N-hydroxy-pipecolic acid-mediated signal transduction and resistance requires DAWDLE

Abstract

Plant systemic acquired resistance (SAR) is a pathogen-induced, long-distance immune response that primes uninfected tissue against future pathogen attacks. N-hydroxy-pipecolic acid (NHP) is a mobile signal required to initiate and amplify SAR signaling in different plant species. However, the key regulators involved in activating NHP-mediated signaling and systemic resistance remain unclear. In this study, we identified an Arabidopsis (Arabidopsis thaliana) mutant, dawdle (ddl), that exhibits a compromised systemic resistance phenotype upon NHP treatment and pathogen infection. Transcriptome profiling revealed that the DDL mutation significantly reduces the expression of NHP-responsive genes in both local treated and distal untreated leaves. Many of these NHP-responsive genes are associated with PAMP-triggered immunity, salicylic acid (SA) and NHP metabolism, SA signaling, and SAR, indicating that DDL is required for an effective NHP response and the activation of systemic transcriptional reprogramming. In addition to mediating NHP-mediated signal transduction, DDL is critical for enhancing PR1 gene expression during the pathogen challenge. Metabolite profiling indicated that the DDL mutation reduces free SA levels in Pst-inoculated tissues while enhancing NHP and SA accumulation in distal, untreated leaves, suggesting that DDL may regulate both biosynthesis and homeostasis of these metabolites. Moreover, loss of DDL decreases plant sensitivity to exogenous NHP, but not to SA, indicating that DDL may specifically regulate NHP perception or signal transduction. Our findings suggest that DDL plays a role in NHP-mediated systemic transcriptional reprogramming and systemic resistance, as well as in gene expression regulation during secondary pathogen challenge.

The Arabidopsis gene DAWDLE activates systemic acquired resistance by regulating immune-related genes induced by N-hydroxy-pipecolic acid.

Introduction

SAR is a long-distance defense response after primary pathogen infection, enhancing resistance in uninfected tissue against a wide range of secondary pathogen infections. To establish a SAR response, a pathogen-induced activation of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) signaling in inoculated tissues is required to generate mobile signals and accumulate defense hormones (Durrant and Dong 2004; Fu and Dong 2013; Kachroo and Kachroo 2020). Many SAR-associated metabolites that serve as mobile signals have been reported, such as salicylic acid (SA) (Métraux et al. 1990; Gaffney et al. 1993), methyl salicylate (MeSA) (Park et al. 2007), azelaic acid (AzA) (Jung et al. 2009), dehydroabietinal (DA) (Chaturvedi et al. 2012), glycerol-3-phosphate (G3P) (Chanda et al. 2011), the reactive oxygen species (ROS), free radical nitric oxide (NO) (Wang et al. 2014; El-Shetehy et al. 2015), extracellular nicotinamide adenine dinucleotide (phosphate) [eNAD(P)] (Wang et al. 2019; Li et al. 2023), pipecolic acid (Pip) (Návarová et al. 2012) and N-hydroxy pipecolic acid (NHP) (Chen et al. 2018; Hartmann et al. 2018).

Among these molecules, the interplay between SA and NHP is crucial for basal immunity and SAR (Hartmann et al. 2018; Yildiz et al. 2021; Löwe et al. 2023). The absence of either NHP or SA only triggers a partial immune response (Löwe et al. 2023). Full establishment of NHP-mediated resistance in systemic leaves usually requires an intact SA biosynthesis and signal transduction pathway (Hartmann et al. 2018; Yildiz et al. 2021; Yildiz et al. 2023). Notably, the NHP-induced transcriptional response requires NONEXPRESSER OF PR GENES 1 (NPR1) and TGA transcription factors even at basal SA levels (Nair et al. 2021; Yildiz et al. 2021; Yildiz et al. 2023), suggesting that both SA biosynthesis and SA signal are essential for NHP-induced systemic resistance. In addition to SA and its signaling, a recent study has shown that ROS-, eNAD(P)-, lectin receptor kinase lectin receptor kinase VI.2 (LecRKVI.2)- and BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)-mediated signaling pathways are involved in NHP-induced systemic resistance (Li et al. 2023). Moreover, the WRKY70 transcription factor, was shown to be an early primary NHP-responsive gene, which is required for NHP-induced systemic resistance, by mediating secondary transcriptional changes, and enhancing ROS generation during PTI (Foret et al. 2024). Despite these recent advances, the regulatory mechanisms underlying NHP-mediated signaling remain largely unknown.

In this study, our goal was to identify critical regulators linking NHP-mediated signals and systemic immunity. We discovered that an Arabidopsis forkhead-associated (FHA) domain protein, DAWDLE (DDL), is required for NHP-induced and pathogen-induced systemic resistance. DDL protein contains an N-terminal nuclear localization signal and a C-terminal FHA domain which contains phospho-binding sites (Chevalier et al. 2009). The N-terminal region of DDL protein is mainly poly-ADP-ribosylated (PARylated) by PARP2 protein (Feng et al. 2015; Song et al. 2015; Feng et al. 2016). The C-terminal FHA domain of DDL protein has also been shown to interact with DICER-LIKE 1 (DCL1) and DICER-LIKE (DCL3) (Zhang et al. 2018), two proteins that regulate small RNA biosynthesis. Previous studies have highlighted the significance of DDL in plant development through small RNA biosynthesis, and in plant immunity (e.g. late PTI responses) via protein PARylation (Morris et al. 2006; Yu et al. 2008; Feng et al. 2016). However, the role of DDL in induced resistance and long-distance signal transduction has not been investigated.

To better understand the role of DDL in NHP-mediated resistance and signal transduction, we characterized the role of DDL in gene expression changes and pathogen resistance in response to NHP or pathogen treatment in both local and distal tissues during SAR. We found that DDL is required for NHP and pathogen-induced systemic immunity. Our transcriptome profiling revealed that proper expression of NHP-responsive genes requires a functional DDL protein in both local, treated leaves and distal, untreated leaves. In addition, we found that DDL is involved in the enhanced expression of PR1 during the pathogen challenge in NHP-treated plants, suggesting that DDL may regulate the events of NHP-mediated signal transduction and pathogen challenge. Taken together, our study reveals that, in addition to its role in basal immunity, DDL is required for NHP-induced transcriptional regulation and systemic resistance.

Results

Arabidopsis DAWDLE is required for NHP-induced systemic resistance

Given that NHP-induced transcriptional reprogramming is indispensable for the activation of SAR in Arabidopsis (Yildiz et al. 2021; Yildiz et al. 2023), we were interested in identifying the genes that are required for NHP-induced transcription in lower, treated leaves (local response) and subsequently in upper, untreated leaves (systemic response) as a result of long-distance defense activation. We selected the Arabidopsis DDL gene as a candidate for reverse genetic screening based on its known impaired phenotypes in development, PTI, and basal immune responses (Feng et al. 2016).

As reported in a previous study (Feng et al. 2016), we confirmed that three homozygous T-DNA insertion mutants of Arabidopsis DDL gene (i.e. ddl-6, ddl-7, and SALKseq_078790.1) exhibited the mildly stunted growth phenotype and either a significant or partial reduction of DDL gene expression compared to wild-type (WT) Col-0 (Supplementary Fig. S1, A to E). We also found that the ddl-6 and SALKseq_078790.1 mutants showed increased susceptibility to a virulent bacterial strain, Pseudomonas syringae pathovar tomato strain DC3000 (Pst DC3000; Pst), in younger leaves under ambient humidity condition (55–60%) compared to wild-type Col-0, but exhibited wild-type-like susceptibility to Pst under high humidity (86–95%) (Supplementary Fig. S2). These data show that susceptibility in the ddl-6 mutant depends on the humidity conditions and supports previous findings that mutation of the DDL gene results in a moderately impaired basal immune response in Arabidopsis.

To determine whether the DDL gene is required for NHP-induced systemic resistance, we infiltrated the local leaves of Col-0 and two ddl mutants, ddl-6 and SALKseq_078790.1, with sterile distilled water only or 1 mm NHP. Two days later, the distal, untreated leaves of these plants were challenged with 1 × 10⁵ CFU/mL suspension of Pst. At 3 d post inoculation (dpi), the Pst infected leaves of water-treated Col-0 and two ddl mutants showed similar chlorotic symptoms and bacterial multiplication (Fig. 1, A and B; Supplementary Fig. S9A). As expected, the infected leaves of NHP-treated Col-0 plants showed alleviated disease symptoms and reduced Pst growth compared to the infected leaves of water-treated Col-0. In contrast, the infected leaves of two ddl mutants treated with NHP exhibited susceptible symptoms and higher Pst titers compared with NHP-treated Col-0 plants (Fig. 1B; Supplementary Fig. S9A). These data indicate that NHP-induced systemic resistance is impaired in the ddl mutants.

Figure 1.

Arabidopsis DDL is required for NHP and pathogen-induced systemic resistance. A and B) The local lower leaves of 4-wk-old Col-0 and two ddl mutants, ddl-6 and SALKseq_078790.1, were infiltrated with water or 1 mm NHP. Two days later, one distal leaf was inoculated with a 1 × 10⁵ CFU/mL suspension of Pst. The disease symptoms of the representative Pst-infected leaves were photographed at 3 dpi. The quantification of Pst growth in upper infected leaves at 3 dpi is shown in (B). Bars represent the mean ± standard deviation (SD; n = 3 or 4 independent infected plants). C to F The lower leaves of 4-wk-old Col-0, ddl-6, and two DDL complementary lines (proDDL₆₁₈::DDL-EGFP/ddl-6 L2 and L5 plants) were infiltrated with either water, 1 mm NHP in water, 10 mm MgCl₂ (Mock), or a 5 × 10⁶ CFU/mL suspension of Pst, respectively. Two days later, two distal upper leaves of the treated plants were inoculated with a 1 × 10⁵ CFU/mL suspension of Pst. At 3 dpi, the disease symptoms of Pst-infected upper leaves of water and NHP-pretreated plants (C) and Mock and Pst pre-treated plants (E) were photographed. The growth of Pst in infected leaves was quantified at 0 and 3 dpi, as shown in (D) and (F). Bars represent the mean ± SD (n = 3 to 4 independent infected plants). Different letters indicate statistically significant differences in bacterial growth using a two-way ANOVA with a post hoc Tukey's HSD test (P  <  0.05). No significant differences were observed in Pst titers at day 0 across all plant genotypes and treatments, indicated by the same letter a’. White bars in (A), (C) and (E) = 1 cm. The dots on individual bars represent biological replicates for each genotype and treatment, shown from a single independent experiment. These experiments were repeated three times with similar results. CFU, colony-forming unit; dpi, days post-inoculation. The replicate data from independent trials are provided in Supplementary Fig. S9.

To confirm whether the loss of DDL function in Arabidopsis is solely responsible for the compromised NHP-induced systemic resistance, we transformed ddl-6 with a full-length genomic copy of the DDL gene containing the GFP gene fused to the 3′ end, driven by a 618 bp promoter region of the DDL gene (i.e. proDDL₆₁₈::DDL-GFP) (Feng et al. 2016) followed by evaluating vegetative growth and NHP-induced systemic resistance in two independent T3 homozygous lines, L2 and L5. Both complementation lines expressed DDL-GFP protein and displayed wild-type-like vegetative growth (Supplementary Fig. S1, H and I), as well as NHP-induced systemic resistance (Fig. 1, C and D; Supplementary Fig. S9B), demonstrating that DDL-GFP protein rescued the impaired phenotypes of ddl-6 mutant.

In addition, we assessed NHP-induced systemic resistance in another ddl mutant allele, ddl-1, in the Wassilewskija (Ws) background. Because 4-wk-old ddl-1 plants display a severe dwarf phenotype with small leaves (Morris et al. 2006; Feng et al. 2016) (Supplementary Fig. S1, F and G), we irrigated wild-type Ws and ddl-1 with water or 1 mm NHP instead of using the leaf infiltration method. Forty-eight hours after application, three random leaves were inoculated with 1 × 10⁵ CFU/mL suspension of Pst. At 3 dpi, compared to the water-treated Ws plants, two-thirds of Pst-infected leaves of NHP-treated Ws plants showed reduced disease symptoms and lower Pst titers (Supplementary Fig. S3, A and B). In contrast, regardless of whether they received water or NHP treatment, the Pst-infected leaves of ddl-1 exhibited similar bacterial titers and disease symptoms (Supplementary Fig. S3, A and B; Supplementary Fig. S10B). Taken together, our results indicate that loss of DDL function in Arabidopsis leads to a compromised NHP-induced systemic resistance.

DDL is necessary for flg22- and pathogen-induced systemic resistance

To determine whether DDL is also required for the establishment of biological SAR, we applied the bacterial flagellin peptide flg22, which triggers the PTI response, or a bacterial pathogen to induce the SAR response. Three lower leaves of 4 to 5-wk-old Col-0, ddl-6 and two complementation lines of ddl-6 (proDDL₆₁₈::DDL-GFP L2 and L5) were inoculated with either 10 mm MgCl₂ (Mock) or 10 mm MgCl₂ containing a 1 µM flg22 or a 5 × 10⁶ CFU/mL suspension of Pst. At 24 h post-flg22 treatment or 48 h post-Pst inoculation, two upper leaves were challenged with a 1 × 10⁵ CFU/mL suspension of Pst. As expected, infected leaves of Col-0, and two DDL complementation plants treated with flg22 or Pst showed fewer disease symptoms (chlorosis and water soaking) and lower Pst titers at 3 dpi (Supplementary Fig. S3, C and D; Fig. 1, E and F; Supplementary Fig. S9C; Supplementary Fig. S10C), indicating that PAMP and bacterial elicitors trigger systemic resistance in wild-type and complementation lines. Conversely, infected leaves of ddl-6 exhibited susceptible disease symptoms and similar Pst titers despite pretreatment with flg22 or Pst. These results indicate that the DDL gene is also required for flg22-induced systemic resistance and pathogen-induced biological SAR.

DDL is involved in NHP-mediated early signal transduction and local resistance

To assess whether DDL is essential for NHP-induced signal transduction, we quantified the expression patterns of two representative NHP-responsive genes, UDP-DEPENDENT GLYCOSYLTRANSFERASE 76B1 (UGT76B1) and PATHOGENESIS-RELATED GENE 1 (PR1), at 0-, 0.5-, 3-, and 24-h post-treatment (Fig. 2A). To reduce mechanical stress and synchronize NHP application, we conducted the experiment using hydroponically grown Arabidopsis seedlings (Foret et al. 2024). After treating Col-0 seedlings with NHP, the expression level of the UGT76B1 gene was elevated as early as 0.5 h, peaked at 3 h, and then slightly decreased to twice the initial level at 24 h. In contrast, NHP-induced UGT76B1 expression was impaired in the ddl mutants, particularly at early time points (i.e. 0.5 and 3 h). Additionally, DDL mutation also altered NHP-induced PR1 expression at 3- and 24-h post-treatment (Fig. 2A). However, the expression of the DDL gene was not altered in Col-0 at any time points after incubation with NHP (Fig. 2A), suggesting the NHP regulation of DDL may not occur at the transcriptional level. These results suggest that DDL is involved in modulating early NHP-mediated signal responses that affect the magnitude of NHP-dependent transcriptional changes.

Figure 2.

Loss of the DDL gene alters NHP-mediated resistance and gene expression. A) Relative transcript abundance of AtUGT76B1, AtPR1 and AtDDL genes was assessed in 12-d-old Col-0 and ddl mutant seedlings treated with 0.5 mm NHP for 0.5-, 3-, and 24 h. Non-treated seedlings were collected at 0 h as a control set. Each datapoint represents the 2^{mean of (−ΔΔCT)  ±  SEM} of seven biological replicates combined from two independent trials. Four to five seedlings per treatment, genotype, and timepoint were collected as a single biological replicate. Asterisks denote statistical significance as determined by two-tailed Student's t-tests (*, P  <  0.05; **, P  <  0.01; ***, P  <  0.001, ****, P  <  0.0001; NS, not significant). B) NHP-induced local resistance in Col-0 and ddl-6 plants. Three fully expanded leaves of each genotype were sprayed with either water or 0.5 mm NHP. After a 5.5-h incubation at room temperature, the treated leaves were inoculated with 1 × 10⁵ CFU/mL suspension of Pst. The growth of Pst in infected leaves was evaluated at 3 dpi. Bars represent the mean ± SD (n = 15 leaves from 5 independent plants). Different letters indicate statistically significant differences (P  <  0.05) using a two-way ANOVA with a post hoc Tukey's HSD test. The dots on individual bars represent biological replicates for each genotype and treatment, shown from a single independent experiment. These experiments were repeated twice (A) and three times (B) with similar results. CFU, colony-forming unit. The replicate data from independent trials are provided in Supplementary Fig. S10.

To investigate whether the involvement of DDL in the early NHP signaling associated with local immune responses, we assessed Pst growth in the leaves that that were treated with water or NHP for a short period. We sprayed water or 0.5 mm NHP onto the leaf surface of Col-0 and ddl-6 mutant and then inoculated the same leaves 5.5 h later with a 1 × 10⁵ CFU/mL suspension of Pst. We found that NHP was sufficient to induce local resistance in Col-0 plants while there is a marked reduction in resistance in the ddl-6 mutant (Fig. 2B; Supplementary Fig. S10A), though resistance was not entirely lost. These results suggest that there is an alteration of the early NHP signaling in ddl mutants, resulting in impaired immune activation.

DDL and SA biosynthesis are required for NHP-induced systemic resistance

An intact SA biosynthesis pathway is required for NHP-induced systemic resistance establishment (Hartmann et al. 2018). Thus, we hypothesized that DDL and SA biosynthesis may operate in the same pathway to activate NHP-induced systemic resistance. To test this, we generated a double mutant line disrupting both the DDL and the ISOCHORISMATE SYNTHASE1 (ICS1) genes, which encodes a SA biosynthesis enzyme, by crossing the ddl-6 and sid2-2 mutants (Supplementary Fig. S4, A and B). Similar to the ddl-6 mutant, the delayed growth was also observed in ddl-6 sid2-2 mutant, suggesting that impaired development dominantly results from DDL mutation. The extent of NHP-induced systemic resistance was assessed in Col-0, the single mutants ddl-6 and sid2-2, and the double mutant ddl-6 sid2-2 (Fig. 3, A and B; Supplementary Fig. S11A). As expected, NHP-induced systemic immunity was strongly exhibited in the Col-0 plants but was largely impaired in ddl-6, sid2-2, and ddl-6 sid2-2 mutants. Notably, the mutation of both the DDL and SA biosynthesis genes did not lead to a synergistic or additive compromise of systemic immunity, suggesting that SAR regulated by DDL and SA may operate within the same signaling pathway.

Figure 3.

DDL and SA biosynthesis function within the same pathway during SAR, with DDL potentially acting upstream of SA signaling. A and B) NHP-induced systemic immunity in Col-0, ddl-6, sid2-2, and ddl-6 sid2-2 plants. The lower leaves of 4-wk-old plants were infiltrated with water or 1 mm NHP followed by 48 h incubation, then the upper leaves were inoculated with 1 × 10⁵ CFU/mL suspension of Pst. At 3 dpi, the disease symptoms of Pst-infected upper leaves were photographed as shown in (A). The growth of Pst in upper infected leaves was quantified as shown in (B). White bar = 1 cm. Bars represent the mean ± SD (n = 4 independent infected plants). C) SA-induced local resistance in Col-0, ddl-6, and npr1-1 plants. Three fully expanded leaves of each genotype were sprayed with either 0.5% ethanol or 0.5% ethanol containing 0.5 mm SA. After a 5.5-h incubation at room temperature, the leaves were inoculated with 1 × 10⁵ CFU/mL suspension of Pst. At 3 dpi, the Pst growth in infected leaves was evaluated. Bars represent the mean ± SD (n = 12 leaves from 4 independent plants). Different letters indicate statistically significant differences (P  <  0.05) using a two-way ANOVA with a post hoc Tukey's HSD test. The dots on individual bars represent biological replicates for each genotype and treatment, shown from a single independent experiment. All experiments were repeated three times with similar results. EtOH, ethanol; SA, salicylic acid; CFU, colony-forming unit. The replicate data from independent trials are provided in Supplementary Fig. S11.

To test whether DDL is required for SA-mediated signaling transduction, we evaluated SA-mediated resistance in the fully expanded leaves of Col-0 and ddl-6 plants by spraying 0.5 mm SA and subsequently inoculating the same leaves with Pst. Because NPR1 is a key regulator of SA signaling and NHP-mediated immunity (Cao et al. 1997; Yildiz et al. 2021), we included the npr1-1 mutant in our experiment (Supplementary Fig. S4C). Notably, exogenous application of SA markedly induced local resistance in both Col-0 and ddl-6, but not in npr1-1 leaves (Fig. 3C; Supplementary Fig. S11B). Our results therefore suggest that, unlike the essential role of NPR1 in the SA signal pathway, DDL is dispensable for SA-induced resistance and likely operates upstream of SA in NHP-mediated signal transduction.

The accumulation of NHP, SA and their derivatives is altered in ddl-6 plants

The accumulation of NHP, SA, and their glycosylated derivatives modulates SAR activation (Hartmann and Zeier 2019; Huang et al. 2020; Holmes et al. 2021). We hypothesized that DDL is required for the accumulation of one or more of these derivatives. To test this hypothesis, we applied ultra-performance liquid chromatography-tandem mass spectrometry (UPLC)-MS/MS analysis to quantify the accumulation of NHP metabolites [i.e. free NHP and NHP-glucoside (NHP-Glc)] and SA metabolites [i.e. free SA and SA-glucoside (SA-Glc)] to determine whether metabolite accumulation differed in ddl-6 plants compared to Col-0 plants following Pst infection. First, we evaluated each metabolite's retention times and MS2 spectra from a standard solution or extracts of NHP-treated Arabidopsis leaves. These data showed that the MS/MS fragmentation patterns of NHP and SA from extracts of NHP-treated Arabidopsis leaves were consistent with the pattern from the standard solution (Supplementary Fig. S5; Table 1).

Table 1.

MRM conditions of the Sciex QTRAP 6500+ system

Compound	Retention time (min)	Precursor (m/z)	Product (m/z)	Component Type	DP (V)	EP (V)	CE (V)	CXP (V)
NHP	2.8	146	128	Quantifier	35	10	13.5	15
	2.8	146	100	Qualifier	35	10	16	12
NHP-Glc	8	308	146	Quantifier	20	10	30	20
	8	308	128	Qualifier	20	10	30	20
SA	13.15	137	93	Quantifier	−25	−9	−22	−12
	13.15	137	65	Qualifier	−25	−9	−39	−9.5
SA-Glc	10.8	299	137	Quantifier	−20	−10	−30	−20
	10.8	299	93	Qualifier	−20	−10	−30	−20
SA-d4 IS	13.3	141	97	Quantifier	−33	−10	−22	−13
	13.3	141	69	Qualifier	−33	−10	−40	−7

The MRM ion pairs and the applied voltages are shown. The peak area of quantifiers in pairs was calculated to plot all LC-MS/MS results, except the peak area of the qualifier was used for NHP. DP: Declustering Potential; EP: Entrance Potential; CE: Collision Energy; CXP: Collision Cell Exit Potential. NHP, N-hydroxy-pepcolic acid; NHP-Glc, NHP-glucoside; SA, salicylic acid; SA-Glc, SA-glucoside; SA-d4, deuterium labeled Salicylic acid.

Subsequently, we analyzed the metabolite accumulation of local leaves of Col-0 and ddl-6 plants treated with 10 mm MgCl₂ (Mock), or 10 mm MgCl₂ containing a 5 × 10⁶ CFU/mL suspension of Pst, along with distal, untreated leaves of the same plants at 48 h post-treatment (Fig. 4A). As expected, the levels of NHP, NHP-Glc, free SA, and SA-Glc were significantly elevated in Pst-treated Col-0 leaves compared to Mock-treated Col-0 leaves (Fig. 4B). A similar accumulation pattern of NHP, NHP-Glc, and SA-Glc was observed in ddl-6 leaves treated with Pst compared to Mock. However, Pst-induced free SA levels were significantly lower in treated ddl-6 leaves compared to Col-0 (Fig. 4B). These data indicate that the DDL mutation results in a lower SA accumulation in Pst inoculated leaves and this association with the increased susceptibility of the ddl mutants following Pst infection (Supplementary Fig. S2).

Figure 4.

DDL mutation changed the accumulation of free SA levels in local treated and distal untreated leaves of pst-inoculated plants. A) Experimental design of metabolite analysis. Three leaves of 4-wk-old Col-0 and ddl-6 plants were infiltrated with either 10 mm MgCl₂ (Mock), or a 10 mm MgCl₂ containing 5 × 10⁶ CFU/mL suspension of Pst, respectively. After 48 h of Pst inoculation, the local, treated leaves and distal, untreated leaves were harvested for metabolite analysis using UPLC-MS/MS. B and C) The relative abundance of NHP, free SA, NHP-Glc and SA-Glc were analyzed in treated and untreated leaves of Mock and Pst inoculated Col-0 and ddl-6 plants. The MRM ion pairs and retention time of each metabolite are shown in Table 1. The bars represent the mean ± SD of 22 biological replicates (dots on the bars), which were combined from six independent trials showing similar trends. Different letters indicate statistically significant differences (P  <  0.05) using a two-way ANOVA with a post hoc Tukey's HSD test.

Unexpectedly, the accumulation of NHP, SA, and their glycosylated derivatives in the distal untreated leaves was higher in the ddl-6 mutant than those in Col-0 following local Pst treatment (Fig. 4C). We found that this may be due to higher endogenous levels of these molecules accumulating in the distal, untreated leaves of mock-treated ddl-6 mutants compared to Col-0. These data indicate that the regulation of metabolite synthesis, accumulation and/or turnover is altered in systemic tissues of ddl-6 mutant compared to Col-0. Nevertheless, this result suggests that the altered accumulation of NHP and SA derivatives in systemic tissues of ddl-6 mutant at 48 h post Pst infection is still insufficient to activate the downstream signal or limit Pst growth during SAR.

NHP-mediated transcriptional reprogramming depends on the DDL gene

Our metabolic data suggest that SAR deficiency in ddl-6 may stem from defective NHP sensing or NHP-mediated downstream signaling, rather than impaired biosynthesis or transmission of NHP molecule. Previous studies have reported that NHP applied exogenously to Arabidopsis wild-type leaves activates SAR-associated gene transcription in systemic foliage, but this transcription is compromised in SAR-deficient mutants (Yildiz et al. 2021; Yildiz et al. 2023; Foret et al. 2024), indicating that NHP-mediated transcriptional reprogramming is essential for SAR establishment. Therefore, we hypothesized that the compromised SAR in the ddl-6 mutant may result from the impaired NHP-mediated gene expression regulated in local, treated leaves, leading to weakened signal propagation to distal, untreated leaves.

To test this hypothesis, we compared the transcriptome profiles of the local Col-0 and ddl-6 leaves infiltrated with water or 1 mm NHP and distal untreated leaves of the same plants after 24 h of treatment using RNA sequencing analysis (RNA-seq). Principal component analysis (PCA) revealed distinct gene expression patterns in NHP and water treatments in local, treated and distal, untreated leaves of Col-0, whereas ddl-6 mutant exhibited only minor changes between two treatment (Supplementary Fig. S6). There was a notable change in gene expression patterns in the water infiltrated ddl-6 leaves compared to Col-0 (Fig. 5; Supplementary Table S1). These mRNA profiling data support the hypothesis that NHP-mediated gene expression is altered in ddl-6 mutant. They also reveal that DDL mutation affects global gene expression unlinked to NHP-mediated processes.

Figure 5.

Mutation of the DDL gene alters Arabidopsis NHP-mediated gene expression in local treated leaves and distal untreated leaves. Lower leaves (i.e. local leaves) of 4-wk-old Col-0 and ddl-6 were infiltrated with water or 1 mm NHP. After incubation for 24 h, the local treated and distal untreated leaves were harvested for RNA extraction and RNA-Seq analysis. Three biological replicates per treatment and genotype were used for RNA-Seq. A) The heatmap displaying the expression pattern of differential expression genes (DEG) in either Col-0 and ddl-6 under water and NHP treatment. These DEGs are grouped into four clusters based on their expression patterns using hierarchical clustering analysis. The z-score normalization was performed using Log2-transformed TPM (transcripts per million) values. Experimental setup of the RNA-Seq are detailed in Supplementary Table S1. B) The box plots representing the expression pattern of each cluster as categorized in (A). Boxplots show the median (center line), interquartile range (box limits), and whiskers that extend to the smallest value not less than Q1 − 1.5 × IQR and the largest value not greater than Q3 + 1.5 × IQR. Data points beyond these limits are plotted as outliers. Sample sizes for each plot and group are provided in Supplementary Table S1. The value of ΔNHP-Water indicates the difference between the mean of Log2 TPM of NHP- and water- treated samples. The FDR values denote the comparison of average gene expression between water and NHP treatment in Col-0 or ddl-6 using the Wilcoxon Test. FDR values approaching zero are displayed as 0. C) The lollipop charts displaying the top 10 enriched GO biological processes in different clusters, filtered by the level of significance [−log10(FDR)]. Inside the bar charts, the digits denote the values of [−log10(FDR)]. The complete results of the GO enrichment analysis of each gene cluster are shown in Supplementary Table S2.

To identify NHP responsive genes that require DDL function, we analyzed differential gene expression in local, treated leaves and distal, untreated leaves of Col-0 and ddl-6 plants following water or NHP treatment (Col-0_NHP/Col-0_Water and ddl-6_NHP/ddl-6_Water) (Supplementary Table S1). A statistical comparison of differentially expressed genes was conducted by filtering for false discovery rate (FDR) values below 0.05. Upregulated genes were defined as those values with Log₂ fold-change (Log₂ FC) ≥ 1 (2-fold increase), while downregulated genes were defined as Log₂ FC ≤ −1 (2-fold decrease) (Fig. 5A; Supplementary Fig. S7; Supplementary Table S1).

Col-0 exhibited 1,807 NHP-upregulated and 1,676 NHP-downregulated genes in local, treated leaves, whereas ddl-6 showed only 41 NHP-upregulated and 28 NHP-downregulated genes. Similarly, in distal, untreated leaves, 2,662 NHP-upregulated and 1,516 NHP-downregulated genes were observed in Col-0, while the ddl-6 mutant exhibited only 31 NHP-upregulated and 40 NHP-downregulated genes (Supplementary Fig. S7; Supplementary Table S1). These results indicate that the number and identity of NHP-responsive genes in local, treated, and distal, untreated leaves of ddl-6 were significantly reduced compared to Col-0 plants, suggesting that the DDL gene is essential for proper NHP-mediated gene expression and signal transduction.

Mutation of the DDL gene alters the NHP-responsive expression of biotic stress-associated genes

Next, we conducted hierarchical clustering and gene ontology (GO) enrichment analysis to elucidate the expression patterns of NHP-responsive genes in local, treated leaves and distal, untreated leaves of Col-0 and ddl-6 plants, as well as to determine the association of biological processes (BP), molecular function (MF), and cellular component (CC). The NHP-responsive genes were categorized into four groups (Groups A to D), based on their gene expression patterns (Fig. 5A; Supplementary Table S1).

Group A is predominantly composed of NHP-upregulated genes, with few NHP-downregulated genes in Col-0 and a small number of NHP-upregulated genes in ddl-6 (Fig. 5A; Supplementary Fig. S8; Supplementary Table S1). The genes of Group A were upregulated by NHP an average of 4.9-fold in local treated leaves and 2.2-fold in distal untreated leaves of Col-0, but only 1.7-fold and 1.2-fold in ddl-6, respectively (Fig. 5B). GO analysis showed that overrepresented biological processes of Group A genes included defense response to bacterium and fungus, plant-type hypersensitive response, cellular response to hypoxia, protein phosphorylation, response to salicylic acid, SAR, pattern recognition receptor signaling pathway, etc. (Fig. 5C; Supplementary Table S2). Similar to Group A, Group B is primarily composed of NHP-upregulated genes, with a few NHP-downregulated genes in Col-0 and NHP-up or downregulated genes in ddl-6 (Fig. 5A; Supplementary Fig. S8; Supplementary Table S1). The genes in Group B showed an average 3.4-fold and 3.9-fold NHP-upregulation in local treated leaves and distal untreated leaves of Col-0 while only 1.2-fold change in NHP-regulation in ddl-6 mutant (Fig. 5B). Compared to Group A, Group B has more NHP-upregulated genes in distal untreated leaves of Col-0 than in local treated leaves, suggesting a predominant role of Group B genes in the NHP-mediated systemic response (Supplementary Fig. S8). GO analysis revealed that Group B genes participate in cellular response to hypoxia, response to salicylic acid, hydrogen peroxide, jasmonic acid and oxidative stress, priming of cellular response to stress, systemic acquired resistance, camalexin biosynthetic process, etc. (Fig. 5C; Supplementary Table S2).

In contrast to Groups A and B, Groups C and D predominantly consist of NHP-downregulated genes in local, treated and distal, untreated leaves of Col-0 (Fig. 5A, Supplementary Fig. S8; Supplementary Table S1). Group C genes exhibited an average 2-fold NHP downregulation in distal untreated leaves of Col-0, while no significant fold-change was observed between water and NHP treatment in local, treated leaves of Col-0 or in local, treated and distal, untreated leaves of ddl-6 (Fig. 5B). The genes in Group C are associated with biological processes such as response to light stimulus, cold, chloroplast organization, carbohydrate metabolism, circadian rhythm, and fatty acid biosynthesis (Fig. 5C; Supplementary Table S2). In Group D, 92.4% and 25.6% of genes in local treated and distal untreated leaves of Col-0 were downregulated by NHP, with fold-changes of 2.4 and 1.6, respectively (Supplementary Fig. S8). These genes are involved in water transport, response to auxin, plant-type cell wall loosening, photosynthesis, and cell differentiation (Fig. 5C; Supplementary Table S2).

Consistent with previous studies (Yildiz et al. 2021; Yildiz et al. 2023; Foret et al. 2024), our transcriptome data indicate that most NHP-upregulated genes are associated with the response to biotic stress. In contrast, NHP-downregulated genes are involved in responses to photosynthesis, cell development, and light stimuli. Notably, the distal, untreated leaves of the ddl-6 mutant exhibited a reduced response to local NHP signaling 24 h post-treatment, significantly altering the expression of NHP-responsive genes related to immunity, stimuli, and photosynthesis in this mutant. Together, these results suggest that compromised systemic immunity in the ddl-6 mutant following NHP treatment in local tissues may result from the reduced number of NHP-upregulated genes associated with plant immunity and the magnitude of their expression.

DDL is required for NHP-induced gene expression associated with SAR response in distal untreated leaves

Pretreatment with NHP in local tissues triggers transcriptional reprogramming and strengthens the plant's defensive capacity in both locally treated and distal untreated leaves against subsequent pathogen attacks (Yildiz et al. 2021). This enhanced capacity includes increased sensitization to pathogen or SA stimuli, elevated phytoalexin biosynthesis, altered SA/NHP metabolism, and enhanced signal transduction. Next, we investigated whether the DDL mutation reduces the expression of NHP-upregulated genes associated with specific biological processes, including SAR, the SA-mediated signaling pathway, responses to SA or hydrogen peroxide, PTI response, priming of cellular responses to stress, protein phosphorylation, immune response, and the camalexin biosynthetic process (Fig. 5, A and C; Supplementary Tables S1 and S2). We found that these biological processes were significantly overrepresented among a set of NHP-upregulated genes in both local, treated and distal, untreated leaves of Col-0, but only marginally in ddl-6 plants (Fig. 6A).

Figure 6.

Loss of DDL reduces NHP-upregulated genes involved in immune responses and SAR-associated biological processes. A) NHP-upregulated genes associated with SAR, SA signaling, immune responses, PTI response, camalexin biosynthesis, protein phosphorylation and priming response were enriched in treated and untreated Col-0 but reduced in the ddl-6 mutant. B) The heatmap displaying gene expression patterns in the biological categories from A, in treated and untreated leaves of Col-0 and ddl-6 mutants under water and NHP treatments. Different biological categories are represented by various color legends. The complete results of transcriptome dataset and the GO enrichment analysis of each gene cluster are shown in Supplementary Tables S1 and S2, respectively.

Among these NHP-upregulated genes, the genes involved in pattern recognition receptor signaling pathway included STROMAL CELL-DERIVED FACTOR 2-LIKE PROTEIN PRECURSOR (SDF2), ENDOPLASMIC RETICULUM-LOCALIZED DNAJ FAMILY 3B (ERDJ3B), PATTERN-TRIGGERED IMMUNITY (PTI) COMPROMISED RECEPTOR-LIKE CYTOPLASMIC KINASE 1 (PCRK1), RECEPTOR LIKE PROTEIN 23 (RLP23), and LECRK-VI.2 while the genes involved in MAPK cascades included MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE 5 (MAPKKK5), MAP KINASE KINASE 9 (MKK9), MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3) (Fig. 6B). For instance, SDF2 and ERDJ3B form a complex with a luminal-binding protein BiP and required for proper accumulation of pattern-recognition receptor (PRR) EFR (Nekrasov et al. 2009). PCRK1, encoding a receptor-like cytoplasmic kinase, positively contributes to plant immunity and a full PTI response, including callose deposition and ROS burst (Sreekanta et al. 2015). AtRLP23, encoding a leucine-rich repeat receptor protein, form a complex with suppressor of brassinosteroid insensitive 1 (BRI1)-associated kinase (BAK1)-interacting receptor kinase 1 (SOBIR1) and BAK1 to recognize the conserved 20-amino-acid fragment of necrosis and ethylene-inducing peptide 1-like proteins (nlp20) to activate an immune response (Albert et al. 2015; Albert et al. 2019). LECRK-VI.2 positively regulates PTI response by interacting with FLAGELLIN SENSING2 (FLS2) (Huang et al. 2014) as well as being associated with BAK1 to regulate eNAD(P) signaling and NHP-mediated systemic immunity (Wang et al. 2019; Li et al. 2023). MPK3 and MPK6 proteins positively regulate the induction of systemic acquired resistance through a positive feedback loop involving WRKY33, AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 (ALD1), and the accumulation of pipecolic acid (Pip) (Wang et al. 2018). Additionally, MAPKKK5 is phosphorylated by MPK6 to enhance MPK3/6 activation and promote plant immunity (Bi et al. 2018). Except for BAK1 and MAPKKK5 (Fig. 6B), the NHP-induced transcript levels of these genes were significantly reduced in the distal, untreated leaves of the ddl-6 mutant, suggesting that NHP-enhanced pathogen recognition and early signal transduction were attenuated by this mutation.

At the level of transcription factors, we found that NHP-mediated upregulation of WRKY and TGA transcription factors in distal untreated leaves was weakened by DDL mutation, including WRKY38, WRKY46, WRKY54, WRKY70, TGA1, TGA3, and TGA5 (Fig. 6B). NHP-induced systemic resistance and transcriptional reprogramming requires the function of TGA2/5/6 and are largely dependent on TGA1/4 (Yildiz et al. 2023). WRKY70 is required for NHP-induced systemic immunity, gene expression, and flg22-induced ROS production (Foret et al. 2024).

Moreover, we found that the NHP-induced genes involved in SA and NHP metabolism, SA perception, immune response, priming response and camalexin biosynthesis were suppressed in the ddl-6 mutant compared to Col-0 (Fig. 6B). Supporting our transcriptome data, RT-qPCR results indicated that the selected genes associated with the aforementioned biological processes were strongly upregulated in the distal, untreated leaves of Col-0 plants. In contrast, their induction was diminished or attenuated in the ddl-6 mutant (Fig. 7). These findings demonstrate that DDL is essential for the full activation of the NHP-induced systemic immune response, including early pathogen detection, signal transduction, transcriptional regulation, and the biosynthesis and homeostasis of defense-related metabolites.

Figure 7.

DDL is required for NHP-induced SAR-associated gene expression in distal and untreated leaves. Relative transcript abundance of NHP-upregulated genes selected from Fig. 6B in water or 1 mm NHP treated leaves of 4-wk-old Col-0 and ddl-6. Three lower leaves of all genotypes were infiltrated with water or 1 mm NHP. One day later, the distal, untreated leaves were harvested for RT-qPCR analysis. The relative expression was calculated by normalizing the Δcycle threshold (CT) values of all samples to that of water-treated Col-0. Bars represent the 2^{mean of (−ΔΔCT)  ±  SEM} of three biological replicates for Col-0 and four biological replicates for ddl-6. Different letters indicate statistically significant differences (P  <  0.05) using a two-way ANOVA with a post hoc Tukey's HSD test. These experiments were repeated three times with similar results.

DDL is involved in PR1 induction by the combined NHP treatment and Pst challenge

Application of a high concentration of NHP to local tissue significantly upregulated PR1 transcript levels in distal, untreated, and pathogen-free leaves, and these levels were further elevated following bacterial pathogen infection (Yildiz et al. 2021), indicating that NHP-induced defense priming in distal tissues regulates PR1 gene expression. To determine if DDL is required for this response, we examined PR1 expression levels in ddl-6 mutant after bacterial pathogen challenge in plants pre-treated with NHP. After infiltrating water or NHP into local leaves, the distal leaves were inoculated with a 5 × 10⁶ CFU/mL suspension of Pst (Fig. 8A). Transcript levels of the PR1 gene were examined at 8 and 24 hpi. At 8 h post Pst inoculation, NHP pretreatment significantly enhanced PR1 expression in Col-0 compared to water pretreatment; however, this effect was not observed in the ddl-6 mutant (Fig. 8B). Notably, the differential regulation of Pst-induced PR1 expression by NHP pretreatment between Col-0 and the ddl-6 mutant was no longer observed at 24 hpi. These results suggest that DDL modulates signaling events during both the establishment of NHP-mediated systemic response (Fig. 7) and the response to a secondary bacterial challenge (Fig. 8B).

Figure 8.

DDL is involved in the induction of PR1 gene expression in response to Pst challenge in NHP-primed plants. A) Experimental design of NHP-inducible defense priming assay. B) Evaluation of PR1 gene expression in plants infiltrated with water or NHP following a bacterial elicitor rechallenge. Three lower leaves (local) of 4-wk-old Col-0 and ddl-6 plants were infiltrated with water or 1 mm NHP as the primary (1°) treatment. After 24 h incubation, the upper leaves (distal) were inoculated with a 5 × 10⁶ CFU/mL suspension of Pst in 10 mm MgCl₂ as the secondary (2°) followed by harvesting the inoculated leaves at 8 and 24 hpi for RT-qPCR analysis. The relative expression was calculated by normalizing the Δcycle threshold (CT) values of all samples to that of water-treated Col-0 upon Pst inoculation. Each bar represents the 2^{mean of (−ΔΔCT)  ±  SEM} of 13 biological replicates (8 hpi) and 10 biological replicates (24 hpi), respectively, combined from three independent trials. Each trial included at least three biological samples. Different letters on bars indicate statistically significant differences (P  <  0.05) using a two-way ANOVA with a post hoc Tukey's HSD test.

Discussion

Plants require the mobile signal, NHP, to activate systemic immunity (Chen et al. 2018; Hartmann et al. 2018; Schnake et al. 2020; Mohnike et al. 2021; Xu et al. 2025), but how NHP establishes systemic resistance in plants is uncertain. In this study, we discovered a role for the Arabidopsis DAWDLE (DDL) protein in modulating the NHP signaling response, which is essential for systemic immunity induced by NHP, flg22 peptide, and Pst (Fig. 1; Supplementary Fig. S3; Fig. 2; Supplementary Fig. S9; Supplementary Fig. S10).

Our transcriptome data and RT-qPCR analyses suggest that DDL participates in the regulation of NHP-induced transcripts associated with PTI, the biosynthesis and metabolism of defense-related metabolites and SAR response in both local-treated and distal untreated leaves (Figs. 5, 6). A drastic reduction in the mRNA levels of NHP-responsive genes was observed in the systemic leaves of the ddl-6 mutant (Figs. 7, 9A), suggesting that DDL plays a major role in NHP-mediated signal communication between local and systemic tissues. In addition, no additive reduction in NHP-mediated systemic resistance was observed in plants carrying mutations in both DDL and the SA biosynthesis gene, whereas DDL is dispensable for SA-induced resistance (Fig. 3; Supplementary Fig. S11), suggesting that DDL may function in the same NHP-mediated SA pathway and likely acts upstream of SA signaling. Furthermore, DDL is required for modulating Pst-induced free SA accumulation in both inoculated and untreated leaves (Fig. 4), suggesting that DDL contributes to free SA accumulation by regulating either SA biosynthesis or modification. After Pst challenge, the DDL mutation primarily alters PR1 upregulation in NHP-treated plants at 8 hpi (Figs. 8, 9B), suggesting that DDL plays a role in regulating not only NHP-mediated signal transduction but also the response following pathogen challenge. Together, these results suggest that DDL regulates NHP-responsive gene expression in locally treated tissues, thereby facilitating the systemic transmission of NHP-dependent signals to distal leaves.

Figure 9.

The role of DDL in NHP-mediated signal and systemic resistance. This illustration summarizes our findings regarding the impact of Arabidopsis DAWDLE (DDL) on NHP-mediated SAR. A) DDL contributes to NHP-mediated transcriptional reprogramming. Upon exogenous NHP treatment, genes associated with PTI responses (e.g. receptor-like kinases and MAPK signaling), transcription factors, SA signaling, SA and NHP metabolism, immune response, priming response, and SAR are highly upregulated in local-, treated and distal-, untreated leaves of wild-type plants. This NHP-activated transcriptional response significantly enhances the capacity for subsequent signal perception and transmission in systemic leaves, leading to increase resistance to secondary pathogen infection. However, DDL mutation reduces the magnitude of these gene expression in local treated leaves and significantly diminishes the transcriptional response in distal untreated leaves, ultimately exhibiting susceptibility to subsequent pathogen infection. B) DDL is involved in mediating NHP-induced PR1 transcription and in promoting the enhancement of PR1 transcripts upon Pst challenge. Pink-marked DDL: involved in regulation.

DDL participates in NHP-mediated signal transduction and in the elevated expression of PR1 gene in the post-challenge primed state

The establishment of induced resistance (IR) is the sum of direct and primed defense responses (De Kesel et al. 2021). Direct defense responses refer to strong immune activation occurring in the absence of a subsequent pathogen challenge. In contrast, the primary outcome of primed defense responses is a faster and stronger immune activation upon pathogen challenge in IR-treated plants, compared to that in non-IR plants (Martinez-Medina et al. 2016; Mauch-Mani et al. 2017; De Kesel et al. 2021).

Exogenous NHP alone is sufficient to induce transcriptional reprogramming, leading to the upregulation of genes associated with pattern recognition receptors, regulatory components of immune signal transduction, and enzymes involved in defense-related metabolic pathways (Fig. 6) (Yildiz et al. 2021; Yildiz et al. 2023). This response triggers strong immune activation in the absence of pathogens and enhances defense responses upon subsequent pathogen challenge (Hartmann and Zeier 2019; Yildiz et al. 2021). The DDL mutation significantly impaired the NHP-induced systemic response, including transcriptional regulation and defense activation (Fig. 1; Supplementary Fig. S9; Supplementary Fig. S10; Figs. 6, 7). In addition to its involvement in NHP signal transduction (Fig. 2A), DDL regulates the NHP priming effect on enhanced PR1 expression during pathogen infection (Fig. 8B). The impaired NHP-mediated gene regulation in the systemic leaves of the ddl mutant, both before and after Pst challenge, leads to a reduction in Pst- and NHP-induced systemic resistance. Further studies are needed to clarify how DDL regulates gene expression in response to NHP treatment and Pst inoculation. Moreover, a recent study indicated that NHP pretreatment enhances flg22-induced ROS generation, and this enhancement requires a functional WRKY70 transcription factor (Foret et al. 2024). The observation that the DDL mutation reduces the expression of a set of NHP-upregulated genes associated with PTI responses, as well as WRKY70 in distal, untreated leaves (Figs. 6, 7), suggests that DDL may play a role in regulating NHP-enhanced PAMP-induced ROS production or other immune responses, which requires further investigation.

The role of DDL in SA and NHP pathway

Loss of DDL results in reduced sensitivity to exogenous NHP (Fig. 2B), but not to SA (Fig. 3B; Supplementary Fig. S11A), suggesting that DDL functions specifically downstream of NHP signaling. However, mutation of both DDL and SA biosynthesis genes did not lead to a more severe SAR defect, suggesting they may function in the same pathway and DDL potentially acts upstream of SA signaling. This supports our observation that free SA levels were reduced in Pst-inoculated leaves of ddl-6 (Fig. 4B). In addition, the DDL mutation led to enhanced accumulation of SA and NHP in uninoculated distal leaves (Fig. 4C). These results suggest that DDL may play a role in both the biosynthesis and modification of these molecules during development or in response to pathogen infection. Moreover, Col-0 exhibited a greater fold induction (Pst/Mock) of SA, NHP, and their glycosylated derivatives compared to ddl-6. Whether the DDL-mediated induction magnitude of these molecules affects downstream signal activation remains to be clarified.

The possible roles of DDL in regulating NHP-mediated transcriptional reprogramming

There are still several outstanding questions regarding the role of DDL in NHP-mediated gene expression and how NHP regulates DDL. Arabidopsis DDL has been shown to play essential roles in response to Pst when post-translationally modified with poly-ADP-ribosylation and in plant development through the regulation of small RNA biogenesis (Yu et al. 2008; Feng et al. 2016). Given the importance of PARylation of DDL in response to Pst (Feng et al. 2016), we propose that NHP may regulate the PARylation and/or phosphorylation of DDL resulting in the dynamic changes of NHP-induced transcriptional regulation. Previous studies reported that PARylated proteins participate in several gene regulatory pathways (e.g. chromatin modulation and DNA methylation, acting as transcriptional co-regulators) to regulate gene transcription (Kraus and Hottiger 2013). In addition, loss of PARP2 in Arabidopsis leads to impaired Pst-activated PR1 protein accumulation, flg22- and SA-induced resistance (Yao et al. 2021), providing a link between PARylation and induced resistance. On the other hand, while current evidence only suggests the necessity of DDL-mediated small RNA biosynthesis for plant development (Yu et al. 2008), a recent study raises the possibility that small RNA biosynthesis could regulate systemic immunity (Shine et al. 2022). Therefore, it will be interesting to determine whether and how DDL–mediated small RNA pathways regulate NHP–induced systemic responses.

In conclusion, our findings reveal that Arabidopsis DDL is a key component involved in NHP-mediated transcriptional regulation and systemic resistance.

Materials and methods

Plant growth conditions

Arabidopsis plants were grown in a FH-740 plant growth chamber (Taiwan Hipoint, Kaohsiung, Taiwan) equipped with Z190 LED sunlight, under a light intensity (photosynthetic photon flux density, PPFD) of 280–330 µmol·m⁻²·s⁻¹, at 22 °C, 55–60% relative humidity and a 10-h light/14-h dark photoperiod. Seeds were surface-sterilized with 75% ethanol and diluted Clorox bleach, then rinsed five times with sterile double-distilled water (ddH₂O). Sterilized seeds were either directly planted in an 8:1:1 mixture of Jiffy substrates, vermiculite, and perlite, or sown on half-strength (0.5X) Murashige and Skoog (MS) medium containing 1% sucrose, 0.05% (w/v) MES, and 0.8% Phytagel (Sigma, P8169) at pH 5.7 before being transferred to the Jiffy substrate mixture. All plants were fertilized once per week using Hyponex No.2 fertilizer, and irrigated with the appropriate amount of tap water every 2 d. Unless stated otherwise, all experiments were conducted using 4-to 5-wk-old plants.

The following Arabidopsis plants were used in this study: Columbia wild-type (Col-0), a kind gifted from Dr. Mary Beth Mudgett; and Wassilewskija wild-type (Ws; CS28823), npr1-1 (CS3726), sid2-2 [CS16438; (Wildermuth et al. 2001)], and four ddl T-DNA mutants ddl-1 (CS6932; in Ws background) (Morris et al. 2006; Yu et al. 2008), ddl-6 (SALK_045025C), ddl-7 (SAIL_1281_F08) (Feng et al. 2016) and SALKseq_078790.1 obtained from the Arabidopsis Biological Resource Center (Ohio State University, USA). The ddl-6 sid2-2 double mutant was generated by crossing ddl-6 and sid2-2. The homozygous F2 of ddl-6 sid2-2 was identified by PCR using specific primer sets. As described in Yildiz et al. 2021, 905-bp PCR product was amplified in Col-0 but not in sid2-2. F2 and F3 generation was used for SAR assay in this study. The genotype of npr1-1 was via a cleaved-amplified polymorphic sequence (CAPS) marker (Supplementary Fig. S4C), as described in Yildiz et al. 2021. The T-DNA insertion and DDL transcript accumulation in ddl mutants were confirmed using the specific primer sets. All specific primers are listed in Supplementary Table S3. DDL complementary lines were generated in this study as described in the cloning section.

Bacterial strains and growth conditions

Growth conditions of bacterial strains used in this study: Pseudomonas syringae pv. tomato DC3000 (Pst) was grown at 28 °C on nutrient yeast glycerol agar (NYGA) (Kim et al. 2016) containing 100 μg/mL rifampicin; Agrobacterium tumefaciens strain GV3101 (A. tumefaciens GV3101) was grown at 28 °C on Lysogeny broth (LB) medium containing 50 μg/mL rifampicin, 50 μg/mL gentamicin, and 50 μg/mL kanamycin for transformation selection; and Escherichia coli strain DH5 alpha (E. coli DH5 alpha) was grown at 37 °C on LB medium containing 100 μg/mL spectinomycin or 50 μg/mL kanamycin for transformation selection.

Assessment of plant susceptibility to pst under different humidity

To assess the humidity-mediated basal immunity in Col-0 and ddl mutants, the full expanded leaves of 4-wk-old plants were inoculated with a 1 × 10⁵ CFU/mL suspension of Pst [(OD600) = 0.0001] in 10 mm MgCl_2. The inoculated plants were either covered with a lid and sealed with plastic wrap to create a high-humidity environment (86% to 95%) or incubated without a lid in the growth chamber under ambient humidity conditions (55% to 60%). The growth of Pst in inoculated leaves was quantified at 3 dpi, as described in the section on quantification of bacterial growth in planta.

NHP- and SA-induced local resistance assay

In the NHP-induced local resistance assay, the adaxial and abaxial surface of three fully expanded leaves per Col-0 and ddl-6 plants were sprayed with about 600 to 700 μl of water or 0.5 mm NHP (MedChemExpress, HY-N7378), then incubated for 5.5 h. For the SA-induced local resistance assay, three fully expanded leaves per Col-0 and ddl-6 plants were sprayed with 0.5% ethanol, 0.5 mm salicylic acid (Sigma, S7401) in 100% ethanol, then incubated for 5.5 h. The treated leaves were inoculated with a 1 × 10⁵ CFU/mL suspension of Pst using a needless syringe. The growth of Pst in inoculated leaves was quantified at 3 dpi, as described in the section on quantification of bacterial growth in planta.

NHP-, bacterial-and PAMP-induced systemic resistance assays

For NHP-, bacterial-and PAMP-induced systemic resistance by leaf infiltration, leaves were numbered according to the previously described protocol (Mousavi et al. 2013). Three lower leaves (leaves 7 to 9 of Col-0 and DDL complementary lines; leaves 6 to 8 from ddl mutants) of 4- to 5-wk-old plants were infiltrated with autoclaved distilled water or 10 mm MgCl₂ as a control set. For elicitor treatments, 1 mm NHP in water, and 10 mm MgCl₂ containing a 5 × 10⁶ CFU/mL [optical densities at 600 nm (OD600) = 0.005] suspension of Pst, or 1 µM peptide flg22 (QRLSTGSRINSAKDDAAGLQIA; synthesized by GenScript) were infiltrated. Twenty-four or forty-eight hours later, two distal leaves (leaves 11 and 12 of Col-0 and leaves 10 and 11 of ddl mutants) were inoculated with a 1 × 10⁵ CFU/mL suspension of Pst using a needless syringe.

For NHP treatment by irrigation, 4-wk-old Ws and ddl-1 plants were drenched with tap water and dried in new trays 1.5 d before chemical treatment. Ten milliliters of autoclaved water or 1 mm NHP were respectively irrigated onto the soil surface of each pot. At 48 h post chemical treatment, three young leaves of Ws (2 to 3.5 cm leaf size) and ddl-1 (1.5 to 2 cm leaf size) were inoculated with a 1 × 10⁵ CFU/mL suspension of Pst. The growth of Pst in inoculated leaves was quantified at 3 dpi, as described in the section on quantification of bacterial growth in planta.

Quantification of bacterial growth in planta

Unless mentioned otherwise, all Pst inoculated plants were watered and covered with lids to maintain high humidity conditions (86% to 95%). One hour later at day 0, the titer of Pst was quantified in an inoculated leaf of each plant. At 3 dpi, disease symptoms of three Pst -inoculated leaves from each plant were photographed, and Pst growth was quantified. Three leaf discs (5 mm) from one inoculated leaf, or 3 leaf discs pooled from three different inoculated leaves were homogenized in 1 mL of 10 mm MgCl₂ at 0 or 3 dpi. Homogenized samples were diluted and then plated on NYGA medium with 100 µg/mL rifampicin. Plates were then incubated at 28 °C for 2 d to count bacterial colonies. At least 3 plants were used in independent experiments, and each experiment was repeated at least three times.

Assessment of Pst-induced PR1 gene expression under NHP treatment

The priming of bacterial pathogen response was conducted in NHP-infiltrated plants. Three lower leaves of Col-0 and ddl-6 plants were infiltrated with distilled water or 1 mm NHP as the primary treatment. Twenty-four hours later, two upper leaves were inoculated with a 5 × 10⁶ CFU/mL suspension of Pst [(OD600) = 0.005] in 10 mm MgCl₂ as the secondary treatment. Inoculated leaves were harvested at 8 and 24 hpi for RNA extraction and RT-qPCR analysis.

RNA extraction, cDNA synthesis and quantitative RT-PCR

Total RNA was isolated from Arabidopsis leaves using miRNeasy Micro Kit (QIAGEN, 217004). Genomic DNA was removed by RNase-Free DNase Set (QIAGEN, 79254). RNA (2.5 µg) was synthesized to cDNA by Oligo d(T) 18 mRNA primer (NEB, S1316S) and SuperScript IV Reverse Transcriptase (Invitrogen, 18090050). Each cDNA sample was amplified with gene-specific primers as shown in Supplementary Table S3 using 2× qPCRBIO SyGreen Blue Mix Lo-ROX (PCR Biosystems) and the CFX Connect Real-Time PCR System (Bio-Rad). Two technical repeats per sample were carried out for each reaction. The mRNA abundance of AtUBC21 (Ubiquitin-Conjugating Enzyme 21; At5g25760) was used as a reference gene to normalize the expression value. The relative gene expression was determined by comparative Ct method (2−ΔΔCt) (Livak and Schmittgen 2001; Schmittgen and Livak 2008; Taylor et al. 2019). The fold-change was calculated by normalizing each value to that of water- or Mock-treated leaves of Col-0 or untreated Col-0 seedling.

Sample preparation, library construction and sequencing of RNA-Seq

Four lower leaves of 4-wk-old Col-0 and ddl-6 were infiltrated with autoclaved distilled water or 1 mm NHP, respectively. Twenty-four hours later, the lower treated leaves (local) and upper untreated leaves (distal) were harvested for RNA-seq analysis. Each biological replicate consisted of eight treated or eight untreated leaves pooled from two independent plants. Three biological replicates per treatment and genotype were analyzed in RNA-seq. The total RNA was isolated using miRNeasy Micro Kit (Qiagen, 217004). Genomic DNA removal was carried out by RNase-Free DNase Set (Qiagen, 79254). Purified RNA was quantified at OD260 nm by using a ND-1000 spectrophotometer (Nanodrop Technology, USA) and then qualified by using a Bioanalyzer 2100 (Agilent Technology, USA) with RNA 6000 lab chip kit (Agilent Technologies, USA). Purified RNA samples were submitted to Welgene Biotech for library preparation and sequencing. Briefly, all RNA sample preparation procedures were performed according to Illumina's official manual. The library was constructed by SureSelect XT HS2 mRNA Library Preparation kit (Agilent, USA) followed by AMPure XP beads (Beckman Coulter, USA) size selection. All libraries were sequenced using Illumina NovaSeq 6000 (Illumina, USA). FASTQ sequence data (150 bp paired end) were generated using Welgene Biotech's pipeline based on Illumina's basecalling program bcl2fastq v.2.20 (Illumina, USA). All raw sequence reads data in this study were deposited to the Sequence Read Archive (SRA) of National Center for Biotechnology Information (NCBI) database (BioProject accession number: PRJNA1255381).

Leaf extraction for metabolism profiling

The extraction method was modified based on previous publication (Chen et al. 2018; Hartmann et al. 2018). Briefly, the harvested leaves in Eppendorf tubes were frozen in liquid nitrogen and then homogenized to fine powder using 5 mm stainless steel beads in a ball mill (Qiagen, Germany) at 22 Hz for 1 min. A solution of 80% MeOH aqueous with 2 µM deuterated-SA (Salicylic acid-d6; Sigma, 616796) (100 mg sample per 500 μl solution) was added into the tubes for extraction on a rotator at 160 rpm and 4 °C for 10 min. Sample solutions were centrifuged at 13,000 rpm for 10 min and the supernatants were collected. The extraction process was repeated twice. The supernatant solutions were filtered through 0.22 µm polytetrafluoroethylene (PTFE) membrane filters and dried using a refrigerated vapor trap (Savant RVT5105) with SpeedVac Concentrator (Savant SPD121P) for 5 h. The dried samples were rehydrated in ddH₂O (with 1/10 volume of the initial extraction buffer per sample) and vortexed for 3 min. The solutions were filtered using 0.2 µm filters (CHROMAFIL AO-20/3) and subjected to ultraperformance liquid chromatography-triple quadrupole mass spectrometer (UPLC-MS/MS) analysis (Supplementary Methods).

Other methods

Generation of the DDL complementary transgenic line, immunoblot analysis, sample preparation for RT-qPCR and metabolite profiling, data processing of RNA sequencing, and the details of UPLC-MS/MS analysis are described in the Supplementary Methods.

Statistical analyses

Two-tailed Student's t-tests in Microsoft Excel or R were used to determine statistically significant differences between indicated groups based upon the null hypothesis. P values of less than 0.05 were considered statistically significant, and significance levels are denoted in the figure legends (*, P  <  0.05; **, P  <  0.01; ***, P  <  0.001, ****, P  <  0.0001; NS, not significant). One-way analysis of variance (ANOVA) with post hoc LSD was performed by the aov function in the stats package and the LSD.test function from the agricolae package in R to compare statistically significant differences among multiple groups (de Mendiburu 2023). Two-way analysis of variance (Two-way ANOVA) with post hoc Tukey's HSD test was performed to compare statistically significant differences among multiple groups using the aov function in the stats package and HSD.test function in the agricolae package in R.

Letters shown in the figures represent statistically significant differences. All statistical testing results are shown in Supplementary Table S4.

Accession numbers

Sequence data used in this article can be found in The Arabidopsis Information Resource database (http://www.arabidopsis.org) under accession numbers:AT1G19250 (FMO1), AT1G64280 (NPR1), AT1G73805 (SARD1), AT1G74710 (ATICS1), AT2G14610 (PR1), AT2G32680 (AtRLP23), AT2G37620 (ACTIN 1), AT3G11340 (UGT76B1), AT3G20550 (DDL), AT3G26830 (PAD3), AT3G45640 (MAPK3), AT3G56400 (WRKY70), AT3G57260 (PR2), AT3G62250 (UBQ5), AT5G01540 (LECRK-VI.2), AT5G25760 (UBC21), AT5G26920 (CBP60G).

Author contributions

J.-K.H., H.-Y.H., C.-H.H., C.-Y.H., S.-F.H. and Y.-C.C. performed bacterial growth assays. J.-K.H., H.-Y.H., C.-Y.H., S.-F.H., Y.-C.L., and Y.-C.C. performed RT-qPCR analysis. J.-K.H. and Y.-C.C. prepared samples for transcriptome data. J.-Y.O. and Y.-C.L. processed the transcriptome data. H.-Y.H., C.-Y.H., E.-T.H., and Y.-C.C. performed genotyping assays and seed collection. C.-H.H. and C.-Y.H. conducted metabolite profiling. S.-C.C. optimized the protocol of metabolite profiling by LC-MS analysis. Y.-C.C. performed cloning and genetic screening. J.-K.H. and Y.-C.C. designed the research and wrote the manuscript.

Supplementary data

The following materials are available in the online version of this article.

Supplementary Figure S1. Characterization of ddl mutants and DDL complementation lines.

Supplementary Figure S2.  DDL mutants in Arabidopsis are susceptible to Pst under ambient humidity.

Supplementary Figure S3.  DDL is required for NHP and flg22 peptide-induced systemic resistance.

Supplementary Figure S4. Genotyping ddl-6 sid2-2 and npr1-1 mutants.

Supplementary Figure S5. Retention time and MRM chromatograms of NHP, SA, NHP-Glc and SA-Glc in standard solutions and extracts of NHP-treated Arabidopsis leaves.

Supplementary Figure S6. Principal component analysis (PCA) of transcriptomic data.

Supplementary Figure S7. The number of differentially expressed NHP-responsive genes is altered in the ddl-6 mutant compared to wild-type plants.

Supplementary Figure S8. Percentages of NHP-regulated genes in local treated and distal untreated tissues of Col-0 and ddl-6 within various expression groups.

Supplementary Figure S9. The results from the independent trials of bacterial growth assays are shown in Fig. 1.

Supplementary Figure S10. The results from the independent trials of bacterial growth assays are shown in Fig. 2 and Supplementary Fig. S3.

Supplementary Figure S11. The results from the independent trials of bacterial growth assays are shown in Fig. 3.

Supplementary Table S1. Lists showing genes that are differentially expressed genes in treated and untreated leaves of Col-0 and ddl-6 upon NHP treatment.

Supplementary Table S2. GO term analysis of NHP-responsive genes in different gene clusters.

Supplementary Table S3. The list of primers used in this study.

Supplementary Table S4. Statistical analyses in this study.

Supplementary Methods.

Funding

This work was supported by a grant from the National Science and Technology Council (NSTC). MOST (Ministry of Science and Technology) (110-2311-B-001-045-MY3) and NSTC (113-2311-B-001-024).

Data availability

All raw sequence reads of RNA-seq in this study were deposited to the NCBI SRA database (BioProject accession number: PRJNA1255381). All data supporting this study are available within the article and its Supplementary materials.

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

• WRKY70 Gramene: AT3G56400

• WRKY70 Araport: AT3G56400

• DCL3 Gramene: AT3G43920

• DCL3 Araport: AT3G43920

• EFR Gramene: AT5G20480

• EFR Araport: AT5G20480

• PR1 Gramene: AT2G14610

• PR1 Araport: AT2G14610

• MAPKKK5 Gramene: AT5G66850

• MAPKKK5 Araport: AT5G66850

• AtRLP23 Gramene: AT2G32680

• AtRLP23 Araport: AT2G32680

• FMO1 Gramene: AT1G19250

• FMO1 Araport: AT1G19250

• MKK9 Gramene: AT1G73500

• MKK9 Araport: AT1G73500

• PARP2 Gramene: AT4G02390

• PARP2 Araport: AT4G02390

• WRKY33 Gramene: AT2G38470

• WRKY33 Araport: AT2G38470

• ALD1 Gramene: AT2G13810

• ALD1 Araport: AT2G13810

• DCL1 Gramene: AT1G01040

• DCL1 Araport: AT1G01040

• PAD3 Gramene: AT3G26830

• PAD3 Araport: AT3G26830

• TGA1 Gramene: At5g62510

• TGA1 Araport: At5g62510

• TGA3 Gramene: AT1G22070

• TGA3 Araport: AT1G22070

• UBQ5 Gramene: AT3G62250

• UBQ5 Araport: AT3G62250

• WRKY38 Gramene: AT5G22570

• WRKY38 Araport: AT5G22570

• CBP60g Gramene: AT5G26920

• CBP60g Araport: AT5G26920

• PCRK1 Gramene: AT3G09830

• PCRK1 Araport: AT3G09830

• ERdj3B Gramene: At3g62600

• ERdj3B Araport: At3g62600

• GFP Gramene: green fluorescent protein

• GFP Araport: green fluorescent protein

• NPR1 Gramene: AT1G64280

• NPR1 Araport: AT1G64280

• DDL Gramene: AT3G20550

• DDL Araport: AT3G20550