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The ABC transporter ABCG29 is involved in H2O2 tolerance and biocontrol traits in the fungus Clonostachys rosea

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Abstract

For successful biocontrol interactions, biological control organisms must tolerate toxic metabolites produced by themselves or plant pathogens during mycoparasitic/antagonistic interactions, by host plant during colonization of the plant, and xenobiotics present in the environment. ATP-binding cassette (ABC) transporters can play a significant role in tolerance of toxic compounds by mediating active transport across the cellular membrane. This paper reports on functional characterization of an ABC transporter ABCG29 in the biocontrol fungus Clonostachys rosea strain IK726. Gene expression analysis showed induced expression of abcG29 during exposure to the Fusarium spp. mycotoxin zearalenone (ZEA) and the fungicides Cantus, Chipco Green and Apron. Expression of abcG29 in C. rosea was significantly higher during C. rosea–C. rosea (Cr–Cr) interaction or in exposure to C. rosea culture filtrate for 2 h, compared to interaction with Fusarium graminearum or 2 h exposure to F. graminearum culture filtrate. In contrast with gene expression data, ΔabcG29 strains did not display reduced tolerance towards ZEA, fungicides or chemical agents known for inducing oxidative, cell wall or osmotic stress, compared to C. rosea WT. The exception was a significant reduction in tolerance to H2O2 (10 mM) in ΔabcG29 strains when conidia were used as an inoculum. The antagonistic ability of ΔabcG29 strains towards F. graminearum, Fusarium oxysporum or Botrytis cinerea in dual plate assays were not different compared with WT. However, in biocontrol assays ΔabcG29 strains displayed reduced ability to protect Arabidopsis thaliana leaves from B. cinerea, and barley seedling from F. graminearum as measured by an A. thaliana detached leaf assay and a barley foot rot disease assay, respectively. These data show that the ABCG29 is dispensable for ZEA and fungicides tolerance, and antagonism but not H2O2 tolerance and biocontrol effects in C. rosea.

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Acknowledgments

This work was financially supported by the Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences; the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS, Grant Numbers 229-2009-1530 and 229-2012-1288); and the Danish Agency for Science, Technology and Innovation (DSF Grant Number 09-063108/DSF).

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  1. Department of Forest Mycology and Plant Pathology, Uppsala BioCenter, Swedish University of Agricultural Sciences, Box 7026, 75007, Uppsala, Sweden

    Mukesh Dubey, Dan Funck Jensen & Magnus Karlsson

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  1. Mukesh Dubey
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  2. Dan Funck Jensen
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Correspondence to Mukesh Dubey.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Communicated by J. Perez-Martin.

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Fig. S1

Schematic illustration of predicted domain organisation of ABCG29. Amino acid (aa) sequences were analyzed for the presence of conserved domains using simple modular architecture research tool (SMART) and conserved domain search (CDS). Abbreviations: NBD, nucleotide binding domain; TMD, Transmembrane domain; TMS, Transmembrane segments. A TMD consists of six TMS. The bar marks indicate a length of 100 amino acids and refers to total protein length (PPTX 41 kb)

Fig. S2

Schematic representation of deletion cassettes and characterization of mutant strains using PCR and RT-PCR. A: Organisation of abcG29 locus in WT and mutant strains of C. rosea. The abcG29 coding region was replaced by hygB cassette by homologous recombination resulting in generation of ΔabcG29 strains. B: PCR verification of ΔabcG29 strains using primers located in the hygB cassette (HygF/HygR) in combination with primers located upstream and downstream from the deletion cassette (abcG29ko F/abcG29ko R). PCR products of ~ 2.7 kb and ~ 2.5 using primers abcG29ko F/HygR and abcG29ko R/HygF respectively were expected from a correct gene replacement. M, gene ruler DNA ladder mix; 1-9, independent ΔabcG29 strains; 10, WT strain. C: RT-PCR analysis of abcG29 gene expression in WT and ΔabcG29 strains using abcG29 specific primers. M, gene ruler DNA ladder mix; 1, WT; 2-6, independent ΔabcG29 strains. Primer combinations used for PCR and RT-PCR are given above the images. The small arrow heads indicate the location of primers used to construct the deletion cassette and analysis of mutants using PCR. The large arrow heads indicate the size of amplified PCR products. Abbreviations: LB, left border; RB, right border (PPTX 613 kb)

Fig. S3

Expression analysis of abcG5 (A), and zhd101 (B) in C. rosea WT and ΔabcG29 strains mycelia treated with ZEA (2 h post treatment). Relative expression levels for abcG5 and zhd101 in relation to β-tubulin were calculated from the Ct values and the primer amplification efficiencies using the formula described by Pfaffl (2001). Error bars represent standard deviation based on 5 biological replicates. Same letters indicate no statistically significant differences (P > 0.05) within experiments based on the Tukey–Kramer test (TIFF 795 kb)

Table S1

Taxonomy of fungal species mentioned in this paper (DOCX 12 kb)

Table S2

List of primers used in this study (DOCX 17 kb)

Table S3

Growth rate of C. rosea WT and ΔabcG29 strains on Czapek-dox (CZ) medium and CZ medium amended with toxin, fungicides, and cell wall, osmotic or oxidative stress compounds (DOCX 18 kb)

Table S4

Antagonistic interactions between C. rosea and F. graminearum, F. oxysporum f. sp. radicis lycopersici or B. cinerea. A: Growth rate of F. graminearum (Fg), F. oxysporum f. sp. radicis lycopersici (Forl) or B. cinerea (Bc) during in vitro antagonistic interactions with C. rosea WT or ΔabcG29 strains. B: Growth rate of C. rosea WT and ΔabcG29 strains during in vitro antagonistic interactions with F. graminearum (Fg), F. oxysporum f. sp. radicis lycopersici (Forl) or B. cinerea. C: Biomass (mycelial dry weight in mg) of C. rosea WT and Δ abcG29 strains in culture filtrates of F. graminearum (Fg) or F. oxysporum f. sp. radicis lycopersici (Forl) 5 days post inoculations (DOCX 21 kb)

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Dubey, M., Jensen, D.F. & Karlsson, M. The ABC transporter ABCG29 is involved in H2O2 tolerance and biocontrol traits in the fungus Clonostachys rosea . Mol Genet Genomics 291, 677–686 (2016). https://doi.org/10.1007/s00438-015-1139-y

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