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

Abstract

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.

dsRNA-induced gene silencing in Moniliophthora perniciosa, the causal agent of witches’ broom disease of cacao

“…This data is in agreement with the stability of gfp silencing during growth described above and further supports the conclusion that silencing is maintained during long periods. Recently, Pungartnik et al (2009) demonstrated the effects of ROS in the M. perniciosa biology, thus, the silencing of MpPRX1 will be very important to validate several hypothesis raised in this work.…”

High-Resolution Transcript Profiling of the Atypical Biotrophic Interaction betweenTheobroma cacaoand the Fungal PathogenMoniliophthora perniciosa

“…For instance, it will be important to determine how biotrophy is maintained for so long in M. perniciosa and how cacao senescence signals the end of this fungal stage. Given that nutrient availability and stress conditions seem to be tightly associated with M. perniciosa virulence and development (Alvim et al, 2009;Pungartnik et al, 2009;Thomazella et al, 2012;Argôlo Santos Carvalho et al, 2013), cacao nutritional status (e.g., carbon starvation) and/or specific plant molecules (e.g., ROS) possibly orchestrate WBD infection. It will be relevant to establish to what extent and how the alterations identified in cacao metabolism/physiology correlate with direct fungal action.…”

Carbon source-induced changes in the physiology of the cacao pathogen Moniliophthora perniciosa (Basidiomycetes) affect mycelial morphology and secretion of necrosis-inducing proteins

“…Recently, in-depth studies of this fungus have been published: M. perniciosa was found to produce hormones and alter endogenous auxin and salicylic acid levels in infected cocoa leaves (Kilaru et al, 2007), as well as necrosis-inducing proteins during infection, i.e., Mp-NeP1 and Mp-NeP2, which are differentially secreted during the course of infection in planta ; Rincones et al (2008) found that genes expressed in the biotrophic phase of M. perniciosa are under catabolite and nitrogen repression. Glycerol seems to play several roles in M. perniciosa biology; Santos et al (2008) showed that M. perniciosa's resistance to stress depends on glycerol, and Pungartnik et al (2009) showed that small amounts of hydrogen peroxide in glycerol medium induce the formation of clamp connections in vitro and that basidiospores are the most resistant life form of this fungus when compared to mono-and dikaryotic cells.…”