Conidial Anastomosis Tube Fusion Research Articles

Ca2+ Signalling Differentially Regulates Germ-Tube Formation and Cell Fusion in Fusarium oxysporum.

Fusarium oxysporum is an important plant pathogen and an emerging opportunistic human pathogen. Germination of conidial spores and their fusion via conidial anastomosis tubes (CATs) are significant events during colony establishment in culture and on host plants and, hence, very likely on human epithelia. CAT fusion exhibited by conidial germlings of Fusarium species has been postulated to facilitate mitotic recombination, leading to heterokaryon formation and strains with varied genotypes and potentially increased virulence. Ca2+ signalling is key to many of the important physiological processes in filamentous fungi. Here, we tested pharmacological agents with defined modes of action in modulation of the mammalian Ca2+ signalling machinery for their effect on germination and CAT-mediated cell fusion in F. oxysporum. We found various drug-specific and dose-dependent effects. Inhibition of calcineurin by FK506 or cyclosporin A, as well as chelation of extracellular Ca2+ by BAPTA, exclusively inhibit CAT induction but not germ-tube formation. On the other hand, inhibition of Ca2+ channels by verapamil, calmodulin inhibition by calmidazolium, and inhibition of mitochondrial calcium uniporters by RU360 inhibited both CAT induction and germ-tube formation. Thapsigargin, an inhibitor of mammalian sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), partially inhibited CAT induction but had no effect on germ-tube formation. These results provide initial evidence for morphologically defining roles of Ca2+-signalling components in the early developmental stages of F. oxysporum colony establishment—most notably, the indication that calcium ions act as self-signalling molecules in this process. Our findings contribute an important first step towards the identification of Ca2+ inhibitors with fungas-specific effects that could be exploited for the treatment of infected plants and humans.

Differential Physiological Prerequisites and Gene Expression Profiles of Conidial Anastomosis Tube and Germ Tube Formation in Colletotrichum gloeosporioides

The conidia of a hemibiotrophic fungus, Colletotrichum gloeosporioides, can conventionally form a germ tube (GT) and develop into a fungal colony. Under certain conditions, they tend to get connected through a conidial anastomosis tube (CAT) to share the nutrients. CAT fusion is believed to be responsible for the generation of genetic variations in few asexual fungi, which appears problematic for effective fungal disease management. The physiological and molecular requirements underlying the GT formation versus CAT fusion remained underexplored. In the present study, we have deciphered the physiological prerequisites for GT formation versus CAT fusion in C. gloeosporioides. GT formation occurred at a high frequency in the presence of nutrients, while CAT fusion was found to be higher in the absence of nutrients. Younger conidia were found to form GT efficiently, while older conidia preferentially formed CAT. Whole transcriptome analysis of GT and CAT revealed highly differential gene expression profiles, wherein 11,050 and 9786 genes were differentially expressed during GT formation and CAT fusion, respectively. A total of 1567 effector candidates were identified; out of them, 102 and 100 were uniquely expressed during GT formation and CAT fusion, respectively. Genes coding for cell wall degrading enzymes, germination, hyphal growth, host-fungus interaction, and virulence were highly upregulated during GT formation. Meanwhile, genes involved in stress response, cell wall remodeling, membrane transport, cytoskeleton, cell cycle, and cell rescue were highly upregulated during CAT fusion. To conclude, the GT formation and CAT fusion were found to be mutually exclusive processes, requiring differential physiological conditions and sets of DEGs in C. gloeosporioides. This study will help in understanding the basic CAT biology in emerging fungal model species of the genus Colletotrichum.

Quorum sensing-mediated inter-specific conidial anastomosis tube fusion between Colletotrichum gloeosporioides and C. siamense

Many plant pathogenic filamentous fungi undergo fusion of conidia through conidial anastomosis tubes (CATs), which is believed to facilitate horizontal gene transfer between species. We discovered a remarkable inter-specific CAT fusion between two important plant fungal pathogens Colletotrichum gloeosporioides and C. siamense. In an invitro assay, under no selection pressure, the inter-specific CAT fusion was preferred with higher frequency (25% ± 5%) than intra-specific CAT fusion (11% ± 3.6%). Different stages of CAT fusion viz. CAT induction, homing, and fusion were observed during this inter-specific CAT fusion. The CAT fusion was found to be higher in absence of nutrients and under physiological stresses. This CAT fusion involved a quorum sensing phenomenon, wherein the CAT induction was dependent on conidial density and the putative quorum sensing molecule was extractable in chloroform. Movement of nuclei, mitochondria, and lipid droplets were observed during the CAT fusion. Post CAT fusion, the resulting conidia gave rise to putative heterokaryotic progenies with variable colony characteristics as compared to their parental strains. Few heterokaryons showed variable AFLP banding pattern compared to their parental strains, thereby suggesting a possible genetic exchange between the two species through CAT fusion. The heterokaryotic progenies exhibited varied fitness under different stress conditions. Our study illustrated a possible role of inter-specific CAT fusion in generation of genetic and phenotypic diversity in these fungal pathogens.

Live-cell imaging of conidial anastomosis tube fusion during colony initiation in Fusarium oxysporum.

Fusarium oxysporum exhibits conidial anastomosis tube (CAT) fusion during colony initiation to form networks of conidial germlings. Here we determined the optimal culture conditions for this fungus to undergo CAT fusion between microconidia in liquid medium. Extensive high resolution, confocal live-cell imaging was performed to characterise the different stages of CAT fusion, using genetically encoded fluorescent labelling and vital fluorescent organelle stains. CAT homing and fusion were found to be dependent on adhesion to the surface, in contrast to germ tube development which occurs in the absence of adhesion. Staining with fluorescently labelled concanavalin A indicated that the cell wall composition of CATs differs from that of microconidia and germ tubes. The movement of nuclei, mitochondria, vacuoles and lipid droplets through fused germlings was observed by live-cell imaging.

Evidence for tryptophan being a signal molecule that inhibits conidial anastomosis tube fusion during colony initiation in Neurospora crassa

Mycelial interconnectedness achieved by hyphal fusion has been hypothesized to facilitate the distribution and sharing of nutrients between different parts of a mycelium, especially when nutrients are heterogeneously distributed in the environment. However, the link between environmental nutrient availability and hyphal fusion is little understood. Here, we report that amino acids and extracellular pH regulate conidial anastomosis tube (CAT) fusion during colony initiation in Neurospora crassa. Quantitative analyses revealed that low extracellular pH and certain amino acids, particularly tryptophan, inhibit CAT fusion. Conidial germination was also inhibited by tryptophan but this inhibition was mitigated by the presence of other amino acids. This provides evidence for tryptophan having a role as a signal molecule that regulates CAT fusion. Tryptophan acts intracellularly because two amino acid permease mutants (Δmtr and Δaap-20) exhibited resistance against tryptophan-mediated inhibition of CAT fusion. Tryptophan and low pH did not significantly affect vegetative hyphal fusion in mature colonies, indicating that the latter is regulated in a different manner to 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.

Germling fusion via conidial anastomosis tubes in the grey mould Botrytis cinerea requires NADPH oxidase activity

In many filamentous ascomycete species, the early steps of colony development include fusion between germinating vegetative spores (conidial germlings). Often these fusion events are mediated by specialized hyphal structures, so-called conidial anastomosis tubes (CATs). Here, we show that germling fusion in the grey mould Botrytis cinerea is mediated by hyphal structures possessing the typical features of CATs. Formation of these structures is delayed when spores are germinating on complex media compared to growth on poor substrates. Fusion frequency is also influenced by the growth conditions of the precultures from which spores were obtained. During germination on hydrophobic plant surfaces, which induce pathogenic development, CAT formation is significantly suppressed. Screening of existing B. cinerea gene knockout mutants identified strains lacking the NADPH oxidase BcNoxA or the potential Nox regulator BcNoxR as fusion deficient, suggesting a potential role of reactive oxygen species (ROS) signalling in CAT formation and fusion.

Nuclear Dynamics, Mitosis, and the Cytoskeleton during the Early Stages of Colony Initiation in Neurospora crassa

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.

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.

Conidial anastomosis tubes in filamentous fungi

Conidial anastomosis tubes (CATs) can be recognized in 73 species of filamentous fungi covering 21 genera, and develop in culture and in host-pathogen systems. They have been shown to be morphologically and physiologically distinct from germ tubes in Colletotrichum and Neurospora, and under separate genetic control in Neurospora. CATs are short, thin, usually unbranched and arise from conidia or germ tubes. Their formation is conidium-density dependent, and CATs grow towards each other. MAP kinase mutants of Neurospora are blocked in CAT induction. Nuclei pass through fused CATs and are potential agents of gene exchange between individuals of the same and different species. CAT fusion may also serve to improve the chances of colony establishment.