The conidial coin toss: A polarized conidial adhesive in Colletotrichum graminicola.

Colletotrichum graminicola is an economically significant fungal pathogen of maize. The primary infective conidia of the fungus, falcate conidia, are splash-dispersed during rain events. The adhesion of the falcate conidia triggers germination and is required for the development of infection structures. Falcate conidia are capable of immediate adhesion upon encountering the substrate. We report that rapid adhesion in C. graminicola is polarized, with a single-sided strip of adhesive material running the length of a single side (or face) of the conidium between the tips. This strip of adhesive is co-localized with dynamic transverse actin cables, and both the adhesive strip and actin cables are formed after liberation of the conidium from its conidiogenous cell but prior to adhesion to the infection court. Orientation of conidia upon contact with substrate determines whether they will rapidly adhere, and those which do not initially adhere can be induced to do so by applying force to reorient or "flip" the conidia. We propose that C. graminicola possesses an adhesive mechanism resulting in an adhesion efficiency of approximately 50% upon initial contact with substrata, and that an increase in adhesion efficiency can be induced by disturbance.

Localization of NPFxD motif-containing proteins in Aspergillus nidulans.

During growth, filamentous fungi produce polarized cells called hyphae. It is generally presumed that polarization of hyphae is dependent upon secretion through the Spitzenkörper, as well as a mechanism called apical recycling, which maintains a balance between the tightly coupled processes of endocytosis and exocytosis. Endocytosis predominates in an annular domain called the sub-apical endocytic collar, which is located in the region of plasma membrane 1-5 µm distal to the Spitzenkörper. It has previously been proposed that one function of the sub-apical endocytic collar is to maintain the apical localization of polarization proteins. These proteins mark areas of polarization at the apices of hyphae. However, as hyphae grow, these proteins are displaced along the membrane and some must then be removed at the sub-apical endocytic collar in order to maintain the hyphoid shape. While endocytosis is fairly well characterized in yeast, comparatively little is known about the process in filamentous fungi. Here, a bioinformatics approach was utilized to identify 39 Aspergillus nidulans proteins that are predicted to be cargo of endocytosis based on the presence of an NPFxD peptide motif. This motif is a necessary endocytic signal sequence first established in Saccharomyces cerevisiae, where it marks proteins for endocytosis through an interaction with the adapter protein Sla1p. It is hypothesized that some proteins that contain this NPFxD peptide sequence in A. nidulans will be potential targets for endocytosis, and therefore will localize either to the endocytic collar or to more proximal polarized regions of the cell, e.g. the apical dome or the Spitzenkörper. To test this, a subset of the motif-containing proteins in A. nidulans was tagged with GFP and the dynamic localization was evaluated. The documented localization patterns support the hypothesis that the motif marks proteins for localization to the polarized cell apex in growing hyphae.

Aspergillus nidulans flippase DnfA is cargo of the endocytic collar and plays complementary roles in growth and phosphatidylserine asymmetry with another flippase, DnfB.

Endocytosis 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.

F-actin localization dynamics during appressorium formation in Colletotrichum graminicola.

Appressoria are essential penetration structures for many phytopathogenic fungi. Here F-actin localization dynamics were documented during appressorium formation in vitro and in planta in Colletotrichum graminicola Four discernible stages of dynamic F-actin distribution occurring in a programmed order were documented from differentiation of appressoria to formation of penetration pores: (stage A) from germ tube enlargement to complete expansion of the appressorium; (stage S) septation occurs; (stage L) a long period of low F-actin activity; (stage P) the penetration pore forms. The F-actin subcellular localization corresponded to each stage. A distinct redistribution of actin cables occurred at the transition from stage A to stage S. The in planta assays revealed that F-actin also assembled in invasive hyphae and that actin cables might play an essential role for penetration-peg development. The F-actin localization distribution may be used as a subcellular marker to define the developmental stages during appressorium formation.

Live Cell Imaging of Actin Dynamics in the Filamentous Fungus Aspergillus nidulans.

Hyphal cells of filamentous fungi grow at their tips in a method analogous to pollen tube and root hair elongation. This process, generally referred to as tip growth, requires precise regulation of the actin cytoskeleton, and characterizing the various actin structures in these cell types is currently an active area of research. Here, the actin marker Lifeact was used to document actin dynamics in the filamentous fungus Aspergillus nidulans. Contractile double rings were observed at septa, and annular clusters of puncta were seen subtending growing hyphal tips, corresponding to the well-characterized subapical endocytic collar. However, Lifeact also revealed two additional structures. One, an apical array, was dynamic on the face opposite the tip, while a subapical web was dynamic on the apical face and was located several microns behind the growth site. Each was observed turning into the other over time, implying that they could represent different localizations of the same structure, although hyphae with a subapical web grew faster than those exhibiting an apical array. The subapical web has not been documented in any filamentous fungus to date, and is separate from the networks of F-actin seen in other tip-growing organisms surrounding septa or stationary along the plasmalemma.

Neurospora crassa ASM-1 complements the conidiation defect in a stuA mutant of Aspergillus nidulans.

Aspergillus nidulans StuA and Neurospora crassa ASM-1 are orthologous APSES (ASM-1, PHD1, SOK2, Efg1, StuA) transcription factors conserved across a diverse group of fungi. StuA and ASM-1 have roles in asexual (conidiation) and sexual (ascospore formation) development in both organisms. To address the hypothesis that the last common ancestor of these diverse fungi regulated conidiation with similar genes, asm-1 was introduced into the stuA1 mutant of A. nidulans. Expression of asm-1 complemented defective conidiophore morphology and restored conidia production to wild type levels in stuA1. Expression of asm-1 in the stuA1 strain did not rescue the defect in sexual development. When the conidiation regulator AbaA was tagged at its C-terminus with GFP in A. nidulans, it localized to nuclei in phialides. When expressed in the stuA1 mutant, AbaA::GFP localized to nuclei in conidiophores but no longer was confined to phialides, suggesting that expression of AbaA in specific cell types of the conidiophore was conditioned by StuA. Our data suggest that the function in conidiation of StuA and ASM-1 is conserved and support the view that, despite the great morphological and ontogenic diversity of their condiphores, the last common ancestor of A. nidulans and N. crassa produced an ortholog of StuA that was involved in conidiophore development.

Effects of Sodium Alginate and Calcium Chloride on Fungal Growth and Viability in Biomass-Fungi Composite Materials Used for 3D Printing.

To combat climate change, one approach is to manufacture products from biomass-fungi composite materials instead of petroleum-based plastics. These products can be used in packaging, furniture, and construction industries. A 3D printing-based manufacturing method was developed for these biomass-fungi composite materials, eliminating the need for molds, and enabling customized product design. However, previous studies on the 3D printing-based method showed significant shrinkage of printed samples. In this paper, an approach is proposed to reduce the shrinkage by incorporating ionic crosslinking into biomass-fungi composite materials. This paper reports two sets of experiments regarding the effects of sodium alginate (SA) and calcium chloride (CaCl2) on fungal growth and fungal viability. The first set of experiments was conducted using Petri dishes with fungi isolated from colonized biomass-fungi material and different concentrations of SA and CaCl2. Fungal growth was measured by the circumference of fungal colonies. The results showed that concentrations of SA and CaCl2 had significant effects on fungal growth and no fungal growth was observed on Petri dishes with 15% CaCl2. Some of these Petri dishes were also observed under confocal microscopy. The results confirmed the differences obtained by measuring the circumference of fungal colonies. The second set of experiments was conducted using Petri dishes with biomass-fungi mixtures that were treated with different concentrations of SA and exposure times in a CaCl2 (crosslinking) solution. Fungal viability was measured by counting colony-forming units. The results showed that the addition of the SA solution and exposure times in the crosslinking solution had statistically significant effects on fungal viability. The 2SA solution was prepared by dissolving 2 g of SA in 100 mL of water, the 5SA solution was prepared by dissolving 5 g of SA in 100 mL of water, and the crosslinking solution was prepared by dissolving 5 g of CaCl2 in 100 mL of water. The results also showed that fungal viability was not too low in biomass-fungi mixtures that included 2SA solution and were exposed to the crosslinking solution for 1 min.

Using fimbrin to quantify the endocytic subapical collar during polarized growth in three filamentous fungi.

Filamentous fungi produce specialized cells called hyphae. These cells grow by polarized extension at their apex, which is maintained by the balance of endocytosis and exocytosis at the apex. Although endocytosis has been well characterized in other organisms, the details of endocytosis and its role in maintaining polarity during hyphal growth in filamentous fungi is comparatively sparsely studied. In recent years, a concentrated region of protein activity that trails the growing apex of hyphal cells has been discovered. This region, dubbed the "endocytic collar" (EC), is a dynamic 3-dimensional region of concentrated endocytic activity, the disruption of which results in the loss of hyphal polarity. Here, fluorescent protein-tagged fimbrin was used as a marker to map the collar during growth of hyphae in three fungi: Aspergillus nidulans, Colletotrichum graminicola, and Neurospora crassa. Advanced microscopy techniques and novel quantification strategies were then utilized to quantify the spatiotemporal localization and recovery rates of fimbrin in the EC during hyphal growth. Correlating these variables with hyphal growth rate revealed that the strongest observed relationship with hyphal growth is the distance by which the EC trails the apex, and that measured endocytic rate does not correlate strongly with hyphal growth rate. This supports the hypothesis that endocytic influence on hyphal growth rate is better explained by spatiotemporal regulation of the EC than by the raw rate of endocytosis.

Advancing the Rose Rosette Virus Minireplicon and Encapsidation System by Incorporating GFP, Mutations, and the CMV 2b Silencing Suppressor.

Plant infecting emaraviruses have segmented negative strand RNA genomes and little is known about their infection cycles due to the lack of molecular tools for reverse genetic studies. Therefore, we innovated a rose rosette virus (RRV) minireplicon containing the green fluorescent protein (GFP) gene to study the molecular requirements for virus replication and encapsidation. Sequence comparisons among RRV isolates and structural modeling of the RNA dependent RNA polymerase (RdRp) and nucleocapsid (N) revealed three natural mutations of the type species isolate that we reverted to the common species sequences: (a) twenty-one amino acid truncations near the endonuclease domain (named delA), (b) five amino acid substitutions near the putative viral RNA binding loop (subT), and (c) four amino acid substitutions in N (NISE). The delA and subT in the RdRp influenced the levels of GFP, gRNA, and agRNA at 3 but not 5 days post inoculation (dpi), suggesting these sequences are essential for initiating RNA synthesis and replication. The NISE mutation led to sustained GFP, gRNA, and agRNA at 3 and 5 dpi indicating that the N supports continuous replication and GFP expression. Next, we showed that the cucumber mosaic virus (CMV strain FNY) 2b singularly enhanced GFP expression and RRV replication. Including agRNA2 with the RRV replicon produced observable virions. In this study we developed a robust reverse genetic system for investigations into RRV replication and virion assembly that could be a model for other emaravirus species.

The Antidepressant Sertraline Induces the Formation of Supersized Lipid Droplets in the Human Pathogen Cryptococcus neoformans.

The prevalence and increasing incidence of fungal infections globally is a significant worldwide health problem. Cryptococcosis, primarily caused by the pathogenic yeast Cryptococcus neoformans, is responsible for approximately 181,000 estimated deaths annually. The scarcity of treatments and the increasing resistance to current therapeutics highlight the need for the development of antifungal agents which have novel mechanisms of action and are suitable for clinical use. Repurposing existing FDA-approved compounds as antimycotic therapeutics is a promising strategy for the rapid development of such new treatments. Sertraline (SRT), a commonly prescribed antidepressant, is a broad-spectrum antifungal agent with particular efficacy against C. neoformans. However, the effect of SRT on fungal physiology is not understood. Here, we report that SRT induces the formation of supersized lipid droplets (SLDs) in C. neoformans, and in Candida albicans, Saccharomyces cerevisiae, and Aspergillus fumigatus. SLDs were not induced in C. neoformans by treatment with the antifungal fluconazole (FLC), consistent with SRT and FLC acting differently to perturb C. neoformans physiology. The formation of SLDs in response to SRT indicates that this compound alters the lipid metabolism of C. neoformans. Moreover, the SRT-induced enlargement of LDs in other fungal species may indicate a common fungal response to SRT.

acon-3, the Neurospora crassa ortholog of the developmental modifier, medA, complements the conidiation defect of the Aspergillus nidulans mutant.

Aspergillus nidulans and Neurospora crassa are ascomycetes that produce asexual spores through morphologically distinct processes. MedA, a protein with unknown function, is required for normal asexual and sexual development in A. nidulans. We determined that the N. crassa ortholog of medA is acon-3, a gene required for early conidiophore development and female fertility. To test hypotheses about the evolutionary origins of asexual development in distinct fungal lineages it is important to understand the degree of conservation of developmental regulators. The amino acid sequences of A. nidulans MedA and N. crassa ACON-3 shared 37% identity and 51% similarity. acon-3 is induced at late time points of conidiation. In contrast, medA is constitutively expressed and MedA protein localizes to nuclei in all tissue types. Nonetheless, expression of acon-3 using its native promoter complemented the conidiation defects of the A. nidulans ΔmedA and medA15 mutants. We conclude that the biochemical activity of the medA orthologs is conserved for conidiation.

Raman Characterization of Fungal DHN and DOPA Melanin Biosynthesis Pathways.

Fungal melanins represent a resource for important breakthroughs in industry and medicine, but the characterization of their composition, synthesis, and structure is not well understood. Raman spectroscopy is a powerful tool for the elucidation of molecular composition and structure. In this work, we characterize the Raman spectra of wild-type Aspergillus fumigatus and Cryptococcus neoformans and their melanin biosynthetic mutants and provide a rough "map" of the DHN (A. fumigatus) and DOPA (C. neoformans) melanin biosynthetic pathways. We compare this map to the Raman spectral data of Aspergillus nidulans wild-type and melanin biosynthetic mutants obtained from a previous study. We find that the fully polymerized A. nidulans melanin cannot be classified according to the DOPA pathway; nor can it be solely classified according to the DHN pathway, consistent with mutational analysis and chemical inhibition studies. Our approach points the way forward for an increased understanding of, and methodology for, investigating fungal melanins.

Aspergillus nidulans striatin (StrA) mediates sexual development and localizes to the endoplasmic reticulum.

Striatin family proteins have been identified in animals and fungi and are considered to be scaffolding proteins. In fungi striatin orthologs have been associated with sexual development and virulence to plants. In this study, we characterized the functions and localization of the striatin ortholog, StrA, in Aspergillus nidulans. deltastrA strains showed multiple defects in conidium germination, mycelial radial growth, production of diffusible red pigment, and reduced conidiation. The most striking phenotype is the production of abnormally small cleistothecia that are defective in ascosporogenesis. Over-expression of strA enhanced cleistothecium development and increased the production of Hülle cells in shaking liquid cultures. In addition, we generated strains expressing StrA::eGFP under the endogenous promoter. By co-labeling with FM4-64 and co-localization with nuclear localized StuA(NLS)::DsRed or CxnA (an endoplasmic reticulum marker), we determined that StrA mainly localizes to endoplasmic reticulum and the nuclear envelope.

RNAi screen of endoplasmic reticulum-associated host factors reveals a role for IRE1alpha in supporting Brucella replication.

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 IRE1alpha(-/-) 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 IRE1alpha, and other ER-associated genes uncovered in our screen, mediate Brucella replication by promoting autophagosome biogenesis.

Current views on endocytosis in filamentous fungi.

Filamentous fungi grow by adding cell wall and membrane exclusively at the apex of tubular structures called hyphae. Growth was previously believed to occur only through exocytosis at the Spitzenkörper, an organised body of secretory macro- and microvesicles found only in growing hyphae. More recent work has indicated that an area deemed the sub-apical collar is enriched for endocytosis and is also required for hyphal growth. It is now generally believed that polarity of filamentous fungi is achieved through the balancing of the processes of endocytosis and exocytosis at these two areas. This review is an update on the current progress and understanding surrounding the occurrence of endocytosis and its spatial regulation as they pertain to growth and pathogenicity in filamentous fungi.

Identification of toxic mold species through Raman spectroscopy of fungal conidia.

We use a 785 nm shifted excitation Raman difference (SERDS) technique to measure the Raman spectra of the conidia of 10 mold species of especial toxicological, medical, and industrial importance, including Stachybotrys chartarum, Penicillium chrysogenum, Aspergillus fumigatus, Aspergillus flavus, Aspergillus oryzae, Aspergillus niger, and others. We find that both the pure Raman and fluorescence signals support the hypothesis that for an excitation wavelength of 785 nm the Raman signal originates from the melanin pigments bound within the cell wall of the conidium. In addition, the major features of the pure Raman spectra group into profiles that we hypothesize may be due to differences in the complex melanin biosynthesis pathways. We then combine the Raman spectral data with neural network models to predict species classification with an accuracy above 99%. Finally, the Raman spectral data of all species investigated is made freely available for download and use.

Molecular origin of the Raman signal from Aspergillus nidulans conidia and observation of fluorescence vibrational structure at room temperature.

Successful approaches to identification and/or biological characterization of fungal specimens through Raman spectroscopy may require the determination of the molecular origin of the Raman response as well as its separation from the background fluorescence. The presence of fluorescence can interfere with Raman detection and is virtually impossible to avoid. Fluorescence leads to a multiplicity of problems: one is noise, while another is "fake" spectral structure that can easily be confused for spontaneous Raman peaks. One solution for these problems is Shifted Excitation Raman Difference Spectroscopy (SERDS), in which a tunable light source generates two spectra with different excitation frequencies in order to eliminate fluorescence from the measured signal. We combine a SERDS technique with genetic breeding of mutant populations and demonstrate that the Raman signal from Aspergillus nidulans conidia originates in pigment molecules within the cell wall. In addition, we observe unambiguous vibrational fine-structure in the fluorescence response at room temperature. We hypothesize that the vibrational fine-structure in the fluorescence results from the formation of flexible, long-lived molecular cages in the bio-polymer matrix of the cell wall that partially shield target molecules from the immediate environment and also constrain their degrees of freedom.

The role of actin, fimbrin and endocytosis in growth of hyphae in Aspergillus nidulans.

Filamentous 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 microm 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.

Aspergillus nidulans ArfB plays a role in endocytosis and polarized growth.

Filamentous fungi undergo polarized growth throughout most of their life cycles. The Spitzenkörper is an apical organelle composed primarily of vesicles that is unique to filamentous fungi and is likely to act as a vesicle supply center for tip growth. Vesicle assembly and trafficking are therefore important for hyphal growth. ADP ribosylation factors (Arfs), a group of small GTPase proteins, play an important role in nucleating vesicle assembly. Little is known about the role of Arfs in filamentous hyphal growth. We found that Aspergillus nidulans is predicted to encode six Arf family proteins. Analysis of protein sequence alignments suggests that A. nidulans ArfB shares similarity with ARF6 of Homo sapiens and Arf3p of Saccharomyces cerevisiae. An arfB null allele (arfB disrupted by a transposon [arfB::Tn]) was characterized by extended isotropic growth of germinating conidia followed by cell lysis or multiple, random germ tube emergence, consistent with a failure to establish polarity. The mutant germ tubes and hyphae that do form initially meander abnormally off of the axis of polarity and frequently exhibit dichotomous branching at cell apices, consistent with a defect in polarity maintenance. FM4-64 staining of the arfB::Tn strain revealed that another phenotypic characteristic seen for arfB::Tn is a reduction and delay in endocytosis. ArfB is myristoylated at its N terminus. Green fluorescent protein-tagged ArfB (ArfB::GFP) localizes to the plasma membrane and endomembranes and mutation (ArfB(G2A)::GFP) of the N-terminal myristoylation motif disperses the protein to the cytoplasm rather than to the membranes. These results demonstrate that ArfB functions in endocytosis to play important roles in polarity establishment during isotropic growth and polarity maintenance during hyphal extension.

Root infection and systemic colonization of maize by Colletotrichum graminicola.

Colletotrichum graminicola is a filamentous ascomycete that causes anthracnose disease of maize. While the fungus can cause devastating foliar leaf blight and stalk rot diseases, little is known about its ability to infect roots. Previously published reports suggest that C. graminicola may infect maize roots and that root infections may contribute to the colonization of aboveground plant tissues, leading to disease. To determine whether C. graminicola can infect maize roots and whether root infections can result in the colonization of aboveground plant tissues, we developed a green fluorescent protein-tagged strain and used it to study the plant root colonization and infection process in vivo. We observed structures produced by other root pathogenic fungi, including runner hyphae, hyphopodia, and microsclerotia. A mosaic pattern of infection resulted from specific epidermal and cortical cells becoming infected by intercellular hyphae while surrounding cells were uninfected, a pattern that is distinctly different from that described for leaves. Interestingly, falcate conidia, normally restricted to acervuli, were also found filling epidermal cells and root hairs. Twenty-eight percent of plants challenged with soilborne inoculum became infected in aboveground plant parts (stem and/or leaves), indicating that root infection can lead to asymptomatic systemic colonization of the plants. Many of the traits observed for C. graminicola have been previously reported for other root-pathogenic fungi, suggesting that these traits are evolutionally conserved in multiple fungal lineages. These observations suggest that root infection may be an important component of the maize anthracnose disease cycle.