Molecular characterization of the NADPH oxidase complex in the ergot fungus Claviceps purpurea: CpNox2 and CpPls1 are important for a balanced host-pathogen interaction.

Reactive oxygen species producing NADPH oxidase (Nox) complexes are involved in defense reactions in animals and plants while they trigger infection-related processes in pathogenic fungi. Knowledge about the composition and localization of these complexes in fungi is limited; potential components identified thus far include two to three catalytical subunits, a regulatory subunit (NoxR), the GTPase Rac, the scaffold protein Bem1, and a tetraspanin-like membrane protein (Pls1). We showed that, in the biotrophic grass-pathogen Claviceps purpurea, the catalytical subunit CpNox1 is important for infection. Here, we present identification of major Nox complex partners and a functional analysis of CpNox2 and the tetraspanin CpPls1. We show that, as in other fungi, Nox complexes are important for formation of sclerotia; CpRac is, indeed, a complex partner because it interacts with CpNoxR, and CpNox1/2 and CpPls1 are associated with the endoplasmatic reticulum. However, unlike in all other fungi, Δcppls1 is more similar to Δcpnox1 than to Δcpnox2, and CpNox2 is not essential for infection. In contrast, Δcpnox2 shows even more pronounced disease symptoms, indicating that Cpnox2 controls the infection process and moderates damage to the host. These data confirm that fungal Nox complexes have acquired specific functions dependent of the lifestyle of the pathogen.

Cordyceps sinensis and its fractions stimulate MA-10 mouse Leydig tumor cell steroidogenesis.

The effects of Cordyceps sinensis (CS) and its extracted fractions on steroidogenesis in MA-10 cells were determined. Different concentrations of CS and 3 fractions of CS (F1, a water-soluble polysaccharide; F2, a water-soluble protein; and F3, a poorly water-soluble polysaccharide and protein) were added to MA-10 mouse Leydig tumor cells with or without human chorionic gonadotropin (hCG), and the production of steroid and the expression of steroidogenic acute regulatory protein (StAR) were examined. The results showed that CS alone (2-10 mg/mL) stimulated MA-10 cell progesterone production in a dose-dependent relationship. Fractions F1 and F3 (2-10 mg/mL) also had significant (P < .05) stimulatory effects on MA-10 cell steroidogenesis with a dose-dependent relationship. However, fraction F2 did not have an effect on MA-10 cells. CS and F3, but not F1, significantly induced more steroid production in hCG-stimulated MA-10 cells (P < .05). As a temporal relationship, F1 and F3 (2 mg/mL) maximally stimulated progesterone production between 1 and 3 hours after stimulation in MA-10 cells. In addition, CS and F3 significantly enhanced MA-10 cell StAR protein expression, which indicates that CS and F3 may use a cyclic adenosine monophosphate signal transduction pathway to activate MA-10 Leydig cell steroidogenesis in a manner to that of luteinizing hormone.

Application of oxygen vectors to Claviceps purpurea cultivation.

The application of a two-phase fermentation system for the production of ergot peptide alkaloids by Claviceps purpurea is described. Perfluorocarbons (PFC) are used as oxygen vectors in Claviceps fermentation for the first time. In shake-flask cultivations, the inclusion of PFC in the medium brings about a five-fold increase in the total alkaloid production and a six-fold increase in the pharmaceutically important component, ergotamine. This rise cannot be correlated with the concentration of the added PFC and it is thought that the enhancement is due to a combination of factors, including the influence of PFC. Other oxygen vectors, such as several hydrocarbons, prove to be poor oxygen carriers in our study. Cultivations with PFC in a bioreactor are reproducible, the maximum total alkaloid and ergotamine production being attained on the 11th and 9th days, respectively. The relatively lower increase in the total alkaloid production in the bioreactor as compared to the shake-flasks is attributed to the unequal oxygen availability in the reactor. Processes with PFC offer the operational advantage of a five-fold reduction in aeration rate.

Cell volume regulation in Claviceps fusiformis. An animal-type Na,K-ATPase operating in a fungus?

Claviceps fusiformis, an ergot pathogen of pearl millet, was equilibrated in media without and with 0.1 mM ouabain (g-strophantin). Highly significant effects of ouabain on the contents of sodium ions and water were found, suggesting that an active transport of sodium ions, similar to that in animal cells, regulates the cell volume of the fungus. The basic role of the sodium pump appears to be to decrease larger cell volumes and to increase the small ones; for this purpose it can even reverse its direction.

Cytochemical localization of polysaccharides in Claviceps paspali ultrastructure during submerged fermentation of alkaloids.

Morphological characteristics of two types of elements in the submerged mycelium of Claviceps paspali are described. Distribution of polysaccharides in the cell wall and cytoplasm was cytochemically determined at the ultrastructural level. Polysaccharide deposition into the cell walls was proportional to the increase in the alkaloid yield. In the cytoplasm, on the other hand, the presence of polysaccharide grains indicated an absence of alkaloid synthesis.