Salicylic acid regulates Arabidopsis microbial pattern receptor kinase levels and signaling

Abstract

In Arabidopsis thaliana, responses to pathogen-associated molecular patterns (PAMPs) are mediated by cell surface pattern recognition receptors (PRRs) and include the accumulation of reactive oxygen species, callose deposition in the cell wall, and the generation of the signal molecule salicylic acid (SA). SA acts in a positive feedback loop with ACCELERATED CELL DEATH6 (ACD6), a membrane protein that contributes to immunity. This work shows that PRRs associate with and are part of the ACD6/SA feedback loop. ACD6 positively regulates the abundance of several PRRs and affects the responsiveness of plants to two PAMPs. SA accumulation also causes increased levels of PRRs and potentiates the responsiveness of plants to PAMPs. Finally, SA induces PRR- and ACD6-dependent signaling to induce callose deposition independent of the presence of PAMPs. This PAMP-independent effect of SA causes a transient reduction of PRRs and ACD6-dependent reduced responsiveness to PAMPs. Thus, SA has a dynamic effect on the regulation and function of PRRs. Within a few hours, SA signaling promotes defenses and downregulates PRRs, whereas later (within 24 to 48 h) SA signaling upregulates PRRs, and plants are rendered more responsive to PAMPs. These results implicate multiple modes of signaling for PRRs in response to PAMPs and SA.

Figure 1.

The flg22 Response Is Enhanced in acd6-1 and the Wild Type after 24 h of BTH Treatment. (A) to (D) Chemical treatment schemes for the indicated panels: (A) for (E), (F), and (H); (B) for (G); (C) for (I); (D) for (J) and (K). In (A), (B), and (D), “on water” indicates that tissue was excised and floated on water to facilitate flg22 uptake. (E) and (F) ROS accumulation after 1 µM flg22 treatment of the indicated plants (n > 6). The times of the ROS accumulation peaks in the wild type (Col) and acd6-1 (a6-1) are is shown in the top right corner (E); – or + indicates the absence or presence of flg22 (F). (G) Callose deposition in leaves of the indicated plant lines (n > 24) as a percentage of the flg22-treated wild type (Col). – or + indicates without or with acd6-1 and absence or presence of flg22. (H) ROS accumulation after 1 µM flg22 treatment in the indicated plants. The graph shows total ROS accumulation after 1 µM flg22 treatment. (I) Callose deposition after mock, 1 μL flg22, or 10 µg/mL chitin treatment in wild-type plants pretreated for 24 h with 100 µM BTH or water as a percentage of chitin-treated plants (n > 30). (J) Total ROS accumulation after 1 µM flg22 treatment in wild-type plants pretreated for 24 h with 100 µM BTH or water (n = 12). (K) ROS accumulation after 1 µM flg22 treatment in wild-type plants pretreated for 24 h with 100 µM BTH or water (n = 6). RLU, relative light units. Error bars in (E), (F), (H), (J), and (K) are sd of data from representative experiments. Error bars in (G) and (I) are se of data from three independent experiments analyzed together. Except for the experiments in (H), which were repeated twice, all experiments were repeated three times with similar results. Letters above bars represent significance groups as determined by the Newman-Keuls multiple comparison test, P < 0.05 or better ([F] to [J]).

Figure 2.

Effects of acd6-1, SA Accumulation, and/or SA Agonist Treatment on PAMP (Co)Receptor Levels. (A) and (B) Chemical treatment and plant tissue collection schemes for the indicated panels: (A) for (C); (B) for (D) to (F). (C) FLS2 and BAK1 protein levels after BTH treatment of the wild type (Col). Leaves were collected 48 h after spray treatment with 100 μM BTH or mock treatment. Total and microsomal fraction (MF) proteins isolated from plants were analyzed by immunoblotting with FLS2 and BAK1 antibodies. (D) FLS2 and BAK1 protein levels in Col and acd6-1 (a6-1). Proteins were extracted and analyzed as in (C). (E) and (F) Effects of sid2-1 and etr1-1 mutations on FLS2 and BAK1 protein levels in acd6-1. Microsomal proteins isolated from Col, acd6-1, sid2-1, acd6-1 sid2-1, etr1-1, and acd6-1 etr1-1 plants were analyzed as in (C). (G) Transcript level of ERF1, an ethylene-responsive marker gene, in acd6-1 relative to the wild type determined by qRT-PCR using three biological repeats. Graphs in (C) to (F) show the mean fold change in receptor levels (normalized to total protein [1], Rubisco [2], or all proteins except Rubisco [3]) of the indicated plants relative to mock (C) or the wild type (Col) ([D] to [F]), quantified from immunoblots using three independent experiments. Dotted lines in (C) and (D) indicate separate comparisons with the respective mock (C) and Col (D) values in the total and microsomal fraction, respectively. Error bars show se. *P < 0.05; letters above bars represent significance groups as determined by the Newman-Keuls multiple comparison test, P < 0.05 or better ([E] and [F]). C, Coomassie blue stained. These experiments were repeated three times with similar results.

Figure 3.

Analysis of Defenses in acd6-1 Single and Double Mutants. (A) and (B) Chemical treatment and plant collection schemes for the indicated panels: (A) for (C) to (H); (B) for (I). (C) MPK levels and MPK activity analysis in the indicated plants, immunodetected in total protein extracts by MPK3, MPK6, and phospho-p44/42 MPK antibodies. The assignment of the phosphorylated MPK3 band was confirmed using acd6-1 mpk3 plants (Supplemental Figure 2). The star indicates a background band. C, Coomassie blue stained. (D) Transcript levels of the flg22-inducible gene At1g51890 in the indicated plants relative to acd6-1 (a6-1) determined by qRT-PCR. (E) Callose deposition in leaves of the indicated plant lines (n > 8) from a representative experiment. (F) Transcript levels of PR1 (an output of SA signaling) in the indicated plants relative to acd6-1 determined by qRT-PCR. In (D) to (F), – or + indicates the absence or presence of acd6-1. These experiments were repeated three times with similar results. (G) Total and free SA levels were quantified from the indicated plant lines. Box plots show the median, the second and third quartiles, which indicate 50% of the data points (open boxes), and the range (vertical lines above and below the boxes; n = 6). Letters above each box represent significance groups as determined by Fisher’s protected least significance measure, a posthoc multiple t test, P < 0.001. (H) Cell death in acd6-1, acd6-1 fls2, and acd6-1 bak1-4 mutants is shown as a percentage of the area of cell death in acd6-1. In different experiments, there was 18 to 26% area of cell death per viewing field in acd6-1. Each genotype was tested at least in two independent experiments. Letters above bars represent significance groups as determined by the Newman-Keuls multiple comparison test (n ≥ 15). Each letter group differs from other letter groups at P < 0.05 or better. In (D), (F), and (H), error bars show se using three or more independent experiments analyzed together. Graphs in (C) show the mean fold change in MPK/phospho-MPK levels (normalized to total protein [1], Rubisco only [2], or all proteins except Rubisco [3]) of the indicated plants relative to acd6-1 quantified from immunoblots using three independent experiments. Letters above bars in (C) to (F) and (H) represent significance groups as determined by the Newman-Keuls test, P < 0.05 or better. (I) FLS2-BAK1 complexes in the wild type (Col) and acd6-1 with water pretreatment and mock (−) or 1 µM flg22 (+) treatment for 10 min. Top, BAK1-containing complexes immunoprecipitated with BAK1 antibody, separated by SDS-PAGE, and subjected to immunoblot analysis with FLS2 and BAK1 antibodies. Bottom, FLS2 and BAK1 protein levels from total proteins of the indicated plants. This experiment was repeated four times with similar results. Complex formation of FLS2-BAK1 in mock treatment was not seen when tissue was incubated overnight in water (Supplemental Figure 3).

Figure 4.

Short-Term Effects of the SA Agonist BTH on Receptor-Dependent Signaling. (A) to (C) Chemical treatment and plant collection schemes for the indicated panels: (A) for (D); (B) for (E) and (F); (C) for (G). (D) Callose deposition 24 h after mock or 100 µM BTH treatment in leaves of the indicated plants (n > 8). – or + indicates the absence or presence of BTH. (E) FLS2 and BAK1 protein levels in total or microsomal fraction (MF) from wild-type (Col) plant leaves 4 h after water, 1 µM flg22, or 100 µM BTH treatment. Graphs show the average fold change in receptor levels (normalized to total protein [1], Rubisco only [2], or all proteins except Rubisco [3]) of the indicated plants relative to mock treatment quantified from immunoblots using three independent experiments. Error bars indicate se. C, Coomassie blue stained. (F) ROS accumulation after flg22 treatment of leaves pretreated for 4 h with BTH or flg22 (n > 10). RLU, relative light units. (G) Callose deposition in leaves infiltrated with water (−), 1 µM flg22 (flg22 +), or 100 µM BTH (BTH +). Callose was detected 20 h after the second treatment and is shown as a percentage of callose in plants (n > 8) treated only with BTH. In (D) to (G), letters above bars represent significance groups as determined by the Newman-Keuls multiple comparison test, P < 0.05 or better. Error bars in (D), (F), and (G) are sd from data from representative experiments. These experiments were repeated three times with similar results.

Figure 5.

Effects of SA Accumulation or the SA Agonist BTH on CERK1, Another PAMP Receptor. (A) to (E) Chemical treatment and plant collection schemes for the indicated panels: (A) for (F); (B) for (G); (C) for (H); (D) for (I); (E) for (J). (F) Effects of acd6-1 and SID2 on CERK1 protein levels. Microsomal proteins isolated from wild-type (Col), acd6-1 (a6-1), sid2-1, and acd6-1 sid2-1 plants were analyzed by immunoblotting with CERK1 antibody. (G) CERK1 protein level after BTH treatment of the wild type (Col). Leaves were collected 48 h after treatment with 100 μM BTH or mock treatment. Proteins were extracted from microsomal fraction and analyzed as in (F). (H) Callose deposition in leaves of the indicated plant lines (n > 24) as a percentage of wild-type (Col) plants treated with 10 µg/mL chitin. – or + indicates without or with acd6-1 and absence or presence of chitin. (I) CERK1 protein levels in microsomal fraction from wild-type (Col) leaves 4 h after water, 1 µM flg22, or 100 µM BTH treatment as in Figure 4E. (J) Callose deposition in leaves infiltrated with 10 µg/mL chitin after pretreatment with water (−) or 100 µM BTH (BTH +). Callose was detected 24 h after the second treatment and is shown as a percentage of callose in plants (n > 24) given mock treatment. Graphs in (F), (G), and (I) show the mean levels of CERK1 (normalized to total protein [1], Rubisco only [2], or all proteins except Rubisco [3]) of the indicated plants relative to Col (F) or mock ([G] and [I]) quantified from immunoblots using three independent experiments. C, Coomassie blue stained. In (F) to (J), error bars show se. *P < 0.05; letters above bars represent significance groups as determined by the Newman-Keuls multiple comparison test, P < 0.05 or better.

Figure 6.

The acd6 Null Mutant Shows Decreased Responses to flg22 and Reduced Receptor Levels. (A) Increased colonization of type III secretion-deficient P. syringae in plants lacking ACD6. The acd6-2 mutant and the wild type (Col) were sprayed with P. syringae pv maculicola ES4326 hrcC⁻ at a dose of OD₆₀₀ = 0.03, and 3 d later, bacteria were enumerated from eight leaf discs per genotype. fls2 was included as a control for increased colonization. Colonization of mutant plants was higher than that seen in the wild type (*P < 0.04, t test). cfu, colony-forming units. (B) to (D) Analysis of acd6-2 in comparison with fls2, bak1-4, and sid2-1. – or + indicates the absence or presence of flg22 in (B) and (C). (B) Total ROS accumulation after 1 µM flg22 treatment (as in Figures 1A and 1E) in the indicated plants (n > 10). RLU, relative light units. (C) Transcript levels of the flg22-induced gene At1g51890 in the indicated plants relative to wild-type (Col) plants 1 h after infiltration with 1 µM flg22 determined by qRT-PCR. (D) and (E) Callose deposition 18 h after 1 µM flg22 (n = 8) (D) or 24 h after 10 µg/mL chitin (n > 24) (E) infiltration in leaves of the indicated plant lines as a percentage of callose in the wild type (Col). (F) FLS2, BAK1, and CERK1 protein levels are low in the membrane microsomal fraction (MF) of acd6-2 relative to wild-type plants. Microsomal fraction proteins isolated from plants were analyzed by immunoblotting with FLS2, BAK1, and CERK1 antibodies. Graphs show the mean fold change in receptor levels (normalized to total protein [1], Rubisco only [2], or all proteins except Rubisco [3]) of acd6-2 relative to the wild type (Col) quantified using immunoblots using three independent experiments. *P < 0.05, which indicates that the acd6-2 values were different from wild-type values. (G) and (H) Chemical treatment schemes for the indicated panels: (G) for (I); (H) for (J). In (G), “on water” indicates that tissue was excised and floated on water to facilitate BTH uptake. (I) ROS accumulation after flg22 treatment of Col or acd6-2 leaves pretreated for 4 h with BTH or flg22 (n > 10). (J) Callose deposition in leaves infiltrated with water (−), 1 µM flg22 (flg22 +), or 100 µM BTH (BTH +). Callose was detected 20 h after the second treatment in plants (n > 8). Error bars in (A), (B), (D), (I), and (J) show sd from one representative experiment. Error bars in (C), (E), and (F) show se of three independent experiments analyzed together. Letters above bars represent significance groups as determined by the Newman-Keuls multiple comparison test, P < 0.05. These experiments were repeated three times with similar results.

Figure 7.

BAK1 and CERK1 Form Complexes with ACD6 in ACD6-HA Plants. Left, ACD6-containing complexes immunoprecipitated with HA matrix, separated by SDS-PAGE, and subjected to immunoblot analysis with BAK1 and CERK1 antibodies. Right, protein levels from microsomal proteins. The wild type (Col) was used as a negative control. This experiment was repeated three times with similar results.