CPK28 is a modulator of purinergic signaling in plant growth and defense

Abstract

Extracellular ATP (eATP) is a key signaling molecule that plays a pivotal role in plant growth and defense responses. The receptor P2K1 is responsible for perceiving eATP and initiating its signaling cascade. However, the signal transduction mechanisms downstream of P2K1 activation remain incompletely understood. We conducted a comprehensive analysis of the P2K1 interactome using co-immunoprecipitation-coupled tandem mass spectrometry, leading to the identification of 121 candidate proteins interacting with P2K1. In silico analysis narrowed down the candidates to 47 proteins, including Ca²⁺-binding proteins, ion transport-related proteins, and receptor kinases. To investigate their involvement in eATP signaling, we employed a screening strategy based on changes in gene expression in response to eATP in mutants of the identified interactors. This screening revealed several Ca²⁺-dependent protein kinases (CPKs) that significantly affected the expression of eATP-responsive genes, suggesting their potential roles in eATP signaling. Notably, CPK28 and CPK6 showed physical interactions with P2K1 both in yeast and plant systems. Calcium influx and gene expression studies demonstrated that CPK28 perturbed eATP-induced Ca²⁺ mobilization and some early transcriptional responses. Overexpression of CPK28 resulted in an antagonistic physiological response to P2K1-mediated eATP signaling during both plant growth and defense responses to the necrotrophic pathogen Botrytis cinerea. Our findings highlight CPK28, among other CPKs, as a modulator of P2K1-mediated eATP signaling, providing valuable insights into the coordination of eATP signaling in plant growth and immunity.

INTRODUCTION

Apart from a metabolic energy source, ATP serves as a signaling molecule when released from cells. Extracellular ATP (eATP) has been primarily described as a danger signal, or damage-associated molecular pattern (DAMP) in plant defense (Jeter et al., 2004; Kim et al., 2006; Song et al., 2006; Torres et al., 2006; Choi et al., 2014; Tanaka and Heil, 2021). Choi and Tanaka et al. identified the first eATP receptor in plants, known as P2K1 (Choi et al., 2014), which perceives eATP and triggers a defense response—enhancing resistance against various plant pathogens (Gouget et al., 2006; Bouwmeester et al., 2011; Bouwmeester et al., 2014; Balagué et al., 2017; Tripathi et al., 2018; Kumar et al., 2020; Jewell et al., 2022). Current evidence suggests that important secondary messengers, Ca²⁺and reactive oxygen species (ROS), mediate the signaling events following eATP perception by P2K1 (Jeter et al., 2004; Demidchik et al., 2009; Tanaka et al., 2010; Choi et al., 2014; Gilroy et al., 2016; Tripathi et al., 2018; Marcec et al., 2019). Additionally, eATP has been found to play a role in regulating plant growth and development, although the involvement of P2K1 in these processes has not been fully understood (Tang et al., 2003; Tonón et al., 2010; Zhu et al., 2020; Xu et al., 2023). Recent research has identified eATP-responsive genes through transcriptomics (Jewell et al., 2019), leading to the development of a reliable tool for monitoring eATP signaling responses (Sowders and Tanaka, 2023). Despite advancements in understanding the cellular responses downstream of P2K1-activation (i.e., Ca²⁺, ROS, transcriptional reprogramming, defense response), the signal transduction mechanisms between P2K1 and the activation of these downstream events have been slow to emerge.

Previous studies have investigated the protein-protein interactions of P2K1, to gain a better understanding of the biochemical mechanisms of P2K1-mediated eATP signaling. For example, it has been demonstrated that P2K1 directly phosphorylates the NADPH oxidase, RBOHD, upon eATP addition, leading to the regulation of ROS production, which in turn affects stomatal immunity against the bacterial pathogen Pseudomonas syringae in guard cells (Chen et al., 2017). Another study showed that P2K1 phosphorylates PAT5/9 in response to eATP, which in turn S-acylate P2K1. The S-acylation of P2K1 dampens its activity, negatively impacting bacterial immunity to P. syringae (Chen et al., 2021). Moreover, P2K1 has been found to phosphorylate mevalonate kinase (MVK), which is required for eATP-responsive Ca²⁺ influx and resistance to P. syringae (Cho et al., 2022). Recently, the Raf-like protein kinase, INTEGRIN-LINKED KINASE 5 (ILK5), also known as BHP1 (Hayashi et al., 2017), was identified as an additional phosphorylation target of P2K1 (Kim et al., 2023). Phosphorylation of ILK5 by P2K1 initiates a MAPK cascade, leading to the phosphorylation of the MAP kinase kinase MKK5, which eventually phosphorylates the MAP kinase MPK3/MPK6 that contributes to eATP-mediated bacterial immunity (Kim et al., 2023). It has also been shown that eATP signaling through P2K1 regulates the levels of jasmonic acid (JA) signaling, a key hormone involved in plant defense responses (Tripathi et al., 2018; Jewell et al., 2019).

It is worth noting that P2K1 is not the only purinergic receptor involved in eATP responses in plants. Another purinergic receptor, P2K2, has been identified as a second receptor for eATP in Arabidopsis (Pham et al., 2020). Interestingly, P2K1 can phosphorylate P2K2 in vitro, and loss of P2K2 leads to a reduction in eATP-mediated defense responses. However, it remains unclear whether P2K2 acts as a coreceptor with P2K1 or independently participates in the perception and signal transduction of eATP. Moreover, P2K2 or another yet-identified signaling component may be the missing link connecting P2K1 to eATP-regulated growth responses.

In this study, we aimed to gain further insights into the interactome of P2K1 and identify potential interacting proteins involved in eATP signaling. To this end, we performed co-immunoprecipitation-coupled tandem mass spectrometry (Co-IP/MS) analysis, followed by a series of screening steps based on functional annotations and analysis of changes in eATP-responsive gene expression in mutants of the candidate interactors. Finally, we identified several Ca²⁺-dependent protein kinases (CPKs) that potentially play significant roles in eATP signaling. Notably, CPK28 and CPK6 exhibited physical interactions with P2K1 among the candidate interactors. Calcium influx and gene expression studies revealed CPK28 exert a mostly negative impact on eATP signaling. Subsequent plant growth and pathogen assays provided additional support for CPK28 as a modulator of the eATP response. Together, our study suggests that CPK28 acts as a putative negative modifier fine-tuning eATP responses in both plant growth and defense.

MATERIALS AND METHODS

Plant materials

In our experiments, we utilized wild-type Arabidopsis plants of the Columbia-0 ecotype (Col-0), as well as dorn1-3 mutant plants (Choi et al., 2014), expressing aequorin (AEQ) (Knight et al., 1991). Mutant germplasms of Arabidopsis thaliana were obtained from the Arabidopsis Biological Resource Center (https://abrc.osu.edu). The stock numbers were listed in Table S1. The oxP2K1 plants (35S::P2K1t-GFP/35S::AEQ in the dorn1-3 background) have been previously described (Sowders et al., 2023). The “oxCPK28 #1” plants (35S::CPK28-YFP/35S::AEQ in the Col-0 background) (Monaghan et al., 2015) were generously shared with us by Dr. Jacqueline Monaghan at Queen’s University in Kingston, ON, Canada. The GFP-LTI6B plants were obtained from the ABRC (Stock #: CS84726). Transgenic lines generated in this study (oxCPK28 #2, #3, and #4; and co-overexpression of P2K1 and CPK28 lines, co-ox #1, #2, and #3) were generated by transferring UB10::CPK28-GFP into pTAT3::GUS in the Col-0 background (Sowders and Tanaka, 2023) or oxP2K1 Arabidopsis plants, respectively, using the floral dip transformation method (Clough and Bent, 1998). The transgenic lines are also described in Table S1.

All seeds were subjected to surface sterilization before being planted on the growth medium. The medium consisted of ½ strength Murashige & Skoog (MS) medium (Murashige and Skoog, 1962) supplemented with vitamins (Caisson, MSP09), 1% sucrose (w/v), 1% Daishin agar (w/v) (RPI), and buffered with 0.05% MES-KOH (w/v) at a pH of 5.7, set in a square petri dish. After surface sterilization, the seeds underwent a cold stratification treatment in darkness at 4°C for 3 days, after which they were grown vertically in a growth chamber. The growth chamber was maintained at a temperature of 22°C, with a 12-h photoperiod and a light intensity of 100 μmol photons m⁻² s⁻¹. For the preparation of the ATP stock solution (100 mM), ATP (Sigma-Aldrich) was dissolved in 50 mM MES buffer. The solution’s pH was adjusted to 5.7 using KOH, and it was subsequently filter sterilized. The solution was stored in aliquots at −20 °C indefinitely.

Plant treatments and paraformaldehyde cross-linking for co-immunoprecipitation

A subset of 7-day-old seedlings grown on solid MS media was aseptically transferred to 6-well plates, with each well containing 2 mL of liquid MS media (5 seedlings/well). These plates were then incubated for an additional 7 days. Subsequently, seedlings (approximately 10 g per treatment) were transferred to 50 mL conical centrifuge tubes. The tubes were filled with water containing 1 mM ATP or plain water to treat the seedlings for 3 or 20 minutes. After this treatment, the samples were chemically cross-linked with 0.5% paraformaldehyde in 1X PBS. Vacuum infiltration was performed for 5 min, followed by a 10-min incubation at room temperature. To stop the cross-linking reaction, 1.25 M glycine was added to a final concentration of 125 mM glycine, and the seedlings were incubated for an additional 10 min at room temperature. Next, the seedlings were rinsed once with water, patted dry on paper towels, snap-frozen in liquid nitrogen, and stored at −80°C until use.

Co-immunoprecipitation-coupled mass spectrometry (Co-IP/MS)

Seedlings were ground to a fine powder in a liquid nitrogen-cooled mortar and pestle with sand to aid grinding. After grinding, the tissue was quickly suspended and thoroughly vortexed after the addition of ice-cold 20 mL buffer H (50 mM HEPES, pH 7.5, 250 mM sucrose, 15 mM EDTA, 5% (w/v) glycerol) supplemented with 1 mM DTT, 1X HALT protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific), 50 μM MG132, and 10 nM calyculin (Heese et al., 2007). These extracts were centrifuged at 10,000 x g for 10 min at 4°C and the supernatants were filtered through a 70 μm cell strainer into new 50 mL centrifuge tubes on ice. The pellet was resuspended in 5 mL buffer H, followed by centrifugation, and filtered to combine with the 1st fraction. Microsomal pellets were obtained by centrifugation at 100,000 x g for 30 min at 4°C. The pellets were thoroughly resuspended in 0.75 mL buffer H supplemented with 0.5% IGEPAL CA-630 from a freshly prepared 10% stock solution. After resuspending the microsomal pellets, the extracts were diluted to a final volume of 1.5 mL with buffer H without IGEPAL.

For immunoprecipitation, 0.1 mL GFP-Trap magnetic agarose beads (ChromoTek) that had been pre-washed thrice with ice-cold buffer H were used. The beads were mixed with the extract and rotated end-over-end at 4°C for 30 min. Following incubation, the beads were washed thrice with buffer H, and the proteins were eluted by heating beads in 50 μL of 2x Laemmli buffer (Laemmli, 1970) at 95°C for 15 min. The magnetic beads were separated from the extracts, and 50 μL of the eluate was loaded onto the SDS-PAGE resolving gel, electrophoresed to a distance of 0.5 cm, excised, washed thrice for 5 min in Milli-Q water, and then shipped in 1% acetic acid to the Southern Alberta Mass Spectrometry facility at the University of Calgary for trypsin digestion and subsequent mass spectrometry analysis.

Mass spectrometry data analysis

The mass spectrometry data were returned to us as a Scaffold (.scf) file and analyzed using Scaffold software version 4 (Searle, 2010). Protein matches were accepted at 99% protein identification threshold, 95% peptide identification threshold, and a minimum of 5 identified peptides. Protein abundance difference between P2K1-GFP and GFP-LTI6B samples were accepted if the protein was identified in P2K1 but not LTI immunoprecipitates, or if the protein was more abundant in P2K1 samples (Fisher’s exact test Benjamini-Hochberg corrected P<0.05). In an effort towards the MIAPE compliance of our interactomics data, we have supplied the .scf file (Supplementary Dataset 1) as received from the Southern Alberta Mass Spectrometry facility.

Mating-based split-ubiquitin yeast two-hybrid system

Candidate interactors were amplified with Gateway^™ recombination sites and inserted into the pDONR-ZEO entry vector. Subsequently, they were cloned into both the mating-based split ubiquitin system (mbSUS) destination vectors pMETYC (Cub-ProtA-LexA-VP16 (Cub-PLV); bait) and pXN22 (Nub-HA; prey), which have been previously described (Grefen et al., 2007). Transformation of yeast strains THY.AP4 (MATa; ade2/hiS5/leu2/trp1/ura3, lexA::ADE2/lexA::HIS5/lexA::lacZ) and THY.AP5 (MATα; ade2/hiS5/leu2/trp1) with pMETYC and pXN22 constructs, respectively, was performed using polyethylene glycol-mediated transformation, following the protocol described previously (Obrdlik et al., 2004). The transformed bait and prey strains were selected on Clontech (Mountain View, California, USA) dropout media lacking leucine (“-L”) or tryptophan (“-T”), respectively. The bait and prey strains were then mated and selected on dropout media lacking leucine and tryptophan (“-LT”), as previously described (Horaruang and Zhang, 2017). To assess the interactions between bait and prey, the mated yeast was grown in -LT liquid culture overnight. The culture was pelleted, resuspended in sterile water to a final OD₆₀₀ of 0.5, and then plated on selective media in a series of six 5-fold dilutions. Interactions were examined on three increasingly selective dropout media (i) lacking leucine, tryptophan, and uracil (“-LTU”), (ii) total dropout media (lacking leucine, tryptophan, uracil, adenine, and histidine; “-LTUAH”) supplemented with 0.5 μM methionine, and (iii) -LTUAH with 5 μM methionine. The use of increasing concentrations of methionine in the selection media was aimed at reducing bait expression, as the pMETYC bait construct is controlled by the methionine suppressible MET25 promoter. This additional stringency in bait expression served to investigate the dynamics of bait-prey interactions in the mbSUS assay. The KAT1-Cub and KAT1-Nub homodimer was used as the positive control condition (Horaruang and Zhang, 2017). CPK5 was chosen as a negative control since it was not found in the Co-IP/MS data, and CERK1 was used as another negative control for surveying P2K1 interactions, as previously described (Chen et al., 2017).

Co-immunoprecipitation in Nicotiana benthamiana

Full-length genes of P2K1 and interactor candidates were cloned into the pGWB14 (Nakagawa et al., 2007) or pUBC vector (Blatt and Grefen, 2014), which contained C-terminal fusions of 3xHA or GFP, respectively. These constructs were then transformed into Agrobacterium tumefaciens GV3101 carrying pMP90 (Koncz and Schell, 1986). Agroinfiltration of pre-reproductive leaves of N. benthamiana was performed using combinations of pGWB14 and pUBC constructs, along with the HC-Pro host suppression cassette, as previously described (Zhao et al., 2005; Wydro et al., 2006). Three days after infiltration, leaves from five to eight N. benthamiana plants were pooled and harvested in liquid N₂ and ground to a fine powder using mortar and pestle. The tissue was then homogenized in 15 to 20 mL of freshly prepared ice-cold “buffer H” (described above), supplemented with 0.5% (w/v) polyvinylpyrrolidone average molecular weight 40,000 (PVP-40), 1 mM sodium molybdate, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μM calyculin, and 1X HALT^™ protease/phosphatase inhibitor cocktail. The homogenate was transferred to a 50 mL tube and centrifuged at 8,000 x g for 20 min at 4 °C. The supernatant was passed through a sterile 40 μm filter into a fresh 50 mL tube on ice, and the pellet was discarded. To enrich the microsomal fraction, 30 mM MgCl₂ was added to the filtered supernatant and gently mixed to facilitate pelleting of the bilayer at lower centrifugal forces, following a modified protocol previously described (Diesperger et al., 1974). The supernatant was then centrifuged again at 12,000 x g for 15 min at 4 °C to induce microsomal pelleting. The resulting supernatant was decanted, and the microsomal pellet was gently resuspended in 1 mL of resuspension buffer (buffer H with 0.5% IGEPAL) on ice. The resuspended microsomal fraction was left on a rocking shaker for 10 min. Twenty microliter of Pierce^™ anti-HA magnetic beads (Thermo Fisher) were washed twice with 300 μL of buffer H. An aliquot of the resuspended microsomal pellet was saved for electrophoresis, and the remaining microsomal fraction was loaded into a 1.5 mL microcentrifuge tube containing 20 μL of prewashed anti-HA magnetic beads. The tube was then incubated on a rotisserie shaker at 4 °C for 45 min. The supernatant was removed, and the beads were rinsed and resuspended thrice with 500 μL of resuspension buffer. Immunoprecipitated proteins were eluted with 150 μL of 1X Laemmli loading buffer (Laemmli, 1970) and boiled at 90 °C for 5 min before electrophoresis.

Protein samples were separated on 10% acrylamide SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membrane using Towbin buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol, 0.1% (w/v) sodium dodecyl sulfate (SDS), pH 8.3) at 4 °C for 70 min at 90 V. After transfer, the membrane was vigorously washed with sterile water thrice and then blocked in tris-buffered saline plus 0.1% v/v TWEEN^® 20 (TBST) with 4% bovine serum albumin (BSA) overnight at 4 °C. The following day, the membranes were washed twice with TBST for 5 min each and then incubated with the appropriate primary monoclonal antibody, mouse αGFP (1:500; Santa Cruz Biotech, Santa Cruz, CA, USA) or mouse αHA (1:1000; Sigma Aldrich, St Louis, MO, USA), in TBST plus 2% BSA for 2 h at room temperature with gentle agitation. After incubation with the primary antibody, the membrane was thoroughly washed with TBST thrice under vigorous agitation for 15 min each. Finally, the membrane was incubated with the rabbit αMouse horseradish peroxidase (HRP)-conjugated secondary antibody (1:1500; Jackson ImmunoResearch, West Grove, PA, USA) at room temperature in TBST plus 2% BSA for 30 min. The membrane was then washed thrice with TBST for 15 min each and briefly incubated with the HRP substrate (SuperSignal^™ west pico PLUS chemiluminescent substrate; Thermo Fisher) according to the manufacturer’s direction. The membrane was imaged using a CCD camera (G: BOX; SYNGENE, Cambridge, UK) in darkness.

Cytosolic calcium measurement

The above-described germplasm expressing aequorin was initially germinated on ½ MS media. Three days after germination, the seedlings were carefully transferred to a 96-well plate. Each well contained 50 μL of coelenterazine buffer (2mM 2-ethane sulfonic acid (MES) pH 5.7, 10 mM CaCl₂, 10 μM coelenterazine). The plate was then placed in darkness and left overnight. The following day, the plates were loaded into the GloMax^® Navigator plate reader (Promega, Madison, WI, USA). Subsequently, the seedlings in the plates were treated with 50 μM ATP (MES-buffered to pH 5.7) using the onboard injector. Luminescence was recorded for 2 min following treatment, and the calcium influx was determined using the method described previously (Tanaka et al., 2013).

RT-qPCR

Seven-day-old seedlings were transferred to a liquid medium (½ MS) and allowed to equilibrate in the growth chamber overnight. On the following day, the plants were treated with either the liquid medium alone or the liquid medium supplemented with ATP (added to a final concentration of 500 μM ATP) for 30 min. Subsequently, the plants were harvested and rapidly frozen in liquid N₂, followed by storing in a −80 °C freezer. To homogenize the frozen tissue, we utilized the Mini-Beadbeater^™ (BioSpec Products), following the recommended protocols provided by the manufacturer. For RNA isolation, we employed the Quick-RNA isolation kit (Zymo Research) by the manufacturer’s instructions. The concentration of RNA was determined using an Eppendorf μCuvvete^® G1.0 on the BioPhotometer^® D30 (Eppendorf). To initiate cDNA synthesis, 1μg of RNA was used with the iScript kit (Bio-rad). Subsequently, the cDNA was diluted 10-fold with sterile deionized water, and 2 μL of the diluted solution was utilized in a 20 μL SYBR Green reaction mix (SsoAdvanced; Bio-rad). All reactions were performed in a CFX96 thermocycler (Bio-Rad). The expression of the target genes was normalized to PP2A and SAND (Czechowski et al., 2005; Zhang et al., 2015), which yielded comparable results. Statistical analysis of gene expression was conducted using a two-way ANOVA and a Tukey (or Dunnett’s) post hoc test in GraphPad Prism V.8, as indicated for each of the individual experiments.

Histochemical GUS staining

The transgenic eATP responsive promoter reporter line pTAT3::GUS (Sowders and Tanaka, 2023) was transformed with UB10::CPK28-GFP in the pUBC (Grefen et al., 2010) cassette. The transformants were selected based on BASTA resistance and PCR resulting in GUS reporter lines expressing UB10::CPK28-GFP (oxCPK28). To compare the expression patterns of eATP markers between wildtype (Col-0) and oxCPK28 lines, 7-day-old plants were treated with 500 μM ATP for 2 h. Subsequently, histochemical GUS staining and imaging were performed as recently described (Sowders and Tanaka, 2023).

Botrytis cinerea infection and chlorosis measurement

Arabidopsis plants were sewn on MS medium and transferred to soil 7 d post-germination and grown at 22 °C with an 8-h photoperiod. At 5 w post-germination, leaves were detached from each of the Arabidopsis genotypes and placed on two pieces of circular filter paper saturated with sterile water in a 90 mm circular petri dish for the Botrytis cinerea infection assay. A strain of Botrytis cinerea isolated from tomato in 2009 and propagated on ½ V8 agar medium was gifted by Shari Lupien at the USDA-ARS in Pullman WA, USA. For the detached leaf assay, a scraping of sporulating B. cinerea from the agar plate was collected and suspended in ½ potato dextrose liquid medium. The spore concentration was determined using a hemocytometer and adjusted to a final concentration of 5 × 10⁵ spores per mL. Then, 5 μL of the spore solution was dropped on the adaxial surface of detached Arabidopsis leaves. The petri dish with 4 – 7 Arabidopsis leaves, including one uninoculated leaf, was sealed, placed in a tray, covered with plastic film, and incubated at 22 °C. To record infection progress, images were recorded every 24 h for 7 d. To measure chlorosis in the detached leaf assay, ImageJ was used to filter the yellow spectrum as described (Laflamme et al., 2016), and measure the area of the isolated yellow hue. The relative or percent chlorosis was determined by comparing the yellow area with the total leaf area. To compare chlorosis in the B. cinerea infection assay across different Arabidopsis genotypes, hierarchical clustering with Heatmapper (Babicki et al., 2016), and statistical analysis by ANOVA with Tukey post hoc test were used.

Growth measurements

Rosette and petiole measurements were taken from 6 w-old Arabidopsis grown under the condition of 100 μmol photons m⁻² s⁻¹ at 22 °C and with an 8-h photoperiod. Root growth was measured with ImageJ (1.53q) from scanned (800 dpi; EPSON V600) images of Arabidopsis seedlings vertically sown and grown on ½ MS media or ½ MS plus 500 μM ATP medium (1% agar) in a Conviron (CMP6010) growth chamber at 22 °C with 100 μmol photons m⁻² s⁻¹ and with a 12-h photoperiod. Root penetration was scored as yes/no 5-d post germination on ½ MS or ½ MS plus 500 μM ATP medium (1% agar) in 6-well plates. The plants were grown on horizontal plates at 22 °C with 100 μmol photons m⁻² s⁻¹ and with a 12-h photoperiod.

RESULTS

Identification of P2K1-interactor candidates

To identify potential interactors of P2K1, we performed co-immunoprecipitation-coupled tandem mass spectrometry (Co-IP/MS) analysis of trypsin-digested proteins that were immunoprecipitated with P2K1-GFP in Arabidopsis microsomal fractions. Enrichment of peptides with P2K1-GFP compared to negative control, GFP-LTI6B, was determined by Fisher’s exact test Benjamini-Hochberg (P < 0.05) (Supplementary Dataset 1). The -log₁₀ (p-value) was plotted over log₂ fold-change of peptide spectral matches (P2K1-GFP / GFP-LTI6B). The resulting volcano plot revealed 121 proteins significantly enriched with P2K1-GFP (Fig. 1A). Among these enriched proteins were proteins previously shown to interact with P2K1 such as RBOHD and ILK5(BHP1) (Chen et al., 2017; Kim et al., 2023), which provided support for the dataset. We further analyzed the P2K1-interactome using hierarchical clustering with Heatmapper (Average linkage; Euclidean) to compare proteins immunoprecipitated with P2K1-GFP at different time points (0, 3, 20 min) after eATP addition (Fig. 1B). The majority (~85%) of coimmunoprecipitated proteins were found in the highest abundance 3 min after ATP addition (Fig. 1B). Proteins coimmunoprecipitated at 0 min, 20 min after ATP addition, or evenly across treatments accounted for approximately 5% of the total proteins each (Fig. 1B). To gain insights into the functional properties of the P2K1-interactor candidates we referred to ARAPORT11 annotations. This analysis revealed 6 proteins matching for Ca²⁺-binding proteins (Fig. 1C), 11 proteins for ion transporters (Fig. 1D), and 30 for receptor kinases (Fig. 1E). These 47 proteins were considered putative P2K1-interacting candidates of interest.

FIGURE 1. Identification of P2K1-interactor candidates.

(A) Co-immunoprecipitation-coupled tandem mass spectrometry (Co-IP/MS) analysis of trypsin-digested P2K1-GFP immunoprecipitates from Arabidopsis microsomes. Arabidopsis plants expressing P2K1-GFP were treated with mock or 500 μM ATP for 3 or 20 min and compared to the negative control GFP-LTI6B. Statistical analysis of peptide enrichment with P2K1-GFP was performed by comparing all P2K1-GFP samples (regardless of treatment) to the null protein GFP-LTI6B, using Scaffold software (v.4) with default parameters (Fisher’s exact test Benjamini-Hochberg correct P<0.05). n = 3 replicates. P2K1, RBOHD and ILK5 were highlighted as known interactors of P2K1. (B) Hierarchical clustering (Heatmapper) was employed to compare putative interacting proteins with P2K1-GFP at different time points with and without ligand (eATP) presentation. Ctrl = GFP-LTI6B. Functional and subcellular localization predictions for P2K1-interactor candidates were carried out using SUBA4 and ARAPORT11 annotations, which identified (C) Ca²⁺-dependent kinases, (D) ion transporters, and (E) receptor kinases.

Analysis of changes in eATP-responsive gene expression in mutants of the candidate interactors

To narrow the pool of P2K1-interacting candidates, we screened T-DNA insertional mutant germplasms for candidate interactors, specifically Ca²⁺-dependent kinases (CPKs) and receptor kinases (RKs). Our screening was based on their impact on perturbances in eATP-responsive gene expression (Fig. 2). Based on genes identified in our recent characterization of eATP-responsive gene expression (Jewell et al., 2019), we measured the fold change (log₂) of ATP-RESPONSIVE 1 (ATPR1), ATP-RESPONSIVE 2 (APTR2), and CML39 (Fig. 2). To determine mutants with significantly different expression patterns, we compared the fold change of eATP-responsive genes in the respective mutant to that of Col-0 using ANOVA Dunnett post hoc analysis (P<0.05). Since the induction of eATP-responsive genes, ATPR1, ATPR2, and CML39, is dependent on the functional P2K1 receptor (Jewell et al., 2019), the expression of these genes in response to eATP was significantly lower (P<0.0001) in the P2K1 mutant dorn1-3 compared to Col-0 (Fig. 2). Fold-induction of all three eATP-responsive genes, ATPR1, ATPR2, and CML39, respectively, was significantly reduced in cpk5 (P = 0.0002; P < 0.0001; P < 0.0001), cpk6 (P < 0.0001; P < 0.0001; P < 0.0001), cpk11 (P < 0.0001; P < 0.0001; P = 0.0075), cpk5/6/11 (P < 0.0001; P < 0.0001; P < 0.0001), feronia (P < 0.0001; P < 0.0001; P < 0.0001), hpca1 (P = 0.0007; P < 0.0001; P < 0.0001), ios1-2 (P < 0.0001; P < 0.0001; P < 0.0001), ios1-3 (P = 0.0083; P < 0.0001; P < 0.0001), pld-delta (P = 0.0025; P < 0.0001; P < 0.0001), and rlk902–1 (P = 0.0019; P = 0.0066; P = 0.0321). Both ATPR1 and CML39 were significantly different from Col-0 in bhp1(ilk5) (P = 0.0314; P = 0.0298). ATPR2 and CML39 fold-change was reduced in cpk5/6 (P = 0.0167; P < 0.0001), bark1 (P < 0.0001; P = 0.0115), and perk15 (P < 0.0001; P = 0.0003). ATPR1 induction was affected in lecrk-i.7 (P = 0.0020) and lecrk-iv.1 (P = 0.0139) mutants. ATPR2 was the only gene with a significant change in eATP-induction in cpk27–1 (P < 0.0001), cpk28–1 (P = 0.0004), lik1-1 (P = 0.0002), sif2 (P = 0.0262), and At1g53440 (P = 0.0070), compared to Col-0. Finally, only CML39 expression was impacted by mutation in At5g10290 (P<0.0001) and rlk902–2 (P=0.0050). Taken together, mutations in 5 CPKs and 12 RKs significantly impacted eATP-responsive gene expression in at least one of ATPR1, ATPR2, or CML39 (Fig. 2). Considering the higher proportion of CPKs (71%) compared to RKs (43%) that affected eATP signaling responses, we decided to focus on the subset of CPKs derived from the P2K1-interactome for further interaction validation and characterization.

FIGURE 2. Evaluating the impact of P2K1-interactor candidates on downstream eATP responses.

To assess the influence of P2K1-interactor candidates on eATP signaling, T-DNA insertional knockout mutants of the candidates were screened. The expression levels of eATP-responsive genes, ATPR1, ATPR2, and CML39 were evaluated following eATP treatment. Gene expression data are presented as the log₂ fold change of eATP-treated expression relative to mock-treated expression of the same genotype. Statistical analysis was performed using ANOVA Dunnett post hoc test (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001), comparing each genotype to Col-0. The experiment comprised 4 biological replicates and 2 technical replicates and was repeated at least twice with mutants that exhibited significant differences from Col-0.

CPK28 and CPK6 physically interact with P2K1 in yeast and planta

To validate physical interactions between P2K1 and CPK candidates, we employed the mating-based based split-ubiquitin system (mbSUS) assay, a membrane protein adapted yeast-2-hybrid technique (Obrdlik et al., 2004). In this assay, P2K1 and LTI6B (negative control) were used as bait proteins, while CPK3/5/6/9/11/27/28/31 were tested as prey proteins. Positive interactions were indicated by yeast growth on media lacking leucine, tryptophan, uracil, adenine, and histidine (“-LTUAH”). The bait protein, expressed under the methionine (Met) repressible MET25 promoter, was subjected to limited abundance through the addition of excess Met, increasing stringency for yeast growth (“-LTUAH + 5 μM Met”). CPK6, CPK27, CPK28, and CPK31 prey showed growth when mated with P2K1 bait—but CPK6 appeared the strongest with growth in the presence of excess methionine (Fig. 3A).

FIGURE 3. CPK28 and CPK6 physically interact with P2K1 in yeast and in planta.

(A) Evaluation of P2K1 and CPK interactions in yeast using the mating-based Split-Ubiquitin System (mbSUS). P2K1 (upper panel) and the negative control LTI6B (lower panel) were used as bait proteins to evaluate CPK interactions in yeast. The selectivity for bait-prey interactions was determined on dropout media lacking Leucine, Tryptophan, Uracil, Adenine, and Histidine (-LTUAH) or -LTUAH with 5 μM methionine (Met) compared to the least selective media, -LTU. Mated yeast cells were serially diluted in a 6 five-fold dilution series and spotted on each plate. Images were captured after 3 days of growth. Positive interactions were indicated by any growth on -LTUAH or -LTUAH plus 5 μM Met media. Bait protein expression was suppressed by excess Met. The experiments were repeated 5 times with consistent results. The KAT1 homodimer served as a standard control (grey arrow). P2K1 self-association and interaction with RBOHD were additional positive controls. Strong interactions growing on -LTUAH plus Met were marked with a green arrow, while weak interactions growing mostly on -LTUAH (lacking Met) were indicated with a yellow arrow. (B) Co-immunoprecipitation of P2K1-CPK interactions in Nicotiana benthamiana. P2K1-HA and CPK-GFP were transiently expressed in N. benthamiana leaves. Immunoprecipitations were performed for P2K1-HA, followed by Western blot analysis with anti-GFP (upper) or anti-HA (lower) antibodies for protein extract (input) and HA-immunoprecipitated (IP:HA) samples to evaluate P2K1 interactions with CPK5, CPK28, CPK6, and CPK31. As CPK5 was not detected in the Co-IP/MS data, it was used as a negative control CPK for comparison. The black bar on each blot indicates the position of a protein standard ladder corresponding to 85 kD. CPK5-GFP, 90 kD; CPK28-GFP, 87 kD; CPK6-GFP, 89 kD; CPK31-GFP, 85 kD. The experiments were performed 4 times with consistent results.

After considering our screening of P2K1-interactor candidates up to this point, we chose to further investigate CPK6 and CPK28, since they both impacted eATP responsive gene expression (Fig. 2) and interacted with P2K1 in yeast (Fig. 3A). As a next step, we employed N. benthamiana as a transient expression system to validate P2K1-CPK interactions in planta by co-immunoprecipitation. P2K1-HA and CPK-GFP were agroinfiltrated in N. benthamiana leaves, and protein extracts were immunoprecipitated using HA antibodies. The results showed that both CPK28-GFP and CPK6-GFP, but not CPK5-GFP or CPK31-GFP, interacted with P2K1-HA in N. benthamiana (Fig. 3B, Fig. S1). As oxCPK28 plants were readily accessible at this stage, and initial experiments with these plants revealed a notable impairment in the eATP response (as demonstrated below), we decided to focus on CPK28 for further investigation.

CPK28 perturbs eATP-mediated Ca²⁺ mobilization and downstream responses

To evaluate eATP signaling responses, we utilized luminescence-based measurements of cytosolic Ca²⁺ influx using aequorin in plants expressing 35S::AEQ (Knight et al., 1991). In Col-0, we observed a characteristic Ca²⁺ influx pattern following ATP treatment (Fig. S2A), consistent with previous reports (Tanaka et al., 2010). The absence of ATP-induced Ca²⁺ influx in the P2K1 mutant dorn1-3/35S::AEQ indicated that the Ca²⁺ response to eATP requires the P2K1 receptor (Fig. S2). Meanwhile, hyperresponsive Ca²⁺ influx was observed in the overexpression line oxP2K1t (expressing a C-terminal truncated P2K1) as previously demonstrated (Sowders et al., 2023). To investigate the impact of CPK28 on eATP-induced Ca²⁺ mobilization, we measured the Ca²⁺ response in oxCPK28 plants (35S::CPK28-YFP/35S::AEQ) (Monaghan et al., 2015). These plants are referred to as oxCPK28 #1, herein. We observed no difference in mean or total Ca²⁺ influx in oxCPK28 in response to eATP (Fig. S2). Unfortunately, after several attempts to cross cpk28–1 mutants with 35S::AEQ plants we were unable to identify any homozygous cpk28–1 mutants expressing 35S::AEQ.

Instead, to further assess the impact of CPK28 on eATP signaling, we evaluated the expression of eATP-responsive genes in Col-0, cpk28-1, and oxCPK28 #1 (Fig. 4A). Expression of 4 out of 6 eATP-responsive genes (TAT3, ATPR1, CML39, VQ29) tested did not show a significant difference from Col-0 in cpk28–1. However, when comparing the eATP-responsive gene expression in CPK28 overexpressing plants (oxCPK28 #1) to Col-0, we found that oxCPK28 #1 exhibited significantly lower expression in 5 of 6 ATP-responsive genes (WRKY46, TAT3, ATPR1, ATPR2, and CML39) following ATP-treatment (Fig. 4A). These results suggest a semi-redundant effect of CPK28, which may influence eATP signaling downstream of P2K1—presumably through direct physical interaction with P2K1. To provide a more detailed picture of CPK28’s influence on eATP-responsive gene expression, we employed the recently described eATP-reporter GUS line pTAT3::GUS #12.1 (Sowders and Tanaka, 2023) and introduced UB10::CPK28-GFP to overexpress CPK28 in the reporter line (UB10::CPK28-GFP/pTAT3::GUS). We found that GUS staining in the three independent lines of UB10::CPK28-GFP/pTAT3::GUS (#2, #8, and #11) showed significantly less intensity compared to pTAT3::GUS/Col-0 plants, where GUS staining saturated the primary root tip after eATP-treatment (Fig. 4B, 4C). These findings indicate that CPK28 overexpression suppresses eATP-responsive gene expression. Specifically, oxCPK28 suppressed eATP-induced expression of TAT3 in the root vasculature, but not the distal root tip—suggesting a potential tissue-specific impact of CPK28 on P2K1-mediated eATP signaling.

FIGURE 4. Suppression of eATP-responsive gene expression in oxCPK28.

(A) Expression levels of eATP-responsive genes in Col-0, cpk28–1, and oxCPK28 #1. All gene expression values were normalized to PP2A. n = 4 reps (15 seedlings/rep). Fold change (FC) represents the relative expression of ATP-treated plants compared to mock-treated plants. The gene expression experiments were repeated thrice with consistent results. (B and C) GUS staining of pTAT3::GUS #12.1 and UB10:CPK28/pTAT3::GUS lines (#2, #8, #11) after 2 h of mock or ATP treatment. Images were captured (B) and quantification was performed (C) using ImageJ relative to ATP-treated pTAT3::GUS #12.1. n = 8. Statistical analysis was conducted using two-way ANOVA with Tukey post hoc test. Samples with different letters indicate significant differences (P<0.05). GUS staining experiments were repeated thrice with consistent results. Scale bar = 50 um. Error bars = SEM.

Overexpression of CPK28 in oxP2K1 plants partially complements eATP hypersensitivity

We hypothesized that if CPK28 mostly negatively impacts eATP signaling through physical interaction with P2K1, we would expect a decrease in eATP hypersensitivity in oxP2K1 plants overexpressing CPK28. To test this hypothesis, we transformed oxP2K1 (35S::P2K1_t-GFP/35S::AEQ/dorn1-3) with UB10::CPK28-GFP resulting in UB10::CPK28-GFP/35S::P2K1_t-GFP/35S::AEQ/dorn1-3 plants. Herein, plants of this genotype are referred to as “co-ox” (Fig. 5; Table S1). In eATP-induced calcium influx assays, the co-ox lines displayed a similar pattern of peak shift (Fig. 5A) as oxCPK28 (Fig S1A). The peak of Ca²⁺ influx in the co-ox lines occurred approximately 27 sec after ATP treatment, compared to around 19 sec in oxP2K1 (Fig. 5A). Moreover, both the mean and total Ca²⁺ influx in response to ATP were significantly reduced in co-ox plants (line #1, #2, and #3) compared to oxP2K1 (Fig. 5B, 5C), indicating that overexpression of CPK28 in oxP2K1 indeed impacted eATP-responsive Ca²⁺ mobilization. Subsequently, we evaluated the eATP-responsive gene expression of WRKY46 and TAT3 in co-ox plants (Fig. 5D). The fold change (FC) of WRKY46 and TAT3, respectively, showed a significant reduction in each co-ox #1 (FC = 58 ± 3.2, FC = 4.5 ± 0.4), co-ox #2 (FC = 44 ± 4.4, FC = 1.5 ± 0.2), and co-ox #3 (FC = 65 ± 6.1, FC = 2.6 ± 0.6) compared to oxP2K1(FC = 110 ± 8.4, FC = 11 ± 0.9). Additionally, we confirmed that the introduction of UB10::CPK28-GFP did not impact the expression of P2K1 in the co-overexpression lines (Fig. S3), suggesting that the reduction in eATP signaling responses observed in co-ox plants could be attributed to a direct protein-protein interaction between CPK28 and P2K1. Overall, the Ca²⁺ influx and gene expression data demonstrated that overexpression of CPK28 in oxP2K1 plants partially alleviated the eATP hypersensitivity phenotype, as indicated by reduced Ca²⁺ influx and decreased fold-induction of eATP-responsive genes. Together, these findings suggest some negative role for CPK28 in the eATP signaling pathway.

FIGURE 5. Complementation of eATP hypersensitivity through CPK28 overexpression in oxP2K1t plants.

(A, B, C) Calcium measurements were conducted using aequorin luminescence in Arabidopsis genotypes expressing 35S::AEQ. The measurements were taken for 80 sec after treatment with ATP (100 μM). (A) The mean luminescence of aequorin was recorded at individual time points during the 80-sec period. (B) The mean luminescence of aequorin across all time points (0–80 sec) was determined. (C) The area under the curve (AUC) representing the total calcium influx over the 80-sec period was calculated for each genotype. The analysis included 8 to 12 seedlings per genotype. The calcium experiments were replicated four times, yielding consistent outcomes. (D) Gene expression analysis was performed to examine the expression of eATP-responsive genes in seven different genotypes: Col-0, oxP2K1t, oxCPK28 #1, oxCPK28 #2, co-ox #1, co-ox #2, and co-ox #3. Co-ox = co-overexpressing CPK28 and P2K1. oxCPK28 #2 refers to UB10::CPK28-GFP/pTAT3::GUS #2. A detailed description of the plant germplasms can be found in Table S1. Gene expression was normalized to PP2A. Each analysis involved 15 seedlings per replication, and a total of four replications were conducted (n = 4 reps). Fold change (FC) represents the relative expression of ATP-treated plants compared to mock-treated plants. The gene expression experiments were repeated three times, yielding similar results. Statistical analysis was carried out using one-way ANOVA followed by a Tukey post hoc test. Samples with different letters indicate significant differences. Error bars = SEM.

CPK28 and P2K1 have opposing impacts on plant growth

To gain further insights into the physiological relationship between CPK28, P2K1, and eATP signaling, we examined the plant growth morphologies of Arabidopsis CPK28- and P2K1-related genotypes: cpk28–1, dorn1-3, oxP2K1, oxCPK28 #1, oxCPK28 #2, co-ox #1, co-ox #2, and co-ox #3. We measured the rosette diameter and petiole length in each genotype (Fig. 6), which revealed an inverse correlation between P2K1 and CPK28 in relation to plant growth. The average rosette diameter of oxP2K1 (7.76 ± 0.71 cm), dorn1-3 (8.06 ± 0.50cm), and cpk28–1 (7.17 ± 0.23cm) genotypes was significantly smaller (mean±SD) at 6 weeks post-germination, compared with Col-0 (8.80 ± 0.31cm), while oxCPK28 #1 (9.67 ± 0.40cm), and oxCPK28 #2 (9.80 ± 0.75cm) exhibited significantly larger rosette diameters (Fig. 6). The overexpression of CPK28 in oxP2K1 (co-ox) complemented the reduced rosette growth phenotype. The rosette diameters of co-ox #1 (8.90±0.68cm) and co-ox #2 (8.91 ± 0.49cm) were significantly different from oxP2K1, but not from Col-0 (Fig. 6). Meanwhile, the correlation between P2K1 and CPK28 and petiole length was less pronounced.

FIGURE 6. Plant growth morphology in transgenic CPK28- and P2K1-related Arabidopsis germplasm.

Rosette diameter and petiole length were measured at 6 weeks post-germination in Col-0, cpk28–1, dorn1-3, oxP2K1t, oxCPK28 #1 oxCPK28 #2, co-ox #1, co-ox #2, and co-ox #3 plants. Co-ox = co-overexpressing CPK28 and P2K1. A detailed description of the plant germplasms can be found in Table S1. n = 11 plants. Statistical analysis of rosette diameter and petiole length was conducted using ANOVA followed by Tukey post hoc test. Genotypes with different letters indicate significant differences. Scale bar = 1 cm. Error bars = SD.

Given that eATP has been shown to influence root growth responses in previous studies (Tang et al., 2003; Tonón et al., 2010; Zhu et al., 2020), we conducted measurements on the root growth of cpk28–1 and oxCPK28 #1 when grown on ATP-containing media (Fig. S4A). Interestingly, cpk28–1 exhibited significantly shorter roots when grown on ATP-containing media (Fig. S4A), which could suggest that the loss of CPK28 lessens regulatory constraint on P2K1—thus increasing sensitivity to eATP-mediated root growth regulation.

Recent evidence has proposed that eATP signaling can contribute to increased cell wall accumulation in the primary root tip (Sowders et al., 2023). Here, we found that ATP treatment also had a positive effect on the agar penetration of roots in Arabidopsis seedlings (Fig. S4B), presumably due to enhanced cell wall rigidity in the root tip. Consistent with the previous experiments, we observed an inverse relationship between P2K1 and CPK28 in the agar penetration assay. Notably, the cpk28–1 mutants exhibited a significantly higher rate of agar penetration compared to oxCPK28 #1 (Fig. S4B). Taken together, these data further demonstrate the impact of CPK28 on P2K1-dependent eATP-regulated responses.

The P2K1-CPK28 relationship in plant response to the necrotrophic pathogen Botrytis cinerea

To investigate the effect of the P2K1-CPK28 module on eATP-mediated defense priming, we employed the Botrytis cinerea detached leaf infection assay. After inoculating Col-0, cpk28–1, dorn1-3, oxP2K1, oxCPK28 #1, oxCPK28 #2, co-ox #1, co-ox #2, and co-ox #3 with B. cinerea spores, the infection progress was monitored at different hours post inoculation (hpi). Interestingly, we found an inverse relationship between P2K1 and CPK28 in terms of chlorotic lesion development (Fig. 4). Previous work has shown eATP signaling mediated by P2K1 promotes basal immunity (Bouwmeester et al., 2011; Balagué et al., 2017; Kumar et al., 2020; Jewell et al., 2022). Interestingly, our finding revealed that overexpression of P2K1 led to significantly more chlorosis caused by B. cinerea infection compared to Col-0 and the P2K1 mutant, dorn1-3 (Fig. 4). We speculate that this increase in chlorosis is due to heightened eATP-responsive signaling in oxP2K1 plants. Interestingly, the loss of CPK28 in cpk28–1 mutants also displayed an increase in chlorotic response, like oxP2K1. Conversely, plants overexpressing CPK28 alone (oxCPK28 #1, #2), or co-overexpressing CPK28 in oxP2K1 (co-ox #1, #2, #3), exhibited significantly reduced chlorosis upon inoculation with B. cinerea (Fig. 4). These findings could suggest that the interaction dynamic between P2K1-CPK28 affects responses to the necrotrophic fungus B. cinerea by modifying eATP signal amplitude.

DISCUSSION

Recent studies have shed light on the mechanism by which P2K1 activates downstream signaling pathways. For example, P2K1 has been found to phosphorylate RBOHD, MVK, PAT5/9, and ILK5, thereby regulating eATP signaling and bacterial immunity (Chen et al., 2017; Cho et al., 2022; Kim et al., 2023). Although these discoveries have contributed to our understanding of purinergic signaling in plants since the identification of the P2K1 receptor a decade ago (Choi et al., 2014), additional signaling components are required to obtain a comprehensive understanding of the eATP signaling mechanism.

The objective of this study was to expand our knowledge of eATP signal transduction between P2K1 and the activation of downstream events associated with growth and defense responses. We generated a P2K1-interactomics dataset (summarized in Table S4) to share with the purinoreceptor signaling community as a valuable resource for gaining an in-depth understanding of plant purinergic signaling. Through our Co-IP/MS, we identified 121 potential interactions with P2K1 (Fig. 1A, Table S3). Further analysis revealed three main categories: Ca²⁺-binding proteins, ion transporters, and receptor kinases (Fig. 1C – 1E). The investigation of the impact of P2K1-interacting proteins on eATP-responsive gene expression (Fig. 2) revealed that mutations in 5 CPKs (cpk5, cpk6, cpk11, cpk27, cpk28) and 12 RKs (lecrki.7, lecrkiv.1, feronia, ios1, rlk902, hpca1, sif2, perk15, bark1, lik1, At5g10290, At1g53440) had a significant impact on the expression of at least one of the eATP responsive genes, namely ATPR1, ATPR2, or CML39 (Fig. 2).

CPKs and RKs are protein kinases involved in signaling pathways that regulate various physiological processes. RKs are typically membrane-associated kinases responsible for transducing external signals, often working in conjunction with other RKs to activate or attenuate signaling activity. In some cases, post-translational modifications lead to the membrane association of CPKs, as observed in CPK5, CPK6, and CPK28 (Lu and Hrabak, 2013; Matschi et al., 2013; Saito et al., 2018). While CPKs are generally considered downstream players in receptor signaling pathways, there are a few instances where they directly interact with transmembrane receptors. For example, CPK5 phosphorylates the transmembrane chitin receptor LYK5 (Huang et al., 2020), and CPK3 interacts with the transmembrane receptor-like pseudokinase GHR1 to regulate stomatal closure (Sierla et al., 2018). These previous observations provide support for our finding based on protein-protein interaction studies, which indicate direct interactions between CPK6 and CPK28 with P2K1 (Fig. 3).

CPKs impart various effects on plant growth, development, and responses to abiotic and biotic stresses, primarily through the phosphorylation of their target proteins (Wei et al., 2014; Monaghan et al., 2015; Atif et al., 2019; Huang et al., 2020; Zhao et al., 2021; Ding et al., 2022). While the first study characterizing CPK28 in Arabidopsis revealed its involvement in plant stem elongation and vascular development (Matschi et al., 2013), it has been shown its crucial role in immune signaling (Monaghan et al., 2014) and abiotic stress responses (Ding et al., 2022). CPK28 negatively regulates immune signaling through the phosphorylation of the receptor-like cytoplasmic kinase BIK1, leading to BIK1 degradation and modulation of immune signaling (Monaghan et al., 2014; Bredow and Monaghan, 2021). In this study, we found that overexpression of CPK28 alleviates eATP-hypersensitivity in oxP2K1 (Fig. 5). Meanwhile, it has been shown that CPK28 is transcriptionally activated by eATP (Choi et al., 2014). Together, the two observations could suggest CPK28 is part of a negative feedback loop, where CPK28 dampens P2K1 activity to prevent continuous eATP signaling responses. However, further work dissecting the P2K1-CPK28 interaction mechanism will be required to resolve this hypothesis.

High expression of P2K1 in the oxP2K1 plants resulted in stunted plant growth, whereas overexpression of CPK28 promoted plant growth, namely in rosette development (Fig. 6). Interestingly, loss of CPK28 in plants exhibited a stunted growth phenotype like that of oxP2K1 plants. Complementation of the stunted growth phenotype in oxP2K1 was observed by co-overexpressing CPK28 in oxP2K1 (Fig.7), supporting our hypothesis of CPK28’s mostly negative impact on P2K1 activity. Additionally, loss of CPK28 (cpk28–1) resulted in increased chlorotic sensitivity in the B. cinerea-infected leaves, which aligned with the observation in oxP2K1 (Fig. 7). Meanwhile, oxCPK28, loss of P2K1 (dorn1-3), and co-ox plants exhibited less chlorotic development in response to B. cinerea (Fig. 7). Considering the physical interaction between P2K1-CPK28 (Fig. 3), as well as the impact of CPK28 overexpression on eATP-induced Ca²⁺ burst (Fig. 4), transcriptional responses (Fig. 4, 6), and plant growth (Fig. 6)—the B. cinerea infection assay further supports CPK28 as a moderator of P2K1-mediated signaling activity (Fig. 7). Another possibility is that, as part of a receptor-complex, CPK28-mediated regulation of BIK1 turnover (Monaghan et al., 2014), which may affect turnover or activity of P2K1. In either case, these findings demonstrate that the P2K1-CPK28 module plays a pivotal role in eATP-regulated plant growth and defense against the necrotrophic pathogen B. cinerea. Further, considering the yield trait-related impacts of CPK28 on purinergic signaling were most pronounced in above ground (Fig. 6, 7) compared to below ground tissues (Fig. S4), we propose a model in which CPK28 is a major regulator of eATP signaling in rosette leaves, but a minor regulator in roots.

FIGURE 7. Botrytis cinerea infection assay in transgenic CPK28- and P2K1-related Arabidopsis germplasm.

Detached leaves of Col-0, cpk28–1, dorn1-3, oxP2K1t, oxCPK28 #1 oxCPK28 #2, co-ox #1, co-ox #2, and co-ox #3 plants were inoculated with Botrytis cinerea. Co-ox = co-overexpressing CPK28 and P2K1. (A) Images of the infection progress were captured at 24 h intervals post-inoculation (hpi). Scale bar = 1 cm. Chlorosis areas were determined by measuring the yellow-hued region. (B) Hierarchical clustering of chlorosis levels among the genotypes was performed using Heatmapper with default parameters. (C) Statistical analysis of the chlorotic area at different hours post inoculation (hpi) within and between genotypes was conducted using two-way ANOVA followed by Tukey post hoc test. Genotypes different letter assignments indicate significant differences. n = 5 – 7 plants. Four independent experiments were done with consistent results.

It is important to address the increased yellowing (chlorosis) observed in oxP2K1 in our present study, which deviates from previous reports. Typically, plant defense mechanisms against necrotrophic pathogens, i.e., B cinerea are to reduce necrotic lesions, as demonstrated by heightened plant immunity via P2K1 (Tripathi et al., 2018). This variation is likely due to the difference of B. cinerea strains used. Fordyce et al. (2018) investigated various B. cinerea strains and noted that some strains exhibited a negative correlation between lesion size and the intensity of yellowing, suggesting that enhanced chlorosis may contribute to the suppression of disease symptoms resulting from B. cinerea infection. The authors further supported this with a GWA study, linking the yellow color trait (chlorosis) to genes associated with defense responses. Additionally, other studies have shown that mutants deficient in jasmonic acid biosynthesis (aos) and phytoalexin production (cyp79b2/cyp79b3) failed to develop the chlorotic zone (as reported by Rowe et al., 2010, and Pavicic et al., 2021). This implies that chlorosis is a component of the defense response against specific strains of B. cinerea. In our current study, we employed a locally isolated B. cinerea strain from tomatoes (provided by a USDA group in WA). This strain differs from the one used in the previously published study, which was isolated from grapes, as reported by Rowe and Kliebenstein (2007). Therefore, the observed differences can be attributed to the use of distinct strains. Further investigation is needed to elucidate the mechanism by which chlorosis contributes to defense against necrotrophic pathogens.

The discovery of CPK28 as a modifier of activity downstream of P2K1 has significant implications as it provides insights into the coordination between plant growth and defense responses mediated by eATP signaling. It suggests that P2K1 integrates eATP signaling to regulate both plant growth and defense, with CPK28 acting as a modulator that dampens these responses. However, it remains unclear whether there is tissue-specific subfunctionalization of either P2K1 activity or its regulation by CPK28. One limitation of our study is that we investigated the physiological impacts of the P2K1-CPK28 module through transgenic overexpression, and it is important to consider that a native promoter-driven study could reveal a cell type-specific role for the modulation of P2K1 by CPK28. CPK28 serves as a rate-limiting factor in PTI signaling and acts as a catalyst for plant growth. Regarding BIK1, modulation of BIK1 protein levels by CPK28 fine-tunes the amplitude of PTI signaling (Monaghan et al., 2014). While the exact nature of the molecular mechanism for CPK28’s regulation of P2K1 is unclear, it is plausible that early eATP-induced Ca²⁺ influx similarly activates CPK28-mediated turnover of P2K1 as BIK1. Moreover, recent findings have highlighted the importance of an intrinsically disordered region in the P2K1’s C-terminal tail for regulating eATP-mediated growth responses (Sowders et al., 2023). S-acylation of P2K1 also plays a crucial role in mediating eATP-induced autophosphorylation and protein turnover (Chen et al., 2021). Thus, modification in the C-terminal tail and S-acylation status could be mechanisms for CPK28’s modulation of P2K1 activity. Investigating these mechanisms in future studies will provide a more detailed biochemical understanding.

In conclusion, our research provides valuable insights into the interactome of P2K1 and identifies CPK28 as a modifier of P2K1-dependent eATP signaling, primarily in rosette leaves. This study enhances our understanding of the signal transduction mechanisms underlying eATP-mediated growth and defense responses in plants. Further investigations into the regulatory mechanisms and downstream targets of P2K1 and CPK28 will contribute to a more comprehensive understanding of eATP signaling and its significance in plant biology.

Supplementary Material

Fig S1

FIGURE S1. Whole blots used in figures Selected areas are used in Figure 3.

Fig S3

FIGURE S3. Baseline expression levels in CPK28- and P2K1-overexpressing plants. RT-qPCR measurement of CPK28 and P2K1 expression levels in Col-0, oxP2K1 (35S::P2K1_t-GFP/35S::AEQ/dorn1-3), oxCPK28 #1 (35S::CPK28-YFP/35S::AEQ) oxCPK28 #2, #3, and #4 (UB10::CPK28-GFP/pTAT3::GUS #2, #8, and #11, respectively), co-ox #1, #2, and #3 (UB10::CPK28-GFP/35S::P2K1_t-GFP/35S::AEQ/dorn1-3). n = 3 replicates. Approximately 15 seedlings per replicate. Expression was compared between genotypes by one-way ANOVA Tukey post hoc. Samples with different letters are significantly different.

Fig S4

FIGURE S4. Effects of CPK28 on root growth and agar penetration in ATP media (A) Seedings were grown vertically on mock or ATP (500 μM) media and root growth was measured at 4 days post germination (dpg). n = 8 −12 seedlings. (B) Seedlings were grown horizontally on the same media as in (A), and the percentage of seedlings with roots penetrating agar was determined at 120 h post-germination. Each well contained 20–40 seedlings. n = 3 replicates. Co-ox = co-overexpressing CPK28 and P2K1. Two-way ANOVA followed by Tukey post hoc analysis was used to compare sample groups. Samples with different letters indicate significant differences. Error bars = SEM. A graphical description of the relationship between eATP, P2K1, and CPK28 is shown for both experiments.

Supinfo2

TABLE S1. Plant materials

TABLE S2. Primers used in this study

TABLE S3. P2K1 interactome

TABLE S4. P2K1 interactome screening summary

Supinfo1

SUPPLEMENTARY DATASET 1. Interactomics data

Figure S2

FIGURE S2. CPK28 tunes eATP-mediated Ca²⁺ mobilization Calcium measurements (A-D) were taken based on aequorin luminescence in Arabidopsis genotypes expressing 35S::AEQ for 80 sec after ATP treatment (100 μM). (A) Mean aequorin luminescence at individual time points in the respective genotypes for 80 sec after treatment. (B) The area under the curve (AUC) was calculated for each of the genotypes as the sum of total calcium influx over 80 sec. (A-B) Internally duplicated from Fig. 5A and 5B. (C-D) Mean aequorin luminescence across all time points between (C) 20 – 30 sec and (D) 50 – 60 sec after treatment. n = 8 – 12 seedlings per genotype. The calcium experiments were repeated four times with similar results. Statistical analysis was done with one-way (B-D) ANOVA plus Tukey post hoc test. Samples with different letters are significantly different (P<0.05). Error bars = SEM.

DATA STATEMENT

Extended data sets from this study are included in the supplementary materials. Other data and materials supporting the findings of this study are available from the corresponding author [kiwamu.tanaka@wsu.edu] upon reasonable request. The plant germplasms generated in this study will be made available upon request and shared in compliance with the relevant governing bodies.