Plasma Membrane Integrity During Cell–Cell Fusion and in Response to Pore-Forming Drugs Is Promoted by the Penta-EF-Hand Protein PEF1 inNeurospora crassa

Abstract

Plasma membrane damage commonly occurs during cellular growth and development. To counteract these potentially lethal injuries, membrane repair mechanisms have evolved, which promote the integrity of the lipid bilayer. Although the membrane of fungi is the target of important clinical drugs and agricultural fungicides, the molecular mechanisms which mediate membrane repair in these organisms remain elusive. Here we identify the penta-EF-hand protein PEF1 of the genetic model fungus Neurospora crassa as part of a cellular response mechanism against different types of membrane injury. Deletion of the pef1 gene in the wild type and different lysis-prone gene knockout mutants revealed a function of the protein in maintaining cell integrity during cell-cell fusion and in the presence of pore-forming drugs, such as the plant defense compound tomatine. By fluorescence and live-cell imaging we show that green fluorescent protein (GFP)-tagged PEF1 accumulates at the sites of membrane injury in a Ca 2+-dependent manner. Sitedirected mutagenesis identified Ca 2+-binding domains essential for the spatial dynamics and function of the protein. In addition, the subcellular localization of PEF1 revealed that the syncytial fungal colony undergoes compartmentation in response to antifungal treatment. We propose that plasma membrane repair in fungi constitutes an additional line of defense against membrane-disturbing drugs, thereby expanding the current model of fungal drug resistance mechanisms. KEYWORDS membrane repair; penta-EF-hand protein; cell fusion; Neurospora crassa; antifungal drug

Ca2+ Signalling Differentially Regulates Germ-Tube Formation and Cell Fusion in Fusarium oxysporum

“…Ca 2+ signalling has been proposed to regulate the interaction between these three proteins, leading to exocytosis of the chemoattractant and/or receptor during hyphal fusion [59,60]. In N. crassa, PEF1, a protein involved in membrane plugging during injury, was shown to be dependent on extracellular Ca 2+ for its membrane recruitment [61]. Extracellular Ca 2+ chelation with EGTA also impaired septal plugging in response to wounds, independent of PEF1.…”

The grey mould Botrytis cinerea employs multiple mechanisms to protect itself from antifungal plant metabolites

“…We studied the pore-forming activity of α-tomatine on B. cinerea. B05.10 transformants were used that overexpress PEF1-GFP, whose homolog in N. crassa is mobilized to damaged sites at the membrane in response to Ca 2+ influx and thereby acts as a marker for membrane damage (Schumann et al, 2019). Comparable to observations in N. crassa, the BcPEF1-GFP signal was mainly cytoplasmic with some accumulation at the endoplasmic reticulum around nuclei in the absence of αtomatine (Fig.…”

“…Specifically, the attention was focused on the GT28 glycosyl transferase family and the RTA1 gene family, for which multiple members of each family were strongly upregulated by α-tomatine. Also, the PEF1 gene was studied that was earlier reported to contribute to membrane damage mitigation in response to αtomatine in N. crassa (Schumann et al, 2019). The ABC transporter gene BcAtrT was not included in further studies, as it appeared to be a pseudogene with a single nucleotide insertion that causes a frameshift, resulting in a protein that only contains 6 (instead of 12) transmembrane domains.…”

Bitter and sweet make tomato hard to (b)eat

“…Besides such passive tolerance, fungi can actively repair membrane damage inflicted by α‐tomatine. Exposure of Neurospora crassa to α‐tomatine triggered the recruitment of the membrane repair protein PEF1 to the lysing point at the membrane, and deletion of pef1 increased the sensitivity of N. crassa to α‐tomatine and other pore‐forming drugs (Schumann et al ., 2019). Furthermore, fungi can actively export exogenous toxic compounds through ATP‐binding cassette (ABC) transporters.…”