Warm temperature suppresses plant systemic acquired resistance by intercepting the N-hydroxypipecolic acid immune pathway

Climate warming influences disease development by targeting critical components of the plant immune system, including pattern-triggered immunity (PTI), effector-triggered immunity (ETI) and production of the central defence hormone salicylic acid (SA) at the primary pathogen infection site. However, it is not clear if and/or how temperature impacts systemic immunity. Here we show that pathogen-triggered systemic acquired resistance (SAR) in Arabidopsis thaliana is suppressed at elevated temperature. This was accompanied by global downregulation of SAR-induced genes at elevated temperature. Abolished SAR under warmer conditions was associated with reduced biosynthesis of the SAR metabolite N-hydroxypipecolic acid (NHP) in Arabidopsis and other plant species, as demonstrated by downregulation of NHP biosynthetic genes (ALD1 and FMO1) and NHP precursor pipecolic acid (Pip) levels. Although multiple SAR signals have been shown previously, exogenous Pip was sufficient to restore disease protection at elevated temperature, indicating that heat-mediated SAR suppression is due to Pip-NHP downregulation. Along with ALD1 and FMO1, systemic expression of the SA biosynthetic gene ICS1 was also suppressed at warm temperature. Finally, we define a transcriptional network controlling thermosensitive NHP pathway via the master transcription factors CBP60g and SARD1. Our findings demonstrate that warm temperatures impact not only local but also systemic immunity by impinging on the NHP pathway, providing a roadmap towards engineering climate-resilient plant immune systems.


Main Text
Warming global temperatures due to climate change pose serious threats to the natural environment and human civilization (Altizer et al., 2016;Deutsch et al., 2018;Cavicchioli et al., 2019;Delgado-Baquerizo et al., 2020).In agricultural systems, increasing average temperatures are predicted to significantly reduce yields of the world's major crops (Zhao et al., 2017;Chaloner et al., 2021;Singh et al., 2023).As many crop varieties are bred to be grown outside the native growth ranges of their wild relatives, it is important to understand the impact of elevated temperatures on plant growth, development, immunity and phenology.Together with other environmental factors, temperature profoundly affects various aspects of plant physiology, ranging from growth and development (Quint et al., 2016 Environmentally compromised immune signaling is associated with increased plant disease development, as postulated as the plant disease triangle (Stevens, 1960;Colhoun, 1973 Currently, the impact of elevated temperature on plant systemic immunity is underexplored.Although previous studies have shown that increased temperatures have a negative effect on several aspects of the plant immune system that occur in the local/primary sites of infection (like PTI, ETI and SA production), it is unknown if and how elevated temperature affect NHP-mediated plant immunity and the SAR response.In our study, we show that elevated temperature suppresses SAR-associated NHP biosynthetic gene expression and

Warm temperature affects systemic acquired resistance in Arabidopsis plants
To determine if SAR is affected by temperature, lower leaves of Arabidopsis Col-0 plants were inoculated with the virulent bacterial pathogen Pst DC3000 as the primary challenge.Two days after primary infection, upper systemic leaves were inoculated with the pathogen Pst DC3000 as the secondary challenge.As shown in Figure 1a, primary pathogen infection expectedly lowered the systemic bacterial counts after secondary pathogen challenge at 23°C but not at 28°C in Col-0 plants.This indicates that Arabidopsis SAR due to local Pst DC3000 infection is effective at normal but not at elevated temperature, suggesting that temperature regulates plant systemic immunity.(A, B) Lower leaves of four-week-old Arabidopsis Col-0 plants were infiltrated with 0.25 mM MgCl2 (mock) and Pst DC3000 (OD600 = 0.02) in (A) or Pst DC3000/AvrRpt2 (OD600 = 0.02) in (B).Plants were then incubated at either 23°C or 28°C.Two days after primary local inoculation, upper systemic leaves were infiltrated with Pst DC3000 (OD600 = 0.001), and plants were incubated again at their respective temperatures (23°C or 28°C).Bacterial numbers (upper panels) and symptom expression photos (lower panels) were taken at 3 days post-inoculation (dpi) of systemic tissues.Data show the mean log CFU Pst DC3000/mL (± S.D. In addition to virulent pathogens, avirulent pathogens that induce local ETI can also induce SAR (Cameron et al., 1994;Zeier, 2021).We therefore tested whether SAR activated by the avirulent strain Pst DC3000/AvrRpt2, which activates RPS2-dependent immunity in Col-0 plants (Kunkel et al., 1993;Bent et al., 1994), is also impacted by warm conditions.As expected, SAR was induced at normal temperature (23°C) after primary Pst DC3000/AvrRpt2 infection (Figure 1b).Strikingly, systemic Pst DC3000/AvrRpt2 levels remained similar compared to mock treatment at elevated temperature (28°C), indicating that even ETI-induced SAR protection is also negatively affected in Arabidopsis Col-0 plants at higher temperature.
To further understand global SAR immune signaling, we analyzed a previously generated Arabidopsis SAR transcriptome after virulent P. syringae infection (Hartmann et al., 2018).We interfaced the SAR-induced (SAR+) genes from that study with our previously published temperature-regulated transcriptome (Kim et al., 2022) and categorized Arabidopsis SAR+ genes into downregulated or upregulated genes at elevated temperature.As shown in Figure 1c, 1037 SAR+ genes were downregulated, while 151 SAR+ genes were upregulated at elevated temperatures.This demonstrates that a significant majority of temperature-regulated SAR genes exhibit downregulated expression at elevated temperature, consistent with loss of systemic protection against secondary infection at 28°C (Figures 1a-b).Overall, our collective results indicate the Arabidopsis SAR induced by both virulent and avirulent pathogens are negatively impacted by warm temperatures.

The N-hydroxypipecolic acid pathway is downregulated by elevated temperature
To determine the mechanism of how temperature regulates systemic immunity, we measured expression levels of genes required for biosynthesis of NHP, which is an immune-activating metabolite crucial for SAR (Hartmann et al., 2018;Chen et al., 2018).As shown in Figure 2a, transcript levels of ALD1 and FMO1 in local (primary) leaves of Arabidopsis Col-0 plants at 1 day post-infection with Pst DC3000 were lower at 28°C than at 23°C.In agreement, pathogen-induced levels of the NHP precursor metabolite Pip were also lower at the warmer temperature (Figure 2b).Similar trends in warm temperature-suppression of NHP biosynthetic gene expression was observed in tomato and rapeseed plants (Figures 2c-d).Finally, as shown in Figure 2e, systemic (uninfected) leaves of Arabidopsis plants at 2 dpi exhibited lower ALD1 and FMO1 gene expression levels at 28°C compared to those at 23°C.Taken together, these results indicate that elevated temperature impacts NHP biosynthesis in various plants species, which is associated with loss of SAR at higher temperatures.Altogether, these results demonstrate that the SA biosynthetic pathway is suppressed at higher temperature similarly as NHP.This suggests that mutual amplification of these two important SAR-associated metabolites is negatively affected when temperatures increase.

Local or systemic application of exogenous Pip restores Arabidopsis immune priming at elevated temperature
Having observed that Pip levels were suppressed at elevated temperature, we hypothesized that exogenous supplementation with Pip may restore disease protection at 28°C if Pip production is the rate-limiting step at elevated temperature.We infiltrated leaves of Arabidopsis plants with mock or 1 mM Pip and then infected the same leaves with Pst DC3000.
As shown in Figure 4a, pathogen levels were reduced after Pip treatment compared to mock treatment at 23°C, expectedly indicating that Pip is sufficient to induce immune priming.
Remarkably, Pip-induced disease protection was maintained at 28°C (Figure 4a), which confirms that Pip-NHP production is the rate-limiting step in SAR at elevated temperature.To determine if elevated temperature also affects systemic Pip-mediated disease protection, we irrigated Arabidopsis roots with mock or 1 mM Pip two days before infiltrating the leaves with Pst DC3000.Similar to the results with Pip-induced disease protection by leaf infiltration, we also observed reduced pathogen levels after Pip root irrigation at both 23°C and 28°C (Figure 4b).These results indicate that the temperature-suppression of the Pip-NHP pathway is governed at the level of biosynthesis and not systemic transport.

CBP60g and SARD1 control temperature-vulnerability of the NHP-SAR pathway
In addition to chemical supplementation, we next investigated if we could restore systemic immune responses genetically at warm temperatures.We recently showed that CBP60g and its functionally redundant paralog SARD1 control the temperature-sensitivity of plant basal resistance and pathogen-induced SA biosynthesis (Kim et al., 2022).We therefore investigated whether CBP60g and SARD1 also control temperature-sensitive systemic defence responses.As shown in Figure 3c-d Consistent with this, constitutive expression of the functionally redundant SARD1 gene in 35S::SARD1 plants also led to restored expression of ALD1 and FMO1 at warm temperature (Figure 5d-e).Taken together, a major mechanism by which higher temperatures target plant systemic immunity and NHP biosynthesis is through the expression of the master immune transcription factor genes CBP60g and SARD1.

Discussion
In this study, we showed that pathogen-induced SAR and the SAR-activating NHP Overall, changing climatic factors like elevated temperature have a broad and significant impact on the plant immune system, not only at local sites of infection (PTI, ETI, SA) but also on systemic immune priming in distal sites via the central SAR metabolite NHP.In this study, we determined that elevated temperature regulates SAR by influencing NHP pathway.We further demonstrated that this temperature-regulation of NHP-mediated SAR is also controlled by the temperature-sensitive master regulators CBP60g and SARD1, reminiscent of their roles in local immune responses (Kim et al., 2022).Not only does temperature affect the plant's ability to directly defend against pathogen attacks but also the plant's immune preparedness for future infections.Our discoveries advance our understanding of how the plant immune landscape is regulated by a changing environment.This foundational mechanistic knowledge of the plant disease triangle is important to inform strategies in mitigating the negative impacts of warming temperatures on plant health and to provide a molecular roadmap towards engineering climateresilient plants.

Plant systemic immunity and SAR assays
Four-week-old Arabidopsis plants were covered with plastic domes to increase humidity and open the stomata 24 hours before infiltration, Plants were infiltrated with 0.25 mM MgCl2 (mock), Pst DC3000 (OD600=0.02)or Pst DC3000/AvrRpt2 (OD600=0.02) in their lower leaves (Xin et al., 2013;Xin et al., 2018).Following infiltration, plants were further incubated in environmentally growth chambers at either 23°C or 28°C with identical relative humidity (60%) and lighting conditions (12h light/12 dark).For SAR disease assays, upper systemic leaves were or infiltrated with Pst DC3000 (OD600 = 0.001).Bacterial levels were quantified 3 days after systemic infiltration based on a previously published protocol (Huot et al., 2017;Kim et al., 2022).Briefly, in planta bacterial extracts were plated on rifampicin-containing LM media and log colony forming units (CFUs) cm -2 were calculated.

Pip treatment and protection assay
Four-week-old Arabidopsis plants were infiltrated with mock solution or 1 mM Pip (Sigma) in their lower leaves.In parallel to direct Pip infiltration into leaves, plants were irrigated with 1 mL of mock solution or 1 mM Pip (Sigma) for root inoculations.Following infiltration or irrigation, plants were further incubated in environmentally growth chambers at either 23°C or 28°C with identical relative humidity (60%) and lighting conditions (12h light/12 dark).After two days, the Pip-treated leaves (for local Pip infiltration) or upper systemic leaves (for root irrigation) were further infiltrated with Pst DC3000 (OD600 = 0.001).Bacterial levels were quantified 3 days after systemic infiltration as stated in the previous section.

Gene expression analyses
Pathogen-infected leaves and upper systemic leaves at either 23°C or 28°C were harvested at 1 or 2 days after local infection, respectively.Tissues were flash-frozen in liquid  1.

Pip metabolite extraction and quantification
Pathogen-infected leaves at 23°C or 28°C were harvested at 1 day after infection.Pip extraction and quantification were performed based on previous reports with slight modifications (Yao et al., 2023).Approximately 100 mg leaf tissue was frozen and ground in liquid nitrogen, and Pip was extracted at 4℃ for 1h in 600 µL ice-cold extraction buffer (80% methanol in water, 0.1 g/L butylated hydroxytoluene).The extraction step was repeated twice, and a 1.2-mL supernatant was speed-dried in a vacuum centrifugal concentrator (Beijing JM Technology).
The pellet was resuspended in 240 µL 30% methanol solution and diluted 10 times for quantification.Pip level was quantified using the AB SCIEX QTARP 5500 LC/MS/MS system.
Selected ion monitoring (SIM) was conducted in the positive ES channel for Pip (m/z 130.0>84.0),which was done using a 20V collision energy and a 50V declustering potential.
The instrument control and data acquisition were performed using Analyst 1.6.3software (AB SCIEX), and data processing was performed using MultiQuant 3.0.2software (AB SCIEX).Pip was separated with an ACQUITY UPLC BEH Amide column (1.7 µm, 2.0 x 100 mm, Waters) using the method in Supplementary Table 2. Pip levels were quantified by calculating the area of each individual peak and comparing it to standard curves.Reported Pip concentration was normalized by sample fresh weight (FW) in gram.
Pip production.Because it has been shown that the NHP immune pathway is redundantly controlled by the master transcription factors CALMODULIN-BINDING PROTEIN 60-LIKE G (CBP60g) and SAR-DEFICIENT 1 (SARD1) (Sun et al., 2015; Sun et al., 2018; Huang et al., 2020), we further found that warm temperature-suppressed plant systemic immunity is restored by constitutive CBP60g/SARD1 gene expression or exogenous Pip application.Collectively, our study indicates that CBP60g and SARD1 control the temperature-vulnerability of Arabidopsis systemic immunity by regulating Pip/NHP biosynthesis.

Fig. 1
Fig. 1 Arabidopsis SAR is suppressed at elevated temperature.
) and individual points (n=16 from 4 independent experiments) analyzed with two-way ANOVA and Tukey's Multiple Comparisons test.Statistical differences of means are denoted by different letters.(C) Transcriptome analysis using SAR+ genes from Hartmann et al. (2018) interfaced with temperature-regulated genes from Kim et al. (2022).The number of SAR+ genes that are downregulated and upregulated at elevated temperature are shown (fold change cutoff > 2).

Fig. 2
Fig. 2 Plant NHP biosynthetic gene expression and Pip production are suppressed at elevated temperature.(A-B) Leaves of four-week-old Arabidopsis Col-0 plants were infiltrated with 0.25 mM MgCl2 (mock) or Pst DC3000 (OD600 = 0.001).Plants in were then incubated at either 23°C or 28°C.(A) ALD1 and FMO1 transcript levels and (B) Pip levels of pathogen-inoculated tissues were measured at 1 day post-inoculation (dpi).(C) Leaves of four-week-old tomato cultivar Castlemart plants were infiltrated with 0.25 mM MgCl2 (mock) or Pst DC3000 (OD600 = 0.001).SlALD1 transcript levels of pathogen-inoculated tissues were measured at 1 dpi.(D) Leaves of 4-to 5-week-old rapeseed cultivar Westar plants were infiltrated with 0.25 mM MgCl2 (mock) or Pst DC3000 (OD600 = 0.0001).BnaALD1 and BnaFMO1 transcript levels of pathogen-inoculated tissues were measured at 1 dpi.(E) Lower leaves of four-week-old Arabidopsis Col-0 plants were infiltrated with 0.25 mM MgCl2 (mock) or Pst DC3000 (OD600 = 0.02).Plants in were then incubated at either 23°C or 28°C.ALD1 and FMO1 transcript levels in upper systemic tissues were measured at 2 dpi.Data show the means (± S.D.) and individual points (n=3 to 4) analyzed with two-way ANOVA and Tukey's Multiple Comparisons test.Statistical differences of means are denoted by different letters.Experiments were performed at least two times with reproducible results.

Fig. 3
Fig. 3 Systemic SA biosynthetic gene expression is suppressed at elevated temperature.

Fig. 4
Fig. 4 Exogenous Pip treatment restores Arabidopsis immune priming at warm temperature.Four-week-old Arabidopsis Col-0 plants were treated with mock or 1mM Pip solution by leaf-infiltration (A) or rootdrenching (B).Plants were then incubated at either 23°C or 28°C.Two days after Pip treatment, leaves were infiltrated with Pst DC3000 (OD600 = 0.001), and plants were incubated again at their respective temperatures (23°C or 28°C).Bacterial numbers were quantified at 3 days post-inoculation (dpi).Data show the mean log CFU Pst DC3000/mL (± S.D.) and individual points (n=12 from 3 independent experiments in A; n=24 from 6 independent

,
CBP60g and SARD1 transcript levels are induced systemically after local Pst DC3000 challenge at 23°C, but this systemic induction is lost at 28°C.These suggest that the loss of systemic immunity at elevated temperature could be due to significantly decreased CBP60g and SARD1 gene expression.To show a causative role for CBP60g/SARD1 downregulation with NHP immune pathway suppression, we used plants constitutively expressing CBP60g (35S::CBP60g) or SARD1 (35S::SARD1).As shown in Figures a-c, 35S::CBP60g lines restored NHP biosynthetic gene expression (ALD1, FMO1) and Pip levels at 28°C in contrast to the wild-type Col-0 plants.

Fig. 5
Fig. 5 CBP60g and SARD1 control the temperature-vulnerability of NHP-mediated immunity.Leaves of four-week-old Arabidopsis Col-0 and 35S::CBP60g (A-C) or 35S::SARD1 plants (D-E) were infiltrated with 0.25 mM MgCl2 (mock) or Pst DC3000 (OD600 = 0.001).Plants were then incubated at either 23°C or 28°C.(A) ALD1 and (B) FMO1 transcript levels were measured using RT-qPCR at 1 day post-inoculation (dpi) of pathogeninoculated tissues of Col-0 and 35S::CBP60g plants.(C) Pip levels were measured at 1 dpi in the same tissues.(D) ALD1 and (E) FMO1 transcript levels were measured at 1 day post-inoculation (dpi) of pathogen-inoculated tissues of Col-0 and 35S::SARD1 plants.Data show the means (± S.D.) and individual points (n=4 in A-C; n=3 in D-E), with experiments performed three times with reproducible results.Statistical analyses were conducted using two-way ANOVA with Tukey's Multiple Comparisons test, with statistically significant differences of means denoted by different letters.
pathway in Arabidopsis plants are sensitive to elevated temperatures.Local infection with both virulent or ETI-activating avirulent pathogens triggers SAR at normal temperatures but not at elevated temperatures.We showed that SAR regulation by temperature is caused by temperature-sensitive NHP pathway in local and systemic tissues.SAR-induced NHP biosynthetic gene expression and/or Pip levels are downregulated at elevated temperature.Even though multiple SAR signals have been proposed previously (Fu and Dong, 2013; Návarová, et al., 2012; Hartmann et al., 2018; Wang, et al., 2018; Vlot et al., 2021; Zeier, 2021), we show here that exogenous supplementation with the NHP precursor Pip (locally and systemically) was sufficient to restore SAR at elevated temperature.This demonstrates a causative relationship between temperature-suppressed Pip-NHP pathway and SAR.Because NHP primes systemic SA biosynthesis and immunity (Yildiz et al., 2021; Zeier, 2021), we also observed that systemic SA biosynthetic gene expression is downregulated by elevated temperature.SA induction is also important for SAR establishment since SA induction deficient sid2 and SA-insensitive npr1 mutants also have abolished SAR (Fu and Dong, 2013; Huang et al., 2020; Peng et al., 2021).Temperature-suppressed systemic SA pathway agrees with our previous study showing a negative impact of higher temperatures on local pathogeninduced SA biosynthesis and basal resistance (Huot et al., 2017; Kim et al., 2022).However, we found that Pip supplementation alone can rescue SAR at elevated temperature, suggesting that Pip-NHP signaling is sufficient in inducing SAR.We previously showed that CBP60g and SARD1 controls the temperature-vulnerability of local pathogen-induced SA biosynthesis (Kim et al., 2022).Functionally redundant paralogs CBP60g and SARD1 are master transcription factors that directly target the promoters of numerous immunity-related genes (Wang et al., 2009; Zhang et al., 2010; Wang et al., 2011; Sun et al., 2015, Sun et al., 2018), including those important for SA biosynthesis (like ICS1) and NHP biosynthesis (like ALD1 and FMO1).In this study, we further demonstrate that CBP60g and SARD1 control the temperature-regulation of NHP biosynthesis and systemic immunity.Expression of CBP60g and SARD1 in systemic tissues are downregulated at elevated temperature, which is associated with suppressed NHP pathway.Restoring systemic CBP60g or SARD1 expression using constitutively expressing 35S::CBP60g or 35S::SARD1 plants rescues NHP biosynthetic gene expression and Pip levels under warming conditions.
nitrogen and stored at -80°C before total RNA extraction.Gene expression levels were quantified based on a previously published protocol(Huot et  al., 2017; Kim et al., 2022) with slight modifications.RNA was extracted from flash-frozen plant tissues using the Qiagen Plant RNeasy Mini Kit (Qiagen, Toronto, ON) or TRIzol Reagent (Aidlab Biotech, China) according to the manufacturer's protocol.Resulting cDNA was synthesized using qScript cDNA super mix (Quantabio) or M-MLV reverse transcriptase (RT, Vazyme Biotech, China) based on manufacturers' recommendations.Real-time quantitative polymerase chain reaction (qPCR) was performed using PowerTrack SYBR Green master mix (Life Technologies) or iTaq™ Universal SYBR® Green Supermix (Bio-Rad, USA) with approximately 1.5-10 ng of template cDNA.Equivalently diluted mRNA without the qScript cDNA mix were used as negative controls.The resulting qPCR mixes were run using the Applied Biosystems QuantStudio3 platform (Life Technologies) or CFX96 Real-Time PCR Detection System (Bio-Rad, USA).The individual Ct values were determined for target genes and the internal control gene: PP2AA3 for Arabidopsis; SlACT2 for tomato and BnaGDI1 for rapeseed (Huot et al., 2017; Kim et al., 2022; Shivnauth et al., 2023).Gene expression values were reported as 2 −ΔCt , where ΔCt is Cttarget gene-Ctinternal control gene.qPCR was carried out with three technical replicates for each biological sample.Primers used for qPCR are shown in Supplementary Table