Abstract
How the host cells of plants and animals protect themselves against fungal invasion is a biologically interesting and economically important problem. Here we investigate the mechanistic process that leads to death of Penicillium expansum, a widespread phytopathogenic fungus, by identifying the cellular compounds affected by hydrogen peroxide (H2O2) that is frequently produced as a response of the host cells. We show that plasma membrane damage was not the main reason for H2O2-induced death of the fungal pathogen. Proteomic analysis of the changes of total cellular proteins in P. expansum showed that a large proportion of the differentially expressed proteins appeared to be of mitochondrial origin, implying that mitochondria may be involved in this process. We then performed mitochondrial sub-proteomic analysis to seek the H2O2-sensitive proteins in P. expansum. A set of mitochondrial proteins were identified, including respiratory chain complexes I and III, F1F0 ATP synthase, and mitochondrial phosphate carrier protein. The functions of several proteins were further investigated to determine their effects on the H2O2-induced fungal death. Through fluorescent co-localization and the use of specific inhibitor, we provide evidence that complex III of the mitochondrial respiratory chain contributes to ROS generation in fungal mitochondria under H2O2 stress. The undesirable accumulation of ROS caused oxidative damage of mitochondrial proteins and led to the collapse of mitochondrial membrane potential. Meanwhile, we demonstrate that ATP synthase is involved in the response of fungal pathogen to oxidative stress, because inhibition of ATP synthase by oligomycin decreases survival. Our data suggest that mitochondrial impairment due to functional alteration of oxidative stress-sensitive proteins is associated with fungal death caused by H2O2.
Highlights
How the host cells in plants and animals respond to the invasion of fungi in a self-protective process is an important biological problem attracting a wide range of interests
In order to identify the reactive oxygen species (ROS)-sensitive proteins in mitochondria, we developed a procedure for high-purity mitochondrial separations from pathogenic fungi and, for the first time, examined the variations of the mitochondrial sub-proteome of a fungal pathogen under oxidative stress of H2O2
Viability was evaluated by propidium iodide (PI) exclusion, a rapid and simple assay developed for the detection of dead and dying cells
Summary
How the host cells in plants and animals respond to the invasion of fungi in a self-protective process is an important biological problem attracting a wide range of interests. An oxidative burst ( known as the respiratory burst in phagocytes of mammals), during which large quantities of reactive oxygen species (ROS) are generated, is one of the earliest host responses after pathogen attack [2,3,4]. These ROS, mostly superoxide anion and hydrogen peroxide (H2O2), are produced by different host enzyme systems of plant or animal host cells. Unraveling the mechanistic basis of death decisions in fungi is necessary for understanding of the important biological process of fungal invasion
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