Antifungal mechanisms of a plant defensin MtDef4 are not conserved between the ascomycete fungiNeurospora crassaandFusarium graminearum

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Defensins play an important role in plant defense against fungal pathogens. The plant defensin, MtDef4, inhibits growth of the ascomycete fungi,Neurospora crassaandFusarium graminearum, at micromolar concentrations. We have reported that MtDef4 is transported into the cytoplasm of these fungi and exerts its antifungal activity on intracellular targets. Here, we have investigated whether the antifungal mechanisms of MtDef4 are conserved in these fungi. We show thatN. crassaandF. graminearumrespond differently to MtDef4 challenge. Membrane permeabilization is required for the antifungal activity of MtDef4 againstF. graminearumbut not againstN. crassa. We find that MtDef4 is targeted to different subcellular compartments in each fungus. Internalization of MtDef4 inN. crassais energy-dependent and involves endocytosis. By contrast, MtDef4 appears to translocate intoF. graminearumautonomously using a partially energy-dependent pathway. MtDef4 has been shown to bind to the phospholipid phosphatidic acid (PA). We provide evidence that the plasma membrane localized phospholipase D, involved in the biosynthesis of PA, is needed for entry of this defensin inN. crassa, but not inF. graminearum. To our knowledge, this is the first example of a defensin which inhibits the growth of two ascomycete fungi via different mechanisms.

MtDef4 is a small cysteine-rich antifungal plant defensin. It inhibits the growth of the ascomycete fungi Fusarium graminearum and Neurospora crassa at micromolar concentrations. However, it uses different molecular mechanisms for fungal cell entry and growth inhibition of these taxonomically related fungi.

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