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

Kurian, Smija M.; Pietro, Antonio Di; Read, Nick D.

doi:10.1371/journal.pone.0195634

Cited by 28 publications

(67 citation statements)

References 63 publications

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“…They involved short cell protrusions/hyphae that were thinner than germ tubes ( Fig. 2B) and superficially resembled conidial anastomosis tubes (CATs) in other fungi (20)(21)(22). An increase in the incubation time to 32 h did not result in an increase in the number of fusion events (data not shown).…”

Section: Resultsmentioning

confidence: 82%

“…The conidia and germ tubes of N. crassa and many other filamentous ascomycetes undergo cell fusion (self fusion) during colony initiation by means of specialized cell protrusions called conidial anastomosis tubes (CATs) that permit the exchange of organelles and nutrients (24). However, the early onset of frequent cell fusion in A. fumigatus would be detrimental to the assessment of the fitness of mutant strains in a pooled culture, because it facilitates nuclear exchange between germlings (18)(19)(20).…”

Section: Resultsmentioning

confidence: 99%

“…Filamentous fungi commonly undergo constitutive vegetative cell fusion, which permits the exchange of cell components including nutrients, water, signal molecules, and genetic material (18)(19)(20)(21)23). Vegetative cell fusion has been most extensively studied in the model N. crassa (22,23).…”

Section: Discussionmentioning

confidence: 99%

“…The sites of fusion between conidia and between germ tubes were identified for the first time in A. fumigatus. They involved short cell protrusions/ hyphae that were characteristically thinner than germ tubes and superficially resembled conidial anastomosis tubes (CATs) in N. crassa and Fusarium oxysporum (20,21).…”

Section: Discussionmentioning

confidence: 99%

“…A number of issues have prevented this technology from being used with Aspergillus fumigatus, including the fact that it is a haploid organism and that a high-throughput gene knockout methodology was not previously available to generate a suitable library of deletion mutants. In addition, filamentous fungi, unlike yeasts, commonly undergo prolific vegetative cell fusion, which permits the exchange of cell components including genetic material (18)(19)(20) that would ultimately prevent the successful analysis of the fitness of individual genetic isolates in a pooled culture. Vegetative cell fusion is very common and occurs constitutively in diverse filamentous fungi; it has been most extensively investigated in the model Neurospora crassa (19,(21)(22)(23).…”

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confidence: 99%

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Inducible Cell Fusion Permits Use of Competitive Fitness Profiling in the Human Pathogenic Fungus Aspergillus fumigatus

Macdonald

Thomson

Johns

et al. 2019

Antimicrob Agents Chemother

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Antifungal agents directed against novel therapeutic targets are required for treating invasive, chronic, and allergic Aspergillus infections. Competitive fitness profiling technologies have been used in a number of bacterial and yeast systems to identify druggable targets; however, the development of similar systems in filamentous fungi is complicated by the fact that they undergo cell fusion and heterokaryosis. Here, we demonstrate that cell fusion in Aspergillus fumigatus under standard culture conditions is not predominately constitutive, as with most ascomycetes, but can be induced by a range of extracellular stressors. Using this knowledge, we have developed a barcode-free genetic profiling system that permits high-throughput parallel determination of strain fitness in a collection of diploid A. fumigatus mutants. We show that heterozygous cyp51A and arf2 null mutants have reduced fitness in the presence of itraconazole and brefeldin A, respectively, and a heterozygous atp17 null mutant is resistant to brefeldin A.

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Section: Resultsmentioning

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Inducible Cell Fusion Permits Use of Competitive Fitness Profiling in the Human Pathogenic Fungus Aspergillus fumigatus

Macdonald

Thomson

Johns

et al. 2019

Antimicrob Agents Chemother

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A pipeline for rapidly evaluating activity and inferring mechanisms of action of prospective antifungal compounds

Kraut‐Cohen,

Frenkel,

Covo

et al. 2024

Pest Management Science

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BACKGROUNDFungal phytopathogens are a significant threat to crops and food security, and there is a constant need to develop safe and effective compounds that antagonize them. In‐planta assays are complex and tedious and are thus not suitable for initial high‐throughput screening of new candidate antifungal compounds. We propose an in vitro screening pipeline that integrates five rapid quantitative and qualitative methods to estimate the efficacy and mode of action of prospective antifungal compounds.RESULTSThe pipeline was evaluated using five documented antifungal compounds (benomyl, catechol, cycloheximide, 2,4‐diacetylphloroglucinol, and phenylacetic acid) that have different modes of action and efficacy, against the model soilborne fungal pathogen Fusarium oxysporum f. sp. radicis cucumerinum. We initially evaluated the five compounds' ability to inhibit fungal growth and metabolic activity using green fluorescent protein (GFP)‐labeled F. oxysporum and PrestoBlue staining, respectively, in multiwell plate assays. We tested the compounds' inhibition of both conidial germination and hyphal elongation. We then employed FUN‐1 and SYTO9/propidium iodide staining, coupled to confocal microscopy, to differentiate between fungal growth inhibition and death at the cellular level. Finally, using a reactive oxygen species (ROS)‐detection assay, we were able to quantify ROS production in response to compound application.CONCLUSIONSCollectively, the proposed pipeline provides a wide array of quantitative and qualitative data on the tested compounds that can help pinpoint promising novel compounds; these can then be evaluated more vigorously using in planta screening assays. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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Establishment of conidial fusion in the asexual fungus Verticillium dahliae as a useful system for the study of non-sexual genetic interactions

2021

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Cell-to-cell fusion is a fundamental biological process across the tree of life. In filamentous fungi, somatic fusion (or anastomosis) is required for the normal development of their syncytial hyphal networks, and it can initiate non-sexual genetic exchange processes, such as horizontal genetic transfer and the parasexual cycle. Although these could be important drivers of the evolution of asexual fungi, this remains a largely unexplored possibility due to the lack of suitable resources for their study in these puzzling organisms. We thus aimed at the characterization of cell fusion in the important asexual fungus Verticillium dahliae via Conidial Anastomosis Tubes (CATs), which can be useful for the analysis of parasexuality. We optimized appropriate procedures for their highly reproducible quantification and live-cell imaging, which were used to characterize their physiology and cell biology, and to start elucidating their underlying genetic machinery. Formation of CATs was shown to depend on growth conditions and require functional Fus3 and Slt2 MAP kinases, as well as the NADPH oxidase NoxA, whereas the GPCR Ste2 and the mating-type protein MAT1-2-1 were dispensable. We show that nuclei and other organelles can migrate through CATs, which often leads to the formation of transient dikaryons. Their nuclei have possible windows of opportunity for genetic interaction before degradation of one by a presumably homeostatic mechanism. We establish here CAT-mediated fusion in V. dahliae as an experimentally convenient system for the cytological analysis of fungal non-sexual genetic interactions. We expect that it will facilitate the dissection of sexual alternatives in asexual fungi.

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

Cited by 28 publications

References 63 publications

Inducible Cell Fusion Permits Use of Competitive Fitness Profiling in the Human Pathogenic Fungus Aspergillus fumigatus

Inducible Cell Fusion Permits Use of Competitive Fitness Profiling in the Human Pathogenic Fungus Aspergillus fumigatus

A pipeline for rapidly evaluating activity and inferring mechanisms of action of prospective antifungal compounds

Establishment of conidial fusion in the asexual fungus Verticillium dahliae as a useful system for the study of non-sexual genetic interactions

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