Discovery of a novel nucleoside immune signaling molecule 2′-deoxyguanosine in microbes and plants

Graphical abstract

Model of 2′-deoxyguanosine involvement in plant immune response. Endophytic fungus-derived 2-dG directly activates ET signaling, EIT and PTI. Moreover, pathogen invasion induces the expression of the plant 2-dG biosynthesis gene VEN4 and promotes 2-dG accumulation in plant leaves. However, 2-dG-mediated plant resistance against Pst DC3000 is conferred in an ET-, NPR1-, PRR/coreceptor- and P2K1-dependent manner.

Highlights

• •We identified for the first time that nucleoside 2′-deoxyguanosine from endophytic fungal metabolites can effectively stimulate classical immune responses in plants.
• •We demonstrated that 2-dG-induced plant resistance depends on salicylic acid (SA) receptor NPR1 (but not SA), ethylene (ET) signaling, pattern-recognition receptors/coreceptors (PRRs/coreceptors) and the ATP receptor P2K1.
• •We provide the first demonstration that VEN4 is 2-dG biosynthesis gene and involved in pathogen-induced 2-dG accumulation, VEN4 also plays an essential role for plant immunity.

Abstract

Introduction

Beneficial microorganisms play essential roles in plant growth and induced systemic resistance (ISR) by releasing signaling molecules. Our previous study obtained the crude extract from beneficial endophyte Paecilomyces variotii, termed ZNC (ZhiNengCong), which significantly enhanced plant resistance to pathogen even at 100 ng/ml. However, the immunoreactive components of ZNC remain unclear. Here, we further identified one of the immunoreactive components of ZNC is a nucleoside 2′-deoxyguanosine (2-dG).

Objectives

This paper intends to reveal the molecular mechanism of microbial-derived 2′-deoxyguanosine (2-dG) in activating plant immunity, and the role of plant-derived 2-dG in plant immunity.

Methods

The components of ZNC were separated using a high-performance liquid chromatography (HPLC), and 2-dG is identified using a HPLC–mass spectrometry system (LC-MS). Transcriptome analysis and genetic experiments were used to reveal the immune signaling pathway dependent on 2-dG activation of plant immunity.

Results

This study identified 2′-deoxyguanosine (2-dG) as one of the immunoreactive components from ZNC. And 2-dG significantly enhanced plant pathogen resistance even at 10 ng/ml (37.42 nM). Furthermore, 2-dG-induced resistance depends on NPR1, pattern-recognition receptors/coreceptors, ATP receptor P2K1 (DORN1), ethylene signaling but not salicylic acid accumulation. In addition, we identified Arabidopsis VENOSA4 (VEN4) was involved in 2-dG biosynthesis and could convert dGTP to 2-dG, and vne4 mutant plants were more susceptible to pathogens.

Conclusion

In summary, microbial-derived 2-dG may act as a novel immune signaling molecule involved in plant-microorganism interactions, and VEN4 is 2-dG biosynthesis gene and plays a key role in plant immunity.

Introduction

Plants live in complex environments with various potential pathogens and beneficial microorganisms. Plant pathogens directly threaten both the growth and yield of crops. Traditional chemical pesticides are widely used to control crop diseases worldwide, which results in serious contamination in the water, soil and atmosphere, and in an imbalance in the ecosystem [1], [2]. Beneficial endophytes play various indispensable functions in plant growth, development, disease resistance, and stress tolerance [3]. Recently, endophytes have been widely used to control different diseases in various crops due to their environmentally friendly characteristics in biological control, including maize-southern corn leaf blight, rice-sheath blight disease, potato late blight and sweet potato-black rot disease [4], [5], [6], [7], [8], [9]. Moreover, the application of endophytes and their extracts in the development of biopesticides is commonly used for environmentally harmless biological control, and has become a green agricultural trend [10].

To prevent pathogen invasion, plants evolved a series of defense mechanisms to stimulate the innate immune system [11]. Plant innate immunity is divided into pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) [12], [13], which are triggered by the recognition of membrane-localized pattern-recognition receptors/coreceptors (PRRs/coreceptors) to detect microbe- or pathogen- associated) molecular patterns (MAMPs/PAMPs) and intracellular nucleotide-binding (NB) domains and leucine-rich repeat receptors (NLRs) to recognize characteristic type III effectors (T3Es) [14], [15], respectively. Bacterial flagellin 22 (flg22) is recognized by membrane surface localized receptor FLAGELLIN SENSING 2 (FLS2) to activate the plant immune response [16], [17], including increased cytoplasmic Ca²⁺ levels, reactive oxygen species (ROS) bursts, mitogen-activated and Ca²⁺-dependent protein kinase (MPK and CDPK) activation, and large-scale activation of the transcription of disease resistance genes [18], [19], [20], [21]. In general, the above-described defense reactions are also indicators of plant immune elicitor identification.

In recent years, the induction of plant immunity by microorganisms through the release of various immune elicitors has become a focus. According to their chemical properties, plant immune elicitors can be classified as carbohydrates, glycopeptides, lipids or lipopeptides, protein, salicylic acid (SA), N-hydroxypipecolic acid (NHP), jasmonic acid (JA) and ethylene (ET) and other small-molecule metabolites [22], [23], [24]. Nucleotide metabolism plays a critical role in plant development or protecting plant against to biotic/abiotic stresses [25]. Cyclic GMP (cGMP) is an essential secondary messenger involved in various bioactivities, including seed germination, root growth, stomatal closure, pollen tube growth and pathogen defense in plants [26], [27]. In addition, a common electron carrier nicotinamide adenine dinucleotide (NAD) was recently reported to be involved in plant development and responses to pathogen infection [28], [29], [30]. Similarly, adenosine-5′-triphosphate (ATP) provides energy for life activities in all organisms, and extracellular ATP (eATP) has been proved to be a damage-associated molecular pattern (DAMP) signaling molecule involved in plant immunity [31], [32], [33], [34]. Moreover, recent research suggests that guanosine tetraphosphate metabolism also plays a role in plant immunity [35].

Our previous study demonstrated that the crude extract from the beneficial endophyte Paecilomyces variotii, termed ZNC, which could enhance plant disease resistance at 100 ng/ml [36]. Here, we established a sensitive and precise method based on a HPLC system to isolate immunoreactive components in ZNC. Furthermore, we identified 2′-deoxyguanosine (2-dG) as the immunoreactive component in ZNC through the use of a LC–MS system and a series of immunologic activity assays. We then found that 2-dG induced classical immune responses, including ROS production, callose accumulation and MPKs phosphorylation, and upregulated the expression of plant immunity-associated genes. Further mutant analysis revealed that 2-dG-induced plant immunity requires salicylic acid (SA) receptor NPR1 (but not SA), ethylene (ET) signaling, pattern-recognition receptors/coreceptors (PRRs/coreceptors) and the ATP receptor P2K1. Previous study suggests that VEN4 is a homologous gene of deoxynucleoside triphosphate triphosphohydrolase (SAMHD1) [37]. Here, we further proved that VEN4 can hydrolyze dGTP to 2-dG in vitro. Furthermore, we provide the first demonstration that VEN4 is involved in pathogen-induced 2-dG accumulation. We also provide genetic evidence showing that VEN4 positively regulates plant immunity. These findings provide a novel perspective that 2-dG is a key immune signaling molecule in plant–microbe interactions and that VEN4 is essential for plant immunity.

Materials and methods

Experimental materials and chemical treatment

Ethanol, acetonitrile, methanol, acetic acid, and ammonium acetate were purchased from Fisher Scientific (Springfield, USA). Deionized water was purified by a Milli-Q system. 2-dG (purity ≥ 99%,Sigma-Aldrich®) was purchased from Sigma-Aldrich the official website (CAS No.: 312693-72-4). ZNC solution (5.63 mg/mL) was donated by Shandong Pengbo Biotechnology Co., Ltd. and stored in 20% ethanol. In our study, 4-week-old Arabidopsis plants were pre-sprayed with 2-dG solution (100 ng/ml) or other chemical substances, respectively. Next, Pst DC3000 inoculation, RNA extraction and other experiments were conducted at indicated times.

Preliminary separation of ZNC

The ZNC solution was filtered through a 0.22-μm econofilter. The separation of ZNC was performed on an LC-20A system (Shimadzu, Kyoto, Japan) equipped with a UV detection device. In brief, the sample was loaded on a Venusil XBP C18 (4.6 × 100, 5 μm) column. The mobile phases were 10 mmol/L ammonium acetate (A) and methanol (B). The gradient elution was optimized as follows. The composition of mobile phase B was started at 1%, maintained at 1% for 2 min, increased to 8% over 7 min, increased to 20% over 21 min, decreased to 1% over 5 min and maintained at 1% for 5 min for re-equilibration at a flow rate of 0.5 ml/min. The loading quantity of the sample was 100 µL, the detection wavelength was 260 nm, and the column temperature was maintained at 35 °C. The fractions collected inline were numbered 1–7, the samples were lyophilized at −80 °C, and then re-dissolve them.

Purification of disease-resistant components in ZNC-4

After bioactivity determination, the ZNC component labeled 4 had obvious disease-resistance activity and was then further purified using the following methods. After centrifugation, the supernatant was collected and passed through a 0.22-μm filter. In addition, the liquid chromatography column was replaced with a Venusil XBP C18 (L) column (4.6 × 250 mm, 5 μm). The gradient elution was as follows: 0–6 min, 1% B; 6–15 min, linear gradient 1–4% B; 15–25 min, linear gradient 4–8% B; 25–40 min, linear gradient 8–20% B; 40–47 min, 20% B; 47–57 min, linear gradient 20–1% B; and 57–65 min, 1% B. The other conditions were the same as before optimization. The purified samples were named 4–1 to 4–4 and were lyophilized at −80 °C, and then re-dissolve them.

Identification and quantification of disease-resistant active components by UPLC–MS

After the disease resistance experiment, 4–2 exerted a better disease resistance effect than the other fractions. An analysis of 4–2 was performed on an UltiMate3000 system (Thermo Fisher Scientific, USA) coupled with a Hypersil BDS C18 column (2.1 × 100 mm, 3 µm) pumped at 35 °C. Acetonitrile (B) and aqueous solution containing 0.1% acetic acid (A) eluted at a flow rate of 0.3 ml/min under isocratic conditions (80% A and 20% B). Online ESI–MS was conducted with a TSQ Quantis™ Access MAXQ Mass Spectrometer (Thermo Fisher Scientific, USA). The MS analysis was performed in the positive-ion mode with the following instrument parameters: spray voltage, 3500 V; vaporizer temperature, 300 °C; sheath gas, 35 psi; auxiliary gas pressure, 2 psi; capillary temperature, 350 °C; tube lens offset, 70 °C; and collision pressure 1.5 psi. Full scan and Q1MS were used for the scan events. The full-scan mass mode covered the range from 100 to 400 m/z. The monoisotopic m/z of the main possible compound read from the mass spectrum was used to retrieve the theoretical structure in the database. The peak assignment was realized by matched degree evaluation between the monoisotopic m/z and theoretical structure database [38]. The external standard and 4–2 were then analyzed under the SRM mode for quantitative analyses. The instrument parameters were as follows: parent mass, 268.1; product mass, 152.1; collision energy, 17; and the others were the same as those in the full scan mode. Data processing was performed using Thermo Xcalibur software. The relative amounts of all components were calculated by comparing the peak area of each component with that of the external standard [38].

Detection of the 2-dG content

Plants powder that ground under liquid nitrogen (0.1 g) were mixed with 3 ml water, and ultrasonicated at 250 W for 30 min at room temperature, and then centrifuge at 8000 r/min for 10 min. Trichloromethane (1.5 ml) was added to the supernatant, and the aqueous phase was absorbed after shaking at room temperature for 30 min. After repeated extraction, 0.0150 g of insoluble cross-linked polyvinylpyrrolidone (PVPP) was added to the aqueous phase solution extracted from trichloromethane. The resulting mixture was shook up and down for 30 s, rested for 10 min at room temperature. and centrifuged at 8000 r/min for 10 min. The supernatant was filtered through a 0.22-μm microporous membrane for HPLC–MS-MS analysis. A Thermo Fisher TSQ Quantum Access MAX liquid chromatography system was used in this research. The separation of analyses was achieved on a Venusil XBP C18(L) column (100 mm × 4.6 mm i.d., 5 μm). The temperature of the column oven was 40 ℃. The mobile phase consisted of water with 0.1% formic acid (A) and methanol (B). The gradient elution was as follows: 0–0.5 min, 20% B; 0.5–1 min, linear gradient 20–35% B; 1–3 min, 35% B; 3–3.5 min, linear gradient 35–55% B;3.5–5.5 min, 55% B; 5.5–6 min, linear gradient 55–20% B; and 6–11 min, 20% B. The mobile-phase flow rate was maintained at 0.3 ml/min, and the injection volume was 10 μL. The electrospray ionization (ESI) source was operated in the positive-ion mode using the following conditions: ion spray voltage, 3500 V; capillary temperature, 300 °C; vaporizer temperature, 350 °C; sheath gas pressure, 35 arb; aux gas pressure, 10 arb; discharge current, 4.0 μab; and tube lens value, 83 v. We selected MRM mode for the quantitative analyses.

Plant material and growth environment

The Arabidopsis thaliana ecotypes used in this experiment have a Columbia-0 (Col-0) background, two transfer DNA insertion lines SALK_023714 (ven4-1) and SALK_077401 (ven4-2) were previously reported [39]. Prof. Cyril Zipfel, Prof. Xiufang Xin and Prof. Dongqin Chen provided bbc, fec, rbohd, dorn1-3 and pat5 mutant seeds [40], [41], [42]. The following methods were used for seed surface sterilization: the seeds were incubated with 75% ethanol for 1 min; 5% NaClO solution was added for 15 min; and the seeds were then washed 5–6 times with double-distilled water. The seeds were sown on 1/2-strength Murashige and Skoog (MS) medium. After 2 d of vernalization at 4 °C, the seeds were transferred to constant temperature greenhouses for germination under the following conditions: 70% humidity, 22 °C, and a 12-h light/12-h dark cycle. Seven-day-old seedlings were transplanted into vermiculite for continuous growth and irrigated with 1/2 MS nutrient solution. Twenty-eight-day-old Arabidopsis was used for bacterial inoculation experiments, ROS detection, RNA sequencing and other experiments.

ROS detection assays

Nitroblue tetrazolium (NBT) was used for superoxide anion (O⁻) detection, and 3,3-diaminobenzidine (DAB) was used as indicators of hydrogen peroxide (H₂O₂), respectively. Dynamic detection of ROS with a luminol-based method was conducted based on previous reports, with some minor modifications [43]. Twenty-eight-day-old Arabidopsis leaves were punched by a cork borer (diameter = 5.5 mm), and then incubated for 12–16 h under continuous light. Sterile distilled water was replaced with a reaction solution containing 20 μg/ml (w/v) horseradish peroxidase ((Sigma) and 40 μg/ml (w/v) luminol ((Sigma) with 100 ng/ml 2-dG. Luminescence signal was measured using a microplate reader (PerkinElmer, USA). ROS level was also detected by using 1 μM H₂DCFDA solution, and the fluorescence signal was detected 15 min later with a confocal microscope (LSM880). Excitation wavelength = 488 nm and emission wavelengths = 501–550 nm.

Callose detection

Solution A is prepared by adding deionized water (52%), lactic acid (20%), phenol (20%) and 8% glycerol (v/v) into a beaker. Following 0–100 ng/ml 2-dG treatments for 24 h, the harvested leaves were cultured in solution B (solution A: ethanol = 2:1) and vacuum for 0.5 h. The above mixture was then incubated at 60 °C and mixed every 10 min. Remove the leaves and wash them 3 times with deionized water. Clean leaves are placed in 0.01% aniline blue staining solution (150 mM K₂HPO₄, PH = 9.5). The fluorescence signal was detected using a confocal microscope (LSM880). Excitation wavelength = 405 nm and emission wavelengths = 410–480 nm.

Pathogen cultivation and inoculation

Pst DC3000 was cultured at 28 °C, and bacterial solution with OD₆₀₀ = 0.001 was dissolved by 10 mM magnesium chloride. The Pst DC3000 suspension was infiltrated into 28-day-old Arabidopsis leaves by a needleless syringe. The inoculation of plants with P. infestans (YWK196), Xanthomonas oryzae pv. oryzicola (Xoc), and Xanthomonas oryzae pv. oryzae (Xoo) was performed as previously described [44], [45], [46].

Bacterial growth assay

Arabidopsis leaves were harvested after inoculation with Pst DC3000 for 36 h, and 3–6 leaves per group were packed into three 10 ml sterile centrifugal tubes and weighed separately. Subsequently, the leaves were placed into 75% ethanol solution for surface disinfection. Sixty seconds later, the leaves were rinsed three times in sterile distilled water. Sterile absorbent paper was then used to absorb excess water from the leaf surface. Finally, the leaves were ground to liquid homogenate with 1 ml of sterile distilled water. The homogenate was continuously diluted (1/10) and plated onto PSA medium (with 50 μg/ml rifampicin) for colony counting and calculation of the bacterial population growth at 28 °C. In addition, a growth suppression assay of Pst DC3000 was performed as follows. Briefly, the Pst DC3000 suspension (OD ₆₀₀= 1) was diluted (1:10) 4–6 times and cultured in PSA medium containing 0–200 ng/ml 2-dG, and the growth of bacterial colonies was recorded after 24 h. For liquid PSA medium, the OD₆₀₀ was detected and recorded at different time points.

RNA extraction for quantitative analysis

RNA was extracted using TRIzol reagent (Cwbio, China). RNA (0.5 μg) was reverse transcribed into cDNA using ReverTra Ace® qPCR RT Master Mix with a gDNA Remover Kit (Toyobo, Japan). For all target genes, ∼5–20 ng of cDNA template was used for qPCR analysis, and both ACTIN2 and PP2AA3 were used as reference genes. Twenty-microliter reactions were performed using the UltraSYBR Mixture reagent (Cwbio, China) and a ABI7500 real time system. Genes expression levels were calculated using previously described methods [47].

Detection of MPK phosphorylation

Four-week-old Arabidopsis leaf discs were used for the detection of MPKs phosphorylation. Briefly, Arabidopsis leaf discs were placed into 12-well plates (12 discs per well) containing 1 ml 1/2 MS liquid medium, incubated overnight, and then transformed into new 12-well plates with 1/2 MS liquid medium added with 0, 1, 10, 100, or 200 ng/ml 2-dG. Leaf samples were ground to homogeneous powder and immediately homogenized in protein extraction buffer (0.05 M Tris, pH = 7.4, 0.025 M glycerophosphate, 0.01 M NaF, 0.001 M sodium orthovanadate, 150 mM NaCl, 5‰ (V/V) Tween-20, protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail 2 (Sigma)). The resultant homogenate was collected in a sterile centrifugal tube and centrifuged for 30 min at 4 °C. The supernatant was collected and prepared for SDS–PAGE. An immunoblot analysis of phosphorylated MPKs was performed with a primary antibody (anti-phospho-p44/42 MPKs (1:2000, Sigma)) and a secondary antibody (peroxidase-conjugated goat anti-rabbit IgG (1:15000, Sigma)).

Analysis of RNA-sequencing data

Three biological replicates of RNA sequencing samples were extracted from 28-day-old Arabidopsis plants after 100 ng/ml 2-dG treatment for 0, 2 and 24 h. RNA samples were selected for RNA sequencing based on quality (RIN ≥ 7). According to previous research [44], [48], nine libraries were constructed and sequenced by Wuhan Genomic Institution (Shenzhen, China). Raw data was mapped to the latest Arabidopsis reference genome and uploaded to NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/PRJNA833157) with a BioProject PRJNA833157. RESM software was used to calculate matched reads and for their normalization them to RPKM for gene expression analysis. In this study, the significance of the differential gene expression was defined by the BGI bioinformatics service based on the absolute value of og2 ratio ≥ 1 and P ≤ 0.05. A heatmap was used to observe gene expression patterns.

Quantification of the SA content and ethylene biosynthesis rates

The extraction and HPLC analysis of SA were based on previous studies, with some slight improvements. Leaf samples (0.1–0.5 g) were frozen and ground to a fine powder in a mortar with liquid nitrogen, and transformed into a 1.5 ml centrifugal tube with 1 ml of extracting solution containing 89% MeOH, 10% H₂O and 1% CH₃COOH (v/v). The samples were agitated for 2–16 h at 4 °C and centrifuged at 12000 × g 15 min and 4 °C for 15 min, and the supernatant was then transferred into a new 1.5-ml centrifugal tube. The precipitate from the previous step was recovered, and the previous step was repeated. The two supernatants were merged into the same centrifuge tube. An HPLC method described in previous studies was used in the present research [49]. For ET analysis, the ET content was measured by gas chromatography [50]. Briefly, after 2-dG treatment for 2 h, 28-day-old Arabidopsis plants were harvested and placed in a 68 ml bottle. Two hours later, a 1 ml gas sample was injected into a cold trap and quantified using a gas chromatograph.

Detection of VEN4 nucleotidase activity

The coding sequence of VEN4 was amplified by PCR and fused to pET30a expression vector using homologous recombination, the primers in table S1. His-tagged VEN4 was expressed in E.coli (BL21) and purified using Ni affinity chromatography. VEN4 (≈0.12 μg) protein was inoculated with 1 mM dGTP for 3 h in the reaction solution, including 20 mM Tris-HCl (pH 7.8), 50 mM NaCl, 5 mM MgCl₂, 0.5 mM TCEP. The reaction mixture was analysed by anion-exchange HPLC refer to the previous research [51]. The production of 2-dG was used to quantify VEN4 nucleotide activity.

Results

Isolation and identification of the immunoreactive component of ZNC

To trace the elicitor-active component of ZNC, a reversed phase liquid chromatography separation method was established based on the physicochemical properties of ZNC. According to the retention time and peak shape, ZNC was divided into seven fractions using high-performance liquid chromatography online sampling system (Fig. 1A). The fractions collected were numbered 1–7. However, only fraction ZNC-4 enhanced plant resistance to pathogens (Fig. 1B). To achieve better separation efficiency, fraction 4 was separated and purified with a longer column. Moreover, the mobile phase elution procedure was adjusted appropriately. ZNC-4 was ultimately separated into four fractions, and the purified samples were collected and named ZNC-4-1 to ZNC-4-4 (Fig. 1C). Additionally, the bacterial population was significantly decreased after infiltration with ZNC-4-2 compared with that after infiltration with the other fractions and was similar to that after infiltration with ZNC (Fig. 1D), indicating that ZNC-4-2 was an elicitor-activity candidate fraction of ZNC.

Fig. 1.

The elicitor-active component of ZNC was identified as 2′-deoxyguanosine. (A) HPLC chromatogram of ZNC, ZNC is divided into seven fractions (ZNC1-7) (B) Bacterial population in Col-0 plant leaves (n = 6) after ZNC1-7 treatment. Col-0 plant leaves were pre-sprayed with the seven fractions (ZNC1-7) at 100 ng/mL, and then inoculated with Pst DC3000 2 h later, bacterial population was enumerated at 3 dpi. All data are representative of three biological experiments. (C) Fraction 4 from ZNC was further separated to and purified with a longer column by HPLC. (D) Bacterial population in Col-0 plant leaves (n = 6) after four components (ZNC-4-1 to ZNC-4-4) treatment 4. Col-0 plant leaves were pre-sprayed with the four fractions (ZNC-4-1 to ZNC-4-4) at 100 ng/mL, and then inoculated with Pst DC3000 2 h later, bacterial population was enumerated at 3 dpi. All data are representative of three biological experiments. (E) Examples of mass spectra of 4–2 compounds under the full scan mode with a zoomed-in spectrum of the main ions [M + H]⁺m/z = 268.13 and m/z = 152.11. (F) Examples of mass spectra of 2-Dg in the full scan mode with a zoomed-in spectrum of the main ions. (G) Examples of mass spectra of 4–2 compounds under the SRM mode; only ions with a parent at m/z 268.13 and unique product fragments at m/z 152.1 were selected. (H) Disease symptoms in Col-0 plants (n = 6) after 0, 1, 10, 100 or 200 ng/ml 2-dG treatment. Col-0 plant leaves were pre-sprayed with 0–200 ng/ml 2-dG treatment, and then inoculated with Pst DC3000 2 h later, bacterial population was enumerated 3 d after inoculation with Pst DC3000. (I) Bacterial colonization in Col-0 plant leaves (n = 6) after 0, 1, 10, 100 or 200 ng/ml 2-Dg treatment. Col-0 plant leaves were pre-sprayed with 0–200 ng/ml 2-dG, and then inoculated with Pst DC3000 2 h later, bacterial population was enumerated 3 d after inoculation with Pst DC3000. The error bars represent the means ± SEMs. Different letters represent significant differences compared with control (0 ng/ml 2-Dg treatment), as determined by two-way ANOVA (P < 0.05). The bacterial growth assay is repeated for three times with similar results.

Subsequently, ZNC-4-2 was analyzed by UPLC-ESI-MS_n under a full scan mode to identify its main compounds. After optimization of the mass and chromatographic conditions, the positive electrospray ionization (ESI) mode was used for the qualitative analysis of ZNC-4-2 (Fig. 1E). The main ions were detected at m/z 268.13 and 152.11, and the monoisotopic m/z of the main possible compound read from the mass spectrum was used to retrieve the theoretical structure from a database. This result was consistent with 2′-deoxyguanosine (2-dG) (purity ≥ 99%), which has [M + H]⁺ values of m/z 268.07 and 152.10 (Fig. 1F). These results indicate that 2-dG is most likely the immunoreactive component of ZNC. Furthermore, the 2-dG content in ZNC was measured under the selective reaction monitoring (SRM) mode. In brief, only the compounds with the same retention time as 2-dG of 0.85 min (Fig. S1A and S1B) were allowed to be analyzed by MS. Simultaneously, only ions with a parent at m/z 268.13 and unique product fragments at m/z 152.1 were selected for area integration (Fig. 1G and Fig. S1C) [52]. In addition, we quantified 2-dG in the SRM mode by establishing a standard curve (Fig. S2), and the content of 2-dG in ZNC was 63.5 μg/ml (1.12% in ZNC).

To further detect the optimal concentration of 2-dG for improving plant disease resistance, the following experiments were performed. We observed obvious yellow water-soaked lesions in Col-0, however, with the increase of 2-dG concentration, the phenotype of disease symptom gradually weakened. In addition, 2-dG significantly decreased bacterial population even at 10 ng/ml, and further decreased that at 100 ng/ml 2-dG, whereas 200 ng/ml 2-dG treatment could not further reduce the bacterial population (Fig. 1H and 1I), which suggested that 100 ng/ml 2-dG is a suitable concentration for the following experiments. Interestingly, Pseudomonas syringae pv. tomato (Pst) DC3000 grew similarly in culture solution or on culture medium (Fig. S3), suggesting that the 2-dG-mediated inhibition of Pst DC3000 multiplication was plant dependent. In addition, 2-dG enhanced rice resistance to Xanthomonas oryzae pv. oryzicola (Xoc) and Rhizoctonia solani (Fig. S4), indicating that 2-dG has potential control effect on various plant diseases.

2-dG is a special immune elicitor with both disease prevention and treatment functions

Our previous study showed that ZNC effectively protects plants against bacterial invasion at 100 ng/ml [36]. To further analyze whether the activation of plant immunity by nucleosides or bases is universal, four bases and three nucleosides were used to compare their ability to activate plant immunity with 2-dG at 100 ng/ml, and ZNC was used as a positive control. We observed that 2-dG significantly induced ROS bursts, upregulated the relative expression of the ICS1 and pathogen-related 1 (PR1) and enhanced plant resistance to pathogens, however, the other four bases and three nucleosides had weaker or no ability to do so (Fig. 2A-2D). Although guanine also induce plant immunity, the activity of guanine in activating plant immune response was lower than that of 2-dG according our results (Fig. 2A-2D). In addition, another part of our recent work also showed that guanine enhance rice resistance to sheath blight [53]. In addition, we also compared the ability of 2-dG and ATP to enhance plant resistance, surprisingly, ATP failed to reduce the bacterial population at concentrations ranging from 1 ng/ml to 200 ng/ml (Fig. S5). These results showed that 2-dG may be a special nucleoside molecule capable of activating plant resistance.

Fig. 2.

2′-deoxyguanosine is a high-activity elicitor compared with SA, Cu²⁺, flg22 and ZNC. (A) Detection of ROS accumulation through DAB and NBT staining. Hydrogen peroxide (top) and superoxide staining (bottom) were performed in Arabidopsis leaves (n = 5) treated with H₂O (control) or with ZNC, 2-dG, guanine (Gua), thymine (Thy), 3′-deoxyadenosine (3-Da), guanosine (Guas), uridine (Uri), 2′-deoxycytidine (2-De), adenosine (Ade), or cytidine (Cyt) at 100 ng/ml for 2 h. (B) and (C) The transcript level of PR1 and ICS1 were conducted using qRT–PCR. Col-0 leaves were treated with H₂O (control) or with ZNC, 2-dG, guanine (Gua), thymine (Thy), 3′-deoxyadenosine (3-Da), guanosine (Guas), uridine (Uri), 2′-deoxycytidine (2-De), adenosine (Ade), or cytidine (Cyt) at 100 ng/ml. The expression of PR1 and ICS1 were conducted using qRT–PCR at 2 hpt. (D) Bacterial population in Col-0 plants (n = 6) at 3 dpi. Col-0 plant leaves were pre-sprayed with H₂O (control) or with ZNC, 2-dG, guanine (Gua), thymine (Thy), 3′-deoxyadenosine (3-Da), guanosine (Guas), uridine (Uri), 2′-deoxycytidine (2-De), adenosine (Ade), or cytidine (Cyt) at 100 ng/ml, and then inoculated with Pst DC3000 2 h later, bacterial population was enumerated 3 d after inoculation with Pst DC3000. (E) Bacterial population in Col-0 plants (n = 6) at 3 dpi. Col-0 leaves were pre-sprayed with 2-dG (100 ng/ml), ZNC (100 ng/ml), Cu²⁺ (100 μM, 6355 ng/ml) or flg22 (1 μM, 2272 ng/ml) at the indicated times, and then inoculated with Pst DC3000 2 h later, the bacterial population was enumerated 3 d. (F) Bacterial population in Col-0 plants (n = 6) at 3 dpi. Col-0 leaves were pre-inoculated with Pst DC3000 at indicated times and then treated with 2-dG, ZNC, Cu²⁺ or flg22 at the indicated times, and then inoculated with Pst DC3000 2 h later, bacterial population was enumerated 3 d after inoculation with Pst DC3000. (G) Bacterial population in Col-0 plants (n = 6) at 3 dpi. Col-0 leaves were pretreated with 10, 100 or 1000 ng/ml SA, Cu²⁺, flg22, ZNC and 2-dG at indicated times, and then inoculated with Pst DC3000 2 h later, bacterial population was determined at 3 dpi. Different letters indicate statistically significant differences compared with the 0 ng/ml 2-dG treatment, as determined by two-way ANOVA (P < 0.05). The error bars represent the means ± SEMs. The bacterial growth assay is repeated for three times with similar results.

We then further conducted the following experiments to compare its effectiveness with that of previously reported plant elicitors, including ZNC, Cu²⁺ and flg22 [6], [18], [36], [54]. On the one hand, we observed that only 2-dG significantly decreased the bacterial population after or before inoculation with Pst DC3000 at 48 h or 24 h (Fig. 2E and 2F). Additionally, we found that the bacterial population after the 100 ng/ml 2-dG treatment had a similar function as those after the 1000 ng/ml SA, ZNC, Cu²⁺ and flg22 treatments in the prevention of pathogens (Fig. 2G). In addition, we also compared the rate of ROS production induced by 2-dG and flg22, we observed that flg22 stimulates ROS production faster than 2-dG, however, the peaks of ROS induced by 2-dG and flg22 were similar (Fig. S6). Accordingly, we conclude that the activity of 2-dG immune-active at lower concentrations is higher than the other compounds.

2-dG stimulates the classic immune response in plants but not animals

To determine whether 2-dG can directly induce the classical plant immune response, we further examined ROS accumulation, callose deposition and MAPKs phosphorylation in Arabidopsis leaves after infiltration with 2-dG at 0–200 ng/ml or at different times. DAB and NBT staining showed that the area of deeper blue/brown deposition in leaves was proportional to the 2-dG concentration (Fig. 3A). In addition, 100 ng/ml 2-dG triggered sustained ROS production within 2 h (Fig. 3B). However, a fluorescence intensity analysis showed that ZNC and 2-dG failed to induce an ROS burst in 3D4/21 (porcine alveolar macrophage) cells (Fig. S7). For detecting callose deposition, aniline blue staining revealed that the staining fluorescence intensity gradually increases with increasing 2-dG concentration (0 ng/ml-200 ng/ml) (Fig. 3C and 3D). We also observed that 2-dG significantly activated MAPKs phosphorylation (Fig. 3E). These results indicated that 2-dG can directly trigger the classical plant immune response.

Fig. 3.

2′-deoxyguanosine triggers classical immune responses in plants. (A) 2-dG induced ROS accumulation in plant leaves. DAB (top) and NBT staining (bottom) were detected in Col-0 leaves pre-treated with 0 (control), 1, 10, 100 or 200 ng/ml 2-dG 2 h later (n = 5). (B) ROS production was detected by a luminol-horseradish peroxidase-based approach. Col-0 plant leaf discs were pre-treated with 100 ng/ml 2-dG or H₂O (Control), ROS production was detected at indicated times. (C) Fluorescence intensity of the image in (D). Different letters represent significant differences compared with control (0 ng/ml 2-dG treatment), as determined by two-way ANOVA (P < 0.05). (D) 2-dG induced callose deposition in Arabidopsis leaves (n = 10). Col-0 plant leaves were pre-treated with 0 (control), 1, 10, 100 or 200 ng/ml 2-dG, aniline blue staining is used to detect callose content at 24 hpt. Scale bar = 100 µm. (E) MAPK phosphorylation in Col-0 plant leaves after 100 ng/ml 2-dG treatment or H₂O (Control) for indicated times. Coomassie (CBB) staining indicates equal total protein in each lane. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2-dG treatment upregulates plant disease-resistance genes transcription level

To better reveal which disease resistance signaling pathways were activated by 2-dG in plants, an RNA sequencing transcriptome analysis of 28-d-old Arabidopsis leaves exposed to 2-dG treatment for 2 h and 24 h was performed (See transcriptome analysis data in Table S2). Nine samples with three replicates were used for RNA sequencing. More than 22.7 million reads were obtained from each sample, and the average rate of genome mapping was 99.02%. We also compared the expression of different genes between 2-dG treatment for 2 h and 24 h and Venn diagram showed the number of differentially expressed genes (Fig. 4A), respectively.

Fig. 4.

Transcriptome profiling indicates that 2′-deoxyguanosine upregulates the transcription of pathogen resistance-related genes. (A) The number of differentially expressed genes in different functional categories after the pretreatment of Arabidopsis leaves with 100 ng/ml 2-dG for 2 h and 24 h. (B) Heatmap analysis of the expression of various genes, including ET pathway genes and other disease resistance genes after the pretreatment of Col-0 plant leaves with 100 ng/ml 2-dG for 2 h and 24 h. (C) qRT–PCR analysis of ET signaling and other resistance marker genes expression. Col-0 plant leaves were pre-sprayed with 100 ng/ml 2-dG, the expression of ET signaling and other resistance marker genes were conducted using qRT–PCR at 2 hpt. The error bars represent the means ± SDs. (D) Heatmap analysis of ET biosynthesis genes expression after the pretreatment of Col-0 plant leaves with 2-dG for 2 h and 24 h. The light blue squares represent ethylene synthesis precursors. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2-dG-activated plant immunity depends on SA- independent NPR1

To further verify whether the 2-dG-induced plant defense is dependent on the SA signaling pathway, SA-deficient ics1 and SA receptor npr1 mutants were used for the following experiments. Surprisingly, the bacterial growth at 3 dpi in ics1 plants under 2-dG treatment showed a significant decrease (10-fold) compared with that in ics1 (control) plants (Fig. 5A). In contrast, the bacterial growth at 3 dpi in npr1 plants treated with 2-dG showed a population similar to that in the npr1 (control) plants (Fig. 5A). In this process, 2-dG promoted SA accumulation in Col-0 and npr1 leaves at 2 and 24 hpt. Thus, we hypothesized that 2-dG still induced SA accumulation through a process independent of ICS1. To test this hypothesis, the SA content in ics1, npr1 and Col-0 plants after the control and 2-dG treatments was determined. The results showed that the SA content markedly increased after 2-dG treatment in npr1 and Col-0 plants but was mildly induced in ics1 plants (Fig. 5B). In addition, another SA-deficient NahG transgenic plant was used to test the role of SA in 2-dG-induced resistance [55]. Similarly, 2-dG enhanced NahG transgenic plant resistance to pathogens compared with that of the control plants (Fig. S7). On the other hand, SA marker gene (WRKY18, WRKY72 and PR1) expression showed no significant difference in npr1 plants but still increased 23-, 7- and 55-fold in ics1 plants after 2-dG treatment (Fig. 5C). Based on all the abovementioned results, we conclude that 2-dG-mediated resistance relies on the SA receptor NPR1 rather than SA.

Fig. 5.

2′-deoxyguanosine-enhanced plant immunity to Pst DC3000 depends on NPR1 and ET. (A) Pst DC3000 population in Col-0, sid2/ics1, and npr1 plant leaves (n = 4) after 2-dG treatment. Col-0 sid2/ics1, and npr1 plant leaves were pre-sprayed with H₂O (control) or 100 ng/ml 2-dG, and then inoculated with Pst DC3000 2 h later, bacterial population was measured at 3 dpi. All experiments were biologically replicated at least three times. (B) Measurement of the free SA content in Col-0, sid2/ics1 and npr1 plants leaves (n = 6) after 100 ng/ml 2-dG pretreatment for 2 h and 24 h. (C) qRT–PCR analysis of the expression of WRKY18, WRKY70 and PR1 in sid2/ics1 and npr1 leaves after 100 ng/ml 2-dG or H₂O (control) treatment for 2 h and 24 h. (D) ET production rate were detected after 100 ng/ml 2-dG or H₂O (control) treatment for indicated times in Col-0 plant leaves. (E) qRT–PCR profiles of the expression of PDF1.2 after 100 ng/ml 2-dG or H₂O (control) treatment for indicated times. (F) Pst DC3000 population in Col-0, ein2 and ein3 plants (n = 4). Col-0, ein2 and ein3 plant leaves were pre-sprayed with 100 ng/ml 2-dG or H₂O (control), and inoculated with Pst DC3000 2 h later, bacterial growth of Pst DC3000 was measured at 3 dpi. All data is representative of three biological experiments. (G) 2-dG induced ET accumulation in Col-0, ein2 and ein3 leaves. Col-0, ein2 and ein3 plant leaves were pre-sprayed with 100 ng/ml 2-dG or H₂O (control), ET accumulation was detected at 2 hpt. (H) The expression of ERF5, ERF6 and PDF1.2 were conducted by qRT–PCR in ein2 and ein3 leaves after 100 ng/ml 2-dG or H₂O (control) treatment for 2 h and 24 h. (I) Pst DC3000 population in Col-0, octuple mutant (acs2-1/acs4-1/acs5-2/acs6-1/acs7-1/acs9-1/amiRacs8acs11) and acs2/6 mutant leaves after 100 ng/ml 2-dG or H₂O (control) treatment. Col-0, octuple mutant and acs2/6 mutant leaves were pre-sprayed with 2-dG (100 ng/ml) or H₂O (control), and then inoculated with Pst DC3000 2 h later, bacterial population was measured at 3 dpi. The error bars represent the means ± SEMs. * = P < 0.05, ** = P < 0.01, ***= P < 0.001. The bacterial growth assay is repeated for three times with similar results.

2-dG-stimulated plant resistance depends on the ET signal

The plant hormone ET plays an essential role for plant immunity. Interestingly, both the heatmap and the results from a quantitative real-time PCR analysis showed that the expression of ET biosynthesis (ACS2, ACS7 and ACS8), downstream genes (PDF1.2) were upregulated at 2 hpt or 24 hpt (Fig. 4B, 4C and 4D), which indicates that 2-dG significantly activates ET signaling pathways in plants. To further prove that 2-dG promotes ET biosynthesis, we examined ET emissions in Arabidopsis plants treated with 2-dG. We found that 2-dG-treated plants showed higher ET production rates at 0, 2, 4, 8, 12 and 24 hpt (Fig. 5D), and the expression of PDF1.2 showed a similar trend (Fig. 5E). To investigate whether the ET signaling pathway is required for the 2-dG-induced resistance, we conducted Pst DC3000 inoculation in ein2 and ein3 mutant plants, which are deficient in ET signal transduction [56]. Compared with that in the control, the bacterial growth showed no significant difference after 2-dG treatment in ein2 and ein3 mutant plants (Fig. 5F). In addition, we observed that the ET content strongly increased in Col-0 and ein2 and ein3 mutant plants (Fig. 5G), but PDF1.2, ERF5 and ERF6 expression was compromised in the ein2 and ein3 mutant plants (Fig. 5H), suggesting that 2-dG-induced plant defense needs EIN2 and EIN3. Moreover, 2-dG still enhance plant resistance to pathogen in Col-0 or acs2-1/acs6-1 (double mutant) plant leaves (Fig. 5I); however, 2-dG failed to decreased Pst DC3000 colonization in octuple mutant plant leaves (Fig. 5I) [57], indicating that 2-dG mediates plant defense and requires ET biosynthesis.

PRRs/coreceptors and P2K1 are needed for 2-dG-induced plant immunity

Two major pattern-recognition receptor (PRR)/coreceptor Arabidopsis mutants, bak1/bkk1/cerk1 (bbc) and fls2/efr/cerk1 (fec), are deficient in PRR/coreceptor binding to bacteria-associated molecular patterns [42] and were used to detect whether 2-dG-stimulated plant immunity is dependent on PRRs/coreceptors. Here, we found that 2-dG also induced ETI- and PTI-related genes (Fig. 6A); however, 2-dG failed to enhance plant resistance (Fig. 6B), upregulate ETI- and PTI- related genes (Fig. 6D-6I) or trigger an ROS burst (Fig. 6J) in bbc and fec. Additionally, we observed the similar results in rbohd (Fig. 6B-6I), which is an ROS production deficient plant mutant [58]. These results suggest that 2-dG activates the plant immune response depending on PRRs/coreceptors and RBOHD. P2K1 (DORN1) is an extracellular ATP receptor that induces plant immunity depending on the S-acylation of protein S-acyltransferases (PATs) [40]. 2-dG failed to induce plant immunity in dorn1-3 and pat5 mutant plants (Fig. 6C). This result suggests that the recognition pattern of 2-dG is similar to that of ATP in plants.

Fig. 6.

2′-deoxyguanosine activates plant immunity-required PRRs/coreceptors. (A) Heatmap analysis of PTI- and ETI-related gene expression in response to 2-dG at 2 hpt and 24 hpt. (B) Bacterial population in Col-0, dorn1-3 and pat5 plant leaves. Col-0, dorn1-3 mutant and pat5 plant leaves were pre-sprayed with 2-dG (100 ng/ml) or H₂O (control), and then inoculated with Pst DC3000 2 h later, bacterial population was measured at 3 dpi. The error bars represent the means ± SEMs. * = P < 0.05, ** = P < 0.01, *** = P < 0.001. The bacterial growth assay is repeated for three times with similar results. (C) Pst DC3000 population in Col-0, bbc, fec and rbohd plant leaves. bbc, fec and rbohd plant leaves were pre-sprayed with 2-dG (100 ng/ml) or H₂O (control), and then inoculated with Pst DC3000 2 h later, bacterial population was measured at 3 dpi. The error bars represent the means ± SEMs. * = P < 0.05, ** = P < 0.01, ***= P < 0.001. The bacterial growth assay is repeated for three times with similar results. (D)-(I) Detection of PTI- and ETI-related gene expression in Col-0, bbc, fec and rbohd plants by qRT–PCR. Col-0, bbc, fec and rbohd mutant leaves were pre-sprayed with 2-dG or H₂O (Control), genes expression was conducted at 2 hpt using qRT-PCR. (J) ROS burst were detected through the fluorescent dye H₂DCFDA staining of leaves of Col-0, bbc, fec and rbohd mutant plant, which were pre-treated with 100 ng/ml 2-dG for 4 h. Bar = 25 μm.

VEN4 is 2-dG biosynthesis gene and involved in plant immunity

In humans, SAMHD1 releases 2-dG from dGTP [37]. 2-dG-related biosynthesis genes have not yet been identified in plants. Accordingly, we further identified that whether VEN4d, which is homologous to human SAMHD1 [39], is involved in 2-dG biosynthesis in plant. His-tagged VEN4 were constructed in the Pet30a vector, and induced and purified (Fig. S9). As expect, we observed that His-tagged VEN4 can hydrolyze dGTP to 2-dG (Fig. 7A) in vitro. To further explore the function of plant endogenous 2-dG in plant immunity, we detected the content of 2-dG after Pst DC3000 inoculation. Because substances such as pigments, polysaccharides and proteins can affect the detection of 2-dG in plant leaves, conventional metabolite extraction methods cannot detect 2-dG. Thus, we improved the 2-dG extraction and purification methods, and established a set of high-sensitivity 2-dG detection methods based on LC–MS. Surprisingly, Pst DC3000 inoculation upregulated the expression of VEN4 and increased the accumulation of 2-dG in plant leaves (Fig. 7B and 7C). These results suggest that VEN4 is 2-dG biosynthesis gene and probably involved in plant–pathogen interactions. Furthermore, two ven4 mutant lines ven4-1 and ven4-2 [39] were performed in the following experiments. We observed that both ven4-1 and ven4-2 mutant plants were more susceptible to Pst DC3000 (Fig. 7D), and the content of 2-dG in ven4-1 and ven4-2 was lower than that in Col-0 under control or Pst DC3000 inoculation conditions (Fig. 7E). In addition, 2-dG can significantly complement resistance of ven4-1 and ven4-2 to Pst DC3000 (Fig. 7F). We then infiltrated 2-dG into local leaves and detected the mRNA levels of systemic resistance-associated genes. The results showed that 2-dG induced the expression of systemic resistance-related genes in plant systemic leaves (Fig. 7G). We also observed that local leaves treated with 2-dG significantly enhanced plant systemic leaves resistance (Fig. 7H). Accordingly, we further detected the bacterial populations of Col-0, ven4 mutant plant in systemic leaves after local leaves inoculation with Pst DC3000. The result showed bacterial population of ven4-1 and ven4-2 was approximately 10 times that of Col-0 (Fig. 7I), and Col-0 systemic leaves accumulated the higher levels of systemic resistance-related genes mRNA (Fig. 7J) compared with ven4 mutant plants. Based on the above results, we conclude that VEN4 play a key role in plant immunity.

Fig. 7.

Plant endogenous 2′-deoxyguanosine is essential for plant immunity. (A) The products of reaction upon incubation of VEN4 with dGTP were detected by HPLC. VEN4 and aGTP were co-incubated for 3 h. The red font indicates the retention time of dGTP and 2-dG. (B) Expression of VEN4 after Pst DC3000 inoculation. Col-0 plant leaves were inoculated with Pst DC3000 in 10 mM MgCl₂, or control-treated (10 mM MgCl₂), the expression of VEN4 were detected using qRT-PCR at indicated times. (C) Detection of the 2-dG content after Pst DC3000 inoculation. Col-0 plant leaves were inoculated with Pst DC3000 in 10 mM MgCl₂, or control-treated (10 mM MgCl₂), 2-dG content were detected using LC-MS at indicated times. Different letters indicate significant differences (P < 0.05). (D) Bacterial population in Col-0, ven4-1 and ven4-2 plants (n = 6) at 3 dpi. *** represent significant differences (P < 0.001). (E) Detection of the 2-dG content in Col-0, ven4-1 and ven4-2 plants after Pst DC3000 inoculation 4 h later. Different letters indicate significant differences (P < 0.05). (F) Bacterial colonization in Col-0, ven4-1 and ven4-2 plant leaves (n = 6). Col-0, ven4-1 and ven4-2 plant leaves were pre-sprayed with 100 ng/mL 2-dG or H₂O (Control), and then inoculated with Pst DC3000, bacterial population was measured at 3 dpi. Different letters indicate significant differences (P < 0.05). (G) Expression of plant systemic resistance-related genes in systemic leaves. Three lower leaves from each 28-d-old plant were inoculated with Pst DC3000 in 10 mM MgCl₂, or control-treated (10 mM MgCl₂). After 24 h, the expression of systemic resistance-related genes in upper untreated systemic leaves was detected using RT-qPCR. (H) Three lower leaves from each 28-d-old plant were sprayed with 100 ng/ml 2-dG, or control-treated (H₂O). After 24 h, the upper untreated systemic leaves were inoculated with Pst DC3000. The bacterial population in system leaves was detected at 3 dpi. (I) Bacterial growth in Col-0, ven4-1 and ven4-2 plant systemic leaves (n = 6) at 3 dpi. Three lower leaves from each 28-d-old plant were inoculated with Pst DC3000 in 10 mM MgCl₂, or control-treated (10 mM MgCl₂). After 24 h, the upper untreated systemic leaves were inoculated with Pst DC3000. The bacterial population in system leaves was detected at 3 dpi. (J) Systemic resistance-associated genes expression in Col-0, ven4-1 and ven4-2 systemic leaves. Three lower leaves from each 28-d-old plant were inoculated with Pst DC3000 in 10 mM MgCl₂, or control-treated (10 mM MgCl₂). After 24 h, the expression of systemic resistance-associated genes in upper untreated systemic leaves was detected using qRT-PCR. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Discussion

2-dG is a novel and high-activity plant elicitor from endophytic fungi

During the long-term evolution of endophytes and plants, endophytes produce and provide abundant active substances for plant growth and defense, among other activities [59], [60], [61]. The isolation and identification of active components from endophytes are essential for the development of plant elicitors. Nucleotides are some of the most important molecules in organisms due to their multiple biological functions, such as heredity, conducting different biochemical reactions, and providing or transferring energy. According to our results, we found that the nucleoside or base activation of plant immunity is not a universal phenomenon (Fig. 2A-2D), which indicates that 2-dG might be a special immune signaling molecule. Among nucleotides, ATP, which is reportedly related to plant immunity, is recognized by the receptor-like kinase P2K1 and induces immune responses such as stomatal closure, calcium burst and MPKs activation in plants [32]. However, exogenous ATP failed to induce plant resistance to the pathogen (Fig S5), which was likely rapidly degraded by the plants. On the one hand, 2-dG can activate multiple plant immune responses, including ROS bursts, callose deposition, MAPKs phosphorylation and upregulate the expression of ET-, ETI- and PTI-associated genes. In addition, 2-dG also improved plant resistance to bacteria (Pst, Xoc) and fungi (R. solani) pathogens, indicating that 2-dG has a broad-spectrum ability to activate plant resistance. Regarding the promotion of plant immunity, our results suggest that 2-dG is more effective than ZNC, SA, Cu²⁺ and flg22 (Fig. 2E-2G). Benzothiadiazole (BTH) is a synthetic analog of SA that enhances plant resistance in the field [62], [63]. However, the effective concentration of BTH in promoting plant resistance is 100 μM (13.618 μg/ml) [64], which is approximately 136-fold higher than that of the effective concentration of 2-dG (100 ng/ml). This information proves that 2-dG has high-efficiency in inducing and broad-spectrum activation of plant immunity.

2-dG-induced plant resistance may require a novel signaling pathway

ET plays critical roles in plant–pathogen interactions [65]. The 1-aminocyclopropane-1-carboxylate (ACC) synthase genes (ACSs) is a key rate-limiting step in ET biosynthesis [48], [66]. ACSs are encoded by the ACS gene family, which contains 12 members annotated as AtACS1-AtACS12 in Arabidopsis [67]. Inoculation with Pst DC3000 or Botrytis cinereal strongly upregulated the expression of AtACS2 and AtACS6 [68], [69]. Intriguingly, we found that 2-dG can upregulate the mRNA expression of ET biosynthesis gene (AtACS7 and AtACS8) and markedly increase the ET emission rate (Fig. 4B-4D, 5D). As expected, 2-dG enhanced plant defense also requires ET biosynthesis and signaling (Fig. 5F and 5I), suggesting that ET signaling plays a pivotal role in 2-dG-induced plant defense.

SA also plays a central role in plant–pathogen interactions. Flg22 is recognized by FLS2, which is localized on the plant cell extracellular membrane [54], [70], [71], and then activates SA signaling to enhance plant immune defense, and this process depends on the SA receptor NPR1. Interestingly, 2-dG markedly upregulated the transcriptional levels of PR1, WRKY18 and WRKY70 and promoted plant resistance to pathogens in SA biosynthesis-deficient plants (ics1 and NahG-transgenic plants) (Fig. 5B and S8). These results indicate that 2-dG has an immune signaling pathway different from that stimulated by conventional PAMPs and that NPR1 may have a new function not only as an SA receptor.

2-dG may act as a PAMP/DAMP-induced signaling pathway similar to induced systemic resistance (ISR)

Cell damage can release ATP, which is considered as DAMP [34]. Extracellular ATP can be recognized by P2K1, and this recognition results in the activation of plant immune responses [32]. Similarly, we also found that 2-dG-induced plant resistance also required P2K1 (Fig. S8). In addition, 2-dG directly triggered the production of ROS, callose deposition, and induced MPKs phosphorylation, and these effects correspond to the characteristics of PAMPs/DAMPs in plants. Moreover, 2-dG failed to enhance plant resistance to pathogens in bbc and fec mutants, further indicating that 2-dG-induced plant immunity depends on PTI-associated PRRs/coreceptors. In view of the above findings, 2-dG may belong to PAMP/DAMPs. ISR is induced by beneficial microbes, including bacteria and fungi [72]. In our study, 2-dG was identified as one of the immunoreactive ingredients of ZNC [36]. 2-dG also activated plant immunity (Fig. 1H, 1I and S4). In addition, 2-dG-induced resistance is not dependent on SA, but depends on NPR1 and ET signaling (Fig. 5), similarly to ISR [72], [73].

VEN4 is needed for plant endogenous 2-dG biosynthesis and plant immunity

Numerous studies have reported that SAMHD1 is an HIV restriction factor that reduces the dNTP levels and thus inhibits HIV reverse transcription and its complementary double-strand biosynthesis [51], [74], [75], [76]. In addition, SAMHD1 is beneficial for the drug treatment of leukemia and cancer [77], [78], which indicates that SAMHD1 may be involved in human innate immunity. In plants, VEN4 is reportedly involved in plant chloroplast development, leaf size, and abiotic stress [39], [79]. However, the role of VEN4 in plant endogenous 2-dG accumulation and plant immunity remains unclear. Here, we provided the first demonstration that VEN4, the human SAMHD1 homologous gene, can hydrolyze GTP to 2-dG in vitro and plays an essential role in pathogen-induced endogenous plant 2-dG accumulation and also is involved in pathogen-plant interactions (Fig. 7A, 7C and 7E). Moreover, disease resistance of the two ven4 mutant plants were impaired (Fig. 7D, 7F and 7I). Accordingly, we propose that VEN4 is essential for increasing the pathogen-induced plant endogenous 2-dG levels and plant immunity.

In brief, 2-dG-induced plant immunity requires SA-independent NPR1, ET signaling, PRR/coreceptors and the ATP receptor P2K1. In plant, VEN4 is a 2-dG biosynthesis gene, induced by pathogens and involved in the accumulation of plant endogenous 2-dG, which may be a novel signaling pathway for plant immunity.

Compliance with ethics requirements

This article does not contain any studies with human or animal subjects.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

The following are the Supplementary data to this article: