Plant defense: ARR11 response regulator as a potential player in Arabidopsis

Abstract

Plant growth and response to environmental cues are largely driven by hormones. Salicylic acid (SA)- and jasmonic acid (JA)-mediated defenses have been shown to be effective against different types of attackers. SA-mediated defense is mainly effective against biotrophic pathogens and phloem-feeding insects, whereas JA-mediated defense is effective against necrotrophic pathogens and tissue-damaging insects. Cytokinins (CKs) are classic growth hormones that have also emerged as plant immunity modulators. Evidence pointed out that CKs contribute to the defense responses mediated by SA and JA, acting as hormone modulators of the SA/JA signaling backbone. Recently, we identified in Arabidopsis a type-B response regulator 11 (ARR 11) involved in cytokinin-mediated responses as a novel regulator of the SA/JA cross-talk. Here we investigated plant fitness and resistance against the fungal necrotrophic pathogen Botrytis cinerea in Arabidopsis wild-type Col-8 and defective arr11 mutant following SA, JA, CK single or combined treatment. Our results demonstrated that the CK and SA/JA/CK combination has a positive outcome on plant fitness in both Arabidopsis Col-8 and arr11 mutant,. The triple hormone treatment is efficient in increasing resistance to B. cinerea in Col-8 and this effect is stronger in arr11 mutant. The results will provide not only new background knowledge, corroborating the role of ARR11 in plant-defense related processes, but also new potential opportunities for alternative ways of protecting plants from fungal diseases.

Keywords: ARR11; Arabidopsis; necrotrophic pathogen; plant defense; plant hormones.

Figure 1

Fitness parameters of Arabidopsis Col-8 and arr11 mutant. Leaf area (cm²), dry weight (mg), flowering time (days), and seed production (mg) were analyzed in Col-8 and arr11 plants after single and triple hormone treatment. Error bars represent SEM. Letters indicates a statistically significant difference between treatments and genotypes (two-way ANOVA; p< 0.0001, n= 15). The experiments have been repeated three times with similar results.

Figure 2

Gene expression analysis after hormone treatments. qRT-PCR analysis of ARR11 (A) in leaves of Col-8 and of PDF1.2 (B) in leaves of Col-8 and arr11, that were treated with a mock solution or with SA, MeJA, CK, SA/MeJA/CK. Fold change is relative to the expression in mock-treated plants and normalized to the reference gene PP2AA3. Gene expression analyses was performed 24 h after hormone treatment of 5-week-old plants. Letters indicates statistically significant differences between treatments and mock. One-way ANOVA, p< 0.05 (A); two-way ANOVA, p< 0.0001 (B).

Figure 3

B cinerea bioassay in Col-8 and arr11. Distribution of disease symptoms of leaves of Col-8 and arr11, 3 days after inoculation with B cinerea. The bars indicate the frequency distribution of disease symptoms. Disease rating is expressed in 5 classes: I, from no necrotic lesion visible to lesion smaller than 3 mm; II, lesion between 4 mm to 1 cm; III, lesion bigger than 1 cm; IV, lesion between 1 cm and 1.5 cm; V, necrotic leaf. A black asterisk above the bars indicates significant differences between mock and each treatment (χ²-test, p-value<0.05). The blue asterisk above the bracket indicates significant differences in response to any treatment between arr11 and Col-8.

Figure 4

Detection of ROS and lipid peroxidation in Arabidopsis Col-8 and arr11 leaves. (A) Detection of ROS on Col-8 and arr11 leaves was carried out by using 2’,7’-DCFH₂-DA or buffer (negative technical control). Fluorescence was observed under an LSM 710 confocal microscope with Plan Neofluar 20/1.30 objective. Laser excitations line was used, i.e., 488 for probe detection (green) and 561 nm for chlorophyll autofluorescence (red). Bar corresponds to 50 µm. The representative merged images are shown. (B) The level of TBARS was used to assess lipid peroxidation measuring the absorbance of MDA–TBA complex at 532 and 600 nm. Letters indicate statistically significant difference between different treatments and genotypes (two-way ANOVA, Tukey’s test p-value< 0.0001). Error bars represent means ± SDs (n = 3).