Quick-Freeze, Deep-Etch Electron Microscopy Reveals the Characteristic Architecture of the Fission Yeast Spore

The spore of the fission yeast Schizosaccharomyces pombe is a dormant cell that is resistant to a variety of environmental stresses. The S. pombe spore is coated by a proteinaceous surface layer, termed the Isp3 layer because it comprises mainly Isp3 protein. Although thin-section electron microscopy and scanning electron microscopy have revealed the fundamental structure of the spore, its architecture remains unclear. Here we visualized S. pombe spores by using a quick-freeze replica electron microscopy (QFDE-EM) at nanometer resolution, which revealed novel characteristic structures. QFDE-EM revealed that the Isp3 layer exists as an interwoven fibrillar layer. On the spore cell membrane, many deep invaginations, which are longer than those on the vegetative cell membrane, are aligned in parallel. We also observed that during spore germination, the cell surface changes from a smooth to a dendritic filamentous structure, the latter being characteristic of vegetative cells. These findings provide significant insight into not only the structural composition of the spore, but also the mechanism underlying the stress response of the cell.

Spore Viability and Cell Wall Integrity of Cordyceps pruinosa Treated with an Electric Shock-Free, Atmospheric-Pressure Air Plasma Jet

Atmospheric-pressure Ar plasma jets are known to be detrimental to Cordyceps pruinosa spores. However, it is not clear what kinds of reactive species are more effective with regard to fungal cell death. Herein, we study which reactive species plays pivotal roles in the death of fungal spores using an electric shock-free, atmospheric-pressure air plasma jet, simply called soft plasma jet. Plasma treatment significantly reduced the spore viability and damaged fungal DNA. As observed from the circular dichroism spectra, scanning electron microscope images, and flow cytometric measurements, cell wall integrity was decreased by reactive oxygen and nitrogen species (RONS) from the plasma itself and the plasma-activated water. Consequently, degradation of the spore cell wall allows RONS from the plasma to reach the intracellular components. Such plasma-induced intracellular RONS can attack spore DNA and other intracellular components, as confirmed by electrophoresis analysis and phosphorylated histone measurement. In addition, weakening of the spore cell wall allowed for the loss of intracellular components, which can lead to cell death. Plasma radicals were investigated by measuring the optical emission spectrum of the soft plasma jet, and intracellular reactive oxygen species were confirmed by measuring the fluorescence of 2′, 7′-dichlorodihydrofluorescein-diacetate (H2DCF-DA)-stained spores. The soft plasma jet generated considerable amounts of H2O2 and NOx but a very small number of OH radicals as compared to the atmospheric-pressure Arplasma jet; this indicates that plasma-induced long-lived reactive species (H2O2 and NOx) play an important role in the weakening of spore cell walls and cell death.

Efficacy of potential biocontrol agents for control of Fusarium verticillioides and fumonisin B1 under different environmental conditions

A mycotoxigenic strain of Fusarium verticillioides previously isolated from Malaysian maize kernels and identified morphologically and molecularly was used in the present work. The objectives were (1) to screen the competitiveness of three potential antagonists isolated from Malaysian maize and three other known candidates for control of growth of F. verticillioides in vitro based on interaction scores, growth rates and hyphal area of F. verticillioides, and (2) to examine the best candidates using different spore/cell ratios on milled maize agar at different water activity conditions on relative control of fumonisin B1 (FB1). Three fungi and three bacteria (BCAs 1-6) were examined for antagonistic effects against F. verticillioides in dual-culture assays. These showed that all fungal candidates intermingled with F. verticillioides while all bacterial candidates inhibited F. verticillioides on contact or at a distance, which in turn decreased the growth rates and hyphal area of F. verticillioides significantly. Although BCA1 (Clonostachys rosea 016) did not inhibit growth or hyphal area of F. verticillioides, it was included in FB1 inhibition studies with other bacterial candidates (BCA4, Streptomyces sp. AS1; BCA5, Gram-negative rod; BCA6, Enterobacter hormaechei) because of its established mycoparasitism. The FB1 inhibition studies were conducted on milled maize agar with different spore/cell ratios of pathogen:antagonist mixtures at 0.95/0.98 water activity (aw) and 25 °C for 14 days. FB1 biosynthesis for all treatments was significantly higher at 0.95 than 0.98 aw. Of the four antagonists tested, the best was BCA1 which inhibited FB1 biosynthesis by 73 and 100% at 0.95 and 0.98 aw, respectively. BCA5 was the next best, resulting in 38 and 78% FB1 inhibition at 0.95 and 0.98 aw, respectively. The pathogen:antagonist ratios for BCA1 and BCA5 showed best results at 50:50 and 25:75. These results were discussed in context of using biocontrol agents to minimise fumonisins in maize.

Beyond Animals and Plants: Dynamic Maternal Effects in the FungusNeurospora crassa

Maternal effects are widely documented in animals and plants, but not in fungi or other eukaryotes. A principal cause of maternal effects is asymmetrical parental investment in a zygote, creating greater maternal versus paternal influence on offspring phenotypes. Asymmetrical investments are not limited to animals and plants, but are also prevalent in fungi and groups including apicocomplexans, dinoflagellates, and red algae. Evidence suggesting maternal effects among fungi is sparse and anecdotal. In an experiment designed to test for maternal effects across sexual reproduction in the model fungusNeurospora crassa, we measured offspring phenotypes from crosses of all possible pairs of 22 individuals. Crosses encompassed reciprocals of 11 mating-type "A" and 11 mating-type "a" wild strains. After controlling for the genetic and geographic distances between strains in any individual cross, we found strong evidence for maternal control of sporocarp production, as well as maternal effects on spore numbers and spore germination. However, both parents exert equal influence on the percentage of spores that are pigmented, and size of pigmented spores. We propose a model linking the stage-specific presence or absence of maternal effects to cellular developmental processes: effects appear to be mediated primarily through the maternal cytoplasm, and, after spore cell walls form, maternal influence on spore development is limited. Maternal effects in fungi, thus far largely ignored, are likely to shape species' evolution and ecology. The association of anisogamy and maternal effects in a fungus suggests maternal effects may also influence the biology of other anisogamous eukaryotes.

Bestatin Inhibits Cell Growth, Cell Division, and Spore Cell Differentiation in Dictyostelium discoideum

ABSTRACTBestatin methyl ester (BME) is an inhibitor of Zn2+-binding aminopeptidases that inhibits cell proliferation and induces apoptosis in normal and cancer cells. We have usedDictyosteliumas a model organism to study the effects of BME. Only two Zn2+-binding aminopeptidases have been identified inDictyosteliumto date, puromycin-sensitive aminopeptidase A and B (PsaA and PsaB). PSA from other organisms is known to regulate cell division and differentiation. Here we show that PsaA is differentially expressed throughout growth and development ofDictyostelium, and its expression is regulated by developmental morphogens. We present evidence that BME specifically interacts with PsaA and inhibits its aminopeptidase activity. Treatment of cells with BME inhibited the rate of cell growth and the frequency of cell division in growing cells and inhibited spore cell differentiation during late development. Overexpression of PsaA-GFP (where GFP is green fluorescent protein) also inhibited spore cell differentiation but did not affect growth. Using chimeras, we have identified that nuclear versus cytoplasmic localization of PsaA affects the choice between stalk or spore cell differentiation pathway. Cells that overexpressed PsaA-GFP (primarily nuclear) differentiated into stalk cells, while cells that overexpressed PsaAΔNLS2-GFP (cytoplasmic) differentiated into spores. In conclusion, we have identified that BME inhibits cell growth, division, and differentiation inDictyosteliumlikely through inhibition of PsaA.

Dependence of Stress Resistance on a Spore Coat Heteropolysaccharide in Dictyostelium

ABSTRACT In Dictyostelium, sporulation occurs synchronously as prespore cells approach the apex of the aerial stalk during culmination. Each prespore cell becomes surrounded by its own coat comprised of a core of crystalline cellulose and a branched heteropolysaccharide sandwiched between heterogeneous cysteine-rich glycoproteins. The function of the heteropolysaccharide, which consists of galactose and N-acetylgalactosamine, is unknown. Two glycosyltransferase-like genes encoding multifunctional proteins, each with predicted features of a heteropolysaccharide synthase, were identified in the Dictyostelium discoideum genome. pgtB and pgtC transcripts were modestly upregulated during early development, and pgtB was further intensely upregulated at the time of heteropolysaccharide accumulation. Disruption of either gene reduced synthase-like activity and blocked heteropolysaccharide formation, based on loss of cytological labeling with a lectin and absence of component sugars after acid hydrolysis. Cell mixing experiments showed that heteropolysaccharide expression is spore cell autonomous, suggesting a physical association with other coat molecules during assembly. Mutant coats expressed reduced levels of crystalline cellulose based on chemical analysis after acid degradation, and cellulose was heterogeneously affected based on flow cytometry and electron microscopy. Mutant coats also contained elevated levels of selected coat proteins but not others and were sensitive to shear. Mutant spores were unusually susceptible to hypertonic collapse and damage by detergent or hypertonic stress. Thus, the heteropolysaccharide is essential for spore integrity, which can be explained by a role in the formation of crystalline cellulose and regulation of the protein content of the coat.

Prespore Cell Fate Bias in G1 Phase of the Cell Cycle in Dictyostelium discoideum

ABSTRACT By generating a population of Dictyostelium cells that are in the G1 phase of the cell cycle we have examined the influence of cell cycle status on cell fate specification, cell type proportioning and its regulation, and terminal differentiation. The lack of observable mitosis during the development of these cells and the quantification of their cellular DNA content suggests that they remain in G1 throughout development. Furthermore, chromosomal DNA synthesis was not detectable these cells, indicating that no synthesis phase had occurred, although substantial mitochondrial DNA synthesis did occur in prespore cells. The G1-phase cells underwent normal morphological development and sporulation but displayed an elevated prespore/prestalk ratio of 5.7 compared to the 3.0 (or 3:1) ratio normally observed in populations dominated by G2-phase cells. When migrating slugs produced by G1-phase cells were bisected, each half could reestablish the 5.7 (or 5.7:1) prespore/prestalk ratio. These results demonstrate that Dictyostelium cells can carry out the entire developmental cycle in the G1 phase of the cell cycle and that passage from G2 into G1 phase is not required for sporulation. Our results also suggest that the population asymmetry provided by the distribution of cells around the cell cycle at the time of starvation is not strictly required for cell type proportioning. Finally, when developed together with G2-phase cells, G1-phase cells preferentially become prespore cells and exclude G2-phase cells from the prespore-spore cell population, suggesting that G1-phase cells have an advantage over G2-phase cells in executing the spore cell differentiation pathway.