PpMYB123-mediated proanthocyanidin accumulation alleviates bacterial spot disease in peach

Abstract

Bacterial spot (BS) disease significantly impairs vigor, fruit quality, and yield in peach trees. However, research on this disease remains limited. In this study, peach leaves and fruits were inoculated with the pathogen isolated from infected leaves, triggering a robust accumulation of proanthocyanidins (PA) in both tissues. Further investigation revealed that pathogen inoculation promoted PA accumulation by upregulating PpMYB123, which transactivated the core PA biosynthetic genes PpANR and PpLAR. Notably, the E3 ubiquitin ligase PpPUB23 negatively regulated PpMYB123. However, its transcript levels were significantly suppressed following inoculation, thereby stabilizing PpMYB123 and enhancing PA production. PA conferred dual protection by scavenging excess reactive oxygen species (ROS) and suppressing pathogen growth. Our findings provide molecular evidence for PA-mediated defense against BS disease in peach.

Introduction

Pathogenic infections caused by microbial agents, such as bacteria and fungi, initiate a cascade of physiological disturbances in plants, including reactive oxygen species (ROS) bursts that can lead to cellular damage. This cellular damage impairs essential physiological processes, such as photosynthetic efficiency and biomass accumulation, ultimately reducing crop yield and marketable quality [1–8]. This biotic stress in plants is counteracted by a complex defense system that involves activating resistance (R) genes, producing hormones, such as salicylic acid (SA) and jasmonic acid (JA), and triggering the synthesis of defensive secondary metabolites [9–13].

The phenylpropanoid pathway generates a wide array of specialized metabolites, including lignin, flavonol, anthocyanin, and proanthocyanidins (PAs). These metabolites play vital roles in plant disease resistance. Lignin, a predominant component of secondary cell walls, confers mechanical reinforcement to prevent pathogen penetration and colonization by increasing cell wall rigidity [14–16]. Flavonol, anthocyanin, and PAs possess multiple hydroxyl groups and a special conjugated structure of the benzene ring. This structure enables them to neutralize ROS through electron transfer or metal chelation and reduce oxidative damage to suppress the occurrence of disease [17–21]. PAs in plants exist as polymeric structures with abundant hydroxyl groups, exhibiting strong resistance to degradation. Moreover, PAs collaborate with lignin to reinforce cell walls, thus playing a vital role in disease resistance. The accumulation of PAs has been found to enhance resistance against fungal diseases in poplar [22, 23]. PA enrichment enhances resistance to Colletotrichum gloeosporioides and significantly reduces susceptibility to powdery mildew in grapevine [20, 24, 25].

The biosynthesis of PAs in plants is regulated by two key enzymatic steps: anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). ANR catalyzes the conversion of anthocyanins into epicatechin derivatives, whereas LAR mediates the formation of catechin precursors [26]. This metabolic process is strictly regulated by environmental stresses, such as strong light, cold, drought, and pathogen infection [20, 23, 27–29], as well as hormone signals including SA and JA [30–32]. The PA biosynthesis gene is regulated by the transcription complex formed by MYB transcription factors, bHLH co-activators, and WD40 repeat proteins, with MYB transcription factor playing a key role. AtTT2/AtMYB123 in Arabidopsis directly binds to the AtANR/BAN promoter to drive PA deposition in seed coats [33]. Subsequent studies revealed the conservation of MYB-mediated PA accumulation in various plant species, for example, the involvement of VvMYBPA1, VvMYB5b, and VvMYBPA2 in grape [34–36] and FaMYB9, FaMYB11, and FaMYB5 in strawberry [37, 38].

Peach (Prunus persica) is a commercially vital stone fruit crop, prized for both its ornamental and culinary applications. However, it faces significant threats from bacterial pathogens, compromising its aesthetic value and fruit marketability. Specifically, bacterial spot (BS) disease results in leaf perforations and necrotic fruit lesions; however, its molecular mechanisms remain poorly elucidated. Our study revealed that pathogen inoculation triggered pronounced PA accumulation in peach leaves and fruits through a novel PpPUB23–PpMYB123 regulatory module. This inoculation upregulated the transcription of PpMYB123, promoting PA accumulation through the transcriptional activation of PpANR and PpLAR. The E3 ubiquitin ligase PpPUB23 negatively regulated the function of PpMYB123. However, pathogen inoculation significantly downregulated the expression of PpPUB23, leading to increased PA accumulation. Thus, PAs confer dual protective mechanisms against pathogen inoculation by removing excessive ROS and suppressing pathogen growth.

Results

PA specifically accumulates under pathogenic bacteria inoculation in peach

To investigate the pathogenesis of BS disease in peach, healthy leaves from ‘QBT’ and ‘GHT’ were selected for pathogen inoculation. Phenotypic characterization showed initial necrotic lesions at inoculation sites in both genotypes by 4 days post-inoculation (dpi), with lesion expansion culminating in cell death by 9 dpi (Fig. 1A). To elucidate the transcriptional regulatory networks involved in the disease response, RNA-seq-based transcriptome profiling was performed on leaf tissues collected at 0 (control), 2, and 4 dpi. Differential expression analysis (|log2FC| ≥ 1, FDR < 0.05) identified 1097 commonly upregulated and 1961 commonly downregulated genes at 2 dpi in both genotypes. At 4 dpi, 1185 genes were upregulated and 1723 downregulated. Intersection analysis revealed 1842 consistently differentially expressed genes (DEGs) across both time points, including 660 persistently upregulated and 1182 persistently downregulated genes (Table S3). These DEGs were likely associated with the peach defense response to BS disease. Within the list of up-regulated genes, we identified multiple pathogenesis-related (PR) genes that exhibited significant induction at 2 dpi, including glycosyl hydrolases (PR2), thaumatin (PR5), and peroxidase (PR9) family genes (Fig. S1A). Concurrently, phytohormone profiling revealed a gradual increase in the level of SA, a key defense-related hormone, following pathogen inoculation (Fig. S1B). These observations collectively suggest the activation of a robust defense response at the site of pathogen inoculation.

Figure 1.

PA specifically accumulates in response to pathogenic bacterial inoculation in peach. (A) Pathogen inoculation induces phenotypic changes in the leaves of ‘QBT’ and ‘GHT’. (B) Pathogen inoculation alters the expression profiles of genes involved in the phenylpropanoid biosynthesis pathway. (C) Pathogen inoculation induces phenotypic changes in the leaves of ‘XHJ’. (D) DMACA staining of pathogen-inoculated leaves. (E) Quantification of PA content in pathogen-inoculated leaf tissues. (F) Expression profiles of PpLAR and PpANR in pathogen-inoculated leaf tissues. (G) Pathogen inoculation induces phenotypic changes in the fruits of ‘XHJ’. (H) DMACA staining of pathogen-inoculated fruits tissues. (I) Quantification of PA content in pathogen-inoculated fruit tissues. (J) Expression profiles of PpLAR and PpANR in pathogen-inoculated fruit tissues. Error bars in (E), (F), (I), and (J) represent the standard error (SE) of three biological replicates.

Given that phenylpropanoid-derived secondary metabolites are well known for being vital in plant disease resistance, the expression of phenylpropanoid pathway genes was analyzed. Transcriptome data revealed strong upregulation of key genes in the PA biosynthetic pathway at 2 dpi (Fig. 1B), suggesting PA accumulation in response to pathogenic inoculation. To test this, pathogen inoculation was conducted in leaves and fruits of the peach cultivar ‘XHJ’, which has been widely documented in the literature as a highly suitable system for transient genetic transformation studies [39–41]. Pathogen-inoculated tissues showed significantly greater lesion formation than mock-treated controls (Fig. 1C and G). DMACA staining and spectrophotometric analysis confirmed substantial PA accumulation in both leaves and fruits (Fig. 1D, E, H, I). Additionally, qRT-PCR analysis verified the expression levels of PpANR and PpLAR, which has been reported playing critical roles in PA biosynthesis [42, 43], showing coordinated upregulation in pathogen-inoculated tissues (Fig. 1F and J). Together, these findings provided strong evidence that PA specifically accumulated in peach under pathogenic bacterial inoculation.

PA mitigates the severity of disease symptoms induced by pathogenic bacteria

Plants subjected to abiotic stress or pathogen inoculation accumulated excessive ROS, known to trigger cell death [2, 6]. To investigate this, we performed the DAB staining to detect H₂O₂, a well-characterized ROS, in peach leaves and fruits inoculated with the pathogen. Compared to the H₂O-treated controls, pathogen inoculation significantly induced H₂O₂ accumulation (Fig. S2A and B). We subsequently treated peach leaves and fruits with 10 mM H₂O₂ and found H₂O₂-treated tissues exhibited phenotypes resembling those of pathogen-inoculated tissues, including pronounced cell death (Fig. S2C and D). Fruits developed more extensive necrosis than leaves at the same H₂O₂ concentration, indicating that ROS overaccumulation induced cell death.

As PA exhibit strong antioxidant activity and neutralize ROS, such as superoxide anions and H₂O₂, it was hypothesized that PA accumulation contributed to disease resistance via ROS scavenging. To test this, co-infiltration experiments with exogenous PA and pathogen were performed in tobacco leaves. PA treatment significantly reduced both the severity (Fig. 2A) and area (Fig. 2B) of necrotic lesions, along with reduced H₂O₂ levels in the treated leaves (Fig. 2C). Similar effects were observed in peach leaves, with reduced symptoms (Fig. 2D), necrotic area (Fig. 2E), and H₂O₂ content (Fig. 2F). Consistent results were obtained in peach fruits (Fig. 2G–I). To assess whether PA directly affected pathogen growth, GFP-labeled pathogens were monitored, and PA application significantly reduced fluorescence intensity in inoculated tissues (Fig. 2J). In vitro assays further demonstrated a dose-dependent inhibition of pathogenic bacterial growth (Fig. 2K and L). These findings indicated that PA mitigated disease development by scavenging ROS and suppressing pathogen proliferation.

Figure 2.

PA mitigates disease severity induced by pathogenic bacterial infection. (A) Effect of exogenous PA treatment on pathogen-inoculated tobacco leaves. (B) Impact of exogenous PA treatment on lesion area in pathogen-inoculated tobacco leaves. (C) Exogenous PA treatment modulates H₂O₂ levels in pathogen-inoculated tobacco leaves. (D) Effect of exogenous PA treatment on pathogen-inoculated peach leaves. (E) Impact of exogenous PA treatment on lesion area in pathogen-inoculated peach leaves. (F) Exogenous PA treatment modulates H₂O₂ levels in pathogen-inoculated peach leaves. (G) Effect of exogenous PA treatment on pathogen-inoculated peach fruits. (H) Impact of exogenous PA treatment on lesion area in pathogen-inoculated peach fruits. (I) Exogenous PA treatment modulates H₂O₂ levels in pathogen-inoculated peach fruits. (J) Exogenous PA application modulates fluorescence intensity of GFP-labeled pathogens in inoculated tissues. (K) Exogenous PA application affects pathogen growth under in vitro culture conditions. (L) Exogenous PA application reduces the number of GFP-labeled pathogens under in vitro conditions. Error bars in (B), (C), (E), (F), (H), and (I) represent the SE of three biological replicates.

PpMYB123 plays a crucial role for PA accumulation

In the list of MYB transcription factors upregulated at 2 dpi (Table S4), two MYB genes, PpMYB123 (Prupe.1G405400) and PpMYBPA1 (Prupe.2G192100), were identified, both exhibiting expression patterns similar to PpANR and PpLAR (Fig. 3A). Correlation analysis revealed a significant association between their expression profiles and those of structural genes involved in PA biosynthesis regulation (Fig. S3). Phylogenetic analysis placed these two genes in a distinct clade, separate from MYB regulators of flavonol and anthocyanin biosynthesis, and confirmed their orthology with AtTT2, the master regulator of PA biosynthesis in Arabidopsis (Fig. 3B). Expression analysis in peach leaves (Fig. 3C) and fruits (Fig. 3D) inoculated with pathogenic bacteria showed that both genes were upregulated, with PpMYB123 displaying more pronounced variation, suggesting a key role in PA accumulation under pathogen infection.

Figure 3.

Expression analysis and identification of the PpMYB123 and PpMYBPA1 genes. (A) Transcriptional profiles of PpMYB123 and PpMYBPA1 in peach leaves following pathogen inoculation. (B) Phylogenetic analysis of PpMYB123 and PpMYBPA1 in peach and other plant species. Peach genes are highlighted with colored blocks. Sequences were retrieved from the NCBI database with the following accessions: Prunus persica PpMYB7 (XP_007210006.2), PpMYB12 (ONH94094.1), PpMYB123 (XP_007224456.1), and PpMYBPA1 (XP_007218760.1); Arabidopsis thaliana AtMYB12 (NP182268), AtMYB111 (NP182268), AtTT2 (NP_198405.1), AtMYB113 (NP_176811.1), AtPAP1 (ABB03879.1), and AtPAP2 (NP_176813.1); Vitis vinifera VvMYBF1 (NP_176813.1), VvMYBPA1 (CAJ90831.1), VvMYBPA2 (ACK56131.1), VvMYBA1 (BAD18977.1), and VvMYBA2 (BAD18978.1); Diospyros kaki DkMYB2 (BAI49719.1); Picea mariana PmMBF1 (AAA82943); Malus domestica MdMYB1 (ABK58136.1); Petunia × hybrida PhAN2 (AAF66727.1); Solanum lycopersicum SIMYB12 (NP001234401.1); and Gerbera hybrid GhMYB1 (CAD87007). (C) Expression patterns of PpMYB123 and PpMYBPA1 in peach leaves under pathogen inoculation. (D) Expression patterns of PpMYB123 and PpMYBPA1 in peach fruits under pathogen inoculation. Error bars in (A), (C), and (D) indicate the standard error (SE) of three biological replicates.

Functional validation in tobacco leaves (Fig. S4A) showed that overexpression of PpMYB123 significantly increased PA accumulation (Fig. S4B and C) and upregulated NtANR and NtLAR (Fig. S4D). Transient overexpression in peach leaves also promoted PA accumulation (Fig. S4E–G) and significantly upregulated PpANR and PpLAR (Fig. S4H). Similar results were observed in peach fruits (Fig. S4I–L). These findings indicated that PpMYB123 played a central role in PA accumulation.

PpMYB123-induced PA accumulation alleviates the disease symptoms under pathogenic bacteria inoculation

To further elucidate the pivotal role of PpMYB123 in mitigating pathogen-induced damage, the functional validation approach was employed. In peach leaves, transient overexpression of PpMYB123 led to a significant reduction in lesion size (Fig. 4A and B), accompanied by a marked increase in PA content (Fig. 4C), significant upregulation of PpANR and PpLAR expression (Fig. 4D), and a notable reduction in H₂O₂ content (Fig. 4E). Conversely, silencing of PpMYB123 exacerbated cell death, increased lesion size (Fig. 4F and G), reduced PA content by ~30% (Fig. 4H), significantly downregulated PA biosynthetic genes (Fig. 4I), and elevated H₂O₂ levels (Fig. 4J). Further functional studies of PpMYB123 in peach fruits yielded results consistent with those observed in peach leaves. Overexpression of PpMYB123 mitigated pathogen-induced damage (Fig. 4K and L), resulted in a threefold increase in PA content (Fig. 4M), upregulated PpANR and PpLAR expression by at least twofold (Fig. 4N), and reduced H₂O₂ content by approximately 50% (Fig. 4O). Conversely, PpMYB123 silencing produced an opposite phenotype, significantly exacerbating disease progression (Fig. 4P–T). Therefore, these findings collectively indicated that PpMYB123-induced PA accumulation alleviated pathogen-induced damage in both peach leaves and fruits.

Figure 4.

PpMYB123-induced PA accumulation alleviates disease symptoms under pathogen inoculation. (A) Phenotypes of PpMYB123-overexpressing and control peach leaves following pathogen inoculation; GUS overexpression was used as the control. (B) PpMYB123 expression levels in overexpressing and control peach leaves. (C) PA contents in PpMYB123-overexpressing and control peach leaves. (D) Transcriptional levels of PpANR and PpLAR in PpMYB123-overexpressing and control peach leaves. (E) H₂O₂ contents in PpMYB123-overexpressing and control peach leaves. (F) Phenotypes of PpMYB123-silenced and control peach leaves under pathogen inoculation; the empty pTRV2 vector was used as the control. (G) PpMYB123 expression levels in silenced and control peach leaves. (H) PA contents in PpMYB123-silenced and control peach leaves. (I) Transcriptional levels of PpANR and PpLAR in PpMYB123-silenced and control peach leaves. (J) H₂O₂ contents in PpMYB123-silenced and control peach leaves. (K) Phenotypes of PpMYB123-overexpressing and control peach fruits following pathogen inoculation; GUS overexpression was used as the control. (L) PpMYB123 expression levels in overexpressing and control peach fruits. (M) PA contents in PpMYB123-overexpressing and control peach fruits. (N) Transcriptional levels of PpANR and PpLAR in PpMYB123-overexpressing and control peach fruits. (O) H₂O₂ contents in PpMYB123-overexpressing and control peach fruits. (P) Phenotypes of PpMYB123-silenced and control peach fruits under pathogen inoculation; the empty pTRV2 vector was used as the control. (Q) PpMYB123 expression levels in silenced and control peach fruits. (R) PA contents in PpMYB123-silenced and control peach fruits. (S) Transcriptional levels of PpANR and PpLAR in PpMYB123-silenced and control peach fruits. (T) H₂O₂ contents in PpMYB123-silenced and control peach fruits. Error bars represent the standard error (SE) of three biological replicates.

PpMYB123 directly promotes the expression of PpANR and PpLAR

Previous studies had established that MYB transcription factors directly regulated PA biosynthesis by activating ANR and LAR gene expression [33, 34, 37]. Based on this, it was hypothesized that PpMYB123 might employ a similar mechanism. Yeast one-hybrid (Y1H) assays showed that PpMYB123 physically interacted with the promoter regions of PpANR and PpLAR (Fig. 5A). Cis-element analysis identified conserved MYB-binding sites (MBS, ‘CAACA/TG’) and MYB recognition sequences (MRS, ‘CCGTTG’) within their promoters (Fig. S5A). Electrophoretic mobility shift assay (EMSA) confirmed specific binding of PpMYB123 to an MBS motif (‘CAACTG’) near the start codon (Fig. S5B). Site-directed mutagenesis showed that substituting the core MBS sequence with ‘TTTTTT’ abolished the binding ability of PpMYB123, confirming the critical role of these nucleotides in mediating protein–DNA interaction (Fig. 5B). Functional assays in yeast demonstrated strong transactivation activity of PpMYB123, with its activation domains driving reporter gene expression (Fig. 5C). Dual-luciferase assays further confirmed that PpMYB123 significantly enhanced transcriptional activation of both PpANR and PpLAR promoters (Fig. 5D). These results collectively demonstrated that PpMYB123 directly bound to the promoters of PpANR and PpLAR, promoting their transcription.

Figure 5.

PpMYB123 directly promotes the expression of PpANR and PpLAR. (A) Assessment of the binding ability of PpMYB123 to the PpANR and PpLAR promoters using Y1H. (B) Assessment of the binding ability of PpMYB123 to the MBS in the promoters of PpANR and PpLAR using EMSA. (C) Transcriptional activity analysis of the PpMYB123 gene in yeast. (D) Schematic representation of reporter and effector constructs used in the dual-luciferase assay. (E) Evaluation of PpMYB123-mediated transcriptional activation of PpANR and PpLAR promoters using the dual-luciferase assay. Error bars represent the SE of three biological replicates.

PpPUB23 negatively regulates the function of PpMYB123

To investigate potential post-translational modifications (PTMs) of PpMYB123, we performed a yeast two-hybrid (Y2H) screening and identified PpPUB23, a U-box-type E3 ubiquitin ligase. Y2H and GST pull-down assays confirmed a direct interaction between PpPUB23 and PpMYB123 (Fig. 6A and B). Phylogenetic analysis identified PpPUB23 as an ortholog of Arabidopsis AtPUB23, clustering with homologs from sweet cherry, apple, and pear (Fig. S6A). Domain analysis confirmed a canonical U-box domain at the N-terminus of PpPUB23 (Fig. S6B). Surprisingly, transcriptome profiling revealed significant downregulation of PpPUB23 upon pathogenic bacterial inoculation (Fig. S6C). Dual-luciferase assays showed that PpPUB23 substantially reduced the transcriptional activation of PpANR and PpLAR by PpMYB123 (Fig. 6C), suggesting its role in PpMYB123 degradation via the ubiquitination pathway. To investigate whether PpPUB23 influences the stability of the PpMYB123 protein, we examined the abundance of PpMYB123 protein in PpPUB23-overexpressing and control peach fruits. The results showed that overexpression of PpPUB23 led to a significant reduction in PpMYB123 protein levels compared to the GUS control group (Fig. 6D). In vitro ubiquitination assays further demonstrated that the PpMYB123-GST fusion protein, but not the GST-tagged control, underwent PpPUB23-mediated ubiquitination and subsequent degradation (Fig. 6E). Additionally, treatment with the proteasome inhibitor MG-132 resulted in an accumulation of PpMYB123 protein in peach fruits (Fig. 6F), which was accompanied by a significant up-regulation of PA biosynthetic genes, PpANR and PpLAR (Fig. 6G). Collectively, these results indicate that PpPUB23 modulates PA biosynthesis by facilitating the ubiquitin-dependent degradation of PpMYB123.

Figure 6.

PpPUB23 negatively regulates the function of PpMYB123. (A) Validation of the interaction between PpMYB123 and PpPPUB23 using Y2H. (B) Validation of the interaction between PpMYB123 and PpPUB23 using GST pull-down. (C) Assessment of the effect of PpPUB23 on PpMYB123-mediated transcriptional activation of PpANR and PpLAR promoters using the dual-luciferase assay. (D) Analysis of PpMYB123 protein levels in PpPUB23-overexpressing and control peach fruit. (E) An in vitro ubiquitination assay was employed to demonstrate that PpPUB23 mediates the ubiquitination of PpMYB123. The reaction products were detected using anti-GST, anti-ubiquitin, and anti-His antibodies. (F) Impact of proteasome inhibitor MG-132 on the abundance of PpMYB123 protein. (G) Effect of proteasome inhibitor MG-132 on the expression of PpANR and PpLAR. (H) Phenotypes of PpPUB23-overexpressing and control peach fruits following pathogen inoculation; GUS overexpression served as the control. (I) Expression levels of PpPUB23 in PpPUB23-overexpressing and control peach fruits. (J) PA content in PpPUB23-overexpressing and control peach fruits. (K) Transcriptional levels of PpANR and PpLAR in PpPUB23-overexpressing and control peach fruits. (L) Phenotypes of PpPUB23-silenced and control peach fruits under pathogen inoculation; empty pTRV2 vector was used as the control. (M) Expression levels of PpPUB23 in PpPUB23-silenced and control peach fruits. (N) PA content in PpPUB23-silenced and control peach fruits. (O) Transcriptional levels of PpANR and PpLAR in PpPUB23-silenced and control peach fruits. Error bars in (C), (G), (I), (J), (K), (M), (N), and (O) represent the SE of three biological replicates.

Functional validation in peach fruits was performed, showing that overexpression of PpPUB23 led to increased cell death at inoculation sites (Fig. 6H and I), reduced PA accumulation (Fig. 6J), and suppressed expression of PpANR and PpLAR (Fig. 6K). Conversely, silencing of PpPUB23 reduced lesion size (Fig. 6L and M), enhanced PA accumulation (Fig. 6N), and upregulated PpANR and PpLAR expression (Fig. 6O). These complementary results established that PpPUB23 negatively regulated PpMYB123, likely via ubiquitin–proteasome-mediated post-translational regulation, thereby fine-tuning PA-mediated defense responses in peach.

Discussion

PA mitigates pathogen-induced damage through removing excessive ROS and suppressing pathogen growth in peach

Secondary metabolites, such as lignin, flavonol, anthocyanin, and PA, produced via the phenylpropanoid pathway, were reported to contribute to plant disease resistance [14–21]. In this study, the expression of PA biosynthetic genes PpANR and PpLAR was specifically upregulated in peach leaves following inoculation by the peach BS pathogen (Fig. 1A and B), leading to PA accumulation (Fig. 1C–E), which was also observed in peach fruits (Fig. 1G–J). Exogenous PA application significantly reduced pathogen-induced cell death in tobacco leaves, peach leaves, and fruits (Fig. 2A, D, G), establishing PA as a defense compound. These findings aligned with previous reports on PA-mediated resistance to powdery mildew in grapevine and fungal disease in poplar [20, 22–25], suggesting a conserved PA-based defense mechanism across plant species.

ROS were identified as key signaling molecules in plant stress responses, contributing to resistance against biotic and abiotic stresses and supporting essential physiological functions [5, 44, 45]. However, excessive ROS during the hypersensitive response (HR) caused oxidative damage and cell death [2, 6]. In this study, pathogen inoculation and high-concentration H₂O₂ treatment induced pronounced necrosis in peach leaves and fruits (Fig. 1C and G; Fig. S2), confirming ROS overaccumulation as a key driver of cellular damage, consistent with findings in rice and cucumber [46, 47].

Given PA's exceptional antioxidant capacity, it was hypothesized that PA could mitigate pathogen-induced damage by removing excess ROS. Experimental validation in tobacco leaves, peach leaves, and peach fruits showed that PA treatment significantly reduced H₂O₂ levels at inoculated sites (Fig. 2C, F, I). This ROS-scavenging activity was attributed to PA's unique molecular structure [17–21]. Additionally, both in planta and in vitro assays revealed that PA directly inhibited pathogenic bacterial growth (Fig. 2J and K), possibly due to the combined effect of PA and lignin in strengthening cell walls [22, 23]. In conclusion, it was demonstrated for the first time that PA mitigated pathogen-induced damage in peach by scavenging ROS and directly inhibiting pathogen growth.

Pathogen-induced PpMYB123 activates PA biosynthesis via transcriptional activation of PpANR and PpLAR

The expression of PA biosynthetic genes was primarily regulated by upstream transcription factors, with MYB family members playing a key role. In Arabidopsis, the MYB transcription factor encoded by AtTT2 directly activated AtANR/BAN transcription to promote PA accumulation in seed coats [33]. In fruit crops, MYB regulators identified included VvMYBPA1, VvMYB5b, and VvMYBPA2 in grape [34–36], and FaMYB9/FaMYB11 and FaMYB5 in strawberry [37, 38]. In peach, MYB genes such as MYBPA1, MYB7, and Peace/PpMYB6 were reported to regulate PA accumulation [43, 48, 49]. However, the specific MYBs involved in pathogen-induced defense responses remained unclear.

In this study, we identified two MYB transcription factors, PpMYB123 and PpMYBPA1, from the set of MYBs upregulated at 2 dpi. In contrast to the downregulation of PpMYB7 and the near absence of Peace/PpMYB6 expression, the expression patterns of PpMYB123 and PpMYBPA1 were strongly correlated with those of the PA biosynthetic genes PpANR and PpLAR (Fig. 3A). Phylogenetic analysis revealed that both genes are homologs of Arabidopsis AtTT2 (Fig. 3B). Notably, compared to PpMYBPA1, PpMYB123 showed more pronounced upregulation in both peach leaves and fruits following pathogen inoculation (Fig. 3C). Overexpression of PpMYB123 significantly enhanced PA accumulation in tobacco, as well as in peach leaves and fruits (Fig. S4). In peach leaves and fruits, PpMYB123 overexpression enhanced disease resistance, accompanied by increased PA accumulation and significantly reduced H₂O₂ levels. Conversely, silencing PpMYB123 aggravated cell death symptoms, reduced PA biosynthesis, and elevated H₂O₂ accumulation (Fig. 4). These findings demonstrate that PpMYB123 confers resistance to BS disease by promoting PA accumulation in peach.

The Y1H, EMSA, and Luc/Ren assays collectively demonstrated that PpMYB123 directly binds to the PpANR and PpLAR promoters (Fig. 5). Further investigation revealed that PpMYB123 specifically recognizes and binds to a DNA fragment containing the MBS motif ‘CAACTG’, located proximal to the ATG start codon (Fig. S5), suggesting evolutionary conservation in its DNA-binding site. Transcriptomic analysis showed coordinated upregulation of early biosynthetic genes (EBGs) shared by the anthocyanin, flavonol, and PA pathways, indicating that PpMYB123 may also regulate these common EBGs. Notably, unlike the expression pattern of PpANR and PpLAR, the genes controlling anthocyanin and flavonol biosynthesis were significantly downregulated following pathogen inoculation (Fig. 1B). This distinct expression pattern strongly suggests that metabolic flux is preferentially directed toward PA biosynthesis at the expense of anthocyanin and flavonol production.

PpPUB23–PpMYB123 module-mediated PA accumulation as an immune response to BS disease in peach

PTMs, including phosphorylation and ubiquitination, are crucial for regulating protein stability and function [50–53]. Plant U-box type E3 ubiquitin ligases (PUBs), characterized by their conserved U-box domain and E3 ubiquitin ligase activity, regulate various biological processes, including development and stress responses [54–57]. Recent studies have shown that PUB proteins play a role in plant disease resistance [25, 58–60], but their functions in peach remain unexplored. In this study, we identified PpPUB23, a PUB family protein that interacts with PpMYB123, through Y2H screening. Biochemical assays confirmed their direct physical interaction in vitro (Fig. 6B). Functional characterization revealed that PpPUB23 attenuates the transcriptional activation of PpANR and PpLAR by PpMYB123 (Fig. 6C). Results from the assays in vivo, in vitro, and MG-132 treatment in peach fruits further demonstrate that PpPUB23 modulates the transcriptional activation of PA biosynthetic genes by PpMYB123 through ubiquitin-mediated regulation of its protein stability (Fig. 6D–G). PpPUB23 overexpression reduced PA accumulation in peach fruits, whereas its silencing enhanced PA content (Fig. 6H–O), demonstrating its negative regulatory role in PpMYB123-mediated PA biosynthesis. Notably, pathogen inoculation significantly downregulated PpPUB23 expression (Fig. S6B), presumably relieving its suppression of PpMYB123 and facilitating PA accumulation during defense responses. Pathogen inoculation induced localized cell necrosis at inoculation sites (Fig. 1A), accompanied by up-regulation of PR genes, a marked increase in the level of the defense hormone SA (Fig. S1), and substantial accumulation of ROS (Fig. S2), which represent hallmark manifestations of immune response in plants [61–64]. This study demonstrates that PA accumulation after pathogen inoculation alleviates disease severity, indicating that PA biosynthesis constitutes a defense response. These findings imply that the PpPUB23–PpMYB123 regulatory module-mediated PA accumulation represents a functional component of immune response against BS disease in peach.

Based on these findings, we propose a molecular mechanism by which PpMYB123-mediated PA accumulation alleviates BS disease in peach (Fig. 7). Under normal conditions (left), basal ROS levels prevent oxidative cytotoxicity, with the expression of PpMYB123 and its inhibitory E3 ligase PpPUB23 maintaining steady-state PA accumulation. Upon pathogen inoculation, excessive ROS production in peach leaves and fruits tissues leads to oxidative damage and cell death (right). Pathogen inoculation upregulates PpMYB123 expression PA biosynthetic genes PpANR and PpLAR and promoting PA accumulation. Concurrently, pathogen inoculation suppresses the expression of PpPUB23 (the negative regulator of PpMYB123), thereby amplifying PpMYB123’s regulatory effect on PA accumulation. PA reduces cell death by scavenging excessive ROS and directly inhibits pathogen growth, ultimately alleviating BS disease in peach.

Figure 7.

A proposed model of PpMYB123-mediated PA accumulation alleviating BS disease in peach. Under normal conditions (left), basal ROS levels prevent oxidative cytotoxicity, while the expression of PpMYB123 and its inhibitory E3 ligase PpPUB23 remains at constitutive levels, resulting in steady-state PA accumulation. In contrast, pathogen inoculation (Right) induces excessive ROS production in peach leaves and fruits, leading to oxidative damage and cell death. The pathogen inoculation upregulates PpMYB123 expression, which consequently activates transcription of PA biosynthetic genes PpANR and PpLAR, thereby enhancing PA accumulation at inoculated tissues. Concurrently, the expression of PpPUB23, a negative regulator of PpMYB123, is suppressed, further amplifying PpMYB123-mediated PA biosynthesis. PA contributes to disease resistance by both scavenging excess ROS to reduce cell death and directly inhibiting pathogen growth, ultimately mitigating the BS disease incidence in peach.

Materials and methods

Plant materials and growth conditions

Leaves of the wild peach variety ‘Guanghetao’ (GHT) (Prunus mira) and the cultivated varieties ‘Qiubaitao’ (QBT) and ‘Xiahuangjin’ (XHJ) (Prunus persica) were collected from the fourth to seventh newly formed leaves at the tops of branches. Fruits of XHJ were harvested at the early ripening stage. All cultivars were maintained at the Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, China. The collected leaves and fruits were rinsed with water, disinfected with 75% alcohol, and inoculated with Xanthomonas arboricola pv. pruni (Xap), isolated from peach leaves showing BS disease, using the sterile syringe without the needle and the sterile syringe with the needle respectively according to previous reports [65, 66]. Inoculated samples were incubated at 25°C with 60% humidity under a 16-h/8-h day/night cycle. After collection, samples were immediately frozen in liquid nitrogen and stored at −80°C. Additionally, Nicotiana benthamiana seedlings were cultivated in a controlled chamber as previously described [40].

Quantifying of PA and H₂O₂

The PA content was determined following Zhao et al. [25]. Approximately 1 g of ground sample was placed in a 1.5-ml tube, mixed with 1 ml of 60% methanol, and incubated at 4°C in the dark for 30 min. After centrifugation, 600 μl of supernatant was transferred to a new tube and mixed with 300 μl of DMACA solution (0.1 g DMACA in 100 ml of 1 M HCl in methanol). After 30 min at room temperature, absorbance was measured at 640 nm using an enzyme-linked immunosorbent assay (ELISA) reader (TECAN Infinite M200, Austria). DMACA staining followed a lab-established method [67], and images were captured with a digital camera. Hydrogen peroxide (H₂O₂) was extracted and quantified using an H₂O₂ detection kit (Solarbio Science & Technology, Beijing, China). 3,3′-diaminobenzidine (DAB) staining was performed according to previous study [25]. All experiments included three biological replicates per sample.

Extraction of total RNA and quantitative RT-PCR (qRT-qPCR) analysis

RNA extraction and quantitative real-time PCR (qRT-PCR) experiments were conducted following protocols from a previous study [41]. Approximately 100 mg of samples, ground in liquid nitrogen, were used for total RNA extraction with the HiPure HP Plant RNA Mini Kit (Magen, Guangzhou, China). The extracted RNA was reverse-transcribed into cDNA using the PrimerScript™ RT Reagent Kit with gDNA Eraser (Yeasen, Shanghai, China). Gene expression levels were quantified using the Applied Biosystems™ 7300 Real-Time PCR System (USA), and relative expression was calculated using the 2^−ΔΔCT method, with PpGADPH as the internal reference [39]. Three biological replicates were included per sample. Primer sequences are listed in Table S1.

Library construction and transcriptome analysis

RNA concentration and integrity were assessed using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA). cDNA libraries were constructed with the CloneMiner™ cDNA Library Construction Kit (Thermo Fisher Scientific, Waltham, USA), and paired-end sequencing was performed by Beijing Biomarker Technologies Co., LTD. on the Illumina HiSeq Xten high-throughput sequencing platform. Raw sequencing data were quality-checked, including removal of low-quality reads and adapter sequences, and aligned to the peach reference genome [68] using HISAT2 and Bowtie. Gene expression levels were normalized and quantified using FPKM (Fragments Per Kilobase of transcript per Million mapped reads).

Phylogenetic analysis and protein sequence alignment

Based on the AtMYB111, AtMYB12, AtTT2, AtPAP1, and AtPAP2 protein sequences from Arabidopsis thaliana, homologous genes in phylogenetically diverse species, including Vitis vinifera, P. persica, and Malus domestica, were identified using the National Center for Biotechnology Information (NCBI) database. Retrieved sequences were aligned using CLUSTALX to assess conservation and variability. A phylogenetic tree was reconstructed in MEGA6 using maximum likelihood or neighbor-joining methods to infer evolutionary relationships among these genes across the examined species. Bootstrap analysis with 1000 replicates was conducted to evaluate the robustness of the tree topology. The same approach was applied for PUB genes.

Yeast one-hybrid and electrophoretic mobility shift assay

The Y1H assay was performed as described previously [40]. Briefly, the PpMYB123 coding sequence (CDS) was amplified from ‘GHT’ cDNA and ligated into the pGADT7 vector. Promoter regions of PpANR and PpLAR were amplified from ‘GHT’ genomic DNA and cloned into the pAbAi vector. The recombinant plasmids were co-transformed into the Y1H yeast strain. Protein–DNA interactions between PpMYB123 and the PpANR and PpLAR promoters were validated by selective screening on nutrient-deficient media containing specific Aureobasidin A (AbA) concentrations.

The EMSA experiment was conducted according to the established protocol [69]. Specifically, the PpMYB123 CDS was amplified and cloned into the pET32a expression vector, which was subsequently transformed into Escherichia coli BL21(DE3) competent cells. The His-tagged recombinant protein was expressed under isopropyl β-d-1-thiogalactopyranoside (IPTG) induction and purified using nickel-affinity chromatography. The target DNA probes were labeled with 5-carboxyfluorescein (5-FAM). Experimental conditions were optimized following the manufacturer's instructions for the Chemiluminescent EMSA Kit (Beyotime, Shanghai, China). The purified PpMYB123 protein was incubated with the labeled probes in binding buffer for 30 min at room temperature. The reaction mixtures were resolved on a 6% non-denaturing polyacrylamide gel. Following electrophoresis, the gel was visualized using a multifunctional laser imaging system (Leica, Wetzlar, Germany) to detect protein–DNA complexes based on fluorescence signals. The primers used for vector construction are listed in Table S2.

Y2H and GST pull-down assay

The Y2H screening assay was conducted in accordance with established protocols described in a previous study [39], using a yeast cDNA library derived from peach fruit tissues. Candidate genes identified through primary screening were amplified by PCR using the ‘GHT’ cDNA as the template and directionally cloned into the pGBKT7 bait vector. The resulting recombinant bait vectors were co-transformed with the AD-PpMYB123 prey plasmid into the Y2HGold yeast strain. To validate protein–protein interactions, the transformed yeast cells were plated onto selective media, including double dropout (DDO; SD/–Trp/–Leu) and quadruple dropout (QDO/X/A; SD/–Trp/–Leu/–His/–Ade supplemented with X-α-gal and Aureobasidin A) agar plates, and incubated at 30°C for 3 days to assess colony growth and color development.

The GST pull-down assay was performed according to the methods described in a previous study [40]. The CDS of PpPUB23 was amplified from the cDNA of ‘GHT’ and subsequently cloned into the pGEX-4T expression vector. The recombinant plasmid was then transformed into E. coli BL21 competent cells, and the PpPUB23-GST fusion protein expression was induced by IPTG. The fusion protein was purified using glutathione-sepharose bead affinity chromatography and subsequently co-incubated with the PpMYB123-His fusion protein for in vitro interaction assays. Protein complexes were isolated via GST affinity purification and separated by SDS-PAGE. Immunoblotting was conducted using monoclonal antibodies specific to GST and His epitopes to confirm the physical interaction between the two recombinant proteins. The primers used for vector construction are listed in Table S2.

Dual luciferase expression assay

The PpANR and PpLAR promoter regions were amplified from the ‘GHT’ DNA and cloned into the pGreenII 0800-LUC binary vector. The recombinant constructs were electroporated into Agrobacterium tumefaciens strain GV3101 harboring the pSoup-p19 helper plasmid. Simultaneously, the CDSs of PpMYB123 and PpPUB23 were amplified from the ‘GHT’ cDNA and directionally cloned into the pSAK277 expression vector using restriction enzymes. The resulting constructs were introduced into A. tumefaciens GV3101 competent cells via electroporation. For transient expression assays, Agrobacterium cultures carrying the different constructs were co-infiltrated into the abaxial surface of fully expanded leaves of 3-week-old tobacco plants, following the optimized protocol described in a previous study [39]. At 48 h post-infiltration (hpi), leaf discs were harvested and homogenized in Passive Lysis Buffer. The luminescent signals from firefly luciferase (Luc) and Renilla luciferase (Ren) were quantified using the Dual-Luciferase Reporter Gene Assay Kit (Yeasen, Shanghai, China), following the manufacturer’s instructions. To normalize for transformation efficiency, relative luciferase activity was calculated as the ratio of Luc to Ren. The primers used for vector construction are listed in Table S2.

In vivo protein degradation assay

The in vivo protein degradation assay was performed according to previously established methods [70]. Total proteins were extracted from peach fruits subjected to transient gene overexpression and treatment with the proteasome inhibitor MG132 (50 μM) using a commercial protein extraction kit (Solarbio, Beijing, China). The extracted proteins were separated by SDS-PAGE and subsequently transferred onto PVDF membranes. The membranes were incubated with the PpMYB123-specific primary antibody (Mabstar, Wuhan, China; dilution 1:2000) or the anti-actin antibody (Mabstar, Wuhan, China; dilution 1:2000), followed by detection with the horseradish peroxidase (HRP)-conjugated secondary antibody. Chemiluminescent imaging was carried out using a multifunctional imaging system (FluorChem R, ProteinSimple, USA).

In vitro ubiquitination assay

The in vitro ubiquitination assay was conducted based on an established methodology [70]. The constructed fusion expression vectors PpPUB23-His and PpMYB123-GST were transformed into BL21 strain, and protein expression was induced with IPTG. A 100-μl reaction mixture was prepared containing 2.5 μl of 20× Ubiquitin Activating Enzyme (E1) Solution, 5 μl of 10× Ubiquitin Conjugating Enzyme (E2, human UbcH5b) Solution, 2.5 μl of 20× Mg-ATP Solution, 5 μl of 10× Ubiquitinylation Buffer, along with purified PpPUB23-His and PpMYB123-GST proteins. The reaction was incubated at 37°C for 3 h, followed by separation via SDS-PAGE and transfer onto a PVDF membrane. Ubiquitination status of PpMYB123-GST was detected using an Anti-GST antibody (TransGen Biotech, Beijing, China; dilution 1:2000).

Statistical analyses

All experimental data were statistically analyzed using the Statistical Package for the Social Sciences (SPSS) software (IBM, Chicago, USA). The statistical significance of differences between two groups was assessed using the independent samples t-test, with * and ** indicating statistically significant differences at P < 0.05 and P < 0.01, respectively. Quantitative data were expressed as mean ± standard error of the mean (SEM), based on three independent biological replicates per experimental group.

Author contributions

J.-P.A. and L.Z. planned and designed the experiments. L.Z., D.A., Z.L. and M.L. performed the experiments. C.L. and Q.P. collected experiment samples. D.A., and Z.L. performed the sequence analysis. L.Z. wrote the paper. J.-P.A. and Y.H. revised the manuscript.

Data availability

All data can be found online in the main text and supporting information materials.

Conflicts of interest statement

The authors declare no competing interests.

Supplementary material

Supplementary material is available at Horticulture Research online.