Arabidopsis Defense against the Pathogenic Fungus Drechslera gigantea Is Dependent on the Integrity of the Unfolded Protein Response

Abstract

Drechslera gigantea Heald & Wolf is a worldwide-spread necrotrophic fungus closely related to the Bipolaris genus, well-known because many member species provoke severe diseases in cereal crops and studied because they produce sesterpenoid phytoxins named ophiobolins which possess interesting biological properties. The unfolded protein response (UPR) is a conserved mechanism protecting eukaryotic cells from the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER). In plants, consolidated evidence supports the role of UPR in the tolerance to abiotic stress, whereas much less information is available concerning the induction of ER stress by pathogen infection and consequent UPR elicitation as part of the defense response. In this study, the infection process of D. gigantea in Arabidopsis thaliana wild type and UPR-defective bzip28 bzip60 double mutant plants was comparatively investigated, with the aim to address the role of UPR in the expression of resistance to the fungal pathogen. The results of confocal microscopy, as well as of qRT-PCR transcript level analysis of UPR genes, proteomics, microRNAs expression profile and HPLC-based hormone analyses demonstrated that ophiobolin produced by the fungus during infection compromised ER integrity and that impairment of the IRE1/bZIP60 pathway of UPR hampered the full expression of resistance, thereby enhancing plant susceptibility to the pathogen.

Cyclopaldic Acid, the Main Phytotoxic Metabolite of Diplodia cupressi, Induces Programmed Cell Death and Autophagy in Arabidopsis thaliana

“…Therefore, on the whole, these results demonstrated that CA targeted plasma membrane where it inhibited PMA, leading to transmembrane potential impairment. To visualize the ER structure, leaves from A. thaliana plants expressing the fluorescent protein GFP-tmKKXX, which specifically localizes in the ER, were used [ 28 ]. Results of confocal microscopy showed that the integrity of the continuous ER network was not significantly affected by 10 or 100 μM CA ( Figure 5 d) Hence, this result indicated that ER is not a primary target of CA.…”

“…The chlorophyll content of A. thaliana leaves was estimated as described previously [ 28 ]. One hundred mg of control or CA-treated leaves were collected in a Falcon tube with 5 mL of DMSO.…”

“…Ion leakage assay was performed as described previously [ 28 ]. Two hundred mg of leaves from three-weeks old A. thaliana plants untreated or treated with 10 µM or 100 µM CA, for 6 h or 24 h, were cut into 5 mm strips, and submerged in 30 mL of deionized water, for 2 h, at 25 °C.…”

“…H 2 O 2 was detected in leaves from three-weeks old A. thaliana plants untreated or treated with 10 µM or 100 µM CA, for 6 h or 24 h, by staining with 3,3′-diaminobenzidine tetrahydrochloride (DAB), as described previously [ 28 ]. For each sample, five leaves were submerged in a solution of 10 mM DAB, pH 6.8, containing 0.05% ( w/v ) Tween 20.…”

“…Membrane-lipid peroxidation was estimated in leaves from three-weeks old A. thaliana plants untreated or treated with 10 µM or 100 µM CA for 6 h or 24 h, by the MDA method, as described previously [ 28 ]. One hundred mg of control or CA-treated A. thaliana leaves were homogenized in liquid N 2 , suspended in 500 μL of 0.1% trichloroacetic acid (TCA) and centrifuged at 15,000× g , for 10 min, at 4 °C.…”