Mitogen-activated protein kinases 3 and 6 are required for full priming of stress responses in Arabidopsis thaliana

Abstract

In plants and animals, induced resistance (IR) to biotic and abiotic stress is associated with priming of cells for faster and stronger activation of defense responses. It has been hypothesized that cell priming involves accumulation of latent signaling components that are not used until challenge exposure to stress. However, the identity of such signaling components has remained elusive. Here, we show that during development of chemically induced resistance in Arabidopsis thaliana, priming is associated with accumulation of mRNA and inactive proteins of mitogen-activated protein kinases (MPKs), MPK3 and MPK6. Upon challenge exposure to biotic or abiotic stress, these two enzymes were more strongly activated in primed plants than in nonprimed plants. This elevated activation was linked to enhanced defense gene expression and development of IR. Strong elicitation of stress-induced MPK3 and MPK6 activity is also seen in the constitutive priming mutant edr1, while activity was attenuated in the priming-deficient npr1 mutant. Moreover, priming of defense gene expression and IR were lost or reduced in mpk3 or mpk6 mutants. Our findings argue that prestress deposition of the signaling components MPK3 and MPK6 is a critical step in priming plants for full induction of defense responses during IR.

Figure 1.

Ability of Phytopathogenic Bacteria and Various Compounds to Induce the Primed State and IR Correlates with Capacity to Elicit MPK3 Transcript Accumulation. (A) Effect of phytopathogenic bacteria on MPK3 transcript accumulation. Three lower leaves of Arabidopsis plants were infiltrated with a suspension (10⁶ colony-forming units [cfu] mL⁻¹) of avirulent Pst DC3000 harboring the avirulence gene avrRpt2 or Psp carrying avrB. The bacteria were suspended in 10 mM MgCl₂. Control plants were infiltrated with MgCl₂ in the absence of bacteria. Three days after infection, upper untreated leaves were harvested and analyzed for the abundance of transcripts for MPK3 normalized to those of ACTIN2. Asterisks indicate significant differences (Student's t test, n = 4, P < 0.05). (B) Effect of selected chemical compounds on MPK3 transcript accumulation. Leaves were harvested from Arabidopsis plants 3 d after spray treatment with 300 μM SA, 300 μM 3-hydroxy-benzoic acid (3-OH-BA), 300 μM 4-chloro-SA (4-Cl-SA), or 100 μM BTH. All these compounds were dissolved in a solution of wettable powder carrier (WP). Control plants were treated with wettable powder carrier only or left untreated (control). An aliquot of leaf tissue was used to determine MPK3 transcript accumulation as described in (A). The experiments were performed three times with similar results. The values shown are means + sd (n = 4).

Figure 2.

BTH Induces Accumulation of MPK3 and MPK6 Transcripts and Proteins but Does Not Elicit Dual TEY Motif Phosphorylation. (A) Accumulation of MPK3 transcript and MPK3 protein. Leaves were harvested at various times after treatment of plants with 100 μM BTH (+; dark vertical bars) or a wettable powder carrier control (−; light vertical bars). An aliquot of leaf tissue was used for RNA extraction and quantitative RT-PCR (qRT-PCR) analysis to examine the abundance of MPK3 transcript normalized to that for ACTIN2. Another aliquot of leaf tissue was used for protein extraction and SDS-PAGE followed by immunodetection of MPK3 protein and dual TEY phosphorylation with polyclonal antibodies. Immunodetection of a loaded doubly phosphorylated human ERK2 (pERK2) kinase served as a positive control for blotting and immunodetection. (B) Accumulation of MPK6 transcript and MPK6 protein. The experimental setup and analyses described in (A) were used to assess the abundance of MPK6 transcript and to immunodetect MPK6 and pTEpY. The experiments were performed four times with similar results. The values shown are means + sd (n = 4). Bars above diagrams give light/dark periods. hpt, h post-treatment; WB, protein gel blot. Prior to immunodetection, the blots were stained with Ponceau S to assess whether gel loading was equal. For quantification of immunodetection signals, see Supplemental Figure 1 online.

Figure 3.

Dual TEY Phosphorylation Is Enhanced in BTH-Primed Leaves after Dip Inoculation with Bacteria or Infiltration with Water. (A) Primed plants challenged by dipping leaves into bacterial suspension. Plants were treated with 100 μM BTH (+) or wettable powder carrier (−) for 3 d. Leaves were then challenged by dipping into a suspension of Ps pv maculicola strain ES4326 (5 × 10⁸ cfu mL⁻¹) in 10 mM MgCl₂ containing 0.01% Silwet L-77 (time point zero), harvested at the times indicated, and analyzed for dual phosphorylation of the TEY motif by SDS-PAGE, protein gel blotting, and immunodetection with polyclonal antibodies. (B) Primed plants challenged by water infiltration. The same experimental setup as described in (A) but challenging leaves by infiltration of water. The experiments were done three times with similar results. Prior to immunodetection, the blots were stained with Ponceau S to assess whether gel loading was equal. For quantification of immunodetection signals, see Supplemental Figure 1 online.

Figure 4.

Attenuation of Priming for Potentiated Defense Gene Activation in MPK-Deficient Plants. (A) Attenuated PAL1 induction in an mpk3 deletion mutant and MPK6-silenced plants. Wild-type plants, an mpk3 deletion mutant (Δmpk3), and MPK6 RNAi-silenced plants were pretreated with wettable powder carrier or BTH for 3 d. Leaves were then left untreated or infiltrated with water and assayed for PAL1 expression after 2 h. Asterisks indicate significant differences (Student's t test, n = 4, P < 0.05). (B) Reduced defense gene activation in mpk3 and mpk6 T-DNA mutants. Same experimental setup as in (A) but using mpk3 and mpk6 T-DNA insertion mutants, assaying PR1 gene expression after 24 h, and determining MPK activity 10 min after infiltration using an in-gel kinase assay. Asterisks indicate significant differences (Student's t test, n = 4, P < 0.05). For quantification of immunodetection signals, see Supplemental Figure 1 online. (C) BTH-induced SAR gene expression in mpk3 and mpk6 T-DNA mutants. Wild-type, mpk3, and mpk6 plants were treated with wettable powder carrier (−) or BTH (+). Three days later, leaf tissue was harvested and analyzed for the accumulation of transcript for PR1, PR2, PR5, and GST1. GST1, glutathione S-transferase. The experiments were performed three times with similar results. The values shown are means + sd (n = 4).

Figure 5.

Reduction or Enhancement of Potentiated TEY Phosphorylation and Defense Gene Expression in npr1 or edr1. Wild-type plants or npr1 or edr1 mutants were treated with wettable powder carrier or BTH (100 μM) for 3 d. Leaves were then left untreated or infiltrated with water and assayed for presence of MPK3 and dual phosphorylation of the TEY motif in MPK3 and MPK6 at the 10-min time point or for PAL1 expression at the 2-h time point after infiltration. The experiment was performed three times with similar results. The values shown are means + sd (n = 4). Asterisks indicate significant differences (Student's t test, n = 4, P < 0.05). WB, protein gel blot. Prior to immunodetection, the blots were stained with Ponceau S to assess whether gel loading was equal. For quantification of immunodetection signals, see Supplemental Figure 1 online.

Figure 6.

Attenuation of BTH-IR, SAR, and Infection-Induced Dual TEY Phosphorylation in mpk Mutants. (A) Reduction of BTH-IR. Wild-type, mpk3, and mpk6 plants were pretreated with wettable powder carrier (−) or 100 μM BTH (+). Three days later, leaves of the plants were dip inoculated with a high titer of Pst DC3000 (5 × 10⁸ cfu mL⁻¹). Four days after infection, leaf discs were harvested and analyzed for the amount of bacteria, while another portion of infected leaves was examined for disease symptoms. Note that in some repeats of the experiment, the BTH-IR was completely abolished in mpk3. The experiment was done seven times with similar results. The values shown are means + sd (n = 10 biological replicates). Asterisks indicate significant differences (Student's t test, n = 10, P < 0.05). (B) Attenuation of SAR. Three lower leaves of wild-type, mpk3, and mpk6 plants were infiltrated with a suspension (5 × 10⁵ cfu mL⁻¹) of avirulent Pst DC3000 harboring the avirulence gene avrRpt2 (+). The bacteria were suspended in 10 mM MgCl₂. Control plants were infiltrated with MgCl₂ in the absence of bacteria (−). Three days later, two upper leaves were challenge-infected with a suspension of Pst DC3000 (5 × 10⁵ cfu mL⁻¹) in MgCl₂. Leaf discs were harvested from challenge-infected leaves and analyzed for the amount of bacteria after 3 d. The experiment was performed three times with similar results. The values shown are means + sd (n = 8 biological replicates). Asterisks indicate significant differences (Student's t test, n = 8, P < 0.05). (C) Reduction of induced dual TEY motif phosphorylation. Same experimental setup as in (B) but harvesting aliquots of leaves 2 h after challenge infection with Pst DC3000 to analyze dual phosphorylation of the TEY motif by SDS-PAGE, protein gel blotting (WB), and immunodetection with polyclonal antibodies. Prior to immunodetection, the blot was stained with Ponceau S to assess whether loading was equal. The experiment was done three times with similar results. Note the absence of pTEpY immunodetection bands in the mpk3 and mpk6 mutant. This data confirms that the immunoreactive material detected by the pTEpY antibody is phosphorylated MPK3 and MPK6. For quantification of immunodetection signals, see Supplemental Figure 1 online.