Inducer of CBF Expression 1 (ICE1) Promotes Cold-enhanced Immunity by Directly Activating Salicylic Acid Signaling

Cold stress affects plant immune responses, and this process may involve the salicylic acid (SA) signaling pathway. However, the underlying mechanism by which low temperature signals coordinate with SA signaling to regulate plant immunity remains not fully understood. Here, we found that low temperatures enhanced the disease resistance of Arabidopsis against Pseudomonas syringae pv. tomato (Pst) DC3000. This process required Inducer of CBF expression 1 (ICE1), the core transcription factor in cold-signal cascades. ICE1 physically interacted with Non-expresser of PR genes 1 (NPR1), the master regulator of the SA signaling pathway. Enrichment of ICE1 on the PR1 promoter and its ability to transcriptionally activate PR1 were enhanced by NPR1. Further analyses revealed that cold stress signals cooperate with SA signals to facilitate plant immunity against pathogen attack in an ICE1-dependent manner. Cold treatment promoted interactions of NPR1 and TGA3 with ICE1, and increased the ability of the ICE1–TGA3 complex to transcriptionally activate PR1. Together, our results characterize a previously unrecognized role of ICE1 as an indispensable regulatory node linking low temperature activated- and SA-regulated immunity. Discovery of a crucial role of ICE1 in coordinating multiple signals associated with immunity broadens our understanding of plant–pathogen interactions.

In the SA signaling transduction pathway, NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) has been well documented as one of the SA receptors that perceives and transduces SA signals (Wang et al., 2020;Kumar et al., 2022a).In the resting state, NPR1 exists in the cytoplasm as an oligomer.After induction, NPR1 monomers are released from oligomers and translocated to the nucleus, where they act as cofactors of transcription factors, such as the TGACG motif-binding transcription factors (TGAs), which regulate global transcriptional reprogramming and resistance to a broad spectrum of pathogens (Zhang et al., 1999;Després et al., 2000;Zhou et al., 2000;Johnson et al., 2003;Kumar et al., 2022a).Recently, NPR1 has attracted attention because of its central roles in plant growth and development, as well as in plant immune responses.For instance, it was reported that NPR1 interferes with the binding of ETHYLENE-INSENSITIVE 3 (EIN3), a key transcription factor involved in ethylene (ET) signaling, to the promoter regions of its target genes to mediate antagonism between SA and ET in apical hook formation (Huang et al., 2020).The transcriptional activation activity of EIN3 is enhanced by NPR1 in a process coordinated by SA and ET during the modulation of leaf senescence (Wang et al., 2021;Yu et al., 2021).
Moreover, NPR1 promotes the polyubiquitination and degradation of GID1, the gibberellin receptor, to balance growth and defense (Yu et al., 2022).
NPR1 is also involved in facilitating cell survival during ETI by ubiquitinating key proteins involved in immune responses, such as ENHANCED DISEASE SUSCEPTIBILITY 1 and WRKY transcription factors (Zavaliev et al., 2020).
NPR1 inhibits the transcriptional activation activity of MYC2, the master regulator of jasmonic acid signaling pathway, to suppress pathogen virulence (Nomoto et al., 2021).
The TGA transcription factors are members of the basic leucine zipper (bZIP) family of proteins, which specifically bind to the TGACG-core activation sequence 1 (as-1) to regulate the transcriptional levels of their target genes (Després et al., 2000;Zhou et al., 2000;Johnson et al., 2003).Ten TGA transcription factors are encoded in the Arabidopsis genome, of which TGA1 to TGA7 have been characterized in terms of their ability to interact with NPR1 (Zhang et al., 1999;Després et al., 2000).Among them, TGA3 is mainly required for basal resistance and PR gene expression (Kesarwani et al., 2007;Kumar et al., 2022a), and also integrates with other signals.For instance, TGA3 interacts with ARR2, the cytokinin-activated transcription factor, to promote plant immunity through NPR1/TGA3-dependent SA signaling (Choi et al., 2010).A protein complex of TGA3 and WRKY53 acts on the Cestrum yellow leaf curling virus promoter to achieve synergistic up-regulation of SAassociated gene expression (Sarkar et al., 2018).BRASSINOSTEROID INSENSITIVE2 (BIN2) balances SA-induced immunity and brassinosteroidmediated plant growth through phosphorylating TGA3 to activate gene expression (Han et al., 2022).
Low temperature has significant effects on plant growth and development (Orvar et al., 2000;Wang et al., 2009).The ICE1/C-REPEAT BINDING FACTOR/DRE-BINDING FACTOR 1 (ICE1/CBF/DREB1) pathway plays an essential role in plants' tolerance to low temperature stress, and ICE1 acts as an important transcriptional regulator of cold-responsive gene expression and cold tolerance (Stockinger et al., 1997;Kim et al., 2015).ICE1, a basic helixloop-helix (bHLH) protein in the MYC class, directly binds to the MYC site (CANNTG) in the promoter of CBF3 to induce its expression under cold stress (Gilmour et al., 1998;Chinnusamy et al., 2003).ICE1 also binds to the promoters of other genes encoding regulatory factors such as cold-responsive (COR), calmodulin binding transcription activator 2, mitogen-activated protein kinase 3 (MPK3), MPK4, and high expression of osmotically responsive genes 1 (HOS1), all of which are critical for the cold response (Tang et al., 2020).As an essential component of the cold signaling network, ICE1 functions as a key cross-talk node between low temperature signals and other signals.For instance, HOS1, SUMO E3 ligase (SIZ1), Open Stomata 1 (OST1), MPK3/6, and BIN2 are involved in regulating responses to freezing by maintaining homeostasis of the ICE1 protein under cold stress (Dong et al., 2006;Miura et al., 2007;Ding et al., 2015;Li et al., 2017;Zhao et al., 2017;Ye et al., 2019).
Moreover, ICE1 integrates endogenous and environmental signals to mediate diverse physiological processes, such as seed germination, flowering time, and stomatal development (Lee et al., 2015(Lee et al., , 2017;;Hu et al., 2019;An et al., 2021;An et al., 2022 (Kim et al., 2013;Kim et al., 2017;Wu et al., 2019;Li et al., 2020).Cold temperatures induce expression of the SA signaling-responsive genes PR1, PR2, and PR5, whose encoded products increase plant immunity (Seo et al., 2010).These results suggest that there are broad signaling interactions between low temperature signals and SA responses.Nonetheless, the underlying molecular mechanisms and signaling pathways are unknown.
In this study, we found that the enhancement of plant immune responses by low temperatures largely depends on ICE1, the core transcription factor in the cold signaling pathway.Mechanistic analyses revealed that the ICE1 protein physically interacts with the SA receptor NPR1 and is involved in SA-mediated resistance against Pst DC3000.Further analyses demonstrated that ICE1 directly activates the transcription of PR1 and NPR1 promotes its activities.
We also found that ICE1 interacts with TGA transcription factors to cooperatively stimulate PR1 expression under low temperatures.Taken together, our results demonstrate that the NPR1-TGA3/ICE1 regulatory module acts as a crucial node integrating cold signals and SA signaling in the synergistic regulation of plant immunity.

ICE1 Is Involved in Low Temperature-enhanced Plant Immunity
Low temperature has complicated effects on plant immunity (Yang and Hua, 2004;Huang et al., 2010;Yang et al., 2010;Wigge, 2013).To investigate the underlying relationship between low temperature and plant immunity, we assessed effect of low temperature on plant immune responses against the hemibiotropic pathogenic bacteria, Pseudomonas syringae pv.tomato (Pst) DC3000.For these analyses, we monitored disease symptoms and populations of Pst DC3000 in Arabidopsis Columbia-0 (Col-0) plants that were grown at normal temperature (22°C) for 4 weeks and then exposed to a longterm (3 weeks) or short-term (10 hours) cold treatment (4°C) before infiltration with the pathogen.We observed that the pathogen-related water-soaking symptom, one of the earliest and most common symptoms of phyllosphere bacterial diseases, as well as bacterial proliferation in infected leaves, were largely reduced in Col-0 plants exposed to a short or long cold treatment, compared with those kept at 22°C (Fig. 1 A-D).These results confirmed that both long-and short-term low temperature treatments promote plant immunity against Pst DC3000, consistent with the results of previous studies (Seo et al., 2010;Kim et al., 2017;Wu et al., 2019).
ICE1 is a transcriptional inducer of genes that encode important components of chilling tolerance of Arabidopsis (Chinnusamy et al., 2003;Lee et al., 2005;Benedict et al., 2006;Chinnusamy et al., 2007).At the early stages of cold stress, ICE1 in its phosphorylated form activates the expression of CBFs to enhance plant freezing tolerance (Ding et al., 2015;Ye et al., 2019).
Given the critical role of ICE1 at the early stage of the cold stress response, we hypothesized that ICE1 may be involved in short-term low temperatureenhanced immunity.To test this possibility, we investigated Pst proliferation in Col-0, the loss-of-function ice1-2 mutant, and an ICE1-complemented mutant (ice1-2/ICE1) after short-term low temperature treatment.As shown in Fig. 1E, F, the leaves of Col-0 and ice1-2/ICE1 exhibited clearly enhanced diseaseresistance phenotypes, with less severe water-soaking symptoms and lower pathogen populations after cold treatment, compared with the control.
Together, these results indicate that ICE1 acts as a positive regulator of plant immunity under ambient and cold temperatures.

ICE1 Physically Interacts with NPR1
ICE1 regulates diverse physiological processes through associating with other proteins (Ding et al., 2015;Lee et al., 2015;Hu et al., 2019;Ye et al., 2019;An et al., 2021;An et al., 2022).To explore the underlying molecular mechanism by which ICE1 regulates plant immune responses, we performed yeast twohybrid screening from a pool of key regulatory proteins that act in Arabidopsis immunity, such as proteins that are involving in SA signaling pathway and PTI response.From this screening, a potential candidate, NPR1, displayed strong interaction with ICE1 in the yeast cells and selected as the first potential candidate for further characterization (Fig. 2A).To identify the functional region responsible for the interaction between ICE1 and NPR1, two truncated versions of the proteins were used in yeast two-hybrid assays.As shown in Fig. 2A, deletion of the N-terminal amino acid residues 1-260 of ICE1 (AD-ICE1 261-249 ) did not affect the ICE1-NPR1 interaction, whereas deletion of the C-terminal residues of ICE1 containing the bHLH domain (AD-ICE1 1-260 ) eliminated the interaction completely (Fig. 2A).Additionally, the N-terminal amino acid residues of NPR1 containing the BTB structural domain (BD-NPR1 1-194 ) strongly interacted with ICE1, while the C-terminal fragment (BD-NPR1 178-593 ) did not.These results demonstrate that the bHLH structural domain of ICE1 and the BTB structural domain of NPR1 are necessary for the ICE1-NPR1 interaction.
Bimolecular fluorescence complementation (BiFC) assays were conducted to further confirm the interaction between ICE1 and NPR1.For these assays, NPR1 fused to the C-terminal of YFP (NPR1-cYFP) and ICE1 fused to the Nterminal of YFP (ICE1-nYFP) were co-expressed in Nicotiana benthamiana leaves, and then fluorescence in the leaves was observed under a laser confocal microscope.Almost no YFP fluorescence was observed when NPR1-cYFP was co-expressed with ICE1-nYFP in N. benthamiana leaves at 22°C (Supplemental Fig. S1A), but the fluorescence signal became very strong after induction at 4°C for 4 h (Fig. 2B; Supplemental Fig. S2A), suggesting that the association between NPR1 and ICE1 may depend on low temperature in vivo.To further verify this interaction, we performed a split luciferase assay by fusing these proteins to the C-terminus (cLUC) or Nterminal (nLUC) of luciferase, with LUC fused with β-glucuronidase (GUS) as the negative control.Similarly, very faint LUC activity was detected when NPR1-cLUC was transiently co-expressed with ICE1-nLUC in N. benthamiana, but strong LUC signals were observed after low temperature induction (Fig. 2C; Supplemental Fig. S1B).
Consistently, in a co-immunoprecipitation (Co-IP) assay, ICE1 was immunoprecipitated by anti-GFP agarose beads from Arabidopsis protoplasts co-expressing NPR1-YFP and ICE1-Myc, but not from those co-expressing YFP and ICE1-Myc, and more precipitation products were detected after low temperature induction (Fig. 2D).To further confirm the interaction between ICE1 and NPR1 in vivo, we performed Co-IP assay using stable Arabidopsis transgenic lines both under normal and cold temperature conditions.In line with the results conducted in protoplasts, more NPR1 proteins could be coimmunoprecipitated with ICE1 after cold temperature treatment (Fig. 2E).
Taken together, these results indicate that ICE1 interacts with NPR1 both in vitro and in vivo, and that low temperature potentiates the association between ICE1 and NPR1 in planta.

ICE1 Is Required for SA-mediated Plant Immunity
Because ICE1 physically associates with the SA receptor NPR1 (Fig. 2), we hypothesized that ICE1 may participate in SA signal transduction.To test this hypothesis, we first detected changes in the transcript levels of ICE1 in response to benzothiadiazole (BTH), a synthetic analog of SA that effectively induces SA responses and is extensively used as an alternative to SA signaling (Huot et al., 2017;Kim et al., 2022).Interestingly, BTH treatment did not affect the transcript level of ICE1, but led to the accumulation of the ICE1 protein to high levels (Supplemental Fig. S3).Next, we examined whether ICE1 is involved in SA-regulated immunity by conducting a BTH protection assay.For this assay, wild-type Col-0, ice1-2, and ice1-2/ICE1 plants were pre-treated with BTH for 24 h before pathogen infiltration, and then their susceptibility to disease caused by Pst DC3000 was evaluated.Compared with the mock pretreated plants, the BTH-pretreated Col-0 and ice1-2/ICE1 plants exhibited greatly reduced disease symptoms on the leaves after Pst DC3000 infection (Fig. 3A and 3B).In contrast, the leaves of ice1-2 displayed severe disease symptoms, i.e., severe water soaking symptoms and strong pathogen growth, after Pst DC3000 infection (Fig. 3A, B).The transcript levels of PR1, PR2, PR5 and WRKY70 were also lower in BTH-pretreated ice1-2 plants (Fig. 3C and Supplemental Fig. S4).These results suggest that ICE1 may be required for effective SA-regulated immunity.PR1, whose expression is activated by the NPR1-TGA regulation module, is a principal output gene of SA-associated immunity (Zhang et al., 1999;Després et al., 2000;Zhou et al., 2000;Johnson et al., 2003).Given that ICE1 interacts with NPR1 and plays an essential role in the SA signaling pathway (Figures 2 and 3), we wondered whether ICE1 directly regulates the transcription of PR1 in a similar manner as TGA transcription factors.ICE1 is a MYC-like bHLH transcriptional activator that binds specifically to the MYC recognition sequences (CANNTG, also known as the E-box) in the CBF3 promoter (Chinnusamy et al., 2003;Tang et al., 2020).We analyzed the promoter of PR1 and found that some MYC recognition sequences exist in the promoter of PR1, 1000 bp upstream of ATG (Supplemental Fig. 5).Hence, we performed electrophoresis mobility shift assays (EMSAs) to determine whether ICE1 could bind directly to the E-box cis-element in the promoter of PR1.As shown in Fig. 3G, the ICE1 protein was able to bind to the E-box in the PR1 promoter (Fig. 3G).To verify these findings, we conducted chromatin immunoprecipitation (ChIP) assays with ICE1-Myc (ICE1-OE 2) plants treated or not with BTH.We observed that ICE1 was enriched in the PR1 promoter region, and BTH promoted its enrichment (Fig. 3H).Next, a dual-luciferase reporter assay was conducted to determine the effects of ICE1 on PR1 transcription.LUC expression driven by the PR1 promoter was significantly activated by ICE1 (Fig. 3I).Taken together, these results indicate that ICE1 is required for effective SA-associated immunity via direct activation of PR1 transcription.

NPR1 Enhances the Ability of ICE1 to Transcriptionally Activate PR1
Having established that ICE1 interacts with NPR1 and positively regulates SA responses through direct binding to the promoter region of PR1, we investigated whether ICE1 functions in a NPR1-relevant manner.To test this possibility, we generated ice1-2 npr1-1 double mutant plants by crossing the ice1-2 mutant with npr1-1, a loss-of-function mutant allele of NPR1 (Col-0 background; (Cao et al., 1997)).Similar to the ice1-2 and npr1-1 single mutants, the ice1-2 npr1-1 double mutant displayed a highly susceptible phenotype to Pst DC3000 (Fig. 4A, B), implying that ICE1 may act in the same pathway as NPR1 to mediate plant immunity.
NPR1 has long been believed to associate with downstream transcription factors, such as TGAs, to trigger their transcriptional activation of PR genes (Zhang et al., 1999;Després et al., 2000;Zhou et al., 2000;Johnson et al., 2003;Kumar et al., 2022a).Thus, we wondered whether NPR1 could promote Next, dual-luciferase reporter assays using Arabidopsis mesophyll protoplasts were conducted to validate the effect of NPR1 on the transcriptional activation activity of ICE1.The effectors consisted of GFP, NPR1, and ICE1 under the control of Pro35S, and the reporter consisted of the PR1 promoter fused to the LUC gene (Supplemental Fig. S6).Interestingly, LUC expression driven by the PR1 promoter was also induced when NPR1 and GFP were co-expressed, implying that basal TGA or other proteins were involved.However, LUC expression was induced to a much higher level when NPR1 and ICE1 were co-expressed than when GFP and ICE1 were coexpressed (Fig. 4E).In contrast, LUC expression driven by the PR1 promoter was compromised by the loss-of-function of NPR1 (Fig. 4F).These results provide further evidence that the ability of ICE1 to activate the transcription of PR1 is stimulated by NPR1.

ICE1 Integrates Low Temperature-enhanced and SA-regulated Immunity
ICE1 is involved in low temperature-activated plant immunity (Fig. 1) and plays a crucial role in the SA signaling pathway (Fig. 3), implying that it may function as a central node linking these two signaling pathways.To test this hypothesis, we assessed disease symptoms, including water-soaking To further explore the underlying molecular mechanism by which ICE1 is synergistically regulated by these two important cues, we determined the amount of ICE1 protein that accumulated in response to a combined low temperature and BTH treatment.Consistent with previous results, ICE1 protein remained stable within 2 hours of 4°C treatment (Ding et al., 2015).
Strikingly, ICE1 accumulated to a much higher level under 6 hours BTH treatment combined with 2 hours of 4°C treatment than in either the low temperature or BTH treatment alone (Fig. 5E-F).

SA-Regulated Immunity
As CBF3 is the canonical target of ICE1, we are wondering if this ICE1mediated immunity involves CBF3 or not.To address this question, we monitored disease symptoms and populations of Pst DC3000 in 4-week-old Col-0 and cbfs mutant plants that were treated with or without short-term (10 h) cold temperature (4°C).We observed that the pathogen-related water-soaking symptom as well as bacterial proliferation showed no significant differences between Col-0 and cbfs mutant leaves at either normal temperature or cold temperature (Supplemental Fig. S7A, B).Expression of PR1 showed no obvious differences in Col-0 and cbfs mutant plants at both normal and low temperature treatments (Supplemental Fig. S7C).These results suggest that CBF genes does not involve in low temperature-enhanced immunity, which is consistent with Li's results that CBF pathway does not play a major role in mediating low-temperature enhancement of disease resistance to Pst DC3000 (Li et al., 2020).
To explore if CBFs involves ICE1-regulated immunity, we conducted the BTH protection assay in wild-type Col-0 and cbfs mutant plants.However, no obvious differences occurred between them either in disease symptoms and pathogen proliferation or PR1 expression, suggesting that CBF genes did not mediate SA-regulated immunity (Supplemental Fig. S7D-F).

ICE1 Physically Interacts with TGA Transcription Factors
TGA transcription factors were confirmed to be interacting proteins of NPR1 in yeast two-hybrid screening assays (Zhang et al., 1999;Zhou et al., 2000), and are believed to be the nuclear targets that bind to the as-1 element required for SA-induced PR1 gene expression (Zhang et al., 1999;Zhou et al., 2000;Johnson et al., 2003;Kesarwani et al., 2007;Ding et al., 2018).To explore the regulatory role of ICE1 in SA signaling comprehensively, we investigated whether ICE1 interacts with TGA transcription factors.The full-length coding sequences (CDSs) of TGA1-TGA7 were fused to pGBK-T7 to construct pGBK-TGAs vectors, and then each one was transformed into the yeast strain with pGAD-ICE1.Interestingly, ICE1 interacted with all tested TGA proteins in yeast (Fig. 6A).Among the seven members of the TGA family in Arabidopsis, TGA3 plays an important role to bind to the as-1 element of PR1 to induce its expression (Kesarwani et al., 2007;Choi et al., 2010;Kumar et al., 2022a).
Hence, we focused on TGA3 in subsequent experiments.
To identify the functional regions responsible for the interaction between and was further enhanced under low temperature (Fig. 7D).Dual-luciferase reporter assays using Arabidopsis mesophyll protoplasts were conducted to validate the effect of TGA3 on the transcriptional activation activity of ICE1.
The effector consisted of GFP, TGA3, and ICE1 under the control of Pro35S, and the reporter consisted of the PR1 promoter fused to the LUC gene (Supplemental Fig. S6).LUC expression driven by the PR1 promoter was induced to higher level when TGA3 and ICE1 were co-expressed than when GFP and ICE1 were co-expressed (Fig. 7E).In contrast, ICE1 induced LUC expression was compromised in the tga3 CR mutant compared with Col-0 (Fig. 7F).These results indicate that the ability of ICE1 to activate the transcription of PR1 is promoted by TGA3.Noticeably, the ability of TGA3 to increase the transcription of PR1 was diminished in the ice1-2 mutant, suggesting that ICE1 also stimulates the transcriptional activation activities of TGA3 (Fig. 7G).
These results support the notion that ICE1 and TGA3 act cooperatively to mediate cold-enhanced plant immunity through increased enrichment on the PR1 promoter to activate its expression in association with SA.
The NPR1-TGA3 complex has been well documented that plays an indispensable role in PR1 expression.Given ICE1 interacts with both NPR1 and TGA3, we questioned whether ICE1 could enhance the transcriptional activities of NPR1-TGA3 module on PR1 expression.To clarify the potential role of ICE1 during the complex, we conducted the dual-luciferase reporter assay.Noticeably, compared with NPR1 and TGA3 were co-expressed, LUC expression driven by the PR1 promoter was induced higher when NPR1, TGA3 and ICE1 were co-expressed (Fig. 7H), implying that ICE1 promotes the function of NPR1-TGA3 in PR1 expression regulating.

ICE1 Does not Affect the Biosynthesis of SA
In Arabidopsis, isochorismate, the precursor of SA, is mainly converted from chorismate by ICS1/SID2 that is induced by pathogen infection (Wildermuth et al., 2001;Peng et al., 2021).AvrPphB Susceptible 3 (PBS3), a member of the GH3 acyl-adenylate/thioester-forming enzyme family, catalyzes the conjugation of isochorismate to glutamate to produce the key SA biosynthetic intermediate, isochorismate-9-glutamate, that can decay into SA spontaneously (Rekhter et al., 2019;Torrens-Spence et al., 2019).To determine whether ICE1-mediated immunity through regulating SA biosynthesis, we tested expression levels of ICS1/SID2 and PBS3 in Col-0 and ice1-2 mutant plants at both normal and cold temperature.As shown in Supplemental Fig. S10, the expression levels of ICS1/SID2 and PBS3 displays no obvious differences between Col-0 and ice1-2 mutant plants at both normal and cold temperature, suggesting that ICE1 did not involving in SA biosynthesis regulating.

DISCUSSION
In this study, we demonstrate a previously uncharacterized function of ICE1 as an essential node bridging cold stimulus and plant immunity.We found that ICE1 is involved in cold-activated immunity as well as SA-regulated immunity, as demonstrated by the fact that the ice1-2 mutant exhibited enhanced susceptibility to pathogen infection, including severe pathogen-related water soaking symptoms and high pathogen proliferation, and decreased expression of PR1 (Fig. 1 and Fig. 3).Most importantly, the coordinated function of coldand SA-enhanced immunity was diminished in ice1-2 mutant plants, suggesting that ICE1 is the crucial regulator linking these two cues (Fig. 5).
The major underlying mechanism is that NPR1 associates with ICE1 to promote its ability to activate PR1 transcription (Fig. 2 and Fig. 4).Additionally, TGA3 works synergistically with ICE1 under low temperature (Fig. 6 and Fig. 7).The relationship between cold stress and immunity has not been fully elucidated in plants (Wigge, 2013;Kim et al., 2017;Wu et al., 2019).The identification of the NPR1-TGA3-ICE1 regulatory module in this study represents a significant step in understanding SA signaling during coldactivated resistance of plants to pathogen attack.
NPR1, the master regulator of SA signaling, is required for basal and systemically acquired resistance in plants (Cao et al., 1997;Kesarwani et al., 2007).Because it lacks a typical DNA-binding domain, NPR1 was long believed to function as a transcriptional coactivator that interacts with other proteins, including TGAs, EIN3, and Heat shock transcription factor 1 (HSFA1), to modulate physiological processes in plants (Zhang et al., 1999;Després et al., 2000;Olate et al., 2018;Huang et al., 2020;Wang et al., 2021;Yu et al., 2021).In terms of plant immune regulation, WRKYs and TGAs are well known to be direct targets of NPR1 and to regulate PR1 gene expression (Zhang et al., 1999;Després et al., 2000;Zhou et al., 2000;Johnson et al., 2003;Wang et al., 2006;Saleh et al., 2015).Interestingly, in the present study, we demonstrate a novel function of ICE1 to activate SA signaling against pathogen infection.ICE1 interacts with NPR1 (Fig. 2) and functions in the same signal pathway with it (Fig. 4).As a MYC-like bHLH transcription factor, ICE1 can bind directly to the MYC recognition sequences of the PR1 gene promoter to activate its expression (Supplemental Fig. S5; Fig. 3G-I), and its transcriptional activation activity is enhanced by NPR1 and TGA3 (Fig. 4C-F and Fig. 7C-F).In addition, ICE1 promotes the transcriptional activation of NPR1-TGA3 complex on expression of PR1 (Fig. 7H).These findings not only provide evidence that ICE1 is an essential component of SA signal pathway at low temperature (Supplemental Fig. S11), but also increase our understanding of the role of the NPR1-ICE1-TGA3 regulatory module in integrating low temperature signal in activating plant immunity.
Recently, increasing attention has been paid to the interactions between low temperature stress and plant immunity.Previous studies have shown that the SA content increases in Arabidopsis plants exposed to cold stress (4°C) for more than 1 week, and that prolonged exposure (3 weeks) to low temperature promotes immunity against the bacterial plant pathogen, Pst DC3000 (Kim et al., 2013).Further study has clarified that low temperature can overcome the calmodulin-binding transcriptional activator 3 (CAMTA3) mediated genes inhibition of the SA pathway (Kim et al., 2017).Similarly, moderate low temperature (16°C) also enhances immunity accompanied by up-regulation of SA biosynthesis and signaling genes (Li et al., 2020).Wu et al. found that exposure to short-term cold stress (4°C for 10 hours) activates SA-dependent immunity as well as H 2 O 2 and callose deposition (Wu et al., 2019).The results of those studies imply that SA may play a crucial role in low temperatureactivated immunity, however, the underlying mechanism remained poorly characterized.In this study, we discovered that ICE1 acts an indispensable regulatory node bridging low temperature cues and the SA signaling pathway.
Our results show that low temperature treatment enhances disease resistance against the bacterial pathogen, Pst DC3000 (Fig. 1A-C), and that ICE1 is required for this modulation (Fig. 1E-H).Interestingly, we found that cold stress coordinates SA signaling to promote immunity, and this synergistic regulation occurs in an ICE1-dependent manner.This is highlighted by the fact that the enhanced immunity caused by low temperature combined with SA signaling was compromised in ice1-2 mutant plants compared with Col-0 plants (Fig. 5A, B).Low temperature has multifaceted influences on the SA signaling pathway, which involves ICE1.For instance, in the Co-IP assays, low temperature facilitated interactions between ICE1 and NPR1 (Fig. 2D, E).
Notably, in the BiFC and LUC assays, the NPR1 and ICE1 interaction only occurred after cold treatment (Fig. 2B, C), consistent with the results of previous studies showing that NPR1 accumulates in response to low temperature and enters the nucleus as a monomer to exert its function as a co-factor (Olate et al., 2018).The enrichment of ICE1-TGA3 on the PR1 promoter and the ability of this complex to transcriptionally activate PR1 were found to be significantly enhanced under low temperature (Fig. 7C-G).These results provide direct evidence for the co-operative regulation of low temperature cues and SA signaling via ICE1, illuminating the relationship between cold stress and SA-regulated immunity.
It has been proposed that NPR1 is present in both an inactive oligomer form and active monomer form and that SA treatment and low temperature promote the conformation change from oligomer to monomer (Mou et al., 2003;Tada et al., 2008;Yu et al., 2022).Although, in normal temperature and absence of SA, the majority of NPR1 proteins are in the oligomer form, some NPR1 proteins are in the monomer form, which may explain why NPR1 can interact with other proteins, including HSFA1, GID1 and EIN3 in normal temperature and absence of SA, however, the interactions can be enhanced by SA and low temperature (Olate et al., 2018;Huang et al., 2020;Yu et al., 2022).
Consistent with these findings, we found that NPR1 could interact with ICE1 in normal temperature and facilitate its transcriptional activation on PR1 expression (Fig. 4).Interestingly, association of NPR1 and ICE1 was enhanced by low temperature (Fig. 2B-E).Low temperature may promote the accumulation of NPR1 monomer in the nuclei, leading to more active NPR1 proteins targeting ICE1.
Temperature is an essential environmental factor affecting plant growth and 20 development, as well as resistance against pathogens.Recent studies have shown that elevated temperatures negatively affect pathogen resistance by suppressing SA production in Arabidopsis (Huot et al., 2017;Kim et al., 2022).
Interestingly, the results of several studies, including ours, show that the SA pathway also plays indispensable role in the defense against bacterial pathogen attack under cold stress conditions (Fig. 5) (Seo et al., 2010;Kim et al., 2013;Kim et al., 2017;Wu et al., 2019;Li et al., 2020).Climate change will have a profound effect on plant immunity, so an in-depth understanding of

Plant Materials and Growth Conditions
The wild-type and mutant Arabidopsis plants used in this study were in the Columbia (Col-0) genetic background.All Arabidopsis plants were soil-grown for four weeks under an artificial growth chamber at 22°C and 60% relative humidity, and 10 h light/14 h dark photoperiod.The ice1-2 (SALK_003155; (Kanaoka et al., 2008)) mutant and npr1-1 (CS3726; (Cao et al., 1994)) mutant have been described.The same cbfs triple mutants with study of Jia et al. (2016) was used in this study (Jia et al., 2016).Seeds of ice1-2/ICE1 were kindly provided by Prof. Shuhua Yang (China Agricultural University).The For the combined cold and BTH treatment, 4-week-old plants sprayed with mock and BTH and kept in a growth chamber with normal temperature (22°C) for 14 h.After then, the low temperature experimental plants were transferred to a low temperature (4°C) growth chamber for 10 h cold treatment, while control plants still kept in the growth chamber with normal temperature (22°C) for additional 10 h.In this way, the plants can suffer from different treatment conditions, such as 24 h mock and 24 h BTH with normal temperature as well as 24 h mock and 24 h BTH with 10 h low temperature before Pst DC3000 inoculation.

Bacterial Disease Assays
Syringe-infiltration was performed in this study.Briefly, Pst DC3000 was cultured in modified Luria-Bertani medium (Xin et al., 2016) containing 100 mg l −1 rifampicin at 28 °C to an OD 600 of 0.8-1.0.Bacteria were collected by centrifugation and re-suspended in 0.25 mM MgCl 2 .Cell density was adjusted to OD 600 = 0.2 (approximately 1 × 10 8 cfu ml −1 ) and further diluted to cell densities of 1 × 10 5 -1 × 10 6 cfu ml −1 .The infiltrated plants were first placed under ambient humidity for 1-2 h to allow water to evaporate, and then placed in high humidity (about 95%; by covering plants with a clear plastic dome) for disease development after the leaves returned to the state before infiltration.
For quantification of Pst DC3000 bacterial populations, leaf disks were taken using a disposable biopsy punch with 4 mm diameter (Integra LifeSciences) and ground in 0.25 mM MgCl 2 .Colony-forming units were determined by serial dilutions and plating on Luria Marine plates containing 100 mg l −1 rifampicin.One biological replicate consists of 12 leaf disks (that is, from 3 leaves of one plant) and at least 3 biological replicates were included in each experiment.Experiments were repeated at least three times.

RNA Extraction and RT-qPCR
Total RNA was extracted by using Trizol reagent (Invitrogen).The RT-qPCR analysis was conducted as described (He et al., 2023;Li et al., 2023).In brief, 1.0 μg DNase-treated RNA was reverse transcribed in a reaction volume of 20 μl containing oligo-(dT)19 primer and Moloney murine leukemia virus reverse transcriptase (Thermo Fisher Scientific).RT-qPCR analysis was then performed with 1.0 μl of 5-fold diluted cDNA using a SYBR premixed Ex Taq kit (Takara), and a Light Cycler 480 Real-Time PCR System (Roche) for the reaction.In each RT-qPCR experiment, at least three biological replicates were used and three technical replicates were performed for each biological replicate.The ACTIN2 (AT3G18780) gene was used as an internal reference gene.
Oligonucleotide probes containing wild type E-box were synthesized and labeled with 5 ' Biotin modification (BGI, China).The unlabeled wild type Ebox served as a competitor, and the mutated E-box modified by biotin served as a negative control.Double-stranded probes were generated by annealing of forward/reverse primers.EMSA was performed using biotin-labeled probes and Chemiluminescent EMSA Kit (Beyotime; GS009) referring to the instructions.For the binding reaction, ICE1-His protein was incubated with binding buffer containing 1 μl of biotin-labeled oligonucleotide in a total volume of 10 μl.Competition experiments were executed using 50-fold and 200-fold unlabeled double-stranded DNA.The DNA-protein complexes were separated on a 6% polyacrylamide gel in 0.5 × Tris-borate-EDTA buffer.The primers used for vector construction are listed in Supplemental Data Set 1.

Yeast Two-Hybrid Screening and Confirmation
The full-length coding sequences (CDS) of NPR1 and TGA were fused to the bait vector pGBKT7 (Clontech, Palo Alto, CA, USA) by NdeI (Thermo Scientific) and EcoRI (Thermo Scientific) using ClonExpress II One Step Cloning Kit (Vazyme) to generate BD-NPR1 and BD-TGA, while the full-length coding sequence of ICE1 was ligated to the prey vector pGADT7 to produce AD-ICE1.To identify structural domains essential for protein interactions, several truncated NPR1 and TGA3 were cloned into pGBKT7 and truncated ICE1 into pGADT7.The yeast two hybrid (Y2H) assays were performed as described previously (Li et al., 2023).The vector pairs were co-transformed into the yeast strain AH109, protein interactions were indicated by the ability of cells to grow on dropout medium lacking Leu, Trp, His, and Ade for 2 days after plating.The primers used for vector construction are listed in Supplemental Data Set 1.

Split Luciferase Complementation Assay
The split luciferase complementation assays were performed as described previously (Fujikawa and Kato, 2007;Chen et al., 2008).The full-length CDS of ICE1 was cloned into the pCAMBIA1300-nLUC vector by BamHI (Thermo Scientific) and SalI (Thermo Scientific) under the control of the 35S promoter using ClonExpress II One Step Cloning Kit (Vazyme).Full-length CDS of NPR1 and TGA3 were cloned into the pCAMBIA1300-cLUC vectors by KpnI (Thermo Scientific) and SalI (Thermo Scientific) using ClonExpress II One Step Cloning Kit (Vazyme), respectively.After co-expression in Nicotiana benthamiana leaves for 48 h，the leaves were sprayed with 1 mM D-luciferin (0.01% Triton X-100) solution and then incubated for 5 min in the dark before detecting.
Luciferase intensity was visualized on Tanon-5200 Chemiluminescent Imaging System (Tanon Science&Technology).The primers used for vector construction are listed in Supplemental Data Set 1.

Bimolecular Fluorescence Complementation Assays
The

Co-immunoprecipitation Assays
To generate proteins fused with YFP or Myc tags, we amplified the full-length CDS of NPR1 and TGA3 and recombined into the intermediate vector pDonor207 using the BP recombination kit (Invitrogen), then cloned into the destination vector pEarleyGate101 with YFP-tagged sequences under the control of Pro35S using the LR recombination kit (Invitrogen) to obtain 35S::NPR1-YFP and 35S::TGA3-YFP constructs, whereas the full-length CDS of ICE1 was inserted into the destination vector Myc-tagged pGWB517 using the same method to get 35S::ICE1-Myc.Arabidopsis protoplasts were transformed with different combinations of plasmids, including 35S::YFP and 35S:: ICE1-Myc, 35S::NPR1-YFP and 35S::ICE1-Myc, 35S::TGA3-YFP and 35S:ICE1-Myc at 22°C for 16 h according to the Sheen laboratory protocol (Sheen, 2001).Total protein was extracted with an IP buffer containing 50 mM MOPs, 5 mM EDTA, 0.2% v/v Triton X-100, 5 mM dithiothreitol, 1 mM PMSF and 1 x complete protease inhibitor cocktail (Roche).Immunoprecipitation experiments with GFP-trap beads were performed according to the manufacturer's protocol.Briefly, cell lysates were incubated with GFP-trap beads (ChromoTek) at 4°C overnight.After incubation, the beads were washed three to five times with IP buffer, and then co-immunoprecipitated proteins were detected by immunoblotting with an anti-Myc antibody (Abmart; 1:10,000).Primers used for vector construction are listed in Supplementary Data Set 1.
For Co-IP assay using stable transgenic seedlings, total proteins were extracted with IP buffer from 35S:HA-NPR1, 35S:ICE1-Myc and 35S:HA-NPR1 35S:ICE1-Myc transgenic Arabidopsis seedlings at both normal temperature and 2 h cold treatments, Col-0 seedlings used as negative control.Proteins immunoprecipitated with Myc-trap beads (ChromoTek) at 4°C overnight, then the beads were washed three to five times with IP buffer.The Co-immunoprecipitated proteins were detected with anti-HA antibody (ZENBIO; 1:10,000).

Transient Transcriptional Activation Assays
The full-length CDS of NPR1 was cloned into the pGreenII 62-SK vector by SacI (Thermo Scientific) and BamHI (Thermo Scientific), while full-length CDS of ICE1, TGA3, and GFP were cloned into the pGreenII 62-SK vector by BamHI (Thermo Scientific) and EcoRI (Thermo Scientific) using T4 DNA ligase (Thermofisher) as effectors, respectively.The promoter sequence of PR1 was amplified by PCR and inserted into the pGreenII 0800-LUC vector (Biovector Science Lab) by SacI (Thermo Scientific) and BamHI (Thermo Scientific) using T4 DNA ligase (Thermofisher) as the reporter (Hellens et al., 2005).Combinations of plasmids were transformed into the Col-0 and/or mutant leaf mesophyll protoplasts according to the Sheen laboratory protocol (Sheen, 2001).Transfected cells were cultured for 16-18 h followed by analysis of relative luciferase (LUC) activity using the Dual-Luciferase Reporter Assay System (Promega), which detects the activities of firefly LUC and the internal control Renilla reniformis (REN).Primers used for vector construction are listed in Supplemental Data Set 1.

STATISTICAL ANALYSIS
Statistical analysis was performed by analysis of variance.The results of statistical analyses are shown in Supplemental Data Set S2.

ACCESSION NUMBERS
Arabidopsis Genome Initiative numbers for the genes discussed in this article are as follows: ICE1, AT3G26744; NPR1, AT1G64280; TGA1, AT5G65210;    .For low-temperature induction, protoplasts were processed at 4°C for 2 h before total protein was extracted.Then total protein was immunoprecipitated with GFP-trap agarose and the co-immunoprecipitated proteins were detected with anti-Myc antibody.Experiments were repeated at least three times with similar trends.(E) Co-IP assay using stable transgenic seedlings.Transgenic plants are growing on a normal temperature chamber for 4 weeks, Col-0 plants used as negative control.For low temperature treatment, plants are moved into another growth chamber with 4°C for 2 h treatment before protein extraction.ICE1-Myc fusion was immunoprecipitated by Myc-trap agarose, and the coimmunoprecipitated protein was detected using an anti-HA antibody.Experiments were repeated at least three times with similar trends.
To further confirm the essential role of ICE1 in SA-regulated immunity, we analyzed the disease symptoms of pretreated Col-0 and ICE1-overexpressing plants (ICE1-OE 1 and ICE1-OE 2).Compared with Col-0 plants, BTH-pretreated ICE1overexpressing plants showed less severe disease symptoms after inoculation with Pst DC3000, accompanied by BTH-induced high transcript levels of PR1 (Fig. 3D-F).
the ability of ICE1 to activate the transcription of PR1.To test this possibility, ChIP assays were conducted with npr1-1 ICE1-Myc and NPR1-HA ICE1-Myc plants treated or not with BTH.As shown in Fig.4C, D, ICE1 was enriched on the PR1 promoter in ICE1-Myc, and BTH promoted its enrichment.However, the high enrichment of ICE1 on the promoter of PR1 was impaired in the background of npr1-1, but enhanced in the NPR1-overexpressing line (Fig.4C, D).These results suggest that the enrichment of ICE1 on the promoter of PR1 is promoted by NPR1.
symptoms and pathogen proliferation, in Col-0, ice1-2, and ice1-2/ICE1 plants treated with BTH for 24 hours with or without a 10-hour low temperature pretreatment.This allowed us to characterize the critical role of ICE1 in integrating low temperature signals and SA signals to induce pathogen resistance.As shown in Fig. 5A, B, in the leaves of Col-0 and ice1-2/ICE1, the water soaking symptoms and pathogen proliferation were alleviated by either BTH or 4°C pretreatment, and significantly diminished by BTH combined with cold treatment, compared with the mock treatment.In contrast, the leaves of ice1-2 showed severe water soaking symptoms and high pathogen proliferation under all the different treatments.The transcript levels of PR1 were also lower in ice1-2 plants compared with Col-0 and ice1-2/ICE1 plants under different conditions (Fig. 5C).We tested the enrichment of ICE1 on the promoter of PR1 in the different treatments.As shown in Fig. 5D, accumulation of ICE1 on the promoter of PR1 was enhanced by the combined low temperature and BTH treatment, compared with either of these treatments alone.Collectively, these results indicate that ICE1 functions as a crucial node by integrating low temperature signals and SA signals to promote plant disease immunity synergistically.
ICE1 and TGAs, different truncated versions of the proteins were used in yeast two-hybrid assays.As shown in Supplemental Fig.S8A, deletion of the N-terminal amino acid residues 1-260 of ICE1 (AD-ICE1261-494 ) did not affect the ICE1-TGA3 interaction, whereas deletion of the C-terminal residues of ICE1 containing the bHLH domain (AD-ICE1 1-260 ) eliminated the interaction completely.Additionally, the N-terminal amino acid residues of TGA3 containing the bZIP domain (BD-TGA3-N) strongly interacted with ICE1, while the C-terminal fragment (BD-TGA3-C) did not.These results demonstrate that the bHLH structural domain of ICE1 and the bZIP structural domain of TGA3 are necessary for the ICE1-TGA3 interaction (Supplemental Fig.S8).The results of the BiFC, split luciferase and Co-IP assays further confirmed that ICE1 interacted with TGA3 in plant cells.For the BiFC assays, full-length CDSs of TGA1 (clade I), TGA2 (clade II), TGA3, and TGA7 (clade III) were fused to the C-terminal yellow fluorescent protein (cYFP) fragment to generate TGA1-cYFP, TGA2-cYFP, TGA3-cYFP, and TGA7-cYFP, respectively, and the full-length CDS of ICE1 was fused to the N-terminal fragment of YFP (nYFP) to produceICE1-nYFP.Truncated TGA3 193-384 -cYFP and ICE1 1-260 -nYFP that could not interact with ICE1 or TGA3 were used as negative controls (Supplemental Fig.S2).When ICE1-nYFP was transiently coexpressed with TGAs-cYFP in leaf cells of N. benthamiana, recombinant YFP fluorescence was observed in the nucleus, whereas no fluorescence signals were detected when either TGA3193-384 -nYFP and ICE1-nYFP or ICE1 1-260 -nYFP and TGA3-cYFP were co-expressed (Fig.6B; Supplemental Fig.S2B).For the split luciferase assay, TGA3 fused to the C-terminus (cLUC) and ICE1 fused to N-terminal (nLUC) of luciferase were co-expressed in N. benthamiana leaves.As shown in Fig.6C, LUC activity could be detected when TGA3 interacted with ICE1.In the Co-IP assay, ICE1 was immunoprecipitated by anti-GFP agarose beads in Arabidopsis protoplasts coexpressing TGA3-YFP and ICE1-Myc, but not in those co-expressing YFP and ICE1-Myc.This result provided further evidence for the interaction between ICE1 and TGA3 in vivo (Fig.6D).Taken together, these results demonstrate that ICE1 physically interacts with the TGA3 transcription factor in planta.ICE1 and TGA3 Works Synergistically during Low Temperatureenhanced ImmunityHaving demonstrated that ICE1 associates with TGA3, we wondered if these two proteins act cooperatively to modulate disease resistance in plants exposed to cold treatment.Hence, we constructed the TGA3-knockout mutant tga3 CR (Supplemental Fig.S9), and then crossed ice1-2 with tga3 CR to obtain ice1-2 tga3 CR double mutant plants.The disease phenotypes of these plants were then determined.As shown in Fig.7A, B, compared with the ice1-2 and tga3 CR single mutants, the ice1-2 tga3 CR double mutant showed a more susceptible phenotype, including severe water soaking symptoms and high pathogen proliferation after inoculation with Pst DC3000 both at normal and low temperatures, indicating a synergistic role of TGA3 and ICE1 in the regulation of plant immunity.ICE1 acts as an essential node integrating SA-regulated and low temperature-enhanced immunity by direct enrichment on the promoter of PR1 (Fig.5).To further delineate the regulatory relationship between ICE1 and TGA3, we conducted ChIP assays to investigate if TGA3 promotes the enrichment of ICE1 on the promoter of PR1 in vivo.For this purpose, tga3 CR ICE1-Myc and TGA3-HA ICE1-Myc seedlings were treated at 4°C for 2 h.Seedlings grown at 22°C served as the control.As shown in Fig.7C, the enrichment of ICE1 on the PR1 promoter was decreased in the background of tga3 CR compared with Col-0.The accumulation of TGA3 on the PR1 promoter was increased in the background of ICE1-Myc-OE plants compared with Col-0, the regulatory role of low temperature on plant immunity is instrumental for revealing the mechanisms of environmental adaptation in plants under cold stress.The results of studies on the temperature regulation of pathogen resistance may provide a theoretical basis for the development of new strategies to improve plants' resistance to pathogens in future agricultural production.In addition, because of their sessile lifestyle, plants are constantly exposed to a variety of abiotic stresses, and endogenous hormone signals often interact with multiple exogenous environmental cues to regulate their resistance to pathogens.Dissecting the regulatory mechanism of the integration of low temperature and SA signals in response to pathogen attack also provides new possibilities for research on the cross-talk between SA signaling and other environmental signals.
35S::GFP-ICE1 (CS68099) line was obtained from the Arabidopsis Resource Center at Ohio State University (http://abrc.osu.edu).To generate the overexpression transgenic plants, the full-length cDNA of ICE1, TGA3 were cloned into the pOCA30 vector by BamHI (Thermo Scientific) and SalI (Thermo Scientific) in the sense orientation behind the CaMV 35S promoter using T4 DNA ligase (Thermofisher), respectively.Primers used for transgenic construction are listed in Supplemental Data Set 1.For CRISPR/Cas9mediated editing of TGA3, one guide RNA was designed by CRISPR-P 2.0 (http://crispr.hzau.edu.cn/CRISPR2/) to target the third exon of TGA3.This guide RNA, driven by the AtU6a promoter, was cloned into the pMH-SA binary vector carrying Cas9 by SpeI (Thermo Scientific) and AscI (Thermo Scientific) using T4 DNA ligase (Thermofisher)(Liang et al., 2016).The detailed information about guide sequence and direct sequencing of PCR products containing targeted sites of TGA3 in Arabidopsis plants are showed in the Supplemental Fig.S9.The genotyping/sequencing primers are listed in Supplemental Data Set 1.For low temperature treatment, plants were grown for 4 weeks at 22°C, followed by 3 weeks or 10 h of 4°C treatment.Plants had roughly 20 rosette leaves and not yet reached to the flowering stage before cold treatment.Control plants were directly inoculated with Pst DC3000, and cold-treated plants were transferred to a cold incubator (4°C, 65% relative humidity, 10 h light/14 h dark photoperiod) for 3 weeks or 10 h treatment before pathogen inoculation.

Figure 2 .
Figure 2. ICE1 interacts with NPR1.(A) Yeast two hybrid assays showing the interactions between ICE1, NPR1, and their truncated versions.Protein interactions were indicated by the growth of yeast cells after 2 days of incubation in dropout medium lacking Leu, Trp, His and Ade.The numbers indicate the positions of amino acids, pGBKT7 (BD) and pGADT7 (AD) were used as negative controls.(B) BiFC assay.Fluorescence was observed in the nucleus of transformed N. benthamiana cells co-expressing ICE1-nYFP (or ICE1 261-494 -nYFP) with NPR1-cYFP or ICE1-nYFP with NPR1 1-194 -cYFP at 4°C for 4 h.No signal was obtained in the negative controls which NPR1-cYFP (or NPR1 1-194 -cYFP) with ICE1 1-260 -nYFP or ICE1-nYFP (or ICE1 261-494 -nYFP) with NPR1 178-593 -cYFP were co-expressed.Nuclei are indicated by DAPI staining.Scale bar = 20 μm.(C) Interaction between ICE1 and NPR1 in split luciferase assay.ICE1-nLUC and NPR1-cLUC were co-expressed in N. benthamiana leaves, the luminescence intensity was detected at 4°C for 4 h after 48 h of incubation by an imaging system.GUS-nLUC and GUS-cLUC were set as a negative control.(D) Co-IP assay.Arabidopsis protoplasts expressing NPR1-YFP with ICE1-Myc, YFP with ICE1-Myc were incubated for 16 h at 22°C.For low-temperature induction, protoplasts were processed at 4°C for 2 h before total protein was extracted.Then total protein was

Figure 4 .
Figure 4. NPR1 promotes the binding and transcriptional activation of ICE1 to PR1. (A) Col-0, ice1-2, npr1-1 and ice1-2 npr1-1 leaves infiltrated with Pst DC3000 (OD600 = 0.0001).Photos were taken after 48 h pathogen inoculation.(B) Bacterial populations in leaves described in (A).Values are displayed as mean ± s.d.(n ≥ 6 biological replicates).(C and D) ChIP-qPCR analysis of the relative enrichment of ICE1 on the promoter regions of PR1.Col-0, ICE1-overexpressing (ICE1-Myc), npr1-1 ICE1-Myc and NPR1-HA ICE1-Myc plants were treated with mock or 100 μM BTH for 6 h and pooled for ChIP assays using anti-Myc antibody, the ACTIN2 untranslated region sequence (pACTIN2) as a negative control.Values are displayed as mean ± s.e.m. (n = 3 biological replicates).(E) Transient transcriptional activity assays showing that NPR1 enhances the transcriptional activation of ICE1 to PR1.Values are displayed as mean ± s.e.m. (n = 3 biological replicates).(F) Transient transcriptional activity assays showing that activation of the PR1 promoter by ICE1 is compromised in the npr1-1 mutant.Values are displayed as mean ± s.e.m. (n = 3 biological replicates).Each biological replicate was from different leaves of more than 60 plants in (E) and (F).Different letters indicate statistically significant differences (two-way ANOVA, P < 0.05).Experiments were repeated at least three times with similar results.

Figure 5 .
Figure 5. ICE1 links low temperature signals and SA pathway to promote plant immunity.(A) Col-0, ice1-2 and ice1-2/ICE1 leaves infiltrated with Pst DC3000 (OD600 = 0.0001) after BTH combined with low temperature pretreating (briefly, 4-week-old plants were sprayed with mock or BTH, and kept them in a growth chamber with normal temperature (22°C) for 14 h.After then, the low temperature experimental plants were transferred to a low temperature (4°C) growth chamber for 10 h cold treatment.).npr1-1 mutant plants was used as a control.Photos were taken after 48 h pathogen inoculation.(B) Bacterial populations in leaves described in (A).Values are displayed as mean ± s.d.(n = 6 biological replicates).(C) RT-qPCR analysis of PR1 expression in leaves described in (A).Values are displayed as mean ± s.e.m. (n = 3 biological replicates).The ACTIN2 gene was used as a control.(D) ChIP-qPCR analysis of the relative enrichment of ICE1 on the promoter regions of PR1.ICE1-overexpressing plants (ICE1-Myc) were treated with 100 μM BTH for 6 hours and 4°C for 2 hours and pooled for ChIP assays using anti-Myc antibody, the ACTIN2 untranslated region sequence (pACTIN2) as a negative control.Values are displayed as mean ± s.e.m. (n = 3 biological

Figure 6 .
Figure 6.ICE1 interacts with TGA transcription factors.(A) Yeast two hybrid assays.Interactions of ICE1 with TGAs were indicated by the ability of yeast cells to grow on dropout medium lacking Leu, Trp, His, and Ade for 2 days after plating.BD and AD were used as negative controls.(B) BiFC assay.Fluorescence was observed in the nuclear compartment of transformed N. benthamiana cells, resulting from the complementation of ICE1-nYFP with TGA1/2/3/7-cYFP.No signal was obtained for the negative controls in which ICE1 1-260 -nYFP with TGA3-cYFP or ICE1-nYFP with TGA3 193-384 -cYFP were coexpressed.Nuclei are indicated by DAPI staining.Scale bar = 20 μm.(C) Split luciferase assay.nLUC-ICE1 and cLUC-TGA3 constructs were co-transformed into N. benthamiana.Luminescence intensity was measured after 48 h of incubation by an imaging system.GUS-nLUC and GUS-cLUC were set as a negative control.(D) Co-IP assay.Arabidopsis protoplasts expressing TGA3-YFP with ICE1-Myc, YFP with ICE1-Myc were incubated for 16 h.Total protein was extracted and then immunoprecipitated with GFP-trap agarose.Co-immunoprecipitated protein was detected with anti-Myc antibody.Experiments were repeated at least three times with similar trends.

Figure 7 .
Figure 7. ICE1 and TGA3 works synergistically to activate expression of PR1 during low temperature stress.(A) Col-0, ice1-2, tga3 CR and ice1-2 tga3 CR leaves infiltrated with Pst DC3000 (OD600 = 0.0001) after 10 hours of 4°C treatment (down), 22°C as control (up).Photos were taken after 48 h pathogen inoculation.(B) Bacterial populations in leaves described in (A).Values are displayed as mean ± s.e.m (n = 4 biological replicates).(C) ChIP-qPCR analysis of the tga3 CR mutation impaired the relative enrichment of ICE1 to the PR1 promoter.Col-0, ICE1-overexpressing (ICE1-Myc) and tga3 CR ICE1-Myc plants were treated for 2 h of 4°C , 22°C as control.(D) ChIP-qPCR analyses of the relative enrichment of TGA3 to the PR1 promoter enhanced by ICE1.Col-0, TGA3-overexpressing plants (TGA3-HA) and TGA3-HA ICE1-Myc (hybridized F1 generation) were treated for 2 h of 4°C, 22°C as control.Treated leaves were pooled for ChIP assays using anti-HA antibody, the ACTIN2 untranslated region sequence (pACTIN2) as a negative control in (C) and (D).(E) Transient transcriptional activity assays showing that TGA3 enhances the transcriptional activation of ICE1 to PR1. (F) Transient transcriptional activity assays showing that activation of the PR1 promoter by ICE1 is compromised in the tga3 CR mutant.(G) Transient transcriptional activity assays showing that activation of the PR1 promoter by ICE1 is diminished in the ice1-2 mutant.Each biological replicate was from different leaves of more than 60 plants in (E-G).(H) Transient transcriptional activity