Multilayered Regulation of Ethylene Induction Plays a Positive Role in Arabidopsis Resistance against Pseudomonas syringae

Abstract

Ethylene, a key phytohormone involved in plant-pathogen interaction, plays a positive role in plant resistance against fungal pathogens. However, its function in plant bacterial resistance remains unclear. Here, we report a detailed analysis of ethylene induction in Arabidopsis (Arabidopsis thaliana) in response to Pseudomonas syringae pv tomato DC3000 (Pst). Ethylene biosynthesis is highly induced in both pathogen/microbe-associated molecular pattern (PAMP)-triggered immunity and effector-triggered immunity (ETI), and the induction is potentiated by salicylic acid (SA) pretreatment. In addition, Pst actively suppresses PAMP-triggered ethylene induction in a type III secretion system-dependent manner. SA potentiation of ethylene induction is dependent mostly on MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) and MPK3 and their downstream ACS2 and ACS6, two type I isoforms of 1-aminocyclopropane-1-carboxylic acid synthases (ACSs). ACS7, a type III ACS whose expression is enhanced by SA pretreatment, is also involved. Pst expressing the avrRpt2 effector gene (Pst-avrRpt2), which is capable of triggering ETI, induces a higher level of ethylene production, and the elevated portion is dependent on SALICYLIC ACID INDUCTION DEFICIENT2 and NONEXPRESSER OF PATHOGENESIS-RELATED GENE1, two key players in SA biosynthesis and signaling. High-order ACS mutants with reduced ethylene induction are more susceptible to both Pst and Pst-avrRpt2, demonstrating a positive role of ethylene in plant bacterial resistance mediated by both PAMP-triggered immunity and ETI.

Figure 1.

Bacterial PAMP- and effector-triggered ethylene induction and its potentiation by SA in Arabidopsis. A, Fourteen-day-old seedlings grown in GC vials were inoculated with Pst, Pst-hrcC⁻, or Pst-AvrRpt2 (final OD₆₀₀ = 0.02). Mock inoculation was used as a control. Ethylene accumulations in the headspace were determined at the indicated times. B, Replot of the data in A as the rates of ethylene production. Ethylene production rates were calculated as the average rates of ethylene production in the intervals of the two adjacent time points. C, Twelve-day-old seedlings grown in GC vials were treated with SA (final concentration of 100 µm). Two days later, they were inoculated with Pst, Pst-hrcC⁻, or Pst-avrRpt2 (final OD₆₀₀ = 0.02). Mock inoculation was used as a control. Ethylene accumulations in the headspace were determined at the indicated times. D, Replot of the data in C as the average rates of ethylene production in the intervals of the two adjacent time points. All data were all collected side by side. Error bars indicate sd (n = 3). FW, Fresh weight.

Figure 2.

RPS2-dependent induction of ethylene biosynthesis in Arabidopsis triggered by the avrRpt2 effector. A, Higher levels of ethylene induction in Arabidopsis inoculated with Pst-avrRpt2 are dependent on the presence of RPS2, the corresponding R gene. Fourteen-day-old wild-type (Col-0) and rps2 mutant seedlings grown in GC vials were inoculated with Pst or Pst-avrRpt2 (final OD₆₀₀ = 0.02). Ethylene accumulations in the headspace were determined at the indicated times. Error bars indicate sd (n = 3). FW, Fresh weight; hpi, hours post inoculation. B, Fourteen-day-old steroid-inducible avrRpt2 transgene (GVG-avrRpt2) in the wild-type Col-0 background (avrRpt2/RPS2), GVG-avrRpt2 in the rps2 mutant background (avrRpt2/rps2), and steroid-inducible vector control were treated with DEX (2 µm). Ethylene accumulations in the headspace were determined at the indicated times. Error bars indicate sd (n = 3). Seedlings were collected at the indicated times for in-gel kinase assays. C, RPS2-dependent activation of MPK3/MPK6 by the avrRpt2 effector. MAPK activities in samples collected in B were determined using 10 µg of total proteins by the in-gel kinase assay with myelin basic protein (MBP) as a substrate.

Figure 3.

Potentiation of Pst-induced ethylene biosynthesis by SA pretreatment is dependent on functional NPR1, and the NahG transgene abolishes this SA effect. Twelve-day-old wild-type (Col-0), npr1, or NahG Arabidopsis seedlings grown in GC vials were treated with SA (+SA; final concentration of 100 µm) or a solvent of SA stock solution (−SA). Two days later, they were inoculated with Pst (final OD₆₀₀ = 0.02). Ethylene accumulations in the headspace were determined at the indicated times. Error bars indicate sd (n = 3). Student’s t test was used to compare mutants and the wild type at the same time point after the same treatment (*, P ≤ 0.05; and **, P ≤ 0.01). FW, Fresh weight; hpi, hours post inoculation.

Figure 4.

SA pretreatment enhances MPK3/MPK6 activation by Pst. Twelve-day-old wild-type (Col-0) Arabidopsis seedlings grown in GC vials were treated with dimethyl sulfoxide (DMSO) solvent (−SA) or SA (+SA; final concentration of 100 µm). Two days later, they were inoculated with Pst, Pst-hrcC⁻, or Pst-avrRpt2 (final OD₆₀₀ = 0.02). Seedlings were collected at the indicated times. MAPK activities were determined using 10 µg of total proteins by the in-gel kinase assay with MBP as a substrate. hpi, Hours post inoculation.

Figure 5.

Arabidopsis MPK3 and MPK6 play essential and overlapping roles in Pst-induced ethylene biosynthesis and SA potentiation. A, Twelve-day-old wild-type (Col-0), mpk3, and mpk6 seedlings grown in GC vials were treated with DMSO solvent (−SA) or SA (+SA; final concentration of 100 µm). Two days later, Pst was inoculated (final OD₆₀₀ = 0.02), and ethylene accumulation in the headspace of the GC vials was measured. B, Twelve-day-old wild-type (Col-0) and chemical genetically rescued mpk3 mpk6 double mutant (MPK6SR line 45) seedlings grown in GC vials were treated with SA (+SA; final concentration of 100 µm) or DMSO, the solvent of SA stock solution (−SA). Two days later, they were inoculated with Pst (final OD₆₀₀ = 0.02) after pretreatment with NA-PP1 (+NA-PP1; 5 µm final concentration) or solvent control (+DMSO) for 30 min. Ethylene accumulations in the headspace were determined at the indicated times. Error bars indicate sd (n = 3). Student’s t test was used to compare mutants and the wild type at the same time point after the same treatment (*, P ≤ 0.05; and **, P ≤ 0.01). FW, Fresh weight; hpi, hours post inoculation.

Figure 6.

SA potentiated ethylene induction in various acs mutants after Pst infection. Twelve-day-old wild-type (Col-0) seedlings and high-order acs mutants were treated with DMSO solvent (−SA) or SA (+SA; final concentration of 100 µm). Two days later, Pst was inoculated (final OD₆₀₀ = 0.02), and ethylene accumulation in the headspace of the GC vials was measured at the indicated times. Error bars indicate sd (n = 3). One-way ANOVA was performed to compare acs mutants and the wild type at the same time point after the same treatment. Lowercase letters above the columns indicate statistically different groups. The two genotypes are considered to produce different amounts of ethylene when two or more time points are significantly different. The allele numbers are omitted for easy labeling. They are acs1-1, acs2-1, acs4-1, acs5-2, acs6-1, acs7-1, acs9-1, and acs11-1. FW, Fresh weight; hpi, hours post inoculation.

Figure 7.

SA potentiation of ethylene induction is associated with enhanced ACS2, ACS6, and ACS7 gene activation. Twelve-day-old wild-type (Col-0) seedlings grown in GC vials were treated with DMSO solvent (−SA) or SA (+SA; final concentration of 100 µm). Two days later, Pst was inoculated (final OD₆₀₀ = 0.02), and seedlings were collected at the indicated times for total RNA preparation. After reverse transcription, transcript levels of ACS2 (A), ACS6 (B), ACS7 (C), and ACS8 (D) genes were quantified by real-time PCR. Expression of the ACS genes was calculated as the percentage of ELONGATION FACTOR-1α (EF1α) transcript. Error bars indicate sd (n = 3). Student’s t test was used to compare plants treated with SA (+SA) and DMSO solvent (−SA) at the same time point after the same treatment (*, P ≤ 0.05; and **, P ≤ 0.01). hpi, Hours post inoculation.

Figure 8.

SA potentiated ethylene induction in high-order acs mutants after Pst-avrRpt2 inoculation. Twelve-day-old wild-type (Col-0) seedlings and acs mutants generated in Dr. A. Theologis’s laboratory were treated with DMSO solvent (−SA) or SA (+SA; final concentration of 100 µm). Two days later, Pst-avrRpt2 was inoculated (final OD₆₀₀ = 0.02), and ethylene accumulation in the headspace of the GC vials was measured at the indicated times. Error bars indicate sd (n = 3). One-way ANOVA was performed to compare acs mutants and the wild type at the same time point after the same treatment. Lowercase letters above the columns indicate statistically different groups. The two genotypes are considered to produce different amounts of ethylene when two or more time points are significantly different. The allele numbers are omitted for easy labeling. They are acs1-1, acs2-1, acs4-1, acs5-2, acs6-1, acs7-1, acs9-1, and acs11-1. hpi, Hours post inoculation.

Figure 9.

Elevated ethylene biosynthesis in response to Pst-avrRpt2 inoculation and SA pretreatment is dependent on functional NPR1 and SID2, and the NahG transgene abolishes this SA effect. Twelve-day-old wild-type (Col-0), npr1, sid2-2, or NahG Arabidopsis seedlings grown in GC vials were treated with SA (+SA; final concentration of 100 µm) or DMSO (−SA). Two days later, they were inoculated with Pst-avrRpt2 (final OD₆₀₀ = 0.02). Ethylene accumulations in the headspace were determined at the indicated times. Error bars indicate sd (n = 3). Student’s t test was used to compare mutants and the wild type at the same time point after the same treatment (*, P ≤ 0.05; and **, P ≤ 0.01). FW, Fresh weight; hpi, hours post inoculation.

Figure 10.

Ethylene is involved in plant resistance against both Pst and Pst-avrRpt2. Five-week-old wild-type (Col-0) and acs mutant Arabidopsis plants grown under a short-day light cycle were used for pathogen assays. Fully expanded leaves (three per plant) were infiltrated with either Pst (A) or Pst-avrRpt2 (B; OD₆₀₀ = 0.001). SA pretreatment was performed by spraying the plants with SA (1 mm) and drenching the soil with SA (1 mm) at the same time for 24 h before pathogen infiltration. Bacterial growth was quantified 3 d post inoculation. Student’s t test was used to compare mutants and the wild type (*, P ≤ 0.05; and **, P ≤ 0.01). CFU, Colony-forming units.

Figure 11.

Model depicting the regulation of ethylene biosynthesis in the Arabidopsis-Pst interaction, which involves PTI, effector-mediated suppression, ETI, and SA-induced potentiation. Three ACS members contribute to the Pst-induced ethylene biosynthesis. Among them, ACS2 and ACS6 are regulated by the stress-responsive MPK3/MPK6 cascade, while ACS7 is regulated by the unidentified signaling pathways. The contribution of ACS8 is uncertain due to the ethylene production by Pst. Even ACS8 is involved; its contribution should be minimal based on the low residual ethylene production in the acs1 acs2 acs6 acs4 acs5 acs9 acs7 acs11 mutant inoculated with Pst. All four ACS genes are activated at the transcriptional level. ACS2 and ACS6 are also regulated at the protein stability level by MPK3/MPK6 phosphorylation. Both the MAPK and unidentified pathways are activated in PTI and ETI. The MPK3/MPK6-regulated ACS2/ACS6 branch is also targeted by the unidentified effectors. This pathway is also the major target of the SA-induced potentiation of ethylene biosynthesis. In addition, induction of ACS7 expression by Pst is potentiated by SA pretreatment, which is similar to ACS2 and ACS6. In contrast, ACS8 is not involved in SA-potentiated ethylene production. ACC, 1-Aminocyclopropane-1-carboxylic acid; ACO, ACC oxidase; FLS2, FLAGELLIN SENSING2; PRRs, pattern-recognition receptors; S-AdoMet, S-adenosylmethionine; TFs, transcription factors.