Necrosis- and ethylene-inducing peptide from Fusarium oxysporum induces a complex cascade of transcripts associated with signal transduction and cell death in Arabidopsis

Abstract

Treatment of Arabidopsis (Arabidopsis thaliana) with a necrosis- and ethylene-inducing peptide (Nep1) from Fusarium oxysporum inhibited both root and cotyledon growth and triggered cell death, thereby generating necrotic spots. Nep1-like proteins are produced by divergent microbes, many of which are plant pathogens. Nep1 in the plant was localized to the cell wall and cytosol based on immunolocalization results. The ratio of chlorophyll a fluorescence (F685 nm/F730 nm) significantly decreased after 75-min treatment with Nep1 in comparison to the control. This suggested that a short-term compensation of photosynthesis occurred in response to localized damage to cells. The concentrations of most water-soluble metabolites analyzed were reduced in Arabidopsis seedlings after 6 h of Nep1 treatment, indicating that the integrity of cellular membranes had failed. Microarray results showed that short-term treatment with Nep1 altered expression of numerous genes encoding proteins putatively localized to organelles, especially the chloroplast and mitochondria. Short-term treatment with Nep1 induced multiple classes of genes involved in reactive oxygen species production, signal transduction, ethylene biosynthesis, membrane modification, apoptosis, and stress. Quantitative PCR was used to confirm the induction of genes localized in the chloroplast, mitochondria, and plasma membrane, and genes responsive to calcium/calmodulin complexes, ethylene, jasmonate, ethylene biosynthesis, WRKY, and cell death. The majority of Nep1-induced genes has been associated with general stress responses but has not been critically linked to resistance to plant disease. These results are consistent with Nep1 facilitating cell death as a component of diseases caused by necrotrophic plant pathogens.

Figure 1.

Effect of F. oxysporum Nep1 on Arabidopsis growth. Nep1 treatment caused necrotic lesions and inhibited cotyledon and root growth. Sterilized Arabidopsis seeds were mixed with Nep1 (20 μg mL⁻¹), 0.001% (v/v) Silwet-L77, 0.3% agarose, 1% Suc in 1× MS media, and plated onto petri dishes. Controls were treated with 0.001% (v/v) Silwet-L77. Seeds were germinated and grown in growth cabinets at 22°C for 5 d under fluorescent lights providing 150 ± 10 μmol m⁻² s⁻¹ PAR with 16 h light or under dark condition. A, Control seedling (left, no Nep1) and Nep1-treated seedling (right). Inset shows necrotic spots on cotyledons. B, Normal stomata. C, Necrotic region on Nep1-treated cotyledon. D, Normal roots with root hairs. E, Damaged root without root hairs from Nep1-treated seedling. F, Normal root tip. G, Abnormal root tip from Nep1-treated seedling. H, Control (left) and Nep1-treated (right), etiolated seedlings. I, Normal cotyledon of etiolated seedling. J, Cotyledon with necrotic regions in Nep1-treated, etiolated seedling. K, Normal root hairs from etiolated control seedling. L, Abnormal root without root hairs in Nep1-treated etiolated seedling. Growth conditions, A to G, 16 h light; H to L, dark.

Figure 2.

Inhibitory effect of F. oxysporum Nep1 on Arabidopsis growth. Sterilized Arabidopsis seeds were mixed with Nep1 (20 μg mL⁻¹), 0.001% (v/v) Silwet-L77, 0.3% agarose, 1% Suc in 1× MS media, and plated onto petri dishes. Controls were treated with 0.001% (v/v) Silwet-L77. Seeds were germinated and grown in growth cabinets at 22°C for 5 d under fluorescent lights providing 150 ± 10 μmol m⁻² s⁻¹ PAR with 16 h light. Nep1 treatment reduced the length of hypocotyl and root, 55% and 93%, respectively. The area of cotyledon was reduced by 79%. Values are the means ± se from 10 independent replicates, with at least six seedlings per replicate.

Figure 3.

Ultrastructure of Arabidopsis chloroplasts and immunolocalization of Nep1. Sterilized Arabidopsis seeds were mixed with Nep1 (20 μg mL⁻¹), 0.001% (v/v) Silwet-L77, 0.3% agarose, 1% Suc in 1× MS media, and plated onto petri dishes. Controls were treated with 0.001% (v/v) Silwet-L77. Seeds were germinated and grown in growth cabinets at 22°C for 5 d under fluorescent lights providing 150 ± 10 μmol m⁻² s⁻¹ PAR with 16 h light prior to sampling for transmission electron microscopy and immunolocalization of Nep1. A, Chloroplasts from a control true leaf. B, Chloroplasts from a Nep1-treated true leaf. Nep1 treatment causes breakdown of chloroplast internal membrane structures and generates lipophilic bodies. C, In control plant, gold particles were not detected. D, Numerous gold particles are present over the plant cell wall/membrane and cytosol. Scale bar = 500 nm. CH, Chloroplast; CW, cell wall; CY, cytosol.

Figure 4.

Chlorophyll a fluorescence image detection after treatment of Nep1. Chlorophyll a fluorescence images were measured from the adaxial surfaces of Arabidopsis cotyledons treated with Nep1. DCMU (200 mm) was used as an electron blocking agent in an electron transport chain of photosystem. Seedlings were grown at 22°C for 7 d under fluorescent lights providing 150 ± 10 μmol m⁻² s⁻¹ PAR with 24 h light. Seven-day-old Arabidopsis seedlings grown in the liquid culture were treated with Nep1 (10 μg mL⁻¹) without Silwet. Control seedlings were treated with an equivalent volume of sterile distilled water. Fluorescence was measured for up to 180 min after treatment. Chlorophyll a fluorescence images were from ratio images of F685/F730, and the graph (bottom) was drawn based on the ratio of F685/F730. Relative fluorescence intensity is given in vertical color scales on the left.

Figure 5.

Pie charts showing the number of genes identified on the Arabidopsis microarray (ATH1, Affymatrix) showing a 2-fold or greater induction (A) or repression (B) in the expression of the transcripts from Arabidopsis seedlings after 30-min treatment of 10 μg mL⁻¹ Nep1. Genes were categorized into functional groups based on the annotation from The Arabidopsis Information Resource and MIPS.

Figure 6.

Confirmation of microarray data by QPCR. Seedlings were grown at 22°C for 7 d under fluorescent lights providing 150 ± 10 μmol m⁻² s⁻¹ PAR with 24 h light. Seven-day-old Arabidopsis seedlings grown in the liquid culture were treated with Nep1 (10 μg mL⁻¹) without Silwet and harvested 30 and 180 min after treatment. Control seedlings were treated with an equivalent volume of sterile distilled water and harvested 0, 30, and 180 min after treatment. In total, 27 genes were selected from each of the expression profiles and functional categories. Genes were tested with cDNA templates from three different biological samples. Numbers inside the graph near the values for 30 min are fold induction from the microarray result. No number means less than 2-fold change in the microarray experiment. y axis, Relative expression; x axis, time after treatment (minutes); white circle, Nep1-treated sample; black circle, control.