7The impact of filamentous fungi on human welfare has never been greater. Fungi are acknowledged as the most economically devastating plant pathogens (1) and are attaining increasing notoriety for their ability to cause life-threatening infections in humans (57,71), and fungal products sustain a billiondollar manufacturing industry (70). The tools available to study filamentous fungi are more sophisticated than ever and include the complete annotated genome sequences of multiple filamentous fungi (12), resources being made available through various functional genomics projects, and advanced bioimaging methods, including high-resolution live-cell imaging (20, 32) and electron tomography (19,50). The increasing impact of filamentous fungi, along with the rediscovery of pseudohyphal growth in yeast (22), has focused attention on the molecular mechanisms underlying hyphal morphogenesis.Attempts to understand hyphal morphogenesis have historically followed two different lines of investigation. Microscopists have defined, with increasing detail, the subcellular organization of the hyphal tip. This led to the description of the Spitzenkörper, an apical cluster of vesicles, cytoskeletal elements, and other proteins, which plays a crucial role in hyphal extension (4). Geneticists have identified gene products required for hyphal morphogenesis by characterizing morphological mutants (51,52). Initial studies in the laboratories of Beadle, Tatum, and colleagues attempted to link morphogenesis to specific biochemical pathways. More recent screens have identified a multitude of signaling and cytoskeletal functions required for hyphal extension (62, 72).In the past few years, comparative genomics efforts have allowed fungal biologists interested in hyphal morphogenesis to exploit the wealth of knowledge about polarized growth in the yeast Saccharomyces cerevisiae. Many informative homologies between filamentous fungi and yeast have been uncovered. Notably, this includes several components of a multiprotein complex termed the polarisome (28), which regulates microfilament formation at polarized growth sites in yeast (61).Perhaps more importantly, several gene products involved in hyphal morphogenesis have been shown to have no homologue outside of the filamentous fungi. This emphasizes the potential novelty of the mechanisms underlying hyphal morphogenesis. In this review, we summarize past efforts to understand hyphal morphogenesis and pose a series of questions designed to focus future efforts in this area. HYPHAL MORPHOGENESIS: A BRIEF OVERVIEWFungal hyphae originate from either a germinating spore or another hypha (i.e., during branch formation). Initially, an axis of polarity is established from a symmetrically expanding spore or hyphal compartment. Subsequently, cell surface expansion is restricted to the specified axis, thereby leading to the formation of a polarized hypha that displays a gradient of expansion that peaks at the tip (2,15,25,30,52). Maintenance of the polarity axis allows hyphae to achieve a linear extens...

SummaryFilamentous fungi are ideal systems to study the process of polarized growth, as their life cycle is dominated by hyphal growth exclusively at the cell apex. The actin cytoskeleton plays an important role in this growth. 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 Aspergillus 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 mm 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. Our data support a novel apical recycling model which indicates a critical role for actin patch-mediated endocytosis to maintain polarized growth at the apex.

Brucella species are facultative intracellular bacterial pathogens that cause brucellosis, a global zoonosis of profound importance. Although recent studies have demonstrated that Brucella spp. replicate within an intracellular compartment that contains endoplasmic reticulum (ER) resident proteins, the molecular mechanisms by which the pathogen secures this replicative niche remain obscure. Here, we address this issue by exploiting Drosophila S2 cells and RNA interference (RNAi) technology to develop a genetically tractable system that recapitulates critical aspects of mammalian cell infection. After validating this system by demonstrating a shared requirement for phosphoinositide 3-kinase (PI3K) activities in supporting Brucella infection in both host cell systems, we performed an RNAi screen of 240 genes, including 110 ER-associated genes, for molecules that mediate bacterial interactions with the ER. We uncovered 52 evolutionarily conserved host factors that, when depleted, inhibited or increased Brucella infection. Strikingly, 29 of these factors had not been previously suggested to support bacterial infection of host cells. The most intriguing of these was inositol-requiring enzyme 1 (IRE1), a transmembrane kinase that regulates the eukaryotic unfolded protein response (UPR). We employed IRE1α−/− murine embryonic fibroblasts (MEFs) to demonstrate a role for this protein in supporting Brucella infection of mammalian cells, and thereby, validated the utility of the Drosophila S2 cell system for uncovering novel Brucella host factors. Finally, we propose a model in which IRE1α, and other ER-associated genes uncovered in our screen, mediate Brucella replication by promoting autophagosome biogenesis.

Surface recognition and penetration are critical steps in the infection cycle of many plant pathogenic fungi. In Magnaporthe oryzae, cAMP signaling is involved in surface recognition and pathogenesis. Deletion of the MAC1 adenylate cyclase gene affected appressorium formation and plant infection. In this study, we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting proteins is the adenylate cyclase-associated protein named Cap1. CAP genes are well-conserved in phytopathogenic fungi but none of them have been functionally characterized. Deletion of CAP1 blocked the effects of a dominant RAS2 allele and resulted in defects in invasive growth and a reduced intracellular cAMP level. The Δcap1 mutant was defective in germ tube growth, appressorium formation, and formation of typical blast lesions. Cap1-GFP had an actin-like localization pattern, localizing to the apical regions in vegetative hyphae, at the periphery of developing appressoria, and in circular structures at the base of mature appressoria. Interestingly, Cap1, similar to LifeAct, did not localize to the apical regions in invasive hyphae, suggesting that the apical actin cytoskeleton differs between vegetative and invasive hyphae. Domain deletion analysis indicated that the proline-rich region P2 but not the actin-binding domain (AB) of Cap1 was responsible for its subcellular localization. Nevertheless, the AB domain of Cap1 must be important for its function because CAP1 ΔAB only partially rescued the Δcap1 mutant. Furthermore, exogenous cAMP induced the formation of appressorium-like structures in non-germinated conidia in CAP1 ΔAB transformants. This novel observation suggested that AB domain deletion may result in overstimulation of appressorium formation by cAMP treatment. Overall, our results indicated that CAP1 is important for the activation of adenylate cyclase, appressorium morphogenesis, and plant infection in M. oryzae. CAP1 may also play a role in feedback inhibition of Ras2 signaling when Pmk1 is activated.

SummaryEndocytosis and exocytosis are strictly segregated at the ends of hyphal cells of filamentous fungi, with a collar of endocytic activity encircling the growing cell tip, which elongates through directed membrane fusion. It has been proposed that this separation supports an endocytic recycling pathway that maintains polar localization of proteins at the growing apex. In a search for proteins in the filamentous fungus Aspergillus nidulans that possess an NPFxD motif, which signals for endocytosis, a Type 4 P-Type ATPase was identified and named DnfA. Interestingly, NPFxD is at a different region of DnfA than the same motif in the Saccharomyces cerevisiae ortholog, although endocytosis is dependent on this motif for both proteins. DnfA is involved in asexual sporulation and polarized growth. Additionally, it is segregated within the Spitzenkörper from another Type 4 P-type ATPase, DnfB. Next, the phosphatidylserine marker GFP::Lact-C2 was expressed in growing hyphae, which revealed that this phospholipid is enriched on the cytosolic face of secretory vesicles. This distribution is affected by deleting either dnfA or dnfB. These findings provide evidence for the spatial and temporal segregation of Type4-ATPases in filamentous fungi, and the asymmetric distribution of phosphatidylserine to the Spitzenkörper in A. nidulans.