scholarly journals Conidial fusion in the asexual fungus Verticillium dahliae

2020 ◽  
Author(s):  
Vasileios Vangalis ◽  
Michael Knop ◽  
Milton A. Typas ◽  
Ioannis A. Papaioannou
Keyword(s):  
Cell Fusion ◽  
Verticillium Dahliae ◽  
Cell Biology ◽  
Map Kinases ◽  
Genetic Interaction ◽  
Pathogenic Fungus ◽  
Growth Conditions ◽  
Parasexual Cycle ◽  
Asexual Fungi ◽  
Fundamental Biological Process

AbstractCell-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. In this study, we report that the spores of the important asexual plant-pathogenic fungus Verticillium dahliae often engage in cell fusion via Conidial Anastomosis Tubes (CATs). 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.

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

2021 ◽  
Author(s):  
Vasileios Vangalis ◽  
Michael Knop ◽  
Milton A. Typas ◽  
Ioannis A. Papaioannou
Keyword(s):  
Cell Fusion ◽  
Verticillium Dahliae ◽  
Cell Biology ◽  
Map Kinases ◽  
Genetic Interaction ◽  
Growth Conditions ◽  
Genetic Interactions ◽  
Parasexual Cycle ◽  
Asexual Fungi ◽  
Fundamental Biological Process

AbstractCell-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.

scholarly journals The Basolateral Polarity Module Promotes Slit Diaphragm Formation in Drosophila Nephrocytes, a Model of Vertebrate Podocytes

2021 ◽  
pp. ASN.2020071050
Author(s):  
Michael Mysh ◽  
John S. Poulton
Keyword(s):  
Nephrotic Syndrome ◽  
Protein Trafficking ◽  
Cell Biology ◽  
Genetic Interaction ◽  
Intercellular Junctions ◽  
Protein Distribution ◽  
Genetic Manipulations ◽  
Apicobasal Polarity ◽  
Apical Polarity ◽  
Polarity Proteins

BackgroundPodocyte slit diaphragms (SDs) are intercellular junctions that function as size-selective filters, excluding most proteins from urine. Abnormalities in SDs cause proteinuria and nephrotic syndrome. Podocytes exhibit apicobasal polarity, which can affect fundamental aspects of cell biology, including morphology, intercellular junction formation, and asymmetric protein distribution along the plasma membrane. Apical polarity protein mutations cause nephrotic syndrome, and data suggest apical polarity proteins regulate SD formation. However, there is no evidence that basolateral polarity proteins regulate SDs. Thus, the role of apicobasal polarity in podocytes remains unclear.MethodsGenetic manipulations and transgenic reporters determined the effects of disrupting apicobasal polarity proteins in Drosophila nephrocytes, which have SDs similar to those of mammalian podocytes. Confocal and electron microscopy were used to characterize SD integrity after loss of basolateral polarity proteins, and genetic-interaction studies illuminated relationships among apicobasal polarity proteins.ResultsThe study identified four novel regulators of nephrocyte SDs: Dlg, Lgl, Scrib, and Par-1. These proteins comprise the basolateral polarity module and its effector kinase. The data suggest these proteins work together, with apical polarity proteins, to regulate SDs by promoting normal endocytosis and trafficking of SD proteins.ConclusionsGiven the recognized importance of apical polarity proteins and SD protein trafficking in podocytopathies, the findings connecting basolateral polarity proteins to these processes significantly advance our understanding of SD regulation.

scholarly journals Hex1, the Major Component of Woronin Bodies, Is Required for Normal Development, Pathogenicity, and Stress Response in the Plant Pathogenic Fungus Verticillium dahliae

2020 ◽  
Vol 6 (4) ◽  
pp. 344
Author(s):  
Vasileios Vangalis ◽  
Ioannis A. Papaioannou ◽  
Emmanouil A. Markakis ◽  
Michael Knop ◽  
Milton A. Typas
Keyword(s):  
Stress Response ◽  
Verticillium Dahliae ◽  
Pathogenic Fungus ◽  
Membrane Integrity ◽  
Fungal Growth ◽  
Growth Physiology ◽  
Cellular Processes ◽  
Ros Homeostasis ◽  
Membrane Bound ◽  
Septal Pores

Woronin bodies are membrane-bound organelles of filamentous ascomycetes that mediate hyphal compartmentalization by plugging septal pores upon hyphal damage. Their major component is the peroxisomal protein Hex1, which has also been implicated in additional cellular processes in fungi. Here, we analyzed the Hex1 homolog of Verticillium dahliae, an important asexual plant pathogen, and we report its pleiotropic involvement in fungal growth, physiology, stress response, and pathogenicity. Alternative splicing of the Vdhex1 gene can lead to the production of two Hex1 isoforms, which are structurally similar to their Neurospora crassa homolog. We show that VdHex1 is targeted to the septum, consistently with its demonstrated function in sealing hyphal compartments to prevent excessive cytoplasmic bleeding upon injury. Furthermore, our investigation provides direct evidence for significant contributions of Hex1 in growth and morphogenesis, as well as in asexual reproduction capacity. We discovered that Hex1 is required both for normal responses to osmotic stress and factors that affect the cell wall and plasma-membrane integrity, and for normal resistance to oxidative stress and reactive oxygen species (ROS) homeostasis. The Vdhex1 mutant exhibited diminished ability to colonize and cause disease on eggplant. Overall, we show that Hex1 has fundamentally important multifaceted roles in the biology of V. dahliae.

scholarly journals Separating myoblast differentiation from muscle cell fusion using IGF-I and the p38 MAP kinase inhibitor SB202190

2015 ◽  
Vol 309 (7) ◽  
pp. C491-C500 ◽  
Author(s):  
Samantha Gardner ◽  
Sean M. Gross ◽  
Larry L. David ◽  
John E. Klimek ◽  
Peter Rotwein
Keyword(s):  
Growth Factor ◽  
Map Kinase ◽  
Cell Fusion ◽  
Muscle Cell ◽  
Map Kinases ◽  
Kinase Inhibitor ◽  
P38 Map Kinase ◽  
Myoblast Differentiation ◽  
Igf I ◽  
Muscle Biology

The p38 MAP kinases play critical roles in skeletal muscle biology, but the specific processes regulated by these kinases remain poorly defined. Here we find that activity of p38α/β is important not only in early phases of myoblast differentiation, but also in later stages of myocyte fusion and myofibrillogenesis. By treatment of C2 myoblasts with the promyogenic growth factor insulin-like growth factor (IGF)-I, the early block in differentiation imposed by the p38 chemical inhibitor SB202190 could be overcome. Yet, under these conditions, IGF-I could not prevent the later impairment of muscle cell fusion, as marked by the nearly complete absence of multinucleated myofibers. Removal of SB202190 from the medium of differentiating myoblasts reversed the fusion block, as multinucleated myofibers were detected several hours later and reached ∼90% of the culture within 30 h. Analysis by quantitative mass spectroscopy of proteins that changed in abundance following removal of the inhibitor revealed a cohort of upregulated muscle-enriched molecules that may be important for both myofibrillogenesis and fusion. We have thus developed a model system that allows separation of myoblast differentiation from muscle cell fusion and should be useful in identifying specific steps regulated by p38 MAP kinase-mediated signaling in myogenesis.

scholarly journals A drug-compatible and temperature-controlled microfluidic device for live-cell imaging

Open Biology ◽  
2016 ◽  
Vol 6 (8) ◽  
pp. 160156 ◽  
Author(s):  
Tong Chen ◽  
Blanca Gomez-Escoda ◽  
Javier Munoz-Garcia ◽  
Julien Babic ◽  
Laurent Griscom ◽  
...  
Keyword(s):  
Cell Biology ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Critical Issue ◽  
Growth Conditions ◽  
Medium Composition ◽  
Live Cell ◽  
Model Systems ◽  
Dynamic Regulation ◽  
Cellular Environment

Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging.

scholarly journals N-linked glycosylation enzymes in the diatom Thalassiosira oceanica exhibit a diel cycle in transcript abundance and favor for NXT-type sites

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Joerg Behnke ◽  
Alejandro M. Cohen ◽  
Julie LaRoche
Keyword(s):  
Cell Biology ◽  
Biochemical Characterization ◽  
Solid Phase ◽  
Transcript Abundance ◽  
Growth Conditions ◽  
Iron Starvation ◽  
Glycosylation Sites ◽  
Glycosylation Pathway ◽  
And Function

AbstractN-linked glycosylation is a posttranslational modification affecting protein folding and function. The N-linked glycosylation pathway in algae is poorly characterized, and further knowledge is needed to understand the cell biology of algae and the evolution of N-linked glycosylation. This study investigated the N-linked glycosylation pathway in Thalassiosira oceanica, an open ocean diatom adapted to survive at growth-limiting iron concentrations. Here we identified and annotated the genes coding for the essential enzymes involved in the N-linked glycosylation pathway of T. oceanica. Transcript levels for genes coding for calreticulin, oligosaccharyltransferase (OST), N-acetylglucosaminyltransferase (GnT1), and UDP-glucose glucosyltransferase (UGGT) under high- and low-iron growth conditions revealed diel transcription patterns with a significant decrease of calreticulin and OST transcripts under iron-limitation. Solid-phase extraction of N-linked glycosylated peptides (SPEG) revealed 118 N-linked glycosylated peptides from cells grown in high- and low-iron growth conditions. The identified peptides had 81% NXT-type motifs, with X being any amino acids except proline. The presence of N-linked glycosylation sites in the iron starvation-induced protein 1a (ISIP1a) confirmed its predicted topology, contributing to the biochemical characterization of ISIP1 proteins. Analysis of extensive oceanic gene databases showed a global distribution of calreticulin, OST, and UGGT, reinforcing the importance of glycosylation in microalgae.

scholarly journals TheBeauveria bassianaGas3 β-Glucanosyltransferase Contributes to Fungal Adaptation to Extreme Alkaline Conditions

2018 ◽  
Vol 84 (15) ◽  
Author(s):  
Zhibing Luo ◽  
Tongbing Zhang ◽  
Pengfei Liu ◽  
Yuting Bai ◽  
Qiyan Chen ◽  
...  
Keyword(s):  
Cell Wall ◽  
Pathogenic Fungus ◽  
Growth Conditions ◽  
Alkaline Ph ◽  
Ph Responsive ◽  
Content Type ◽  
Cell Wall Remodeling ◽  
Alkaline Conditions ◽  
Increased Sensitivity ◽  
Ph Dependent

ABSTRACTFungal β-1,3-glucanosyltransferases are cell wall-remodeling enzymes implicated in stress response, cell wall integrity, and virulence, with most fungal genomes containing multiple members. The insect-pathogenic fungusBeauveria bassianadisplays robust growth over a wide pH range (pH 4 to 10). A random insertion mutant library screening for increased sensitivity to alkaline (pH 10) growth conditions resulted in the identification and mapping of a mutant to a β-1,3-glucanosyltransferase gene (Bbgas3).Bbgas3expression was pH dependent and regulated by the PacC transcription factor, which activates genes in response to neutral/alkaline growth conditions. Targeted gene knockout ofBbgas3resulted in reduced growth under alkaline conditions, with only minor effects of increased sensitivity to cell wall stress (Congo red and calcofluor white) and no significant effects on fungal sensitivity to oxidative or osmotic stress. The cell walls of ΔBbgas3aerial conidia were thinner than those of the wild-type and complemented strains in response to alkaline conditions, and β-1,3-glucan antibody and lectin staining revealed alterations in cell surface carbohydrate epitopes. The ΔBbgas3mutant displayed alterations in cell wall chitin and carbohydrate content in response to alkaline pH. Insect bioassays revealed impaired virulence for the ΔBbgas3mutant depending upon the pH of the media on which the conidia were grown and harvested. Unexpectedly, a decreased median lethal time to kill (LT50, i.e., increased virulence) was seen for the mutant using intrahemocoel injection assays using conidia grown at acidic pH (5.6). These data show that BbGas3 acts as a pH-responsive cell wall-remodeling enzyme involved in resistance to extreme pH (>9).IMPORTANCELittle is known about adaptations required for growth at high (>9) pH. Here, we show that a specific fungal membrane-remodeling β-1,3-glucanosyltransferase gene (Bbgas3) regulated by the pH-responsive PacC transcription factor forms a critical aspect of the ability of the insect-pathogenic fungusBeauveria bassianato grow at extreme pH. The loss ofBbgas3resulted in a unique decreased ability to grow at high pH, with little to no effects seen with respect to other stress conditions, i.e., cell wall integrity and osmotic and oxidative stress. However, pH-dependent alternations in cell wall properties and virulence were noted for the ΔBbgas3 mutant. These data provide a mechanistic insight into the importance of the specific cell wall structure required to stabilize the cell at high pH and link it to the PacC/Pal/Rim pH-sensing and regulatory system.

The parasexual cycle in the entomopathogenic fungus Paecilomyces fumoso-roseus (Wize) Brown and Smith

1984 ◽  
Vol 30 (7) ◽  
pp. 922-926 ◽  
Author(s):  
Guy Riba ◽  
Anne M. Ravelojoana
Keyword(s):  
Entomopathogenic Fungus ◽  
Genetic Recombination ◽  
Agar Medium ◽  
Growth Conditions ◽  
Parasexual Cycle ◽  
Auxotrophic Mutants ◽  
Petri Dishes ◽  
Ultraviolet Treatment

Auxotrophic mutants of Paecilomyces fumoso-roseus obtained by ultraviolet treatment were used to demonstrate parasexual recombination. Formation of hybrid diploids was found to be closely related to growth conditions on artificial media and on host insects. One hundred percent of prototrophic hybrids were isolated from mycosed insects, while no more than 1.5% were recovered from the complete agar medium in the Petri dishes. A few haploids (1%) were obtained from hybrid diploids exposed to haploidizing agents. Some of the haploids were segregants which could only have resulted from genetic recombination.

scholarly journals Biocontrol potential of Trichoderma harzianum in controlling wilt disease of pistachio caused by Verticillium dahliae

2017 ◽  
Vol 57 (2) ◽  
pp. 185-193 ◽  
Author(s):  
Zeinab Fotoohiyan ◽  
Saeed Rezaee ◽  
Gholam Hosein Shahidi Bonjar ◽  
Amir Hossein Mohammadi ◽  
Mohammad Moradi
Keyword(s):  
Verticillium Dahliae ◽  
Trichoderma Harzianum ◽  
Pathogenic Fungus ◽  
Antagonistic Activity ◽  
Wilt Disease ◽  
Dual Culture ◽  
Volatile Metabolites ◽  
Biocontrol Potential

Abstract Verticillium wilt caused by Verticillium dahliae, is one of the most devastating diseases in pistachio orchards in the world including Iran. In search for an effective non-chemical strategy for the management of this disease, we evaluated the biocontrol potential of Trichoderma harzianum isolates obtained from the rhizosphere of healthy pistachio trees in different locations of the Kerman province of Iran against V. dahliae under laboratory and greenhouse conditions. Dual culture tests in the laboratory were conducted in a completely randomized design using 72 T. harzianum isolates. Twenty isolates showed the highest in vitro antagonistic activity. The results indicated that all 20 isolates were capable of inhibiting the mycelial growth of V. dahliae significantly. Among them, isolates Tr8 and Tr19 were the most effective by 88.89% and 85.12% inhibition, respectively. Extracted cell free metabolites of all effective isolates also inhibited the growth of V. dahliae in the culture medium significantly. According to the results, isolates Tr4 and Tr6 inhibited fungal pathogen growth by 94.94% and 88.15% respectively, through production of non-volatile metabolites. In the evaluation of volatile metabolites, isolates Tr5 and Tr4 were the most effective by 26.27% and 24.49% growth inhibition, respectively. Based on the results of the in vitro experiments, the five most effective isolates were selected for evaluation under greenhouse conditions for their biocontrol potential in controlling Verticillium wilt of pistachio. Results of the greenhouse, (in vivo) experiments were positive and indicated that the occurrence of wilt disease in plants treated with the antagonists alone or in combination with pathogenic fungus was lower than in plants inoculated with pathogen alone. The overall results of this study suggest that Trichoderma fungal antagonist may be an effective biocontrol agent for the control of Verticillium wilt of pistachio.

scholarly journals Mitochondrial fission dysfunction alleviates heterokaryon incompatibility-triggered cell death in the industrial filamentous fungus Aspergillus oryzae

2021 ◽  
Author(s):  
Chan Lu ◽  
Takuya Katayama ◽  
Noriko Mori ◽  
Ryota Saito ◽  
Kazuhiro Iwashita ◽  
...  
Keyword(s):  
Cell Death ◽  
Protoplast Fusion ◽  
Cell Fusion ◽  
Aspergillus Oryzae ◽  
Filamentous Fungus ◽  
Cellular Response ◽  
Mitochondrial Fission ◽  
Mitochondrial Fragmentation ◽  
Parasexual Cycle ◽  
Heterokaryon Incompatibility

ABSTRACTIn filamentous fungi, cell-to-cell recognition is a fundamental requirement for the formation, development, and maintenance of complex hyphal networks. Basically, self/compatible individuals within the fungal species are capable of fusing together, a step important for crossbreeding, which results in the formation of viable vegetative heterokaryons. Conversely, the fusion of incompatible individuals does not result in the formation of viable hyphal networks, but it often leads to growth inhibition or cell death. Even though a number of studies have been conducted to investigate such incompatibility, the understanding of the associated molecular mechanism is still limited, and this restricts the possibility of crossbreeding incompatible individuals. Therefore, in this study, the characteristics of compatibility/incompatibility in the industrial filamentous fungus, Aspergillus oryzae, were comprehensively investigated. Protoplast fusion and co-culture assays indicated the existence of a correlation between strain phylogeny and compatibility/incompatibility features. Time-course fluorescence observations were employed to investigate the types of incompatible responses that are induced at different cellular levels upon incompatible cell fusion, which eventually lead to cell death. Propidium iodide-indicated cell death, ROS accumulation, and mitochondrial fragmentation were identified as the major responses, with mitochondrial fragmentation showing the most significant subcellular change immediately after incompatible cell fusion. Furthermore, the deletions of mitochondrial fission-related genes Aofis1 and Aodnm1 in incompatible pairing alleviated cell death, indicating that mitochondrial fission is an important mechanism by which incompatibility-triggered cell death occurs. Therefore, this study provides new insights about heterokaryon incompatibility.IMPORTANCEFor a long time, it was believed that as an asexual fungus, A. oryzae does not exhibit any sexual cycle. However, the fungus has two mating types, indicating the potential for sexual reproduction besides a known parasexual cycle. Therefore, given that viable heterokaryon formation following cell fusion is an important step required for genetic crossing, we explored the mechanism of incompatibility, which restricts the possibility of cell fusion in A. oryzae. Protoplast fusion and co-culture assays led to the identification of various vegetative compatible groups. Mitochondrial fragmentation was found to be the most significant incompatible cellular response that occurred in organelles during incompatible pairing, while the deletion of mitochondrial fission-related genes was identified as a strategy used to alleviate incompatibility-triggered cell death. Thus, this study revealed a novel mechanism by which mitochondrial fission regulates incompatible responses.

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