Active photosynthetic inhibition mediated by MPK3/MPK6 is critical to effector-triggered immunity

Extensive research revealed tremendous details about how plants sense pathogen effectors during effector-triggered immunity (ETI). However, less is known about downstream signaling events. In this report, we demonstrate that prolonged activation of MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MPKs), is essential to ETI mediated by both coiled coil-nucleotide binding site-leucine rich repeats (CNLs) and toll/interleukin-1 receptor nucleotide binding site-leucine rich repeats (TNLs) types of R proteins. MPK3/MPK6 activation rapidly alters the expression of photosynthesis-related genes and inhibits photosynthesis, which promotes the accumulation of superoxide (O2•−) and hydrogen peroxide (H2O2), two major reactive oxygen species (ROS), in chloroplasts under light. In the chemical-genetically rescued mpk3 mpk6 double mutants, ETI-induced photosynthetic inhibition and chloroplastic ROS accumulation are compromised, which correlates with delayed hypersensitive response (HR) cell death and compromised resistance. Furthermore, protection of chloroplasts by expressing a plastid-targeted cyanobacterial flavodoxin (pFLD) delays photosynthetic inhibition and compromises ETI. Collectively, this study highlights a critical role of MPK3/MPK6 in manipulating plant photosynthetic activities to promote ROS accumulation in chloroplasts and HR cell death, which contributes to the robustness of ETI. Furthermore, the dual functionality of MPK3/MPK6 cascade in promoting defense and inhibiting photosynthesis potentially allow it to orchestrate the trade-off between plant growth and defense in plant immunity.

Compared to the well-studied effector recognition, the mechanisms underlying the activation of NLRs and their downstream signaling pathways are still poorly understood [14][15][16][17]. Current findings suggest that the activity of NLRs undergoes multilayered regulation, including self-inhibition, dimerization or oligomerization, epigenetic and transcriptional regulation, alternative splicing, and proteasome-mediated degradation [14][15][16][17]. Despite of the different recognition and activation mechanisms of NLRs and PRRs, ETI and PTI involve a similar set of downstream defense responses, including calcium-mediated signaling, activation of mitogen-activated protein kinases (MAPKs), production of reactive oxygen species (ROS), transcriptional reprogramming, and biosynthesis of antimicrobial compounds [3,4,7,[21][22][23][24][25][26][27][28]. However, the responses during ETI have a longer duration and higher magnitude. As a result, ETI was proposed to be an amplified PTI [7]. Recently, it was proposed that plasma membrane-localized CNLs such as Resistance to Pseudomonas syringae pv maculicola 1 (RPM1), Resistance to Pseudomonas syringae 2 (RPS2), and Resistance to Pseudomonas syringae 5 (RPS5) trigger downstream defense responses similar to that activated by PRRs during PTI, with the exception of different magnitude and duration [12]. In contrast, ETI mediated by nucleus-localized TNLs, including Resistance to Pseudomonas syringae 4/Resistance to Ralstonia solanacearum 1 (RPS4/RRS1) and Resistance to Pseudomonas syringae 6 (RPS6), which is dependent on Enhanced Disease Susceptibility 1 (EDS1), seems to be more associated with transcriptional reprogramming [12]. This notion was supported by the findings that both plasma membrane-localized PRRs and CNL-type RPS2 can activate MPK3/ MPK6 [24]. However, whether TNLs can activate MAPK signaling remains to be determined. Interestingly, EDS1 also contributes to RPS2-conditioned resistance when salicylic acid (SA) biosynthesis is blocked [29], indicating a complicated cross talk between CNL-and TNLmediated resistance.
Recently, two new immune responses were identified, cell cycle repression and chloroplast stromule formation [30,31]. During ETI, the canonical function of cyclin-dependent kinase inhibitor (CKI)-retinoblastoma (RB)-E2F transcription factor in cell cycle progression is repressed and shifted to promote programmed cell death and transcriptional reprogramming [30]. Chloroplast stromules, dynamic tubular projections of chloroplasts, are strongly induced during plant immunity [31]. Some of the stromules were observed to connect chloroplasts with the nucleus, which was proposed to transport pro-defense signals, e.g., chloroplast-generated ROS, into the nucleus to promote transcriptional reprogramming [31]. At present, the mechanism underlying the generation of ROS in chloroplasts is still unclear. We have previously shown that prolonged activation of SA-induced protein kinase (SIPK) and woundinduced protein kinase (WIPK), the orthologs of Arabidopsis MPK6 and MPK3 in tobacco, respectively, inhibits photosynthesis and induces the accumulation of ROS in chloroplasts, which accelerates HR-like cell death in plants under light [32]. Later, chloroplast-originated ROS was implicated in promoting localized cell death in tobacco during nonhost interaction [33]. Because HR cell death can be uncoupled from ETI [34], i.e., host cell death is not the cause for resistance [7], it remains to be determined whether MAPK signaling and chloroplast-originated ROS accumulation contribute to ETI.
In this study, we demonstrate that both CNL and TNL NLR-mediated ETI induce prolonged activation of MPK3/MPK6, which contributes to the rapid and global inhibition of photosynthesis at multiple levels and the generation of ROS in chloroplasts. Loss-of-function of MPK3/MPK6 signaling compromises effector-triggered inhibition of photosynthetic activities, accumulation of ROS in chloroplasts, HR cell death, and pathogen resistance. Furthermore, it was discovered that inhibition of photosynthetic activities and chloroplastic ROS accumulation can mutually enhance each other. Based on these findings, we conclude that MPK3/MPK6-mediated active photosynthetic inhibition is a part of Arabidopsis immune response and plays a positive role during ETI.

MPK3/MPK6 activation induces global down-regulation of genes related to photosynthesis
Inhibition of photosynthesis occurs in plants under a variety of abiotic and biotic stresses [44,51]. At present, it is unclear whether the inhibition is a passive response caused by stresses/ pathogens or a response actively regulated by host signaling pathways, and if so, what the outcomes/functions are of such active inhibition. In our previous study, we found that activation of SIPK and WIPK, two stress-responsive MPKs in tobacco, causes rapid and strong inhibition of CO 2 fixation [32]. To elucidate the underlying mechanism, we utilized the Arabidopsis system and profiled the gene expression in the conditional gain-of-function Arabidopsis GVG-NtMEK2 DD (abbreviated as DD) transgenic plants. In this system, dexamethasone (DEX) treatment induces the expression of NtMEK2 DD , a constitutively active variant of Nicotiana tabacum MAP kinase kinase 2 (NtMEK2), which in turn activates specifically the endogenous MPK3 and MPK6 in Arabidopsis [52,53]. RNA sequencing (RNA-seq) profiling revealed a total of 2,984 genes that were down-regulated (log 2 < −3) and 1,042 genes up-regulated (log 2 > 3) at 6 h after DEX treatment (S1 Table). Genes involved in photosynthesis, including photosynthetic light harvesting, light reaction, electron transport, and dark reaction, were highly enriched in the down-regulated genes (Fig 1A and 1B). The up-regulated genes were mainly enriched in genes involved in plant-pathogen and plant-environment interactions and secondary metabolism (Fig 1C). Up-regulation of defense genes, including those involved in phytoalexin biosynthesis, is consistent with our previous reports [28,[53][54][55][56]. Down-regulation of selected photosynthetic genes was further confirmed by quantitative reverse transcriptionpolymerase chain reaction (RT-PCR). As shown in Fig 1D, expression of genes involved in PSII repair (LQY1 and LTO1), PSII assembly (PAM68), PSII stabilization (PSB32), and transcription in chloroplasts (e.g., SIG1-SIG6) were all decreased drastically after MPK3/MPK6 activation.

MPK3/MPK6 activation causes photosynthetic inhibition and ROS accumulation in chloroplasts
To determine the physiological consequences of the inhibition of photosynthetic genes, we measured PSII activity using chlorophyll fluorescence techniques [59]. As shown in Fig 2A  and 2B, the maximal PSII activity parameter Fv/Fm and effective PSII operating efficiency parameter Y(II) both decreased upon MPK3/MPK6 activation. We next measured the fast chlorophyll fluorescence kinetics, also known as O-J-I-P curve [60]. The J-I rise was lower in DEX-treated DD plants than in ethanol solvent control (Fig 2C), indicating a reduced plastoquinol (PQ) reduction after MPK3/MPK6 activation. We next measured PQ redox status parameter 1-qL. Consistent with a decrease in PSII activity, a more oxidized PQ pool, reflected by the decrease of 1-qL, was detected ( Fig 2D).
NPQ dissipates light energy as heat to protect PSII from photodamage [61,62]. We found that NPQ induction by high light (610 μmol m −2 s −1 ) was not affected at the early stage of MPK3/MPK6 activation but decreased significantly at 18 h and 24 h after DEX treatment ( Fig  2E). The decreased NPQ at 18 h and 24 h is likely to be a consequence of PSII inhibition rather than an active down-regulation of NPQ. The decreased NPQ may accelerate PSII inhibition due to impaired dissipation of light energy.
We next examined PSII inhibition using blue native polyacrylamide gel electrophoresis (BN-PAGE), by which changes of thylakoid membrane photosynthetic complexes can be visualized. There was a decrease in PSII-LHCII super-complexes and an increase in CP43-less PSII core complex in both BN-PAGE ( Fig 2F) and two-dimensional sodium dodecyl sulfate (SDS)-PAGE analyses (S1 Fig), which correlates well with PSII inhibition. Collectively, the changes in photosynthetic parameters and complexes were found to be associated with the down-regulation in mRNA levels of photosynthetic genes after MPK3/MPK6 activation in Arabidopsis ( Fig  1A). Together with our previous report that SIPK/WIPK activation inhibits photosynthetic activities in tobacco [32], we can conclude that inhibition of photosynthesis after the activation of pathogen-responsive MPK3/MPK6 in Arabidopsis or their orthologs in other plant species is a common response in plants.
Photosynthesis inhibition in plants frequently leads to the accumulation of ROS [63,64]. As shown in Fig 2G and S2 Fig, nitroblue   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 −log 10 (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 (log 2 ratio −3) after MPK3/MPK6 activation. Enrichment scores are shown as −log 10 (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   (Fig 2H and S2  Fig). Consistent with our previous report using tobacco, MPK3/MPK6 activation-induced HR-like cell death in Arabidopsis was also light dependent ( Fig 2I). It is worth noting that MPK3/MPK6 activation-induced PSII inhibition is independent of light, although it is delayed in the absence of light (Fig 2A and 2C), suggesting that ROS accumulation in chloroplasts may accelerate PSII inhibition and HR-like cell death in DD plants.

Long-lasting MPK3/MPK6 activation is needed to induce photosynthetic inhibition
Previous studies showed that MPK3/MPK6 activation is transient during PTI and is prolonged during CNL-type RPS2-mediated ETI [24]. To examine the amplitude of MPK3/MPK6 activation in regulating photosynthetic inhibition, DD plants were treated with increasing concentrations of DEX. As shown in Fig 3A, there was a correlation between the level of PSII inhibition and the amplitude of MPK3/MPK6 activation. Prolonged MPK3/MPK6 activation in DD caused a drastic photosynthetic inhibition, as demonstrated by western blot detection of PSII core proteins D1 (Fig 3B), while no photosynthetic inhibition was observed in Columbia-0 (Col-0) plants treated with a 22 amino acids flagellin fragment (flg22), which induced a transient MPK3/MPK6 activation ( Fig 3B and S3 Fig). Moreover, MPK3/MPK6 activationinduced decrease of D1 protein is also independent of light ( Fig 3C), which correlates well with the measured chlorophyll fluorescence in dark (Fig 2A-2C). To further test the duration of MAPK activation in regulating photosynthetic inhibition, we crossed DD transgene into MPK6SR, a chemical-genetically rescued mpk3 mpk6 double mutant system [38,65], to generate DD MPK6SR (genotype: GVG-NtMEK2 DD mpk3 mpk6 P MPK6 :MPK6 YG ). In DD MPK6SR plants, MPK6 can be activated by DEX and inhibited by 4-amino-1-tert-butyl-3-(1'-naphthyl) pyrazolo [3,4-d]pyrimidine (NA-PP1), a specific inhibitor of the sensitized MPK6 YG . As revealed by BN-PAGE analysis, short-term MPK6 activation failed to induce photosynthetic inhibition (Fig 3D), demonstrating that prolonged, but not transient, MAPK activation is required for photosynthetic inhibition. It was noted that photosynthetic inhibition in DD MPK6SR plants was slower in comparison to DD plants after DEX treatment, which may be due to the lack of MPK3, or reduced expression of NtMEK2 DD as a result of gene silencing, or both. Nonetheless, it clearly demonstrates that long-lasting, but not transient, MPK3/MPK6 activation induces photosynthetic inhibition.

ETI, but not PTI, induces prolonged MAPK activation, photosynthetic inhibition, and ROS accumulation in chloroplasts
To determine the involvement of MPK3/MPK6 in photosynthetic inhibition during plant immunity, we first measured the kinetics of photosynthetic parameters in wild-type plants infiltrated with Pseudomonas syringae pv. tomato DC3000 carrying empty vector (Pst-EV), Pst-AvrRpt2, a Pst strain carrying the avirulence effector recognized by RPS2 (AvrRpt2) effector dark or light for 8 h. Cellular O À 2 (G) and H 2 O 2 (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 0 -diaminobenzidine; DD, GVG-NtMEK2 DD ; 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.  gene, or Pst-hrcC − , a Pst strain carrying a mutation in hrcC gene. Pst-AvrRpt2 triggers both PTI and CNL-type RPS2-dependent ETI in Arabidopsis. Due to the lack of functional type-III secretion system, Pst-hrcC − cannot deliver effectors into plant cells and only induces PTI.
Similar to the gain-of-function activation of MPK3/MPK6 in DD plants, Pst-AvrRpt2 induced drastic reductions in Fv/Fm ( Fig 4A and S4 Fig), Y(II) (Fig 4B and S4 Fig), and 1-qL ( Fig 4C). The decreases in Fv/Fm and Y(II) after Pst-EV inoculation were much slower and delayed in comparison to after Pst-AvrRpt2 inoculation (Fig 4A and 4B and S4 Fig). Interestingly, Pst-hrcC − , which only induces PTI, had no effect on any measured chlorophyll fluorescence parameters (Fig 4A-4C and S4 Fig), indicating that PTI is not sufficient to induce photosynthetic inhibition. Consistent with this conclusion, flg22 infiltration failed to induce change in photosynthetic parameters and decrease of D1 protein ( Fig 3B and S4 Fig). In addition, no photosynthetic changes were detected after infiltration of a nonpathogenic strain, P. fluorescens, carrying empty vector [66], while P. fluorescens carrying AvrRpm1, which triggers CNL-type RPM1-mediated ETI, induced strong photosynthetic inhibition (S4 Fig). These results further support that ETI, but not PTI, induces photosynthetic inhibition.

MPK3 and MPK6 are required for both CNL-and TNL-mediated ETI
To determine whether MPK3/MPK6 are required for ETI-induced photosynthetic inhibition and ROS accumulation in chloroplasts, we utilized the newly generated chemical-genetically rescued mpk3 mpk6 double mutant systems [38,65]. Both MPK6SR (genotype: mpk3 mpk6 P MPK6 :MPK6 YG ) and MPK3SR (genotype: mpk3 mpk6 P MPK3 :MPK3 TG ) were tested. As is shown in Fig 5A, Pst-AvrRpt2-induced PSII inhibition was partially impaired in MPK6SR and MPK3SR plants after pretreatment with NA-PP1, a specific inhibitor of the sensitized MPK6 YG and MPK3 TG , demonstrating that MPK3 and MPK6 are required for the fast and drastic PSII inhibition triggered by ETI activation. No differences were observed in MPK6SR and MPK3SR plants pretreated with DMSO (Fig 5B), or mpk3 and mpk6 single mutants (Fig 5C), demonstrating that MPK3 and MPK6 function redundantly in mediating ETI-induced PSII inhibition. 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 (OD 600 = 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 5D and 5F, Pst-AvrRpt2-induced O À 2 accumulation in chloroplasts was delayed in NA-PP1-treated MPK6SR and MPK3SR, which correlates well with the reduced PSII inhibition in NA-PP1-treated MPK6SR and MPK3SR plants (Fig 5A). These results suggest that MPK3/MPK6 are involved in ETI-induced ROS accumulation in chloroplasts. However, although MPK3/MPK6 activation can induce both accumulation of H 2 O 2 and O À 2 (Fig 2G  and 2H and S2 Fig), Pst-AvrRpt2-induced H 2 O 2 accumulation was not affected in MPK6SR and MPK3SR (Fig 5E), possibly due to the complicated enzymatic and nonenzymatic conversion of O À 2 to H 2 O 2 and/or H 2 O 2 decomposition. Although HR-like cell death after the activation of MPK3/MPK6 or their orthologs in tobacco was detailed more than a decade ago [52,76,77], it is still unknown whether pathogeninduced HR cell death requires this MAPK cascade. As a result, we examined HR cell death in MPK3SR and MPK6SR during ETI after Pst-AvrRpt2 inoculation. As shown in Fig 6A and 6B, HR cell death and ion leakage were impaired in NA-PP1-, but not DMSO-, treated MPK6SR and MPK3SR plants. Associated with this, we also observed compromised resistance to Pst-AvrRpt2 in NA-PP1-treated MPK6SR and MPK3SR plants (Fig 6C). Associated with the high titer of Pst-AvrRpt2 growth in Arabidopsis, leaf chlorosis was observed (Fig 6C), consistent with the breach of plant ETI in the loss-of-function mpk3 mpk6 double mutant system. In solvent DMSO-treated controls, the growth of Pst-AvrRpt2 was suppressed, demonstrating an effective ETI (Fig 6C). These results demonstrate that MPK3 and MPK6 function redundantly and are required for ETI.
We also measured PSII inhibition, ion leakage, and bacterial growth in the loss-of-function mpk3 mpk6 double mutant system after inoculation with Pst carrying AvrRpm1, AvrB, and AvrRps4. As shown in S6 Fig, PSII inhibition and ETI were all compromised in NA-PP1-, but not DMSO-, treated MPK6SR and MPK3SR plants. These results suggest that MPK3/MPK6 are essential for both CNL-and TNL-type NLR-mediated PSII inhibition and ETI.
Prolonged, but not transient, MAPK activation induces photosynthetic inhibition ( Fig 3B). As a result, we examined whether long-lasting MAPK activation is essential for ETI. Wild-type MPK6SR and MPK3SR plants infiltrated with Pst-AvrRpt2 were treated with NA-PP1 to inhibit MAPK activity at different times after inoculation. As shown in Fig 6D, NA-PP1 treatment at 12 hpi could still compromise RPS2-mediated ETI, demonstrating that short-term MAPK activation was not sufficient to confer efficient ETI.

Photosynthetic inhibition is essential to ETI
To provide genetic evidence to support the importance of photosynthetic inhibition in ETI, we expressed a plastid-targeted cyanobacterial flavodoxin (pFld) in DD and GVG-AvrRpt2 plants.
Flowering plants do not have flavodoxin [78], and ectopically expressing a cyanobacterial flavodoxin in tobacco confers broad stress tolerance [79,80]. We found that overexpression of pFld in DD and GVG-AvrRpt2 background caused growth retardation (Fig 7A and 7B, and S7  Fig). This is likely a result of the lower efficiency of flavodoxin as an electron carrier in comparison to ferredoxin [78,81]. Nonetheless, we observed that PSII inhibition induced by MPK3/MPK6 activation was impaired in pFld-overexpressing plants. Two independent pFld expression lines were used. Neither line, the induction of DD protein nor the activation of diaminobenzidine; ETI, effector-triggered immunity; MPK, mitogen-activated protein kinase; NA-PP1, 4-amino-1-tert-butyl-3-(1'-naphthyl)pyrazolo [3,4-  MPK3/MPK6 after DEX treatment, was affected by the overexpression of pFld ( Fig 7C). As shown in Fig 7D, expression of pFld impaired MPK3/MPK6 activation-mediated ROS accumulation. Concomitantly, PSII inhibition, disassembly of photosynthetic complexes and HRlike cell death were all delayed (Fig 7E-7G). Expression of pFld also alleviated the photosynthetic inhibition triggered by conditional expression of AvrRpt2 (S7 Fig). To test whether the inhibition of photosynthetic activities in chloroplasts is required for the robustness of ETI, we infiltrated DD, DD/pFld-7, and DD/pFld-8 with Pst-AvrRpt2. In the absence of DEX, DD transgene is not expressed, and these plants can be treated as wild-type control and pFId transgenic plants, respectively. PSII inhibition induced by Pst-AvrRpt2 was greatly delayed in DD/pFld-7 and DD/pFld-8 plants, which was associated with the inhibition of HR cell death (Fig 8A), a compromised resistance (Fig 8B), impaired ROS accumulation (Fig 8C), and delayed disassembly of photosynthetic complexes (S8 Fig). The elevated Pst-AvrRpt2 growth led to chlorosis, a susceptible phenotype (Fig 8B). These results strongly suggest that inhibition of photosynthetic activity is essential to ETI. This notion is further supported by the observation that HR cell death and PSII inactivation is delayed in dark (Fig 7D), in which no ROS accumulation in chloroplasts was observed (Fig 2G and 2H and S5 Fig). In addition, disease resistance to Pst-AvrRpt2 was also greatly compromised in dark or under low light, and Pst-AvrRpt2 grew to higher titers and caused chlorosis, symptoms of susceptibility (Fig 8E and 8F). Altogether, these results suggest that light-dependent ROS accumulation in chloroplasts is an important part of ETI.

Discussion
Decrease in plant photosynthetic activity and global down-regulation of photosynthetic genes have long been associated with plants under biotic stresses [44][45][46][47][48][49][50]. However, it is unclear whether this is a reflection of deterioration of plant health or an active part of plant immunity. Recently, we reported the essential role of MPK3/MPK6 in plant PTI [38]. MPK3 and MPK6 are also activated during ETI [24,25]. However, genetic evidence demonstrating the requirement of MPK3/MPK6 in ETI is still lacking. In this report, we demonstrate that gain-of-function activation of MPK3/MPK6 in Arabidopsis is sufficient to induce active inhibition of photosynthesis and light-dependent ROS accumulation in chloroplasts, two processes that mutually enhance each other under light. Loss-of-function data revealed that MPK3 and MPK6 are essential to effector-triggered photosynthetic inhibition and ROS accumulation in chloroplasts, and eventually ETI. This study highlights the important role of MPK3/MPK6mediated photosynthetic inhibition and ROS accumulation in chloroplasts during ETI, which can explain why plants are more resistant under light than in dark. We propose that active photosynthetic inhibition mediated by the MPK3/MPK6 pathway is one of the key immune responses downstream of NLR activation and contributes to a robust ETI (Fig 8G).

MPK3/MPK6 activation induces light-independent photosynthetic inhibition and light-dependent ROS accumulation in chloroplasts
MPK3/MPK6 activation-induced HR-like cell death and ROS accumulation in chloroplasts are light dependent (Fig 2G-2I). This is also true in a tobacco system [32]. However, MPK3/ MPK6 activation-induced PSII inhibition can be independent of light (Figs 2A-2C and 3C). Ã 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-  We also noticed that PSII inhibition was slower in the absence of light, which could be a result of the lack of ROS generation (Fig 2G and 2H). ROS are known to play an important role in accelerating PSII inhibition by oxidative damage of PSII proteins [82][83][84]. Under light, MPK3/ MPK6 activation-induced PSII inactivation and ROS accumulation in chloroplasts can form a positive feed-forward loop to accelerate the PSII inhibition. Nonetheless, MPK3/MPK6 activation-induced photosynthetic inhibition can occur in the absence of light and be independent of chloroplastic ROS accumulation.

MPK3/MPK6-mediated inhibition of photosynthesis is an important part of both CNL-and TNL-type NLR-mediated ETI
Photosynthetic inhibition is a well-documented phenomenon in plants challenged by pathogens [46][47][48][49][50][85][86][87][88]. However, it was not clear whether photosynthetic inhibition is a programmed part of immune response or merely a side effect caused by pathogen infection. In this study, we provided several lines of evidence suggesting that photosynthetic inhibition is an active defense response and an important part of ETI. First of all, AvrRpt2-induced photosynthetic inhibition requires its immune receptor, RPS2, demonstrating that photosynthetic inhibition is an event downstream of NLR activation. Secondly, prolonged activation of MPK3/ MPK6, an event downstream of NLR activation in ETI [24], induces photosynthetic inhibition. Thirdly, ETI and MAPK signaling-mediated photosynthetic inhibition facilitate ROS accumulation in chloroplasts, which is essential to ETI. Previous studies demonstrate that light is essential for virus-induced HR [32,89]. In this study, we also found an essential role of light in Pst-AvrRpt2-induced HR and plant resistance against Pst-AvrRpt2 (Fig 8D-8F). Thus, photosynthetic inhibition during ETI is actively regulated and is part of the immune response that enhances resistance.
We found that Pst-AvrRps4 also induces prolonged MAPK activation (Fig 4G), although AvrRps4 is sensed by RPS4/RRS1, a TNL-type NLR, which was thought to function in nuclei mainly through transcriptional reprogramming [12]. The requirement of MPK3/MPK6 in both CNL-and TNL-mediated ETI raises a question of how NLR activation leads to prolonged MPK3/MPK6 activation. MPK4 is guarded by CNL-type R protein, suppressor of mkk1 mkk2 (SUMM2), which monitors the phosphorylation status of MPK4 substrates, including MAP kinase kinase kinase 2 (MEKK2), mRNA de-capping protein PAT1, and calmodulin binding receptor-like cytoplasmic kinase 3 (CRCK3) [90][91][92][93]. It remains to be determined whether MPK3 and MPK6 are also protected by CNL-or TNL-type R proteins. In PTI, MPK3/MPK6 activation after PAMP perception by PRRs can be mediated by receptor-like cytoplasmic kinases (RLCKs), which are similar to RLKs but lack an extracellular domain. Arabidopsis RLCK PBS1-like 27 (PBL27) and rice (Oryza sativa) OsRLCK85 were demonstrated to connect chitin perception to MPK3/MPK6 activation [94,95]. It will be interesting to identify the proteins that connect NLRs to MPK3/MPK6, which may reveal the mechanism(s) underlying the prolonged activation of MPK3/MPK6 during ETI.

MPK3/MPK6 cascade regulates the trade-off between growth and defense in plant immunity
The concept of a trade-off between growth and defense has been proposed for many decades [96]. Our current knowledge on the growth-defense trade-off mainly stems from antagonistic cross talk among hormones that promote defense and that promote growth, such as SA-auxin, SA-brassinosteroid (BR), SA-gibberellic acid (GA), jasmonic acid (JA)-Auxin, JA-BR, and JA-GA [97][98][99]. However, how a plant integrates multiple internal and external stimuli to shift the balance between growth and defense remains poorly understood. It is also unclear why these two events are coupled together most of the time. We showed in this report that both events are regulated by the same MAPK signaling pathway. MPK3/MPK6 activation globally down-regulates photosynthetic genes and, in the meantime, up-regulates numerous defenserelated genes (Fig 1A-1C), suggesting that MAPK signaling plays important roles in orchestrating growth and defense in plant immunity. Consistent with the down-regulation of photosynthetic genes, we did observe decreases in CO 2 fixation [32] and photosynthetic inhibition (Fig 2A-2C). Both would have negative impacts on normal plant growth and development. In the meantime, up-regulation of defense genes by the MPK3/MPK6 cascade leads to an increased biosynthesis of defense-related secondary metabolites such as camalexin [53] and indole glucosinolate derivatives [56]. Considering that multiple developmental and environmental signals converge at the MPK3/MPK6 cascade [28,100], we propose that the MPK3/ MPK6 cascade is a key hub in orchestrating the trade-off between growth and defense.
MPK3/MPK6 activation-induced photosynthetic inhibition, as well as its associated ROS accumulation and HR cell death, contribute positively to the robust ETI. Regulation of photosynthetic inhibition by an active signaling cascade demonstrates that the inhibition of photosynthesis is an active defense response in plant immunity triggered by effectors, not a passive consequence of the deterioration of plant fitness caused by pathogen infection. It also reveals a potential mechanism underlying the growth-defense trade-off during plant immunity. Plant ETI is a stronger and more robust form of immune response in comparison to PTI [3,7,8]. In such a case, a robust defense, but not growth, is of high priority. Long-lasting activation of MPK3/MPK6 triggered by pathogen effectors contributes to the robustness of ETI (Fig 8G). It is worth noting that PTI, a weaker form of plant immunity, induces only transient MAPK activation and does not cause photosynthetic inhibition (Fig 3B and S4 Fig), indicating that photosynthetic activities are differently regulated during different forms of immune responses by the same MAPK signaling pathway, depending on its activation kinetics.

Generation of transgenic lines
For generation of DD/pFld and GVG-AvrRpt2/pFld plants, the coding sequence of Fld from cyanobacterium Anabaena sp. PCC 7119 [102] was first optimized to codons preferred in Arabidopsis using OptimumGene algorithm (Genscript) (S9 Fig). After introducing Nde I and Spe I enzyme digestion sites, the Nde I-Fld-Spe I fragment was directly synthesized into pUC57 vector, and then the Nde I-Fld-Spe I fragment was subcloned into pBluescript (pBS) vectors with RbcS signal peptide sequence to generate pBS-RbcS-Fld. The pBS-RbcS-Fld was cut with Xho I and Spe I and subcloned into pBID vector to generate pBID-RbcS-Fld constructs. The pBID-RbcS-Fld construct was then transformed to Agrobacterium tumefaciens GV3101. Finally, the A. tumefaciens GV3101 containing pBID-RbcS-Fld was used to transform DD and GVG-AvrRpt2/RPS2 plants, respectively. Single insertion lines were selected and the expression of Fld was confirmed by immunoblot. F3 homozygous DD/pFld and GVG-AvrRpt2/pFld plants were used for experiments.

Chlorophyll fluorescence measurement
The O-J-I-P curve was measured by using Dual-PAM chlorophyll fluorometer (Walz, Germany) with a built-in fast kinetic protocol. Other chlorophyll fluorescence parameters were measured with the Maxi-version of Imaging-PAM chlorophyll fluorometer (Walz, Germany) or FMS2 (Hansatech, United Kingdom). F o (minimum fluorescence of dark adapted leaves) was measured using weak light (<1 μmol m −2 s −1 ) at a low frequency (2 Hz). For measuring F m (maximum fluorescence yield of dark-adapted leaves), dark-adapted leaves were exposed to a PPFD of approximately 2,700 μmol m −2 s −1 . When performing induction kinetics measurements, the intensity of actinic light was set to 110 μmol m −2 s −1 . For NPQ induction analysis, the intensity of actinic light was set to 610 μmol m −2 s

ROS staining
In vivo H 2 O 2 generation in plants was detected by using DAB as described previously [32]. Twelve-d-old Arabidopsis seedlings after treatment were submerged into a solution containing 1 mg/mL DAB (pH 5.5) for 2 h under growth light. Oxidation of DAB leads to its polymerization and deposition at the site of ROS generation. The seedlings were then boiled in ethanol for 10 min to remove chlorophyll. H 2 O 2 production is visualized as a reddish-brown coloration. In vivo O À 2 production was monitored by NBT staining as described previously [32]. Twelve-d-old Arabidopsis seedlings after treatment were submerged into 10 mM potassium phosphate buffer (pH 7.8) containing 1 mg/mL NBT and 10 mM NaN 3 . To avoid overstaining, the seedlings were stained in dark for 30 min. The seedlings were then boiled in ethanol for 10 min to remove chlorophyll. Reduced NBT was visualized as a dark blue-colored formazam deposit. Single layer mesophyll cells were prepared according to [103] for visualization of H 2 O 2 and O À 2 accumulation in mesophyll cells. Seedlings stained with DAB and NBT were fixed in 3.5% glutaraldehyde for 1 h and then softened with 0.1 M EDTA, pH 9.0, for 2 h at 55˚C. A leaf sample (about 1 mm 2 ) was placed on a glass slide and covered with a cover slide. The leaf sample was stretched into a single cell layer by lightly tapping with the eraser of a pencil. H 2 O 2 and O À 2 accumulation in chloroplasts were imaged with a microscope equipped with a digital camera.

Quantitative RT-PCR analysis
Real-time quantitative PCR (qPCR) was performed as previously described [54]. Total RNA was extracted using TRizol reagent (Invitrogen). After DNase treatment, 1 μg of total RNA was used for reverse transcription. Real-time qPCR analysis was performed using an ABI 7500 real-time PCR machine (Life Technologies). EF-1a was used as an internal control. The primer pairs used for qPCR are listed in S2 Table. Illumina RNA-seq gene expression profiling Total RNA was extracted with TRizol reagent (Invitrogen) from 12-d-old DD seedlings treated with 2 μM DEX for 0 and 6 h, respectively. After DNase treatment, total RNA was purified using RNA clean and concentrator Kit. RNA sequencing libraries were constructed using Tru-Seq RNA library preparation Kit and sequenced using the HiSeq X Ten according to the manufacturer's instructions. Dirty raw reads were filtered out using SONPnuke software. Clean reads were mapped to the Arabidopsis reference genome with BWA and to reference gene sequences with Bowtie. Gene expression levels were calculated using the RPKM method (reads per kb per million reads). The raw Illumina reads generated from RNA-seq experiments were deposited at NCBI Sequence Read Archive (SRP111959).