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.

Carbon source-dependent variation of acquired mutagen resistance of Moniliophthora perniciosa: similarities in natural and artificial systems.

The basidiomycete Moniliophthora perniciosa causes Witches' Broom disease in Theobroma cacao. We studied the influence of carbon source on conditioning hyphae to oxidative stress agents (H(2)O(2), paraquat, 4NQO) and to UVC, toward the goal of assessing the ability of this pathogen to avoid plant defenses involving ROS. Cells exhibited increased resistance to H(2)O(2) when shifted from glucose to glycerol and from glycerol to glycerol. When exposed to paraquat, cells grown in fresh medium were always more resistant. Apparently glycerol and/or fresh media, but not old glucose media, up-regulate oxidative stress defenses in this fungus. For the mutagens UVC and 4NQO, whose prime action on DNA is not via ROS, change of carbon source did not elicit a clear change in sensitivity/resistance. These results correlate with expression of fungal genes that protect against ROS and with biochemical changes observed in infected cacao tissues, where glycerol and high amounts of ROS have been detected in green brooms.

Rapid and efficient protocol for DNA extraction and molecular identification of the basidiomycete Crinipellis perniciosa.

DNA isolation from some fungal organisms is difficult because they have cell walls or capsules that are relatively unsusceptible to lysis. Beginning with a yeast Saccharomyces cerevisiae genomic DNA isolation method, we developed a 30-min DNA isolation protocol for filamentous fungi by combining cell wall digestion with cell disruption by glass beads. High-quality DNA was isolated with good yield from the hyphae of Crinipellis perniciosa, which causes witches' broom disease in cacao, from three other filamentous fungi, Lentinus edodes, Agaricus blazei, Trichoderma stromaticum, and from the yeast S. cerevisiae. Genomic DNA was suitable for PCR of specific actin primers of C. perniciosa, allowing it to be differentiated from fungal contaminants, including its natural competitor, T. stromaticum.

Rapid and efficient protocol for DNA extraction and molecular identification of the basidiomycete Crinipellis perniciosa

DNA isolation from some fungal organisms is difficult because they have cell walls or capsules that are relatively unsusceptible to lysis. Beginning with a yeast Saccharomyces cerevisiae genomic DNA isolation method, we developed a 30-min DNA isolation protocol for filamentous fungi by combining cell wall digestion with cell disruption by glass beads. High-quality DNA was isolated with good yield from the hyphae of Crinipellis perniciosa, which causes witches' broom disease in cacao, from three other filamentous fungi, Lentinus edodes, Agaricus blazei, Trichoderma stromaticum, and from the yeast S. cerevisiae. Genomic DNA was suitable for PCR of specific actin primers of C. perniciosa, allowing it to be differentiated from fungal contaminants, including its natural competitor, T. stromaticum.