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Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan.

ABSTRACT: Chitosan is a natural polymer with antimicrobial activity. Chitosan causes plasma membrane permeabilization and induction of intracellular reactive oxygen species (ROS) in Neurospora crassa. We have determined the transcriptional profile of N. crassa to chitosan and identified the main gene targets involved in the cellular response to this compound. Global network analyses showed membrane, transport and oxidoreductase activity as key nodes affected by chitosan. Activation of oxidative metabolism indicates the importance of ROS and cell energy together with plasma membrane homeostasis in N. crassa response to chitosan. Deletion strain analysis of chitosan susceptibility pointed NCU03639 encoding a class 3 lipase, involved in plasma membrane repair by lipid replacement, and NCU04537 a MFS monosaccharide transporter related to assimilation of simple sugars, as main gene targets of chitosan. NCU10521, a glutathione S-transferase-4 involved in the generation of reducing power for scavenging intracellular ROS is also a determinant chitosan gene target. Ca(2+) increased tolerance to chitosan in N. crassa. Growth of NCU10610 (fig 1 domain) and SYT1 (a synaptotagmin) deletion strains was significantly increased by Ca(2+) in the presence of chitosan. Both genes play a determinant role in N. crassa membrane homeostasis. Our results are of paramount importance for developing chitosan as an antifungal.

SUBMITTER: Lopez-Moya F

PROVIDER: S-EPMC4729629 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan.

Lopez-Moya Federico F Kowbel David D Nueda Maria José MJ Palma-Guerrero Javier J Glass N Louise NL Lopez-Llorca Luis Vicente LV

Molecular bioSystems 20160201 2

Chitosan is a natural polymer with antimicrobial activity. Chitosan causes plasma membrane permeabilization and induction of intracellular reactive oxygen species (ROS) in Neurospora crassa. We have determined the transcriptional profile of N. crassa to chitosan and identified the main gene targets involved in the cellular response to this compound. Global network analyses showed membrane, transport and oxidoreductase activity as key nodes affected by chitosan. Activation of oxidative metabolism ...[more]

PMID: 26694141

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Project description:Purpose: Evaluate and analyze the transcriptional response of N. crassa germinating conidia and determined the main gene functions related with the exposure to chitosan. Identification of main chitosan gene tragets in N. crassa. Methods: From samples in contact with chitosan for 4, 8 and 16h total RNA was isolated from material using TRIzol reagent. Total RNA was washed and then resuspended. For first strand cDNA synthesis, the fragmented poly (A+) RNA was incubated with random hexamers. Superscript II (200 U; Invitrogen) was added to the first cDNA strand synthesis. End-repair was performed by adding T4 DNA ligase buffer T4 DNA polymerase, Klenow DNA polymerase, T4 PNK. Standard Illumina adapters (FC) were ligated to the cDNA fragments using 2× DNA ligase buffer, 1 µL of adapter oligo mix and DNA ligase. The sample was purified in a 2% low-melting point agarose gel, and a slice of gel containing 200-bp fragments was removed and the DNA purified. A aliquot of purified cDNA library was amplified by PCR using the pfx DNA polymerase with genomic primers 1.1 and 2.1 (Illumina). All libraries were sequenced on a HiSeq 2000 platform. Results: A total of 523 N. crassa genes were differentially expressed (log2foldchange ≥ 2; p-value < 0.05) in response to chitosan. Of these, 55.6% (291 genes) were down-regulated and 45.3% (237 genes) up-regulated. This time-course experiment showed a progressive reduction in the number of genes whose expression increased upon exposure to chitosan (142 induced genes at 4h, 119 at 8h and 45 at 16h). In contrast, exposure to chitosan resulted in an increase in the number of genes whose expression levels decreased over time (79, 93 and 207 genes down-regulated at 4, 8 and 16h, respectively). A subset of 22 genes was differentially expressed consistently (log2foldchange ≥ 2; p-value < 0.05) throughout the whole time-course. Expression of 10 N. crassa genes representative of functional categories that were differentially expressed by exposure to chitosan were selected to validate our RNA-seq analysis. Gene expression was evaluated by qRT-PCR following an 8h exposure to chitosan. These genes were involved in the main functions related with the chitosan response. All genes analyzed by qRT-PCR showed an expression pattern consistent with that derived from RNA-seq data analysis. Conclusions: This work provides the first study of the gene expression response of a filamentous fungus (N. crassa) to chitosan. Transcriptomics revealed oxidoreductase activity, membrane homeostasis and microtubule organization as main gene functions differentially expressed.

Project description:An ensemble of genetic networks that describe how the model fungal system, Neurospora crassa, utilizes quinic acid (QA) as a sole carbon source has been identified previously. A genetic network for QA metabolism involves the genes, qa-1F and qa-1S, that encode a transcriptional activator and repressor, respectively and structural genes, qa-2, qa-3, qa-4, qa-x, and qa-y. By a series of 4 separate and independent, model-guided, microarray experiments a total of 50 genes are identified as QA-responsive and hypothesized to be under QA-1F control and/or the control of a second QA-responsive transcription factor (NCU03643) both in the fungal binuclear Zn(II)2Cys6 cluster family. QA-1F regulation is not sufficient to explain the quantitative variation in expression profiles of the 50 QA-responsive genes. QA-responsive genes include genes with products in 8 mutually connected metabolic pathways with 7 of them one step removed from the tricarboxylic (TCA) Cycle and with 7 of them one step removed from glycolysis: (1) starch and sucrose metabolism; (2) glycolysis/glucanogenesis; (3) TCA Cycle; (4) butanoate metabolism; (5) pyruvate metabolism; (6) aromatic amino acid and QA metabolism; (7) valine, leucine, and isoleucine degradation; and (8) transport of sugars and amino acids. Gene products both in aromatic amino acid and QA metabolism and transport show an immediate response to shift to QA, while genes with products in the remaining 7 metabolic modules generally show a delayed response to shift to QA. The additional QA-responsive cutinase transcription factor-1β (NCU03643) is found to have a delayed response to shift to QA. The series of microarray experiments are used to expand the previously identified genetic network describing the qa gene cluster to include all 50 QA-responsive genes including the second transcription factor (NCU03643). These studies illustrate new methodologies from systems biology to guide model-driven discoveries about a core metabolic network involving carbon and amino acid metabolism in N. crassa.

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Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan.

Publications

Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan.

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