 and human arf6, two proteins known to be involved in vesicle formation, endocytosis and actin localization. Disruption of arfB resulted in a loss of hyphal polarity phenotype and delay in endocytosis. Our data supports a novel apical recycling model which indicates a critical role for actin patch mediated endocytosis to maintain polarized growth at the apex.

Fungal hyphae produce numerous secretory vesicles and microvesicles that congregate temporarily at the apex as part of the Spitzenkörper (Spk). One of our main research objectives is to determine where the chitin synthases, presumably transported in microvesicles, are located in the cell and to elucidate their routes of traffic from their sites of synthesis to the Spk. We chose Neurospora crassa, whose genome contains seven chs genes.

GFP labelling in combination with high resolution fluorescence microscopy have allowed us to analyze the distribution and traffic of CHS-3 (class I), CHS-1 (class III) and CHS-6 (class VI) in growing hyphae of N. crassa. At the apex, fluorescence of the three CHS-GFPs concentrated at the Spk. At the subapex, fluorescence was observed in small globular bodies that moved forward until disintegrating in vesicles or groups of vesicles. A closer analysis at the immediate surrounding of the Spk showed the remarkably fast incorporation of clouds of these vesicles into the Spk. In distal regions, the CHS-GFP stains a network of tubular endomembranes, spherical vacuoles and septa. Western analysis of cell fractions obtained by isopicnic sedimentation in linear sucrose gradients (10-65%) revealed in the three transformant strains a unique band of high molecular weight in the fractions with the highest chitin synthase activity and density around 1.13 g/ml, the typical buoyant density of N. crassa chitosomes.

Initially, expression of the fusion genes was kept under the control of the strong promoter ccg1 in the receptive strains, which contained the native copy of the gene. Recently, we used N. crassa strains FGSC 9717 (matA Δmus-51::bar⁺, his-3) and 9718 (mata Δmus-51::bar⁺) and have adapted the strategy used by the Neurospora genome project to endogenously tag the corresponding gene. Using that method we have replaced the native chs1 with a copy of the gene fused to the gfp gene. No apparent localization and traffic differences have been found in comparison with our previous observations. Ongoing current studies include the use of different selective markers and transmission electron microscopy to characterize the various compartments illuminated by the CHS-GFP fusion proteins, which appear to be part of a non-classical secretory pathway.

Richard O'Connell, Hiroyuki Takahara, Jochen Kleemann

Colletotrichum higginsianum causes anthracnose disease on the model plant Arabidopsis thaliana. Host invasion by this hemibiotrophic fungus involves the sequential development of specialized infection structures: host cell entry is mediated by melanized appressoria that breach the plant cuticle and cell wall by elaborating a thin penetration peg; during the initial biotrophic phase, bulbous haustorium-like intracellular hyphae (IH) invade living epidermal cells, inducing expansion and specialization of the host plasma membrane; subsequent entry into the necrotrophic phase coincides with differentiation of filamentous secondary hyphae, which kill host cells ahead of infection. Marked changes in fungal cell wall structure and composition accompany these developmental switches. Our aim is to understand the molecular mechanisms that permit Colletotrichum to proliferate biotrophically inside living plant cells. To discover fungal genes expressed during the biotrophic phase, we first isolated the haustorium-like IH from infected Arabidopsis leaves by a combination of density-gradient centrifugation and fluorescence-activated cell-sorting. A stage-specific cDNA library was generated from the purified IH, and after enrichment for low-abundance transcripts, 4700 clones were sequenced. Biocomputational predictors of signal peptides and transmembrane domains were then used to screen the resulting ESTs for fungal genes encoding soluble secreted proteins, which may function as virulence effectors to promote biotrophy. Using RT-PCR to profile the expression of effector candidates in different fungal cell types and during plant infection, we identified a set of secreted proteins which are specifically expressed or upregulated in IH during the biotrophic phase. These include a homologue of C. lindemuthianum CIH1, Colletotrichum Intracellular Hypha 1, an extracellular glycoprotein localised at the plant-fungal interface which contains two LysM carbohydrate-binding domains. Progress towards the functional characterization of selected candidate effectors will be presented.

Alexandra Brand¹, Scott Shanks², Stephen Milne³, Vanessa Duncan⁴, Cheryl Gale⁵, Neil Gow¹

1University of Aberdeen, Aberdeen, United Kingdom, ²University of Glasgow, Glasgow, United Kingdom, ³University of Exeter, Exeter, United Kingdom, ⁴Novabiotics, Aberdeen, United Kingdom, ⁵University of Minnesota, Minneapolis, United States

Introduction: Eukaryotic cells that grow in a sustained polarised manner, including neurites, root hairs, pollen tubes and fungal hyphae, share the ability to navigate through their environment and make contact with an external target. The components involved in polarity establishment and maintenance have been characterised in budding yeast and are conserved across all eukaryotes. They include small GTPase modules, such as Rsr1 and Cdc42, and a tip-high calcium gradient. We are interested in how this molecular machinery is regulated during tip-steering in response to signals generated by interaction with the environment.

Methods and Results: We developed assays in which the orientation of the hyphal apex can be directed by exposing cells to exogenous electrical fields or growing them on microfabricated surfaces with a patterned topography. The hyphal form of the dimorphic fungus, Candida albicans, responds in a pre-programmed, or tropic, manner to these various external stimuli. Using these tropisms, we have demonstrated that calcium-influx through the Cch1/Mid1 complex and signalling via the Ca²⁺-dependent transcription factor, Crz1, is a requirement for tip re-orientation. In addition, we have shown recently that GTP-GDP cycling of the Rsr1 and Cdc42 GTPase modules is required for normal tropic responses.

Discussion: Our studies suggest that environmental activation of the voltage-activated Ca²⁺-influx channel, Cch1, or its stretch-activated regulator, Mid1, provides upstream directional information that overrides endogenous site-selection signals. This allows the cell polarity machinery to be re-positioned and the establishment of a new axis of growth.

M. Gabriela Roca¹, Michael Freitag², Hsiao-Che Kuo¹, Nick D. Read¹

1University of Edinburgh, Edinburgh, Scotland, United Kingdom, ²Oregon State University, Corvallis, OR, United States

Macroconidia of the filamentous fungus Neurospora crassa form germ tubes that are involved in colony establishment, and conidial anastomosis tubes (CATs) that fuse to form interconnected networks of conidial germlings. Nuclear behavior was analyzed in macroconidia, germ tubes and CATs in strains that express proteins tagged with green fluorescent protein (GFP). Heterokaryons formed by CAT fusion provided a rapid method for imaging multiple labelled fusion proteins, and a means to reduce the potential overexpression of fusion proteins. In heterokaryons in which one of the parent homokaryons expressed H1-GFP, it took >1 h for unlabelled nuclei in the germling to become fluorescent. In contrast, β-tubulin labelled with GFP passed through fused CATs and became incorporated into unlabelled microtubules within ~8 min. Mitosis occurred more slowly in ungerminated macroconidia (~1.5 h) than in germ tubes (~15 min). During CAT homing, nuclei did not enter CATs and mitosis was inhibited. We analyzed a mutant defective in the cytoplasmic dynein pathway (ro-11), which proved to be impaired in nuclear positioning but not in nuclear migration during CAT formation and germination.

Veronica Veses, Andrea Richards, Neil A.R. Gow

C. albicans is a major human fungal pathogen, able to grow as a budding yeast, pseudohyphae and true hyphae. Hyphae present a particular cell cycle organization, different from yeast and pseudohyphal forms and different as well from other mycelial fungus. The extensive vacuolation of the parent yeast cell and the sub-apical regions of the emerging germ tube of C. albicans has been suggested as a possible explanation for the linear kinetics of its hyphal growth. Using fluorescence live-cell video imaging we describe here the different segregation patterns of the vacuole in yeast, pseudohyphae and germ tubes. To analyze how vacuoles are affecting hyphal growth eight tetracycline regulatable mutants involved in different steps of the vacuolar inheritance pathway were generated. Under repressing conditions, vac7, vac8 and fab1 mutants generated mycelial compartments with more symmetrically distributed vacuoles, increased branching frequencies and shorter compartments. Repression of VAC1, VAM2, VAM3 resulted in even greater asymmetries in vacuole inheritance and sparsely branched hyphae. Differences in the organization of the nuclei and microtubules were detected in the sub-apical compartments of the tetracycline-regulated mutants. Under repressing conditions, the vam3∆/TETp-VAM3 mutant also showed differences in the temporal pattern of Tyr19-CDC28 phosphorylation. These data suggest that the asymmetric pattern of vacuole inheritance in C. albicans influences the cell cycle progression in hyphal forms, by modifying the time and the size at which the sub-apical compartments reach the START.

Yvonne Rolke, Paul Tudzynski

An interesting model to study directed growth of fungal hyphae in a pathogen host interaction is represented by the biothrophic ascomycete Claviceps purpurea on rye, because of it`s specific infection pattern. C. purpurea grows down the style following the pollen tube path through the transmitting tissue, establishes a stable host-pathogen interface by tapping the vascular bundles and finally colonizes the entire ovary.

We are interested in the role of signalling factors affecting polarity and differentiation, especially during host colonization. For that reason the highly conserved Rho-GTPases Rac and Cdc42 and a downstream acting PAK-kinase which are well known to be involved in cell polarity are analyzed.

We could show that the functions of Rac and Cla4 proteins in C. purpurea are highly overlapping. Gene deletion of either, rac or cla4 lead to a loss of polarity and sporulation and to a block in infection at the earliest stage. Consistently, a constitutively active Cla4 protein leads to a hypersporulating phenotype with abberant cell morphology. Compared to the phenotype of another small GTPase, Cdc42, which also shows hypersporulation (Scheffer et al., 2005) rac deletion mutants show a more severe and partly inverse phenotype concerning specific features of fungal development.

Based on the identical phenotypes of rac and cla4 deletion mutants and on yeast-two-hybrid analyses we propose that Cla4 is the major downstream partner of Rac in C. purpurea. To substantiate the hierarchical relationship of small GTPases and PAK kinases a macroarray approach was initiated to compare expression profiles of ∆cdc42, ∆rac and ∆cla4 mutants grown in axenic culture. Evaluation of the data corroborated our model.

Phosphorylation assays of MAP kinases and expression studies of genes encoding ROS scavenging and generating enzymes indicate a function of Rac and Cla4 in fungal ROS homoeostasis which could contribute to their drastic impact on differentiation.

Norio Takeshita, Yuhei Higashitsuji, Sven Konzack, Reinhard Fischer

In filamentous fungi hyphal extension depends on the continuous delivery of vesicles to the growing tip. Here, we describe the identification of two cell-end marker proteins, TeaA and TeaR, in Aspergillus nidulans, corresponding to Tea1 and Mod5 in Schizosaccharomyces pombe. Deletion of teaA or teaR caused zig-zag-growing and meandering hyphae, respectively. The Kelch-repeat protein TeaA, the putatively prenylated TeaR protein, and the formin SepA were highly concentrated in the Spitzenkörper, a vesicle transit station at the tip, and localized along the tip membrane. TeaA localization at tips depended on microtubules and TeaA was required for microtuble convergence in the hyphal apex. The CENP-E family kinesin KipA was necessary for proper localization of TeaA and TeaR, but not for their transportation. TeaA and TeaR localization were interdependent. TeaA interacted in vivo with TeaR, and TeaA co-localized with SepA. Sterol-rich membrane domains localized at the tip in teaA and teaR mutants like in wild type, and filipin treatment caused mislocalization of both proteins. This suggests that sterol-rich membrane domains determine cell-end factor destinations and thereby polarized growth.