The fusarium mycotoxin deoxynivalenol can inhibit plant apoptosis-like programmed cell death

Abstract

The Fusarium genus of fungi is responsible for commercially devastating crop diseases and the contamination of cereals with harmful mycotoxins. Fusarium mycotoxins aid infection, establishment, and spread of the fungus within the host plant. We investigated the effects of the Fusarium mycotoxin deoxynivalenol (DON) on the viability of Arabidopsis cells. Although it is known to trigger apoptosis in animal cells, DON treatment at low concentrations surprisingly did not kill these cells. On the contrary, we found that DON inhibited apoptosis-like programmed cell death (PCD) in Arabidopsis cells subjected to abiotic stress treatment in a manner independent of mitochondrial cytochrome c release. This suggested that Fusarium may utilise mycotoxins to suppress plant apoptosis-like PCD. To test this, we infected Arabidopsis cells with a wild type and a DON-minus mutant strain of F. graminearum and found that only the DON producing strain could inhibit death induced by heat treatment. These results indicate that mycotoxins may be capable of disarming plant apoptosis-like PCD and thereby suggest a novel way that some fungi can influence plant cell fate.

Figure 1. Cell types present following a 10 minute 53°C heat treatment.

(a) Cells that are alive have the ability to cleave FDA, and fluoresce under light at a wavelength of 490 nm. (b) Necrotic cells cannot cleave FDA so do not fluoresce and show no evidence of protoplast condensation. (c) In cells that undergo apoptosis-like PCD the protoplast retracts from the cell wall. These cells cannot cleave FDA, so do not fluoresce. As this is a moderate stress, the majority of the cells die via apoptosis-like PCD.

Figure 2. The effect of a high concentration of DON on viability of Arabidopsis suspension cells.

Arabidopsis suspension cells were treated with either water control or 120 ppm DON for 24 and 48 hours. Apoptosis-like PCD increased at 24 and 48 hours. Results are the mean percentage (+/− standard deviations) of cells in a given state, taken from three independent experiments each carried out in triplicate.

Figure 3. The effect of DON on heat and ethanol induced apoptosis-like PCD.

(a) Heat treatment caused a high level of apoptosis-like PCD in control samples. In samples that were treated with DON, apoptosis-like PCD was greatly reduced, while the number of live cells increased. Results are the mean percentage (+/− standard deviations) of cells in a given state, taken from two independent experiments each carried out in triplicate. (b) Ethanol (EtOH) treatment induced apoptosis-like PCD in control samples. In samples that were treated with DON, apoptosis-like PCD was reduced, while the number of live cells increased. Results are the mean percentage (+/− standard deviations) of cells in a given state, taken from two independent experiments each carried out in triplicate.

Figure 4. The effect of CHX on heat-induced apoptosis-like PCD.

Heat treatment caused a high level of apoptosis-like PCD in control samples. In samples treated with CHX, apoptosis-like PCD was greatly reduced, while the number of live cells increased. Results are the mean percentage (+/− standard deviations) of cells in a given state, taken from two independent experiments each carried out in triplicate.

Figure 5. The effect of DON on heat and ethanol induced mitochondrial cytochrome c release.

(a) Cytochrome c (Cyt c) remained associated with the mitochondrial fraction of non-heat-treated control cell samples (+/−DON). However, Cyt c release from the mitochondrial fraction was induced immediately following a 10 minute heat treatment (0 h +/− DON). VDAC/porin remained associated with the mitochondrial fraction in all samples. C = control samples. (b) Cyt c remained associated with the mitochondrial fraction of non-EtOH-treated control cell samples (+/−DON). Mitochondrial Cyt c release occurred in all samples treated with EtOH (+/− DON) at 1 hour and 4 hours post EtOH treatment. VDAC/porin remained associated with the mitochondrial fraction in all samples. C = control samples.

Figure 6. The effect of infection with wild-type and DON-minus strains of F. graminearum on heat-induced apoptosis-like PCD in Arabidopsis cells.

Heat treatment caused a high level of apoptosis-like PCD in control samples and few live cells were observed. Samples that were inoculated with DON-minus mutant (GZT40) spores had decreased viability, and apoptosis-like PCD was increased in comparison to controls. Samples that were inoculated and infected with ‘wild type’ (GZ3639) spores had a much-reduced level of apoptosis-like PCD and cell death in general, while the number of live cells was increased. Results are the mean percentage (+/− standard deviations) of cells in a given state, taken from two independent experiments each carried out in triplicate.