Cold priming on pathogen susceptibility in the Arabidopsis eds1 mutant background requires a functional stromal Ascorbate Peroxidase

ABSTRACT

24 h cold exposure (4°C) is sufficient to reduce pathogen susceptibility in Arabidopsis thaliana against the virulent Pseudomonas syringae pv. tomato (Pst) strain even when the infection occurs five days later. This priming effect is independent of the immune regulator Enhanced Disease Susceptibility 1 (EDS1) and can be observed in the immune-compromised eds1–2 null mutant. In contrast, cold priming-reduced Pst susceptibility is strongly impaired in knock-out lines of the stromal and thylakoid ascorbate peroxidases (sAPX/tAPX) highlighting their relevance for abiotic stress-related increased immune resilience. Here, we extended our analysis by generating an eds1 sapx double mutant. eds1 sapx showed eds1-like resistance and susceptibility phenotypes against Pst strains containing the effectors avrRPM1 and avrRPS4. In comparison to eds1–2, susceptibility against the wildtype Pst strain was constitutively enhanced in eds1 sapx. Although a prior cold priming exposure resulted in reduced Pst titers in eds1–2, it did not alter Pst resistance in eds1 sapx. This demonstrates that the genetic sAPX requirement for cold priming of basal plant immunity applies also to an eds1 null mutant background.

Keywords:

• arabidopsis thaliana
• pathogen
• immunity
• cold
• priming
• ascorbate peroxidases

Short communication

Plants have evolved strategies for improved stress responses based on prior stress experiences. One such strategy that differs from acclimation and adaptation but requires a molecular stress imprint or memory is defined as priming.^1,2 A diverse set of stimuli has been shown for being effective in priming the plant immune system against pathogens.^3,4 This includes abiotic changes and pretreatments with altered environmental conditions as a consequence of activated cross-tolerance. Several short (1.5 h) and repetitive cold (4°C) or heat (38°C) treatments increase the resistance of Arabidopsis thaliana (Arabidopsis) against the hemi-biotrophic, virulent pathogen Pseudomonas syringae pv. tomato DC3000 (Pst).⁵ Improved plant resistance was also observed when the light period the day prior to the Pst infection is extended from 8 h to 16–32 h as a consequence of photoperiod stress.⁶ A 24 h pre-exposure of Arabidopsis to an extended or continuous light phase increases the ability for a strong apoplastic production of reactive oxygen species, boosts pathogen-driven salicylic acid accumulation and signaling, and reduces the capability of Pst for inducing so-called water-soaking leasions.^7,8

Recently, we showed that a 24 h cold exposure (4°C) is sufficient to prime plant immunity for an infection with Pst occurring 5 days later and resulting in reduced bacterial titers in cold pre-treated Arabidopsis plants (accession: Col-0) compared to naïve control plants.⁹ This effect is independent of the plant immune regulator Enhanced Disease Susceptibility 1 (EDS1) and can be observed in the highly susceptible null mutant eds1–2. ⁹ In contrast, cold priming did not lead to reduced bacterial titers when Pst strains delivering the pathogen effector proteins avrRPM1 or avrRPS4 were used.⁹ When detected by the host, these strains initiate strong and robust plant immune responses in the context of effector-triggered immunity (ETI).^10–13 While avrRPS4-triggered ETI is strongly EDS1-dependent, defense activation triggered by the recognition of avrRPM1 is mainly EDS1-independent.¹⁴

EDS1 is part of a small family of nucleocytoplasmic lipase-like proteins.^15–18 Together with its other family members Phytoalexin-Deficient 4 (PAD4) and Senescence-Associated Gene 101 (SAG101), EDS1 forms exclusive heterodimers and functions as a central regulator of ETI, basal immunity, and systemic acquired resistance.^17,19–21 Intracellular immune receptors containing Toll-Interleukin 1 receptor (TIR) domains catalyze ribosylated nucleotide second messengers that specifically bind either to EDS1-PAD4 or to EDS1-SAG101 heterodimers and initiate complex activation.^22–25 Mobilized EDS1 complexes contribute to the activation of pathogen-triggered transcriptional defense reprogramming and cell death, and boost accumulation of immune enhancing metabolites, such as salicylic acid and pipecolic acid derivatives.^20,21,26

As mentioned above, EDS1-dependent signaling is dispensable for cold priming-enhanced Pst resistance. However, functional plastid ascorbate peroxidases (APX) are indispensable.⁹ Two APX isoforms reside in the chloroplasts of Arabidopsis and most tracheophytes: a soluble stromal APX (sAPX) and thylakoid-bound APX (tAPX).^27–29 While tAPX specifically resides in the plastids, sAPX is dual targeted to the chloroplast stroma and the mitochondrial matrix.^30,31 Based on homologies, a further plastid APX-like protein, named TL29, was identified with location to the thylakoid lumen, but does not possess peroxidase activities.^32,33 The interplay of tAPX and sAPX provides two spatial layers for scavenging photosynthesis-related H₂O₂ in the plastid.^27,28 In this context, sAPX and tAPX have mainly redundant functions for photooxidative protection under abiotic stress situations in mature plants.^34–36 However, also distinct roles are reported. Photoprotection in seedlings rather requires sAPX, while tAPX functions in leaves as central regulator of cold priming mediated-repression of core stress-responsive genes during a second cold phase.^35–38 In the priming control, tAPX-mediated suppression of chloroplast NADPH dehydrogenase subunits resulting in less cyclic electron transport provides a source for altered chloroplast-to-nucleus stress signaling.³⁹ While cold priming-reduced Pst susceptibility is significantly weakened in tapx-knockout (KO) lines compared to Col-0, Pst titers are similar in cold-pretreated and control sapx-KO (hereafter: sapx) plants indicating a stronger contribution of sAPX.⁹

To test, whether cold priming-reduced Pst susceptibility requires plastid ascorbate peroxidases also in the background of the null mutant eds1–2, we generated an eds1 sapx line (Figure 1a) using the eds1–2 null mutant and the sapx line (SALK_083737).^9,35,40 We tested EDS1 and sAPX protein abundance in the eds1 sapx line using a plastid APX serum³⁷ and a commercial EDS1 antibody (AS13 2751, Agrisera, Sweden) confirming (in addition to prior genotyping) lack of EDS1 and sAPX in the eds1 sapx line (Figure 1b). Growth and developmental phenotype of 5-week-old plants did not differ between eds1 sapx and parental lines (Figure 1c).

Figure 1. Generation and first analyses of an eds1 sapx double mutant line. (a) The eds1–2 null mutant was crossed with the T-DNA-inserted sapx-knockout line (sapx) to receive an eds1 sapx line. (b) Protein detection of stromal Ascorbate Peroxidase (sAPX) and Enhanced disease Susceptibility1 (EDS1) in leaf extracts of eds1 sapx and corresponding single lines. Ponceau S staining of the rubisco large subunit (rbcL) is shown as loading control. (c) Representative picture of rosettes of 5-week-old plants. Scale bar = 1 cm. (d,e) Pathogen-related immune phenotyping of eds1 sapx line and parental single lines (5-week-old) was verified by leaf syringe infiltration using Pseudomonas syringae pv. tomato DC3000 (Pst) strains (OD₆₀₀ = 0.001 in 10 mM MgCl₂) delivering either the EDS1-independent effector avrRPM1 (d) or the EDS1-dependent effector avrRPS4 (e). Bacteria were re-isolated 3 days post infection (dpi) and colony-forming units per leaf disk area (CFU/cm²) were determined. Bars show mean of log₁₀-transformed CFU/cm² and standard error (n = 18 from 3 independent experiments). Different letters above the bars denote statistically significant differences (Tukey HSD, P < .05).

Next, we analyzed the impact of sAPX for EDS1-dependent and -independent immunity. For this purpose, we infiltrated the eds1 sapx double line either with Pst avrRPM1 or Pst avrRPS4. We could neither detect differences in bacterial titers between the wildtype Col-0 and the sapx nor between eds1–2 and eds1 sapx (Figure 1d, e). The bacterial titers of Pst avrRPM1 determined three days after inoculation were equally ~ 0.5 log₁₀ higher in eds1–2 and eds1 sapx compared to Col-0 (Figure 1d). In contrast, bacterial numbers of Pst avrRPS4 were ~ 3 log₁₀ higher in eds1–2 and eds1 sapx than in Col-0 and sapx (Figure 1e). This demonstrates that under stable conditions sAPX does not affect plant immunity and that eds1 sapx largely resembles the immune phenotype of eds1–2.

Our main aim with this study was to investigate whether sAPX is not only required for cold priming-reduced Pst susceptibility in Col-0 but also in eds1–2. We repeated the cold priming experiments from our recent study⁹ in the exact same way, but compared this time eds1–2 and eds1 sapx (Figure 2a). As shown before,⁹ a 24 h lasting cold exposure reduced the enhanced susceptibility of eds1–2 when the Pst inoculation was performed 5 days later (Figure 2b). Pst titers were lower in cold-primed eds1–2 than in the non-primed control group (Figure 2b). Pst numbers in eds1 sapx were already significantly lower without a pre-cold exposure (Figure 2b), which was similar but not significant between Col-0 and sapx in our earlier study.⁹ The cold priming exposure did not further alter Pst titers in eds1 sapx. This confirms our recent finding, (i) that a prior cold exposure does not alter Pst susceptibility when sAPX is lacking. (ii) It additionally shows, that the requirement of sAPX for cold priming-reduced susceptibility also exists in eds1–2. (iii) It further highlights, that in the absence of sAPX, Pst susceptibility in eds1 sapx is constitutively reduced to the level of cold-primed eds1–2. As outlined above, EDS1 is required for many different pathogen responses, but not the main player in the cold priming signaling cascade. The generated eds1 sapx line provides the opportunity to further analyze the cold priming signaling response on plant immunity in the absence of well-known and strong EDS1-dependent defense responses.

Figure 2. Pst titers in eds1 sapx after prior cold exposure. (a) Experimental design: 4-week-old eds1–2 and eds1 sapx Arabidopsis plants were cold-treated (4°C) for a full light/dark phase (10 h/14 h) of 24 h starting 2.5 h after the onset of light. After 5 d back at normal temperature conditions (day/night: 22°C/20°C), plants were infiltrated with Pst (OD₆₀₀ = 0.001). (b) Bacterial titers of Pst (log₁₀-transformed) in (+) cold-primed and (-) control eds1–2 and eds1 sapx plants were determined 3 days post infection (dpi). Bars represent means of log₁₀-transformed colony-forming units (CFU/cm²) and standard errors calculated from 3 independent experiments (n = 15–18). Different letters above the bars denote statistically significant differences (Tukey HSD, P < .05). Numbers between two bars show the effect size between two means according to Cohen’s d.

Acknowledgments

We thank Petra Redekop (FU Berlin) for comments on the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the German Research Foundation (CRC973/C4) and the FU Berlin.