Fig 1.
Activation of MPK3/MPK6 globally down-regulates photosynthetic genes.
(A) GO enrichment of 2,977 down-regulated genes (log2 ratio ≤ −3) after MPK3/MPK6 activation. RNA-seq analysis was performed using 12-d-old DD seedlings treated with 5 μM DEX for 0 and 6 h under light. Transcript levels were fold changes relative to the 0 h sample. GO analysis was carried out by using DAVID online tool [57,58]. Enrichment scores are shown as −log10 (p-value). (B) Schematic diagram of photosynthetic linear electron flow. Expression levels of genes involved in photosystem assembly and repair are shown as a heat map. (C) GO enrichment of 1,039 up-regulated genes (log2 ratio ≤ −3) after MPK3/MPK6 activation. Enrichment scores are shown as −log10 (p-value). (D) Activation of MPK3/MPK6 induces drastic down-regulation of photosynthesis-related genes. Twelve-d-old DD plants grown in liquid medium were treated with EtOH or 5 μM DEX for 8 h under light. Transcript levels were quantified by real-time PCR and presented as fold changes relative to 0 h samples. Values are means ± SD, n = 3. EF1a was used as internal control, n = 3. The numerical values used to construct panels A–D can be found in S1 Data. See also S1 Table. Cytb6f, cytochrome b6f; DAVID, Database for Annotation, Visualization and Integrated Discovery; DD, GVG-NtMEK2DD; DEX, dexamethasone; EF1a, elongation factor 1a; EtOH, ethanol; Fd, ferredoxin; FNR, ferredoxin-NADP+ reductase; GO, gene ontology; LHCI, light-harvesting complex I; LHCII, light-harvesting complex II; MPK, mitogen-activated protein kinase; OEC, oxygen-evolving complex; PSI, photosystem I; PSII, photosystem II; RNA-seq, RNA sequencing.
Fig 2.
Activation of MPK3/MPK6 induces multilayered photosynthetic inhibition ROS accumulation in chloroplasts.
(A and B) Activation of MPK3/MPK6 induces PSII inhibition. Fv/Fm (A) and Y(II) (B) were measured using soil-grown DD plants spray treated with EtOH (solvent control) or 15 μM DEX for indicated periods of time. Values are means ± SD, n = 8. (C) Changes of O-J-I-P curve induced by MPK3/MPK6 activation. Normalized fluorescence units were shown. Soil-grown DD plants were spray treated with EtOH or 15 μM DEX and kept in dark or light for 12 h. Representative measurements for each treatment were shown. (D) MPK3/MPK6 activation affects PQ pool redox status. Values are means ± SD, n = 8. (E) Activation of MPK3/MPK6 causes decreased NPQ at late stage. After treatment with EtOH or 15 μM DEX for indicated times under light, NPQ induction was carried out by using a light intensity of 610 μmol m−2 s−1 for 7 min and followed by an 8-min relaxation period in dark. Values are means ± SD, n = 6. (F) Activation of MPK3/MPK6 induces a decrease in PSII-LHCII super-complexes and an increase in CP43-less PSII core complex. Twelve-d-old DD plants grown in liquid medium were treated with 5 μM DEX for indicated times. Thylakoid membranes were isolated and solubilized with 1% dodecyl maltoside. Samples equivalent to 8 μg of chlorophyll were loaded to a BN-PAGE. (G and H) Activation of MPK3/MPK6 induces light-dependent accumulation of ROS in chloroplasts. DD plants grown in liquid medium were treated with EtOH solvent control or 5 μM DEX and kept in dark or light for 8 h. Cellular (G) and H2O2 (H) were visualized by NBT and DAB staining, respectively. (I) Light accelerates MPK3/MPK6 activation-induced HR-like cell death. Soil-grown DD plants were first spray treated with EtOH solvent control or 15 μM DEX and then kept in dark or under light for 36 h. The numerical values used to construct panels A–E can be found in S1 Data. See also S1 and S2 Figs. BN-PAGE, blue native polyacrylamide gel electrophoresis; CP43, photosystem II chlorophyll protein of 43 kDa; DAB, 3,3′-diaminobenzidine; DD, GVG-NtMEK2DD; DEX, dexamethasone; EtOH, ethanol; HR, hypersensitive response; LHCII, light-harvesting complex II; MPK, mitogen-activated protein kinase; NBT, nitroblue tetrazolium; NDH, NADH dehydrogenase-like; NPQ, non-photochemical quenching; PQ, plastoquinol; PSI, photosystem I; PSII, photosystem II; ROS, reactive oxygen species; r.u., relative unit.
Fig 3.
The amplitude and duration of MAPK activation regulate photosynthetic inhibition.
(A) Controlling the amplitude of MPK3/MPK6 activation in DD plants by spraying different concentrations of DEX. Soil-grown DD plants were spray treated with different concentrations of DEX. At 9 h after DEX spray, NtMEK2DD expression and MPK3/MPK6 activation were detected by immunoblot analysis using anti-FLAG and anti-pTEpY, respectively. Fv/Fm was measured at 18 h after DEX treatment. Values are means ± SD, n = 6, **P ≤ 0.001. The numerical values used to construct panel A can be found in S1 Data. (B) Transient activation of MPK3/MPK6 after flg22 treatment does not induce a decrease in PSII core D1 protein. Twelve-d-old DD and Col-0 plants grown in liquid medium were treated with 5 μM DEX and 200 nM flg22 for indicated times, respectively. Thylakoid membranes were isolated and solubilized with 1% dodecyl maltoside. Samples equivalent to 2 μg of chlorophyll were loaded to a 12% SDS-PAGE with 6 M urea. Anti-D1 was used to detect D1 abundance. MAPK activation was detected by immunoblot analysis using anti-pTEpY antibody. Ponceau S staining was used to show equal loading. (C) Twelve-d-old DD and Col-0 plants grown in ½ MS agar plates were treated with 5 μM DEX or EtOH (solvent control) and then kept in dark or under light for 12 h. D1 protein abundance in thylakoid membrane preparations was detected by immunoblot analysis using anti-D1 antibody. Ponceau S staining was used to show equal loading. (D) Prolonged, but not transient, MAPK activation induces photosynthetic inhibition. Twelve-d-old DD MPK6SR plants grown in liquid medium were first treated with 5 μM DEX. Seedlings were washed three times with ½ MS medium to remove DEX before the addition of 10 μM NA-PP1. Black bars indicate the durations of DEX or NA-PP1 treatment. Thylakoid membranes were isolated and solubilized with 1% dodecyl maltoside. Samples equivalent to 8 μg of chlorophyll were loaded to a BN-PAGE. See also S3 Fig. BN-PAGE, blue native polyacrylamide gel electrophoresis; CBB, Coomassie brilliant blue; Col-0, Columbia-0; CP43, photosystem II chlorophyll protein at 43 kDa; DD, GVG-NtMEK2DD; DEX, dexamethasone; DNH, NADH dehydrogenase-like; EtOH, ethanol; FLAG, an octapeptide; flg22, a 22 amino acids flagellin fragment; LHCII, light-harvesting complex II; mc, mega-complex; MPK, mitogen-activated protein kinase; MS, Murashige and Skoog medium; NA-PP1, 4-amino-1-tert-butyl-3-(1’-naphthyl)pyrazolo[3,4-d]pyrimidine; pMPK, phosphorylated MPK; PSI, photosystem I; PSII, photosystem II; pTEpY, dually phosphorylated Thr/Glu/Tyr peptide; sc, super-complex; SDS-PAGE, sodium dodecyl sulfate-PAGE.
Fig 4.
ETI induces strong PSII inhibition and prolonged MAPK activation.
(A–C) Photosynthetic parameters are differentially affected by different Pst strains. Four-wk-old Col-0 plants were infiltrated with 10 mM MgCl2 (mock), Pst-EV, Pst-AvrRpt2, or Pst-hrcC− (OD600 = 0.2) and then were kept under light with a transparent lid. Photosynthetic parameters, including Fv/Fm (A), Y(II) (B), and 1-qL (C), were measured at 9 and 18 hpi. Values are means ± SD, n = 5–8, **P ≤ 0.001. (D–F) AvrRpt2-triggered immunity induces drastic PSII inhibition. Four-wk-old soil-grown GVG-AvrRpt2 transgenic plants in Col-0 (AvrRpt2/RPS2) or rps2 mutant background (AvrRpt2/rps2) were spray treated with 15 μM DEX. Photosynthetic parameters, including Fv/Fm (D), Y(II) (E), and 1-qL (F), were measured at indicated times. Values are means ± SD, n = 4–8. The numerical values used to construct panels A–F can be found in S1 Data. (G) ETI mediated by both CNL- and TNL-type NLRs induces prolonged MAPK activation. Four-wk-old Col-0 plants were infiltrated with Pst carrying EV, AvrRpm1, AvrB, AvrRpt2, or AvrRps4 (OD600 = 0.02) for indicated time points. MPK3/MPK6 activation was detected by anti-pTEpY antibody. See also S4 and S5 Figs. AvrB, avirulence protein B; AvrRpm1, avirulence effector recognized by RPM1; AvrRpt2, avirulence effector recognized by RPS2; AvrRps4, avirulence effector recognized by RPS4; CNL, coiled coil-nucleotide binding site-leucine rich repeat; Col-0, Columbia-0; DEX, dexamethasone; ETI, effector-triggered immunity; EV, empty vector; GVG-AvrRpt2, DEX-inducible promoter-driven AvrRpt2; hpi, hours post inoculation; hrcC−, outer membrane type III secretion protein HrcC mutant; MPK, mitogen-activated protein kinase; NLR, nucleotide-binding leucine-rich repeat; OD, optical density; pMPK, phosphorylated MPK; PSII, photosystem II; Pst, Pseudomonas syringae pv tomato; pTEpY, dually phosphorylated Thr/Glu/Tyr peptide; RPS2, Resistance to Pseudomonas syringae 2; TNL, Toll/interleukin-1 receptor-nucleotide binding site-leucine rich repeat.
Fig 5.
MPK3 and MPK6 are required for ETI-induced PSII inhibition and ROS accumulation in chloroplasts.
(A and B) Pst-AvrRpt2–induced PSII inactivation is partially compromised in mpk3 mpk6 double mutant. Four-wk-old soil-grown Col-0, MPK6SR, and MPK3SR plants were first spray treated with NA-PP1 (10 μM) or DMSO solvent control for 2 h and then infiltrated with Pst-AvrRpt2 (OD = 0.2). Values are means ± SD, n = 4, 0.01 ≤ *P ≤ 0.001 and **P ≤ 0.001. (C) Normal Pst-AvrRpt2–induced PSII inactivation in mpk3 and mpk6 single mutants. Four-wk-old soil-grown Col-0, mpk3, and mpk6 plants were directly infiltrated with Pst-AvrRpt2 (OD = 0.2). Fv/Fm and Y(II) were measured at indicated time points. Values are means ± SD, n = 4. The numerical values used to construct panels A–C can be found in S1 Data. (D–F) MPK3 and MPK6 are required for Pst-AvrRpt2–induced , but not H2O2, accumulation. Twelve-d-old Col-0, MPK6SR, and MPK3SR plants grown in liquid medium were first treated with 2 μM NA-PP1 or DMSO (mock) for 1 h. They were then treated with Pst-AvrRpt2 (OD600 = 0.02) for indicated periods of time.
and H2O2 accumulation were detected by NBT and DAB staining, respectively. AvrRpt2, avirulence effector recognized by RPS2; Col-0, Columbia-0; DAB, 3,3′-diaminobenzidine; ETI, effector-triggered immunity; MPK, mitogen-activated protein kinase; NA-PP1, 4-amino-1-tert-butyl-3-(1’-naphthyl)pyrazolo[3,4-d]pyrimidine; NBT, nitroblue tetrazolium; OD, optical density;
, superoxide; Pst, Pseudomonas syringae pv tomato; PSII, photosystem II; ROS, reactive oxygen species.
Fig 6.
Prolonged MPK3/MPK6 activation is essential to ETI.
(A) MPK3 and MPK6 are required for Pst-AvrRpt2–induced HR cell death. Soil-grown Col-0, MPK6SR, and MPK3SR plants were first spray treated with 10 μM NA-PP1 for 2 h and then infiltrated with Pst-AvrRpt2 (OD = 0.02). Photos were taken at 18 hpi. Numbers are ratios of leaves with HR phenotype. (B) MPK3 and MPK6 are required for Pst-AvrRpt2–induced ion leakage. Soil-grown Col-0, MPK6SR, and MPK3SR plants were first spray treated with 10 μM NA-PP1 for 2 h; leaf discs were punched and then infiltrated with Pst-AvrRpt2 (OD = 0.02) by vacuum. Leaf discs were then transferred to GC vials containing 2 μM NA-PP1 or DMSO. Ion leakage was measured as increase in conductivity. Values are means ± SD, n = 4. (C) MPK3 and MPK6 are required for disease resistance against Pst-AvrRpt2. Col-0, MPK6SR, and MPK3SR plants grown in soil were first spray treated with 10 μM NA-PP1 or DMSO and then infiltrated with Pst-AvrRpt2 (OD600 = 0.0005). NA-PP1 or DMSO was sprayed again at 1.5 dpi. Pst-AvrRpt2 growth was quantified at 3 dpi. Values are means ± SD, n = 3, 0.01 ≤ *P ≤ 0.001. (D) Prolonged activation of MPK3/MPK6 is required for ETI-mediated resistance. Col-0, MPK6SR, and MPK3SR plants grown in soil were infiltrated with Pst-AvrRpt2 (OD600 = 0.0005). NA-PP1 was sprayed at indicated time points. Pst-AvrRpt2 growth was quantified at 3 dpi. Values are means ± SD, n = 3, *P ≤ 0.001. The numerical values used to construct panels B–D can be found in S1 Data. See also S6 Fig. AvrRpt2, avirulence effector recognized by RPS2; Col-0, Columbia-0; ETI, effector-triggered immunity; GC, gas chromatography; hpi, hours post inoculation; HR, hypersensitive response; MPK, mitogen-activated protein kinase; NA-PP1, 4-amino-1-tert-butyl-3-(1’-naphthyl)pyrazolo[3,4-d]pyrimidine; OD, optical density; Pst, Pseudomonas syringae pv tomato.
Fig 7.
Expression of pFld impairs MPK3/MPK6 activation-induced ROS accumulation, PSII inhibition, and HR-like cell death.
(A) Phenotype of 4-wk-old pFld transgenic plants in DD background. (B) Immunoblot analysis of pFld expression using anti-Fld antibody. (C) Normal activation of MPK3/MPK6 in pFld transgenic plants. Soil-grown plants were spray treated with 15 μM DEX for indicated periods of time. DD expression and activation of MPK3/MPK6 activation were detected by using anti-FLAG and anti-pTEpY, respectively. (D) Overexpression of pFld suppresses MPK3/MPK6 activation-induced ROS accumulation. Soil-grown DD and DD/pFld plants were infiltrated with EtOH or 5 μM DEX. was visualized by NBT staining. (E–G) Overexpression of pFld suppresses MPK3/MPK6 activation-induced photosynthetic inhibition. Fv/Fm was measured at 24 h (E), HR-like phenotype was recorded at 30 h (F), and disassembly of photosynthetic complexes at 24 h was visualized by BN-PAGE (G). Samples equivalent to 8 μg of chlorophyll were loaded. Values are means ± SD, n = 6, **P ≤ 0.001. The numerical values used to construct panel E can be found in S1 Data. See also S7 Fig. BN-PAGE, blue native polyacrylamide gel electrophoresis; CBB, Coomassie brilliant blue; CP43, photosystem II chlorophyll protein of 43 kDa; DD, GVG-NtMEK2DD; DEX, dexamethasone; EtOH, ethanol; FLAG, an octapeptide; Fld, flavodoxin; HR, hypersensitive response; LHCII, light-harvesting complex II; MPK, mitogen-activated protein kinase; NtMEK2DD, constitutively activated NtMEK2; NBT, nitroblue tetrazolium; NDH, NADH dehydrogenase-like;
, superoxide; pFld, plastid-targeted cyanobacterial flavodoxin; pMPK, phosphorylated MPK; PSI, photosystem I; PSII, photosystem II; pTEpY, dually phosphorylated Thr/Glu/Tyr peptide; ROS, reactive oxygen species.
Fig 8.
Photosynthetic inhibition and light are essential to a robust ETI.
(A) Expression of pFld suppresses Pst-AvrRpt2–induced HR phenotype and PSII inhibition. Soil-grown DD and DD/pFld plants were infiltrated with Pst-AvrRpt2 (OD = 0.02). Photos were taken at 24 hpi. Fv/Fm was measured at 18 hpi. Values are means ± SD, n = 6, **P ≤ 0.001. (B) Expression of pFld compromises disease resistance against Pst-AvrRpt2. Four-wk-old soil-grown DD and DD/pFld plants were infiltrated with Pst-AvrRpt2 (OD600 = 0.0005). Photos were taken at 3 dpi and Pst-AvrRpt2 growth was quantified at 2.5 dpi. Values are means ± SD, n = 3, **P ≤ 0.001. (C) Expression of pFld impairs Pst-AvrRpt2–induced ROS accumulation. Soil-grown DD and DD pFld plants were infiltrated with mock (10 mM MgCl2) or Pst-AvrRpt2 (OD = 0.02). was visualized by NBT staining at 6 hpi. (D) Light accelerates Pst-AvrRpt2–induced PSII inhibition and HR cell death. Four-wk-old Col-0 plants were infiltrated with Pst-AvrRpt2 (OD = 0.02). Plants were kept in dark or under light. Photos were taken at 18 hpi and numbers are ratios of leaves with HR phenotype. Fv/Fm was measured at 12 hpi. Values are means ± SD, n = 7, **P ≤ 0.001. (E and F) Light is essential to plant resistance against Pst-AvrRpt2. Four-wk-old Col-0 plants were infiltrated with Pst-AvrRpt2 (OD = 0.0005). After infiltration, plants were kept under light for 2 h to allow the evaporation of liquid, and then kept in dark or under different light intensities, as indicated. Photos were taken and Pst-AvrRpt2 growth was determined at 3 dpi. Values are means ± SD, n = 3, **P ≤ 0.001. The numerical values used to construct panels A, B, D, and F can be found in S1 Data. (G) Schematic model of MPK3/MPK6 activation-induced PSII inhibition, ROS accumulation in chloroplasts, and the robustness of ETI. AvrRpt2, avirulence effector recognized by RPS2; Col-0, Columbia-0; DD, GVG-NtMEK2DD; dpi, days post inoculation; ETI, effector-triggered immunity; flg22, a 22 amino acids flagellin fragment; FLS2, flagellin-sensitive 2; hpi, hours post inoculation; HR, hypersensitive response; MPK, mitogen-activated protein kinase; NBT, nitroblue tetrazolium; OD, optical density;
, superoxide; PAMP, pathogen/microbe-associated molecular pattern; pFld, plastid-targeted cyanobacterial flavodoxin; PRR, pattern recognition receptor; Pst, Pseudomonas syringae pv tomato; PSII, photosystem II; ROS, reactive oxygen species; RPS2, Resistance to Pseudomonas syringae 2; TTSS, type III secretion system.