M. Gabriela Roca

Role of a mitogen-activated protein kinase pathway during conidial germination and hyphal fusion in Neurospora crassa.

ABSTRACT Mitogen-activated protein (MAP) kinase signaling pathways are ubiquitous and evolutionarily conserved in eukaryotic organisms. MAP kinase pathways are composed of a MAP kinase, a MAP kinase kinase, and a MAP kinase kinase kinase; activation is regulated by sequential phosphorylation. Components of three MAP kinase pathways have been identified by genome sequence analysis in the filamentous fungus Neurospora crassa. One of the predicted MAP kinases in N. crassa, MAK-2, shows similarity to Fus3p and Kss1p of Saccharomyces cerevisiae, which are involved in sexual reproduction and filamentation, respectively. In this study, we show that an N. crassa mutant disrupted in mak-2 exhibits a pleiotropic phenotype: derepressed conidiation, shortened aerial hyphae, lack of vegetative hyphal fusion, female sterility, and autonomous ascospore lethality. We assessed the phosphorylation of MAK-2 during conidial germination and early colony development. Peak levels of MAK-2 phosphorylation were most closely associated with germ tube elongation, branching, and hyphal fusion events between conidial germlings. A MAP kinase kinase kinase (NRC-1) is the predicted product of N. crassa nrc-1 locus and is a homologue of STE11 in S. cerevisiae. An nrc-1 mutant shares many of the same phenotypic traits as the mak-2 mutant and, in particular, is a hyphal fusion mutant. We show that MAK-2 phosphorylation during early colony development is dependent upon the presence of NRC-1 and postulate that phosphorylation of MAK-2 is required for hyphal fusion events that occur during conidial germination.

Cell Biology of Conidial Anastomosis Tubes in Neurospora crassa

ABSTRACT Although hyphal fusion has been well documented in mature colonies of filamentous fungi, it has been little studied during colony establishment. Here we show that specialized hyphae, called conidial anastomosis tubes (CATs), are produced by all types of conidia and by conidial germ tubes of Neurospora crassa. The CAT is shown to be a cellular element that is morphologically and physiologically distinct from a germ tube and under separate genetic control. In contrast to germ tubes, CATs are thinner, shorter, lack branches, exhibit determinate growth, and home toward each other. Evidence for an extracellular CAT inducer derived from conidia was obtained because CAT formation was reduced at low conidial concentrations. A cr-1 mutant lacking cyclic AMP (cAMP) produced CATs, indicating that the inducer is not cAMP. Evidence that the transduction of the CAT inducer signal involves a putative transmembrane protein (HAM-2) and the MAK-2 and NRC-1 proteins of a mitogen-activated protein kinase signaling pathway was obtained because ham-2, mak-2, and nrc-1 mutants lacked CATs. Optical tweezers were used in a novel experimental assay to micromanipulate whole conidia and germlings to analyze chemoattraction between CATs during homing. Strains of the same and opposite mating type were shown to home toward each other. The cr-1 mutant also underwent normal homing, indicating that cAMP is not the chemoattractant. ham-2, mak-2, and nrc-1 macroconidia did not attract CATs of the wild type. Fusion between CATs of opposite mating types was partially inhibited, providing evidence of non-self-recognition prior to fusion. Microtubules and nuclei passed through fused CATs.

Live-cell Imaging of Filamentous Fungi Using Vital Fluorescent Dyes and Confocal Microscopy

Publisher Summary This chapter discusses microscope technologies for imaging living fungal cells at high spatial resolution and reviews the vital fluorescent dyes that are proving useful for analyzing the cell biology of filamentous fungi with the confocal laser-scanning microscope (CLSM). Definitions of important practical aspects that need to be taken into account for optimal live-cell imaging at high spatial resolution with the CLSM and indications of future directions for live-cell imaging of filamentous fungi are described in the chapter. A range of microscope technologies have been developed over the past 20 years that allow the imaging of living cells at high spatial resolution using optical sectioning. These technologies include CLSM , spinning-disk confocal microscopy, two-photon microscopy, and deconvolution microscopy. The advantages and disadvantages of these optical-sectioning methods for imaging living fungal cells are summarized in the chapter. Of these types of microscopies, CLSM has been the most popular, and this chapter focuses on results obtained using this extremely powerful imaging technique.

Oscillatory recruitment of signaling proteins to cell tips promotes coordinated behavior during cell fusion

Cell-cell communication is essential for coordinating physiological responses in multicellular organisms and is required for various developmental processes, including cell migration, differentiation, and fusion. To facilitate communication, functional differences are usually required between interacting cells, which can be established either genetically or developmentally. However, genetically identical cells in the same developmental state are also capable of communicating, but must avoid self-stimulation. We hypothesized that such cells must alternate their physiological state between signal sending and receiving to allow recognition and behavioral changes. To test this hypothesis, we studied cell communication in the filamentous fungus Neurospora crassa, a simple and experimentally amenable model system. In N. crassa, germinating asexual spores (germlings) of identical genotype chemotropically sense others in close proximity, show attraction-mediated directed growth, and ultimately undergo cell fusion. Here, we report that two proteins required for cell fusion, a MAP kinase (MAK-2) and a protein of unknown molecular function (SO), exhibit rapid oscillatory recruitment to the plasma membranes of interacting germlings undergoing chemotropic interactions via directed growth. Using an inhibitable MAK-2 variant, we show that MAK-2 kinase activity is required both for chemotropic interactions and for oscillation of MAK-2 and SO to opposing cell tips. Thus, N. crassa germlings undergoing chemotropic interactions rapidly alternate between two different physiological states, associated with signal delivery and response. Such spatiotemporal coordination of signaling allows genetically identical and developmentally equivalent cells to avoid self-stimulation and to coordinate their behavior to achieve the beneficial physiological outcome of cell fusion.

Nuclear dynamics, mitosis, and the cytoskeleton during the early stages of colony initiation in Neurospora crassa

ABSTRACT Neurospora crassa macroconidia form germ tubes that are involved in colony establishment and conidial anastomosis tubes (CATs) that fuse to form interconnected networks of conidial germlings. Nuclear and cytoskeletal behaviors were analyzed in macroconidia, germ tubes, and CATs in strains that expressed fluorescently labeled proteins. Heterokaryons formed by CAT fusion provided a rapid method for the imaging of multiple labeled fusion proteins and minimized the potential risk of overexpression artifacts. Mitosis occurred more slowly in nongerminated macroconidia (1.0 to 1.5 h) than in germ tubes (16 to 20 min). The nucleoporin SON-1 was not released from the nuclear envelope during mitosis, which suggests that N. crassa exhibits a form of “closed mitosis.” During CAT homing, nuclei did not enter CATs, and mitosis was arrested. Benomyl treatment showed that CAT induction, homing, fusion, as well as nuclear migration through fused CATs do not require microtubules or mitosis. Three ropy mutants (ro-1, ro-3, and ro-11) defective in the dynein/dynactin microtubule motor were impaired in nuclear positioning, but nuclei still migrated through fused CATs. Latrunculin B treatment, imaging of F-actin in living cells using Lifeact-red fluorescent protein (RFP), and analysis of mutants defective in the Arp2/3 complex demonstrated that actin plays important roles in CAT fusion.

Heterokaryon Incompatibility Is Suppressed Following Conidial Anastomosis Tube Fusion in a Fungal Plant Pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits.

The ham-5, rcm-1 and rco-1 genes regulate hyphal fusion in Neurospora crassa

Mutants of Neurospora crassa unable to participate in vegetative hyphal fusion (anastomosis) were isolated and characterized. From this analysis, three genes, rcm-1, rco-1 and ham-5, were identified and shown to be required for hyphal fusion. The rcm-1 and rco-1 genes are homologues of the Saccharomyces cerevisiae SSN6 and TUP1 genes, which encode a dimeric transcription factor in yeast. We demonstrate that in N. crassa the rcm-1 and rco-1 genes are required for hyphal fusion and normal hyphal morphology, and influence both asexual and sexual development. The ham-5 gene encodes a 1686 amino acid protein with two putative WD40 domains, which might participate in protein–protein interactions. ham-5 deletion mutants had a reduced rate of hyphal extension and altered hyphal morphology, and were unable to produce the conidial anastomosis tubes that are required for hyphal fusion during colony initiation.

Live-cell imaging of conidial fusion in the bean pathogen, Colletotrichum lindemuthianum.

Fusion of conidia and conidial germlings by means of conidial anastomosis tubes (CATs) is a common phenomenon in filamentous fungi, including many plant pathogens. It has a number of different roles, and has been speculated to facilitate parasexual recombination and horizontal gene transfer between species. The bean pathogen Colletotrichum lindemuthianum naturally undergoes CAT fusion on the host surface and within asexual fruiting bodies in anthracnose lesions on its host. It has not been previously possible to analyze the whole process of CAT fusion in this or any other pathogen using live-cell imaging techniques. Here we report the development of a robust protocol for doing this with C. lindemuthianum in vitro. The percentage of conidial germination and CAT fusion was found to be dependent on culture age, media and the fungal strain used. Increased CAT fusion was correlated with reduced germ tube formation. We show time-lapse imaging of the whole process of CAT fusion in C. lindemuthianum for the first time and monitored nuclear migration through fused CATs using nuclei labelled with GFP. CAT fusion in this pathogen was found to exhibit significant differences to that in the model system Neurospora crassa. In contrast to N. crassa, CAT fusion in C. lindemuthianum is inhibited by nutrients (it only occurs in water) and the process takes considerably longer.

Vegetative Hyphal Fusion in Filamentous Fungi

The formation of channels between fungal hyphae by self fusion is a defining feature of filamentous fungi and results in the fungal colony being a complex interconnected network of hyphae. During the vegetative phase hyphal fusions are commonly formed during colony establishment by specialized conidial anastomosis tubes (CATs) and then later by specialized fusion hyphae in the mature colony. CAT induction, homing and fusion in Neurospora crassa provides an excellent model in which to study the process of vegetative hyphal fusion because it is simple and experimentally very amenable. Various mutants compromised in hyphal fusion have been isolated and characterized. Although the self-signalling ligand(s) involved in CAT induction and homing has/have not been identified, MAP kinase signalling is downstream of the initial ligand-receptor interaction(s), and has features in common with MAP kinase signalling during mating cell interactions in the budding yeast and during fungal infection structure (appressorium) formation. Hyphal fusion also resembles yeast cell mating and appressorium formation in other ways. Vegetative hyphal fusion between hyphae of different genotypes (nonself fusion) usually results in a form of programmed cell death which normally prevents heterokaryons from developing further. This process in N. crassa is controlled by heterokaryon incompatibility (het) loci. Understanding hyphal fusion in the model fungus, N. crassa, provides a paradigm for self-signalling mechanisms in eukaryotic microbes and might also provide a model for understanding somatic cell fusion in other eukaryotic species.

Colletotrichum lindemuthianum exhibits different patterns of nuclear division at different stages in its vegetative life cycle

Live-cell imaging with fluorescent protein labeling is providing major new insights into nuclear dynamics in filamentous fungi. With this approach we provide a detailed report of nuclear organization and behavior during mitosis in the bean pathogen Colletotrichum lindemuthianum. Nuclear division and nuclear migration were analyzed in ungerminated conidia, conidial germlings and the mature colony. Ungerminated conidia were uninucleate and completion of mitosis was found not to be essential for germ tube formation, conidial anastomosis tube (CAT) formation or fusion. Nuclei in fused conidial germlings exhibited asynchronous mitoses, and nuclear migration through fused CATs occurred after the nuclei had divided. Different patterns of nuclear division were found in vegetative hyphae of the mature colony. Synchronous, parasynchronous and asynchronous patterns of mitosis were observed in apical hyphal compartments at the colony border, while only synchronous and asynchronous mitoses occurred in subapical hyphal compartments. These findings have revealed unexpected diversity in the patterns of mitosis in different cells of C. lindemuthianum.