Prior exposure of Arabidopsis seedlings to mechanical stress heightens jasmonic acid-mediated defense against necrotrophic pathogens

Abstract

Background: Prolonged mechanical stress (MS) causes thigmomorphogenesis, a stress acclimation response associated with increased disease resistance. What remains unclear is if; 1) plants pre-exposed to a short period of repetitive MS can prime defence responses upon subsequent challenge with necrotrophic pathogens, 2) MS mediates plant immunity via jasmonic acid (JA) signalling, and 3) a short period of repetitive MS can cause long-term changes in gene expression resembling a stress-induced memory. To address these points, 10-days old juvenile Arabidopsis seedlings were mechanically stressed for 7-days using a soft brush and subsequently challenged with the necrotrophic pathogens, Alternaria brassicicola, and Botrytis cinerea. Here we assessed how MS impacted structural cell wall appositions, disease symptoms and altered gene expression in response to infection.

Results: The MS-treated plants exhibited enhanced cell wall appositions and jasmonic acid (JA) accumulation that correlated with a reduction in disease progression compared to unstressed plants. The expression of genes involved in JA signalling, callose deposition, peroxidase and phytoalexin biosynthesis and reactive oxygen species detoxification were hyper-induced 4-days post-infection in MS-treated plants. The loss-of-function in JA signalling mediated by the JA-insensitive coronatine-insensitive 1 (coi1) mutant impaired the hyper-induction of defense gene expression and promoted pathogen proliferation in MS-treated plants subject to infection. The basal expression level of PATHOGENESIS-RELATED GENE 1 and PLANT DEFENSIN 1.2 defense marker genes were constitutively upregulated in rosette leaves for 5-days post-MS, as well as in naïve cauline leaves that differentiated from the inflorescence meristem well after ceasing MS.

Conclusion: This study reveals that exposure of juvenile Arabidopsis plants to a short repetitive period of MS can alter gene expression and prime plant resistance upon subsequent challenge with necrotrophic pathogens via the JA-mediated COI1 signalling pathway. MS may facilitate a stress-induced memory to modulate the plant's response to future stress encounters. These data advance our understanding of how MS primes plant immunity against necrotrophic pathogens and how that could be utilised in sustainable agricultural practices.

Keywords: Alternaria brassicicola; Botrytis cinerea; Jasmonic acid; Mechanical stress; Necrotrophic pathogen; Stress priming.

Fig. 1

Effect of a short period of MS to juvenile Arabidopsis seedlings. A TCH-inducible gene expression within 30 and 60 min after 10 s of MS. Relative mRNA expression levels were normalized using β-ACTIN as the housekeeping gene (n = 3). B Petiole length and rosette leaf area in a 10-day old plant subject to 7-days of repetitive MS (touched twice daily for 10 s). A representative image of a 17-day old plant has been displayed and data was averaged from scoring individual plants (n = 15 plants). C Floral stem height and reproductive architecture in MS-treated and control plants. 10-day old plants were subjected to 7-days of MS without any further stimulation and allowed to flower. Phenotypes were scored in a 35-day old plant from the main floral bolt. A representative image has been displayed and the floral stem height defines the average of multiple independent plants (n = 15). D Jasmonic acid (JA) and salicylic acid (SA) levels were quantified in a whole rosette from juvenile plants subject to 7-days of MS. Leaves were harvested 30 min post-MS (n = 10 plants). E Leaves from controls and 7-days MS plants stained with phloroglucinol to highlight lignin accumulation as red colouration (indicated by arrow). Error bars show standard error of biological variation. Statistical significance denoted by letters (A) was determined using ANOVA with the Bonferroni test, and asterisks (B, C, D) determined with student’s t-test (p < 0.05). Scale bars = B, 1 cm; C, 5 cm; E, 1 cm

Fig. 2

Defense response of MS plants to B. cinerea and A. brassicicola infection. The 5th to 7th true leaf from 17-day old plants were inoculated with 5 μL of 3.5 × 10⁴ spores/mL^− 1 of pathogens (n = 15 plants). A Lactophenol-trypan blue was used to stain fungal hyphae and to determine necrotic lesion denoted by circle in leaves from control and MS plants 48 hpi. A representative stained image and the average lesion area are displayed (n = 15 leaves). B Representative images of leaves from MS wild-type plant (WT) and the JA mutant coi1 infected with A. brassicicola (5 dpi). C Necrotic lesions were stained with lactophenol trypan blue and the lesion area (mm²) denoted by circle with arrow was measured to provide a quantitative measurement of pathogen proliferation (n = 15). All data are representative of at least two independent experiments. Error bars show the standard error of biological variation. Statistical significance denoted by letters was determined using ANOVA with the Bonferroni test (P < 0.05). Scale bars = A, 0.2 cm B, 1 cm; C, 0.3 cm

Fig. 3

Changes in cell wall appositions and biosynthetic gene expression in response to A. brassicicola infection. A Leaves from control, infected, MS-treated and MS-infected plants were stained with aniline blue (3-dpi). A higher intensity of callose deposition fluoresces bright blue (indicated by arrow). The mean callose fluorescence intensity quantitatively determined in inoculated leaves. The image is a representation of several leaves (n = 15). The relative gene expression of GSL6 was quantified 5 dpi. B-C The relative expression of PRX71 and PAD3 involved in the synthesis of lignin and the phytoalexin camalexin, respectively 5 dpi. D Leaves from control and MS -infected plants stained with 3,3′-Diaminobenzidine stain (36 hpi) that forms brown precipitation to indicate reactive oxygen species (ROS) accumulation. The relative expression of ROS marker genes (GST1 and RBOHD), was determined 5 dpi. The expression levels of all genes were normalized to the β-ACTIN housekeeping gene (n = 4 biological reps) and are representative of at least two independent experiments. Error bars show standard error. Letters are statistical differences using ANOVA with the Bonferroni test (p < 0.05). Scale bars = A, 50 μm; C, 1 cm

Fig. 4

Regulation of defense gene expression in rosette leaves subject to MS and/or A. brassicicola infection. The 5th through 7th true leaves from control, infected, MS-treated and MS-infected 17-day old plants were inoculated with 5 μL of 3.5 × 10⁴ spores/mL^− 1 of A. brassicicola. Gene expression was quantified 5 dpi. A Gene expression of VSP1, OPR3, ERF3, PAD3, PDF1.2, and PR1 in WT. B Relative expression of VSP1, OPR3, ERF3, PAD3, PDF1.2, and PR1 in coi1–16 mutant compared to the WT. Relative gene expression levels were normalized to the β-ACTIN housekeeping gene. Error bars show standard error of the mean (n = 4 biological replicate). Letters are the statistical difference between treatments with Bonferroni test using a 2-way ANOVA, (P < 0.05). Statistical analysis in the coi1–16 mutant (B) is relative to the WT (A). The experiment was repeated twice, with similar expression patterns obtained

Fig. 5

Analysis of defense gene expression and disease symptoms in naïve cauline leaves challenged with A. brassicicola. Ten days old WT plants MS for 7-days was allowed to grow without further stimulation until 2–3 cauline leaves emerged from the primary floral stem. The 1st and/or 2nd cauline leaves from MS-treated and control plants were inoculated with 5 μL of 3.5 × 10⁴ spores/mL^− 1 of A. brassicicola. A, B The relative expression of pathogen-related defense genes in cauline leaves from control, and MS plants, 2 dpi. C Three days post-inoculation, cauline leaves were stained with lactophenol trypan blue to reveal the necrotic lesion area. Representative cauline leaves are displayed from two experiments showing similar results. Gene expression levels were normalized to the β-ACTIN housekeeping gene. Error bars show the standard error of the mean (n = 4). Letters are statistical differences between treatments with Bonferroni test using ANOVA, P < 0.05. The experiment was performed twice with similar results

Fig. 6

A model showing how prior exposure of juvenile seedlings to a short period of MS can prime gene expression and resistance to subsequent necrotrophic pathogen infection. The first stress encounter induced by repetitive MS (pre-infection) causes: 1) an increase in JA accumulation, 2) the accumulation of defense compounds (e.g. lignin and callose) that promote structural barriers, and 3) altered levels of basal gene expression. The secondary stress encountered by inoculating MS-treated plants with A. brassicicola causes the hyper-induction or contra-regulation of gene expression five-days post-infection that primes resistance to necrotrophic pathogen infection. A genetic perturbation in JA signalling mediated by the coronatine-insensitive 1 (coi1–16) mutant affected the priming of gene expression and rendered MS-treated plants susceptible to infection