2.1 Heat Shock and Thermopriming in Adult Arabidopsis Plants

The first step towards establishing the ATT protocol was to standardize a temperature treatment condition that would be just lethal for unprimed WT plants. We observed that plants that were treated at 45°C for up to 1 h did not show much symptoms in 2 days (Figure 1A,B). Plants that were treated at 50°C for 1 h showed severe stress symptoms within 2 days, and many of them died, but not all (Figure 1B). In contrast, when plants were treated at 50°C for 2 h, all the plants completely collapsed and did not recover (Figure 1B). The plants that were primed for ATT, survived this treatment (discussed below). To have a comparative evaluation of the effect of heat stress, we performed pulse-amplitude-modulation (PAM) chlorophyll fluorometry on the plants on the second day of heat shock. Fluorescence increases due to inefficient photochemistry in stressed plants, and consequently, the Fv/Fm value reduces. In agreement with the visible symptoms, we observed enhanced fluorescence when plants were treated at 45°C or 50°C for 1 h (Figure 1C) with reduced Fv/Fm values (Figure 1D). Fluorescence was not much observed when plants were treated at 50°C for 2 h due to lethality. Thus, a treatment of 50°C for 2 h was used as a heat shock (HS) challenging condition for studying ATT response in WT Arabidopsis plants.

For priming standardization, we exposed the plants at 35°C, 37°C, 40°C, 42°C, and 45°C for 2 h, and after a gap of 2 days, plants were challenged at 50°C for 2 h (Figure 1E,F; Supporting Information S1: Figure 1). We also used unprimed and unstressed plants as controls. We observed that the primary treatment of 35°C was ineffective in giving the protection, and 37°C had a modest ATT response (Figure 1F). In contrast, plants that were treated between 40°C and 45°C for 2 h provided ATT-mediated protection (Figure 1F). PAM chlorophyll fluorometry studies also supported a high level of protection by pretreatment at 40°C–45°C temperature (Figure 1G,H).

2.2 ATT Activation, Retention Time and Booster Effect in Arabidopsis

To study the minimum time required for ATT activation and length of retention, we separated primary induction and challenge by different time gaps and examined the heat tolerance (Figure 2A). We observed that ATT-mediated protection lasts up to 2 days and starts fading away from 3 days onwards (Figure 2B). On the other side, ATT activation was observed when the time gap between primary and secondary was 2 h or more; however, very little protection was observed when only 1 h gap was kept (Figure 2C). Therefore, ATT activation requires about 2 h and lasts up to 2 days in Arabidopsis, under our growth conditions.

To investigate the effect of multiple priming on ATT retention, we primed plants for one to three times with a gap of 2 days and gave HS after 2 to 7 days of the last priming. As shown above, when primary treatment was given once, the ATT lasted for 2 days, however, with one booster, the ATT lasted for 5 days and with three boosters, the ATT lasted for 7 days (Supporting Information S1: Figure 2). Thus, the ATT retention time was incremental with the number of pretreatments applied to plants.

2.3 Pathogen Inoculation Provides Heat Tolerance

The primary aim of this work was to study the extent of interactions between SAR and ATT inductions in plants. We investigated the level of cross-protection between SAR and ATT. We performed primary inoculation with Pseudomonas syringae pv tomato DC3000 (Pst) carrying effector AvrRpt2 (Pst-AvrRpt2) suspended in 10 mM MgCl₂ in three leaves of 4-week-old Arabidopsis plants to induce SAR. Two days later, plants were challenged at 50°C for 2 h. The control set received only 10 mM MgCl₂. We found that compared to the mock-treated plants, primary pathogen-inoculated plants showed a significantly higher level of tolerance to HS (Supporting Information S1: Figure 3). To substantiate our observation further, we induced SAR by several virulent and avirulent pathogens, such as Pst, Psm, Pst-AvrB, and Pst-AvrRpt2. Two days after primary inoculation, plants were subjected to HS at 50°C for 2 h (Figure 3A). We observed that localized inoculation of all these pathogens provided significant protection against HS (Figure 3B). Moreover, as normally observed, a higher level of immunity activation by avirulent pathogens than virulent pathogens, we observed a higher level of protection on the plants that were primed with avirulent pathogens than virulent pathogens (Figure 3B). PAM chlorophyll fluorometry on the plants on the second day of heat shock also supported a higher level of protection by avirulent pathogens than virulent pathogens (Figure 3C,D)

To investigate whether thermopriming can provide enhanced resistance, we pretreated the plants at 40°C or 45°C for 2 h, and 2 or 3 days later challenged with virulent pathogen Psm. In contrast to our expectation, prior heat treatment showed no enhanced resistance compared to the mock-treated plants, and the bacterial load was higher in the plants that had been previously subjected to elevated temperatures (Supporting Information S1: Figure 4).

2.4 Pathogen-Mediated Heat Tolerance Lasts Longer Than Thermopriming-Induced ATT

The results described above showed that a primary pathogen inoculation provides enhanced heat tolerance in plants. Whereas the memory of heat induction lasts 2 to 3 days (Figure 2; Supporting Information S1:Figure 2; Charng et al. 2007), the SAR memory lasts for several days (Singh et al. 2023). To examine the comparative longevity of HS tolerance, we gave primary heat treatment at 40°C for 2 h in one batch of plants and simultaneously inoculated avirulent pathogen Pst-AvrRpt2 in a few leaves of another batch of plants. These plants were challenged with 50°C after the gap of 2 to 4 days. As observed before, heat-prime-induced tolerance lasted only for 2 days (Figure 4A). Comparatively, pathogen-primed plants tolerated HS with a longer gap between priming and challenge (Figure 4A). PAM chlorophyll photometry images (Figure 4B) and Fv/Fm values (Figure 4C) also strongly supported the longer retention of heat tolerance by pathogen priming than heat priming. Further, we observed that Pst-AvRpt2-mediated heat shock protection gradually reduces over days but retains significantly till 10 days postinoculation with pathogen (Supporting Information S1: Figure 5). A similar enhanced protection was observed after primary treatment with the Pst-AvrB pathogen (Supporting Information S1: Figure 6).

2.5 ATT-Responsive Genes Are Upregulated Upon Pathogen-Inoculation

To investigate whether pathogen priming-mediated HS protection is associated with the expression of HSP and HSFs, we monitored their mRNA accumulation by quantitative real-time PCR (qPCR). We inoculated a few leaves of soil-grown 4-week-old Arabidopsis plants with Pst-AvrRpt2 or mock and monitored mRNA accumulation of key ATT regulatory genes in pathogen-free distal tissues. We observed that expression of HSFA2, HSFA3, HSP101, and HEAT-STRESS-ASSOCIATED 32 (HSA32) was significantly higher in systemic leaves of pathogen-treated plants compared to the mock-treated plants (Figure 5A–E). The expression of PR1 was used as control of the experiment, which was also upregulated in systemic leaves, as expected (Figure 5E). ATT regulatory genes also showed enhanced expression in pathogen-inoculated tissues (Supporting Information S1: Figure 7). In agreement with the enhanced heat protection by avirulent pathogens (Figure 3), we observed higher levels of HSFA2, HSP101 and HSA32 expression in systemic tissues upon avirulent pathogen Pst-AvrB inoculation than the virulent pathogen Pst (Supporting Information S1: Figure 8). Altogether, the results supported our observation of enhanced HS protection by pathogen priming.

2.6 Pretreatment With Heat Inhibits SAR Activation by Pathogen

We observed that primary heat treatment does not provide systemic protection against pathogens (Supporting Information S1: Figure 4). To further investigate whether heat treatment affects pathogen-induced SAR, we provided a combination of heat and pathogen for SAR induction. Plants were either heat-treated at 40°C for 2 h or left untreated, and 2 h later, a few leaves were infiltrated with Pst-AvrRpt2 for SAR induction (Figure 6A). We observed SAR-mediated protection in plants that were not heat-treated (Figure 6B). In contrast, a pretreatment at 40°C completely abolished SAR induction in plants (Figure 6B). The disease symptoms developed in these plants also supported the bacterial loads observed (Figure 6B). SAR induction is associated with the accumulation of SA in pathogen-free systemic tissues (Patil and Nandi 2022; Singh et al. 2023). We found that when plants were heat-primed before Pst-AvrRpt2 infiltration, showed reduced accumulation of total and free SA (Figure 6C,D), in pathogen-free systemic tissues, compared to untreated plants. Results demonstrated that heat treatment negatively impacts SAR development. To investigate whether there is any difference in the sequence of heat and pathogen priming on SAR development, we applied heat at 40°C for 2 h before or after primary pathogen inoculation and followed SAR development. We observed that irrespective of sequence, heat priming blocks SAR activation (Supporting Information S1: Figures 9 and 10).

2.7 Heat Treatment Blocks Pathogen-Induced SAR Mobile Signal Generation

SAR induction requires the generation and transport of mobile signals from the infected tissues to distal parts. The mobile signals can be collected as petiole exudates (Pex) from pathogen-inoculated leaves to induce SAR in other plants (Chaturvedi et al. 2008a; Singh et al. 2023; Singh et al. 2013). Pex collected from previously inoculated leaves functions like SAR inducing chemical. To investigate whether heat priming affects SAR mobile signal production, we treated plants at 40°C or left untreated for 2 h and 2 h later infiltrated with Pst-AvrRpt2 and collected petiole Pex. As expected, when Pex from Pst-AvrRpt2-inoculated control plants was applied on naïve WT plants, it provided SAR compared to mock-inoculated plants (Figure 7). However, the Pex from heat-treated plants did not provide SAR (Figure 7). The results suggest that pretreatment of heat impaired the initial steps of SAR activation, which includes generation and transport of SAR mobile signals.

2.8 Heat Treatment Reduces Local Resistance, Cell Death and ROS Production

The results described above showed that a prior heat treatment fails to activate SAR. To ensure that the loss of SAR was not associated with the reduction of bacterial growth of primary inoculation, we monitored the growth of virulent and avirulent pathogens after heat priming. We observed that heat-treated plants supported higher bacterial loads than control plants for virulent (Figure 8A), as well as avirulent pathogens (Figure 8B). Inoculation with avirulent pathogens activates stronger immunity with enhanced accumulation of reactive oxygen species and rapid cell death in the form of hypersensitive response (HR) (Spoel and Dong 2012) Ion leakage from dying and dead cells increases electrical conductivity when floated on distilled water (Roy and Nandi 2017). We observed that heat-priming significantly reduces visible hyper-sensitive (HR) symptoms (Supporting Information S1: Figure 11), cell death (Figure 8C), ion leakage (Figure 8D), and H₂O₂ production (Figure 8E), all of which are in agreement with reduced immunity observed in these plants. Heat treatment also reduced resistance against other virulent pathogen Pst and avirulent pathogen Pst-AvrB (Supporting Information S1: Figure 12). Altogether, our data convincingly established that heat treatment before pathogen inoculation reduces resistance and impairs the ability of SAR induction in the distal tissues.

2.9 The ATT-Defective Mutants Such as hsfa2, hsfa3, and hsp101 Are Defective in SAR Activation

We observed enhanced expression of HS memory-associated genes such as HSFA2, HSFA3, and HSP101 in the systemic leaves of pathogen-primed plants (Figure 5). To investigate whether induction of these HS memory-associated genes has any impact on SAR, we studied SAR development in their mutants. We obtained their T-DNA insertion mutations and confirmed the presence of T-DNA (Supporting Information S1: Figures 13A,B, 14A,B). As expected, soil-grown adult mutant plants did not provide ATT (Supporting Information S1: Figure 13C, 14C). The rsi1 mutant plants, which are specifically defective in SAR development (Singh et al. 2023; Singh et al. 2013) were used as control. As expected, we observed SAR-mediated protection in WT plants but not in rsi1 plants (Figure 9A,B). Intriguingly, we observed that the hsfa2, hsfa3 and hsp101 mutants were also defective in SAR development (Figure 9A,B). The lack of SAR in these mutants is also reflected in the development of symptoms. Disease symptoms in SAR-induced WT plants were much lower than in mock-induced plants. However, no obvious differences in symptoms were observed in hsfa2, hsfa3, hsp101, and rsi1 mutants (Figure 9A,B). The plants in these experiments were not challenged with higher temperatures and were continuously maintained at 22°C. Therefore, our results demonstrated that the ATT-requiring genes like HSFA2, HSP101, and HSFA3 are essential components for SAR induction.

4.1 Plant Materials and Growth Conditions

Wild type and all mutants used in this paper are in Col-0 ecotype background. T-DNA insertion lines for hsfA2-1 (SALK_008978) (Alonso et al. 2003), hsp101-1 (SALK_066374) and hsfA3-1 (SALK_011107C) were obtained from Arabidopsis stock center. Plants were grown on soilrite vermiculite mixture at 22°C with 12h-12h light-dark cycle at 50%–70% humidity as described earlier (Patil and Nandi 2022; Singh et al. 2023; Singh et al. 2013).

4.2 Pathogen Growth and Inoculation

Pathogens used in this study are Pseudomonas syringae pv tomato DC3000 (Pst), P. syringae pv maculicola ES4326 (Psm), Pst carrying AvrRpt2 effector (Pst-AvrRpt2) and Pst carrying AvrB (PstAvrB). All bacterial pathogens were grown in Kings medium at 28°C. Over-night grown cultures were resuspended in 10 mM MgCl₂ with desired dilution just before inoculation. Culture suspension was pressure infiltrated through the abaxial sides of the leaves. SAR experiments were carried out as described earlier (Singh et al. 2023; Singh et al. 2013). Avirulent pathogens such as Pst-AvrRpt2 or Pst-AvrB were used as primary inducers. These pathogens were suspended in 10 mM MgCl₂. The mock-treated plants received only 10 mM MgCl₂. Typically, after two to 3 days, both mock or avirulent pathogen-treated plants were challenged with a virulent pathogen such as Pst or Psm at 5 × 10⁵ cells/ml. SAR by petiole exudates was performed as previously described (Chaturvedi et al. 2008b; Maldonado et al. 2002).

4.3 Heat Treatment on Plants

Plants were grown normally for 4 weeks at 22°C. 1 h before heat treatment, plants were irrigated with water by soil drenching. The pots, along with the tray, were placed in a hot air oven with air circulation at the desired temperature, as mentioned for each experiment. After the treatment, plants were placed back in the growth room. The trays were loosely covered with a plastic dome for 1 day after the heat treatment and photographed at regular intervals. For each treatment or genotype, we took at least two pots with three plants each. All ATT experiments were repeated at least three times with similar results.

4.4 Salicylic Acid Estimation

Salicylic acid (SA) accumulation was estimated by using the Acinetobacter sp. ADPWH_lux-mediated bioluminescence assay as described earlier (Huang et al. 2005; Patil and Nandi 2022). In brief, leaf samples of 50 mg were collected and frozen in liquid N₂ and crushed to homogenized in 0.1 M acetate buffer (pH 5.6, 2.5 µL/mg of tissue), centrifuged at 12 000 rpm for 15 min, and supernatants were collected for the assay. For total SA, 4 units of β-glucosidase (Sigma, USA Cat # G0395) was mixed with 50 µL of the supernatant and incubated at 37°C for 90 min, and another 50 µL aliquot was kept on ice to measure the free SA content. Samples were centrifuged at 12 000 rpm for 15 min. From both the sets 20 µL of supernatant was mixed with 60 µl LB medium and 50 µL of freshly grown Acinetobacter culture (0.4 Od at 600 nm). After 60 min of incubation at 37°C, luminescence was measured in a luminometer (FLUOStar Omega multiplate reader, BMG Labtech).

4.5 Total RNA Isolation and Gene Expression Analysis

Total RNA isolation and cDNA preparation was done using the iScript cDNA Synthesis Kit (#1,70,8891, Biorad, USA). The relative abundance of transcripts was quantified as described previously (Patil and Nandi 2022; Singh et al. 2013). At least three biological replicates were collected for each sample. qPCR was performed using two technical duplicates of each biological sample. The relative abundance of transcripts was normalized using Arabidopsis ACTIN2 (AT3G18780) gene. During the analysis of qPCR data, the average of both technical duplicates was taken as the reading of the biological replicate. Primers used in this study are listed in Supporting Information S1: Table 1 in supplementary DATA.

4.6 Ion Leakage, Cell Death, DAB Staining and Chlorophyll Fluorescence Studies

Four weeks grown WT plants were treated with 40°C for 2 h and transferred to 22°C for 2 h for recovery. Later, both sets of plants were pressure infiltrated with Pst-AvrRtpt2 (1 × 10⁷ CFU/mL) suspended in 10 mM MgCl₂. For the mock, only 10 mM MgCl₂ infiltrated in both treated and nontreated plants. All plants are covered with a dome for 7 h in the dark and kept in the growth room. Hypersensitive response-associated ion-leakage, cell death and DAB staining experiments were carried out as previously described (Giri et al. 2017; Roy et al. 2018; Roy and Nandi 2017). Chlorophyll fluorescence fluorometry was carried out by a Pulse-amplitude modulated (PAM) fluorometry (Heinz Walz, model-Image-CM). For PAM measurement, plants were first kept in the dark conditions for 30 min at 22°C and maximum quantum yield also referred as F_v/F_m ratio was measured in non-stresses and stressed plants via exposing these plants under the saturating pulse of 1 s (ca. 2500 μmol m⁻² s⁻¹) (Aldea et al. 2007; Tsai et al. 2019).