The Fusarium mycotoxin deoxynivalenol elicits hydrogen peroxide production, programmed cell death and defence responses in wheat

Abstract

Fusarium species infect cereal crops worldwide and cause the important diseases Fusarium head blight and crown rot in wheat. Fusarium pathogens reduce yield and some species also produce trichothecene mycotoxins, such as deoxynivalenol (DON), during infection. These toxins play roles in pathogenesis on wheat and have serious health effects if present in grain consumed by humans or animals. In the present study, the response of wheat tissue to DON has been investigated. Infusion of wheat leaves with DON induced hydrogen peroxide production within 6 h followed by cell death within 24 h that was accompanied by DNA laddering, a hallmark of programmed cell death. In addition, real-time PCR analysis revealed that DON treatment rapidly induced transcription of a number of defence genes in a concentration-dependent manner. Co-treatment with DON and the antioxidant ascorbic acid reduced these responses, suggesting their induction may be at least partially mediated by reactive oxygen species (ROS), commonly known to be signalling molecules in plants. Wheat defence genes were more highly expressed in wheat stems inoculated with a DON-producing fungal strain than those inoculated with a DON-non-producing mutant, but only at a late stage of infection. Taken together, the results are consistent with a model in which DON production during infection of wheat induces ROS, which on the one hand may stimulate programmed host cell death assisting necrotrophic fungal growth, whereas, on the other hand, the ROS may contribute to the induction of antimicrobial host defences.

Figure 1

Infiltration of wheat tissue with DON resulted in H₂O₂ production, cell death and DNA laddering. DAB‐stained wheat leaf tissue from 2‐week‐old seedlings showing H₂O₂ production at 6 h after (A) mock infiltration, (B) 100 mg/L DON infiltration and (C) 200 mg/L DON infiltration. Trypan blue‐stained wheat leaf tissue 24 h after (D) mock infiltration, (E) 100 mg/L DON infiltration and (F) 200 mg/L DON infiltration. (G) Genomic DNA laddering in wheat 24 h after infiltration with mock solution shown in the first lane, 100 mg/L DON shown in the second lane, 100 mg/L DON combined with 7 g/L ascorbic acid shown in the third lane and the 1‐kb + DNA size ladder (Invitrogen) in the fourth lane. Further images of tissue from all infiltration treatments are shown in supplementary Fig. S1.

Figure 2

H₂O₂ production and cell death was observed in wheat leaves after inoculation with F. pseudograminearum spores. (A) Wheat leaf tissue was inoculated using a detached leaf assay stained using DAB 7 days later. H₂O₂ was visible in stomata that were in close proximity to spores and also in the spore tips (inset). (B) Trypan blue staining of inoculated leaf tissue showed cell death seemed to follow vascular tissue and was most widespread in stomatal guard cells. Rows of affected stomatal guard cells that surround the primary infection site are indicated by arrows. Further images of cell death in inoculated tissue are shown in supplementary Fig. S2.

Figure 3

(A) Induction of defence gene expression in 2‐week‐old seedlings at 1 day after treatment with 1, 10 and 100 mg/L DON. Columns represent average induction ratios (± SE; n = 3) of gene transcripts in treated compared with mock‐treated and are plotted on a logarithmic scale. (B) Western blot analysis of total protein extracted from wheat tissue using β‐1,3‐glucanase (PR2) and chitinase (PR3) antibodies 2 days after mock treatment, F. pseudograminearum inoculation, or 100 mg/L DON treatment. Lower panel shows Rubisco stained with Ponceau red to show protein loading. The numbers above bands for chitinase and β‐1,3‐glucanase indicate protein levels as a percentage of Rubisco. Total protein (20 µg) was separated on a 4–12% polyacrylamide gradient gel. Protein molecular masses are indicated on the left.

Figure 4

Induction of defence gene expression in 2‐week‐old wheat seedlings (A) 6 h and (B) 24 h after treatment with 100 mg/L DON and a combination of 100 mg/L DON and 7 g/L ascorbic acid. Columns represent average induction ratios (± SE; n = 3) of gene transcripts in treated compared with mock‐treated plants and are plotted on a logarithmic scale. Statistically significant differences in gene induction (Student's t‐test, P < 0.05) resulting from treatments including ascorbic acid are indicated by a star.

Figure 5

(A) Induction of defence gene expression 28 days after inoculation with the DON‐non‐producing F. graminearum Tri5 deletion line (Fg ΔTri5) and wild‐type. Columns represent average induction ratios (± SE; n = 3) of gene transcripts in treated compared with mock‐treated plants and are plotted on a logarithmic scale. Statistically significant differences in gene induction (Student's t‐test, P < 0.05) resulting from treatments are indicated by a star. (B) Fungal biomass 28 days after inoculation with the DON‐non‐producing F. graminearum Tri5 deletion line and wild‐type. Columns represent average amplification of the Fg 18S rRNA gene relative to wheat actin (± SE; n = 3).