Priming of protein expression in the defence response of Zantedeschia aethiopica to Pectobacterium carotovorum

Abstract

The defence response of Zantedeschia aethiopica, a natural rhizomatous host of the soft rot bacterium Pectobacterium carotovorum, was studied following the activation of common induced resistance pathways—systemic acquired resistance and induced systemic resistance. Proteomic tools were used, together with in vitro quantification and in situ localization of selected oxidizing enzymes. In total, 527 proteins were analysed by label-free mass spectrometry (MS) and annotated against the National Center for Biotechnology Information (NCBI) nonredundant (nr) protein database of rice (Oryza sativa). Of these, the fore most differentially expressed group comprised 215 proteins that were primed following application of methyl jasmonate (MJ) and subsequent infection with the pathogen. Sixty-five proteins were down-regulated following MJ treatments. The application of benzothiadiazole (BTH) increased the expression of 23 proteins; however, subsequent infection with the pathogen repressed their expression and did not induce priming. The sorting of primed proteins by Gene Ontology protein function category revealed that the primed proteins included nucleic acid-binding proteins, cofactor-binding proteins, ion-binding proteins, transferases, hydrolases and oxidoreductases. In line with the highlighted involvement of oxidoreductases in the defence response, we determined their activities, priming pattern and localization in planta. Increased activities were confined to the area surrounding the pathogen penetration site, associating these enzymes with the induced systemic resistance afforded by the jasmonic acid signalling pathway. The results presented here demonstrate the concerted priming of protein expression following MJ treatment, making it a prominent part of the defence response of Z. aethiopica to P. carotovorum.

Figure 1

Venn diagrams showing the number of proteins whose expression was induced by the plant activators methyl jasmonate (MJ) and benzothiadiazole (BTH) and by Pectobacterium carotovorum infection. Zantedeschia aethiopica plants received the following treatments: C, distilled deionized water (ddw), BTH (5 μg/mL), MJ (10 mg/mL), followed by inoculation with P. carotovorum (Pc). Differences in expression between the inoculated and uninoculated treatments were calculated (threshold value, ≥2). Data are based on pooled protein samples from three independent experiments.

Figure 2

Level 2 Gene Ontology (GO) classification of all proteins. The identified proteins were classified on the basis of the molecular function term, biological processes term and cellular component term for each protein, from the annotations in the Gene Ontology Annotation (UniProt‐GOA) Database. The GO term distribution was extracted using blast2go.

Figure 3

Clusters of leaf proteins expressed in the different treatments: C, distilled deionized water (ddw); B, benzothiadiazole (BTH; 5 μg/mL); M, methyl jasmonate (MJ; 10 mg/mL); Pc, Pectobacterium carotovorum; BPc, BTH + P. carotovorum; MPc, MJ + P. carotovorum. The pie charts on the right show the number of proteins in different functional classes [Gene Ontology (GO) level 3]. The CLICK (CLuster Identification via Connectivity Kernels) algorithm was used to cluster the annotated proteins on the basis of their expression levels. (a, b) Cluster 1. (c, d) Cluster 2. (e, f) Cluster 3. (g, h) Cluster 4.

Figure 4

In vitro peroxidase (POD) and polyphenol oxidase (PPO) activity of the total soluble protein extracted from Zantedeschia aethiopica leaves. Leaves were treated with distilled deionized water (ddw) (control, C), 5 μg/mL benzothiadiazole (B) or 10 mg/mL methyl jasmonate (M) and, 24 h later, inoculated with Pectobacterium carotovorum (Pc). Proteins were extracted 24 h after inoculation (10 mm sodium acetate, pH 5.6). (a) In vitro enzymatic activity was quantified spectrophotometrically (475 nm) using 3,3′,5,5′‐tetramethylbenzidine (TMB) in the presence of H₂O₂ as the POD substrate and dopa as the substrate for PPO. Bars represent mean POD and PPO activity, based on four biological replicates for each treatment ± SE. Treatments followed by the same letter are not significantly different according to the Tukey–Kramer multiple range test at P < 0.01. (b, c) Sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) (10%) activity gels for PODs using TMB in the presence of H₂O₂ as the substrate (b) and for PPO using dopa as the substrate (c). Each lane contained 30 μg of protein. In all cases, extracted protein was pooled from the leaves of three plants that received the same treatment. Molecular weight (MW) markers are shown at the left of each gel.

Figure 5

Light microscopy images of peroxidase (POD) and polyphenol oxidase (PPO) activity in leaf discs of Zantedeschia aethiopica treated with plant activators: distilled deionized water (ddw) (control), 5 μg/mL benzothiadiazole (BTH) or 10 mg/mL methyl jasmonate (MJ). The discs were inoculated with Pectobacterium carotovorum 24 h later. Enzymatic activity was recorded 24 h after inoculation following incubation with diaminobenzidine (DAB) for the localization of POD activity (a) and dopa for the localization of PPO activity (b). POD or PPO activity was visualized via the accumulation of a polymerized product, as shown by the brown/black ring around the wounded or infected site (bar, 500 μm).