Dataset Information

Screening the Saccharomyces cerevisiae Nonessential Gene Deletion Library Reveals Diverse Mechanisms of Action for Antifungal Plant Defensins.

ABSTRACT: Plant defensins are a large family of proteins, most of which have antifungal activity against a broad spectrum of fungi. However, little is known about how they exert their activity. The mechanisms of action of only a few members of the family have been investigated and, in most cases, there are still a number of unknowns. To gain a better understanding of the antifungal mechanisms of a set of four defensins, NaD1, DmAMP1, NbD6, and SBI6, we screened a pooled collection of the nonessential gene deletion set of Saccharomyces cerevisiae Strains with increased or decreased ability to survive defensin treatment were identified based on the relative abundance of the strain-specific barcode as determined by MiSeq next-generation sequencing. Analysis of the functions of genes that are deleted in strains with differential growth in the presence of defensin provides insight into the mechanism of action. The screen identified a novel role for the vacuole in the mechanisms of action for defensins NbD6 and SBI6. The effect of these defensins on vacuoles was further confirmed by using confocal microscopy in both S. cerevisiae and the cereal pathogen Fusarium graminearum These results demonstrate the utility of this screening method to identify novel mechanisms of action for plant defensins.

SUBMITTER: Parisi K

PROVIDER: S-EPMC6811411 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Screening the <i>Saccharomyces cerevisiae</i> Nonessential Gene Deletion Library Reveals Diverse Mechanisms of Action for Antifungal Plant Defensins.

Parisi Kathy K Doyle Stephen R SR Lee Eunice E Lowe Rohan G T RGT van der Weerden Nicole L NL Anderson Marilyn A MA Bleackley Mark R MR

Antimicrobial agents and chemotherapy 20191022 11

Plant defensins are a large family of proteins, most of which have antifungal activity against a broad spectrum of fungi. However, little is known about how they exert their activity. The mechanisms of action of only a few members of the family have been investigated and, in most cases, there are still a number of unknowns. To gain a better understanding of the antifungal mechanisms of a set of four defensins, NaD1, DmAMP1, NbD6, and SBI6, we screened a pooled collection of the nonessential gene ...[more]

PMID: 31451498

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Project description:Plant defensins are small cysteine-rich antimicrobial proteins. Their three-dimensional structures are similar in that they consist of an ?-helix and three anti-parallel ?-strands stabilized by four disulfide bonds. Plant defensins MsDef1 and MtDef4 are potent inhibitors of the growth of several filamentous fungi including Fusarium graminearum. However, they differ markedly in their antifungal properties as well as modes of antifungal action. MsDef1 induces prolific hyperbranching of fungal hyphae, whereas MtDef4 does not. Both defensins contain a highly conserved ?-core motif (GXCX(3-9)C), a hallmark signature present in the disulfide-stabilized antimicrobial peptides, composed of ?2 and ?3 strands and the interposed loop. The ?-core motifs of these two defensins differ significantly in their primary amino acid sequences and in their net charge. In this study, we have found that the major determinants of the antifungal activity and morphogenicity of these defensins reside in their ?-core motifs. The MsDef1-?4 variant in which the ?-core motif of MsDef1 was replaced by that of MtDef4 was almost as potent as MtDef4 and also failed to induce hyperbranching of fungal hyphae. Importantly, the ?-core motif of MtDef4 alone was capable of inhibiting fungal growth, but that of MsDef1 was not. The analysis of synthetic ?-core variants of MtDef4 indicated that the cationic and hydrophobic amino acids were important for antifungal activity. Both MsDef1 and MtDef4 induced plasma membrane permeabilization; however, kinetic studies revealed that MtDef4 was more efficient in permeabilizing fungal plasma membrane than MsDef1. Furthermore, the in vitro antifungal activity of MsDef1, MsDef1-?4, MtDef4 and peptides derived from the ?-core motif of each defensin was not solely dependent on their ability to permeabilize the fungal plasma membrane. The data reported here indicate that the ?-core motif defines the unique antifungal properties of each defensin and may facilitate de novo design of more potent antifungal peptides.

Project description:BACKGROUND:Despite the latest advancements in metabolic engineering for genome editing and characterization of host performance, the successful development of robust cell factories used for industrial bioprocesses and accurate prediction of the behavior of microbial systems, especially when shifting from laboratory-scale to industrial conditions, remains challenging. To increase the probability of success of a scale-up process, data obtained from thoroughly performed studies mirroring cellular responses to typical large-scale stimuli may be used to derive crucial information to better understand potential implications of large-scale cultivation on strain performance. This study assesses the feasibility to employ a barcoded yeast deletion library to assess genome-wide strain fitness across a simulated industrial fermentation regime and aims to understand the genetic basis of changes in strain physiology during industrial fermentation, and the corresponding roles these genes play in strain performance. RESULTS:We find that mutant population diversity is maintained through multiple seed trains, enabling large scale fermentation selective pressures to act upon the community. We identify specific deletion mutants that were enriched in all processes tested in this study, independent of the cultivation conditions, which include MCK1, RIM11, MRK1, and YGK3 that all encode homologues of mammalian glycogen synthase kinase 3 (GSK-3). Ecological analysis of beta diversity between all samples revealed significant population divergence over time and showed feed specific consequences of population structure. Further, we show that significant changes in the population diversity during fed-batch cultivations reflect the presence of significant stresses. Our observations indicate that, for this yeast deletion collection, the selection of the feeding scheme which affects the accumulation of the fermentative by-product ethanol impacts the diversity of the mutant pool to a higher degree as compared to the pH of the culture broth. The mutants that were lost during the time of most extreme population selection suggest that specific biological processes may be required to cope with these specific stresses. CONCLUSIONS:Our results demonstrate the feasibility of Bar-seq to assess fermentation associated stresses in yeast populations under industrial conditions and to understand critical stages of a scale-up process where variability emerges, and selection pressure gets imposed. Overall our work highlights a promising avenue to identify genetic loci and biological stress responses required for fitness under industrial conditions.

Dataset Information

Screening the Saccharomyces cerevisiae Nonessential Gene Deletion Library Reveals Diverse Mechanisms of Action for Antifungal Plant Defensins.

Publications

Screening the <i>Saccharomyces cerevisiae</i> Nonessential Gene Deletion Library Reveals Diverse Mechanisms of Action for Antifungal Plant Defensins.

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