Reactive oxygen species and autophagy play a role in survival and differentiation of the phytopathogen Moniliophthora perniciosa.

The hemibiotrophic basidiomycete Moniliophthora perniciosa causes "witches' broom disease" in cacao (Theobroma cacao). During plant infection, M. perniciosa changes from mono to dikaryotic life form, an event which could be triggered by changes in plant nutritional offer and plant defense molecules, i.e., from high to low content of glycerol and hydrogen peroxide. We have recently shown that in vitro glycerol induces oxidative stress resistance in dikaryotic M. perniciosa. In order to understand under which conditions in parasite-plant interaction M. perniciosa changes from intercellular monokaryotic to intracellular dikaryotic growth phase we studied the role of glycerol on mutagen-induced oxidative stress resistance of basidiospores and monokaryotic hyphae; we also studied the role of H(2)O(2) as a signaling molecule for in vitro dikaryotization and whether changes in nutritional offer by the plant could be compensated by inducible fungal autophagy. Mono-/dikaryotic glycerol or glucose-grown cells and basidiospores were exposed to the oxidative stress-inducing mutagens H(2)O(2) and Paraquat as well as to pre-dominantly DNA damaging 4-nitroquinoline-1-oxide and UVC irradiation. Basidiospores showed highest resistance to all treatments and glycerol-grown monokaryotic hyphae were more resistant than dikaryotic hyphae. Monokaryotic cells exposed to 1microM of H(2)O(2) in glycerol-media induced formation of clamp connections within 2 days while 1mM H(2)O(2) did not within a week in the same medium; no clamp connections were formed in H(2)O(2)-containing glucose media within a week. Lower concentrations of H(2)O(2) and glycerol, when occurring in parallel, are shown to be two signals for dikaryotization in vitro and may be also during the course of infection. Q-PCR studies of glycerol-grown dikaryotic cells exposed to oxidative stress (10mM H(2)O(2)) showed high expression of MpSOD2 and transient induction of ABC cytoplasmic membrane transporter gene MpYOR1 and autophagy-related gene MpATG8. Expression of a second ABC transporter gene MpSNQ2 was 14-fold induced after H(2)O(2) exposure in glucose as compared to glycerol-grown hyphae while MpYOR1 did not show strong variation of expression under similar conditions. Glucose-grown dikaryotic cells showed elevated expression of MpATG8, especially after exposure to H(2)O(2) and 4-nitroquinoline-1-oxide. During different stages preceding basidiocarp formation MpATG8 and the two catalase-encoding genes MpCTA1 and MpCTT1 were expressed continuously. We have compiled our results and literature data in a model graph, which compares the in vitro and in planta development and differentiation of M. perniciosa with the help of physiological and morphological landmarks.

Low productivity of ribonucleotide reductase in Saccharomyces cerevisiae increases sensitivity to stannous chloride.

Ribonucleotide reductase (RNR) of the yeast Saccharomyces cerevisiae is a tetrameric protein complex, consisting of two large and two small subunits. The small subunits Y2 and Y4 form a heterodimer and are encoded by yeast genes RNR2 and RNR4, respectively. Loss of Y4 in yeast mutant rnr4Delta can be compensated for by up-regulated expression of Y2, and the formation of a small subunit Y2Y2 homodimer that allows for a partially functional RNR. However, rnr4Delta mutants exhibit slower growth than wild-type (WT) cells and are sensitive to many mutagens, amongst them UVC and photo-activated mono- and bi-functional psoralens. Cells of the haploid rnr4Delta mutant also show a 3- to 4-fold higher sensitivity to the oxidative stress-inducing chemical stannous chloride than those of the isogenic WT. Both strains acquired increased resistance to SnCl2 with age of culture, i.e., 24-h cultures were more sensitive than cells grown for 2, 3, 4, and 5 days in liquid culture. However, the sensitivity factor of three to four (WT/mutant) did not change significantly. Cultures of the rnr4Delta mutant in stationary phase of growth always showed higher frequency of budding cells (budding index around 0.5) than those of the corresponding WT (budding index <0.1), pointing to a delay of mitosis/cytokinesis.

Low productivity of ribonucleotide reductase in Saccharomyces cerevisiae increases sensitivity to stannous chloride

Ribonucleotide reductase (RNR) of the yeast Saccharomyces cerevisiae is a tetrameric protein complex, consisting of two large and two small subunits. The small subunits Y2 and Y4 form a heterodimer and are encoded by yeast genes RNR2 and RNR4, respectively. Loss of Y4 in yeast mutant rnr4delta can be compensated for by up-regulated expression of Y2, and the formation of a small subunit Y2Y2 homodimer that allows for a partially functional RNR. However, rnr4delta mutants exhibit slower growth than wild-type (WT) cells and are sensitive to many mutagens, amongst them UVC and photo-activated mono- and bi-functional psoralens. Cells of the haploid rnr4delta mutant also show a 3- to 4-fold higher sensitivity to the oxidative stress-inducing chemical stannous chloride than those of the isogenic WT. Both strains acquired increased resistance to SnCl2 with age of culture, i.e., 24-h cultures were more sensitive than cells grown for 2, 3, 4, and 5 days in liquid culture. However, the sensitivity factor of three to four (WT/mutant) did not change significantly. Cultures of the rnr4delta mutant in stationary phase of growth always showed higher frequency of budding cells (budding index around 0.5) than those of the corresponding WT (budding index <0.1), pointing to a delay of mitosis/cytokinesis.

Sensitivity to Sn2+ of the yeast Saccharomyces cerevisiae depends on general energy metabolism, metal transport, anti-oxidative defences, and DNA repair.

Resistance to stannous chloride (SnCl(2)) of the yeast Saccharomyces cerevisiae is a product of several metabolic pathways of this unicellular eukaryote. Sensitivity testing of different null mutants of yeast to SnCl(2) revealed that DNA repair contributes to resistance, mainly via recombinational (Rad52p) and error-prone (Rev3p) steps. Independently, the membrane transporter Atr1p/Snq1p (facilitated transport) contributed significantly to Sn(2+)-resistance whereas absence of ABC export permease Snq2p did not enhance sensitivity. Sensitivity of the superoxide dismutase mutants sod1 and sod2 revealed the importance of these anti-oxidative defence enzymes against Sn(2+)-imposed DNA damage while a catalase-deficient mutant (ctt1) showed wild type (WT) resistance. Lack of transcription factor Yap1, responsible for the oxidative stress response in yeast, led to 3-fold increase in Sn(2+)-sensitivity. While loss of mitochondrial DNA did not change the Sn(2+)-resistance phenotype in any yeast strain, cells with defect cytochrome c oxidase (CcO mutants) showed gradually enhanced sensitivities to Sn(2+) and different spontaneous mutation rates. Highest sensitivity to Sn(2+) was observed when yeast was in exponential growth phase under glucose repression. During diauxic shift (release from glucose repression) Sn(2+)-resistance increased several hundred-fold and fully respiring and resting cells were sensitive only at more than 1000-fold exposure dose, i.e. they survived better at 25 mM than exponentially growing cells at 25 microM Sn(2+). This phenomenon was observed not only in WT but also in already Sn(2+)-sensitive rad52 as well as in sod1, sod2 and CcO mutant strains. The impact of metabolic steps in contribution to Sn(2+)-resistance had the following ranking: Resting WT cells > membrane transporter Snq1p > superoxide dismutases > transcription factor Yap1p >or= DNA repair >> exponentially growing WT cells.