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

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.

The mycoparasitic fungus Clonostachys rosea responds with both common and specific gene expression during interspecific interactions with fungal prey

“…Putative transformants were selected on a plate containing hygromycin (200 μg/ml). Validation of homologous integration of the deletion cassettes in putative transformants was performed using a PCR screening approach with primer combinations targeting the hph gene (Hyg F/Hyg R) and sequences flanking the deletion cassettes (Appendices and ) as described previously (Dubey, Jensen, & Karlsson, ; Dubey et al., ). RT‐PCR analysis was conducted on WT and gene deletion strains using RevertAid premium reverse transcriptase (Fermentas, St. Leon‐Rot, Germany) and primer pairs specific for the respective genes (Appendix ).…”

“…RT‐PCR analysis was conducted on WT and gene deletion strains using RevertAid premium reverse transcriptase (Fermentas, St. Leon‐Rot, Germany) and primer pairs specific for the respective genes (Appendix ). The PCR‐positive transformants were tested for mitotic stability, and were purified by two rounds of single spore isolation (Dubey et al., , ).…”

“…Colony diameter was measured after 5 days of growth at 25°C in darkness. The concentration of chemicals used for phenotypic analysis in this study was based on our previously published results (Dubey et al., , ) with the exception of ions. The appropriate concentration of ions was selected based on screening of C .…”

“…Antagonistic behaviour of C . rosea WT and deletion strains against F. graminearum or B. cinerea was tested in five biological replicates using an in vitro plate confrontation assay and a culture filtrate assay, as described previously (Dubey et al., , ). In addition, a liquid culture confrontation test was performed in five biological replicates, where conidia (10 5 conidia) from WT and deletion strains were inoculated in 50 ml VM with 0.3% glucose in Erlenmeyer flasks and were allowed to grow for 24 hr at 25°C in darkness.…”

“…rosea and F. graminearum DNA with qPCR using primers specific to β‐tubulin (Dubey et al., ; Reischer, Lemmens, Farnleitner, Adler, & Mach, ). An in vivo bioassay using a sand seedling test for fusarium foot rot disease on barley was performed in five biological replicates, where each replicate included 15 plants, following the procedure described previously (Dubey et al., , ).…”