Zearalenone and ß-Zearalenol But Not Their Glucosides Inhibit Heat Shock Protein 90 ATPase Activity

Abstract

The mycotoxin zearalenone (ZEN) is produced by many plant pathogenic Fusarium species. It is well known for its estrogenic activity in humans and animals, but whether ZEN has a role in plant-pathogen interaction and which process it is targeting in planta was so far unclear. We found that treatment of Arabidopsis thaliana seedlings with ZEN induced transcription of the AtHSP90.1 gene. This heat shock protein (HSP) plays an important role in plant-pathogen interaction, assisting in stability and functionality of various disease resistance gene products. Inhibition of HSP90 ATPase activity impairs functionality. Because HSP90 inhibitors are known to induce HSP90 gene expression and due to the structural similarity with the known HSP90 inhibitor radicicol (RAD), we tested whether ZEN and its phase I metabolites α- and ß-zearalenol are also HSP90 ATPase inhibitors. Indeed, AtHSP90.1 and wheat TaHSP90-2 were inhibited by ZEN and ß-zearalenol, while α-zearalenol was almost inactive. Plants can efficiently glycosylate ZEN and α/ß-zearalenol. We therefore tested whether glucosylation has an effect on the inhibitory activity of these metabolites. Expression of the A. thaliana glucosyltransferase UGT73C6 conferred RAD resistance to a sensitive yeast strain. Glucosylation of RAD, ZEN, and α/ß-zearalenol abolished the in vitro inhibitory activity with recombinant HSP90 purified from Escherichia coli. In conclusion, the mycotoxin ZEN has a very prominent target in plants, HSP90, but it can be inactivated by glycosylation. This may explain why there is little evidence for a virulence function of ZEN in host plants.

Keywords: Arabidopsis; Fusarium; HSP90; glycosylation; radicicol; wheat.

Figure 1

Structures of radicicol, zearalenone (Metzler, 2011), and the zearalenone phase I metabolites α- and ß-zearalenol. Structural differences are highlighted red. Below the structures of zearalenone-14-O-ß-D-glucopyranoside and zearalenone-16-O-ß-D-glucopyranoside. The glucosides of α-zearalenol and ß-zearalenol produced in this study carry the glucose moiety at the analogous position.

Figure 2

Transcriptional response of Arabidopsis thaliana HSP90 genes to zearalenone (ZEN). (A) Expression levels of A. thaliana HSP90 genes after 2 h treatment with ZEN (50 µM) or solvent (control; dimethyl sulfoxide, DMSO) according to microarray data from Werner (2005). The genes HSP90.3 and HSP90.4 cannot be distinguished by the probes on the chip (labeled HSP90.3/4). (B) Quantitative real-time PCR of A. thaliana HSP90.1 after 2 h treatment with ZEN (50 µM) or DMSO. The results are expressed as relative expression to the reference gene ADAPTOR PROTEIN-2 MU-ADAPTIN (AP2M; AT5G46630) and represent the means of four biological replicates ± standard deviation.

Figure 3

Inhibition of purified 6xHIS-tagged yeast Hsp82p ATPase activity by radicicol (RAD), ZEN, zearalenol-α (αZEL), and zearalenol-β (ßZEL).

Figure 4

Inhibition of Arabidopsis thaliana HSP90.1 (A) and Triticum aestivum HSP90-2 (B–D) ATPase activity by RAD, ZEN, zearalenone-14-glucoside (ZEN14G), zearalenone-16-glucoside (ZEN16G), αZEL, α-zearalenol-14-glucoside (αZEL14G), α-zearalenol-16-glucoside (αZEL16G), ßZEL, ß-zearalenol-14-glucoside (ßZEL14G), and ß-zearalenol-16-glucoside (ßZEL16G).

Figure 5

Expression of Arabidopsis thaliana UDP-glucosyltransferase AtUGT73C6 confers radicicol (RAD) resistance to yeast strain YZGA515. Spotting of YZGA515 transformants (three serial 10⁻¹ dilutions) expressing either AtUGT73C6 (upper row) or containing the empty vector. Synthetic complete medium without leucine (SC-LEU) plates without added RAD (left) or with 40 mg/L RAD.

Figure 6

High-performance liquid chromatographic–tandem mass spectrometric (HPLC–MS/MS) determination of RAD and radicicol-glucoside (RADGlc expressing UGT73C6 treated with RAD. (A) HPLC–MS/MS analysis in selective reaction monitoring mode of a culture filtrate of yeast. (B) Enhanced product ion (EPI) MS/MS spectrum of RAD at 30 eV collision energy; the inlay shows the MS spectrum of the deprotonated ions. (C) EPI MS/MS spectrum of RADGlc at 50 eV collision energy; the inlay shows the MS spectrum of the deprotonated ions.