Study of Pear Resistance to Multiple Pathogens Through Mediation of JA/SA Signaling Pathways

TL;DR

This study identifies a gene in pears that helps resist multiple diseases by regulating plant defense pathways.

Contribution

The study identifies and characterizes PbeZFP3, a gene involved in pear resistance to multiple pathogens via JA/SA signaling.

Findings

PbeZFP3 is a C2H2-type transcription factor that enhances resistance to Valsa canker in pear rootstock.

PbeZFP3 overexpression activates jasmonic acid and salicylic acid signaling pathways.

PbeZFP3 may act as a negative regulator against Colletotrichum fructicola infection.

Abstract

Background: Apples and pears, as important economic fruit crops, are frequently threatened by various diseases, including Valsa canker. Given the numerous advantages of disease resistance breeding, the identification of key resistance genes is particularly important. This study aimed to identify the “Duli-G03” (Pyrus betulifolia) resistance gene PbeZFP3 and clarify its regulatory mechanism in disease resistance via JA/SA pathways, providing a theoretical basis for resistant breeding. Results: In this study, we identified a C2H2-type transcription factor, PbeZFP3, in the Valsa canker-resistant rootstock “Duli-G03”. Expression analysis revealed that PbeZFP3 is induced by both Valsa pyri (Vp) and Vp metabolites (VpM). Transient expression in pear and apple fruits and stable expression in suspension cells confirmed that PbeZFP3 positively regulates Valsa canker resistance. Meanwhile, PbeZFP3 not only enhances the resistance of “Duli-G03” cells to Botrytis cinerea infection, but may also act as a negative regulator against Colletotrichum fructicola. The overexpression of PbeZFP3 in “Duli-G03” significantly upregulated the expression of genes related to jasmonic acid (JA) and salicylic acid (SA) signaling. Conclusions: These findings demonstrate that the jasmonic acid (JA) and salicylic acid (SA) signaling pathways are involved in the enhanced Valsa canker resistance conferred by PbeZFP3 overexpression. A deeper understanding of this host resistance mechanism will provide theoretical support for breeding strategies aimed at developing disease-resistant fruit trees.

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1. Introduction

Apple and pear are economic fruit crops which have suffered from the threat of various diseases. Valsa canker, caused by the necrotrophic fungus Valsa species, is one of the most devastating diseases in China, even in Asia [1]. It mainly occurs on fruit trees in the peak fruit stage, harming fruits, leaves, main branches and side branches, resulting in weakening of the tree, declining fruit quality and serious economic losses [2]. In addition, the fungal disease resulting from Colletotrichum fructicola and Botrytis cinerea also frequently occurred in the plantation area of apple and/or pear [3,4]. For the purpose of controlling the outbreak of this disease, chemical, biological and integrated control strategies have been implemented [5,6]. However, these methods are largely limited by the effects of high labor costs, the risk of environmental pollution, and weak prevention. Resistance breeding has long been regarded as the most effective method to improve the stress and disease resistance of fruit trees [7]. However, its progress is limited by long reproductive cycles. Currently, there is an urgent need to screen for the main genes that contribute to disease resistance.

The plant immune system is fundamental to plant survival in natural ecosystems and productivity in crop fields. Substantial evidence supports the prevailing notion that plants possess a two-tiered innate immune system, called pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) [8,9,10]. For successful colonization, pathogens secrete pathogen-associated molecular patterns (PAMPs) onto the apoplast of plant cells and the signals can be recognized by plant membrane-localized pattern recognition receptors (PRRs), and triggers PTI, also known as the horizontal resistance [11]. After that, most pathogens can release effector proteins, another type of pathogenic factor, into plant cells to overcome PTI. However, plant resistant proteins can specifically sense effector signaling and activate a second layer of immune responses, ETI. Subsequently, the local signals are transited to whole plants and activate systematic acquired resistance (SAR). A large number of signaling molecules such as hormones, the accumulation of reactive oxygen species (ROS), salicylic acid (SA), jasmonic acid (JA), and transcription factors (TFs) are involved in the regulation of the above immune molecular networks [12,13].

As central regulators of gene expression, transcription factors (TFs) are involved in the regulation of plant growth, development, environmental adaptation and stress responses via the binding of specific cis-elements or recruiting epigenetic-modification complexes [14]. Previous investigations confirmed that mummer members of TFs play vital and unique roles in plant tolerance against multiple stresses [15]. The first plant-specific C2H2-type ZFP, ZPT2-1 (EPF1), was isolated from Petunia hybrida [16]. Since then, large C2H2-ZFP repertoires have been cataloged in many species, such as Arabidopsis thaliana [17], Solanum lycopersicum [18], Oryza sativa [19], Solanum tuberosum [20], and Nicotiana tabacum [21]. Functional studies have established that C2H2-ZFPs are key players in plant immunity and stress tolerance. The overexpression of the pepper CAZFP1 in transgenic Arabidopsis enhances resistance to Pseudomonas syringae [22]. The constitutive expression of Arabidopsis ZAT7 and ZAT12 improves tolerance to cold, drought and salt stress [23]. Another Arabidopsis C2H2 member, PUX2, confers increased resistance to powdery mildew by modulating salicylic acid signaling [24]. In tomato, SlZF3 controls plant height by directly repressing gibberellin-biosynthesis genes [25]. The apple ZAT10 accelerates leaf senescence through upregulating senescence-associated genes [26].

The purpose of this study was to identify key resistance genes from the Valsa canker-resistant pear rootstock “Duli-G03” (Pyrus betulifolia), characterize the regulatory function of PbeZFP3 against multiple pathogens including Valsa pyri, Botrytis cinerea, and Colletotrichum fructicola, and reveal the molecular mechanism by which PbeZFP3 mediates disease resistance through the jasmonic acid (JA) and salicylic acid (SA) signaling pathways, so as to provide a theoretical basis for disease-resistant breeding of fruit trees.

2. Results

2.1. PbeZFP3 Is a Typical Transcription Factor in the C2H2 Family

To acknowledge the potential functions, we first investigated the basic characteristics of GWHPAAYT030327. Chromosomal mapping and gene-structure analysis showed that it is located on chromosome 17 and the locus is a single-exon gene flanked by 5′- and 3′-UTRs. The ORF sequence encodes a 269-amino acid protein containing a canonical C2H2-type zinc finger motif spanning residues 81-103 (Figure 1A). The sequence BLAST indicated that the predicted protein shares high similarity with Arabidopsis zinc finger protein 3 (ZFP3) and is hence named PbeZFP3. Phylogenetic analysis demonstrated that PbeZFP3 is closely related to Pyrus bretschneideri protein XP_048440037.1 and exhibits higher homology with Rosaceae orthologues than with those from other plant species (Figure 1B). Multiple sequence alignment using DNAMAN revealed that the ZFP3 proteins from pear (PbeZFP3), Arabidopsis (AtZFP3), and apple (MdZFP3) exhibit high sequence similarity and contain multiple highly conserved regions (Figure 1C). Predictions using the online tools DeepLoc 2.1 and UniProt revealed nuclear localization of PbeZFP3 (Figure S1A,B). A PbeZFP3-GFP fusion construct was generated and transiently expressed in Nicotiana tabacum leaves. The results showed that the GFP signals from leaves infiltrated with the Agrobacterium strain carrying 35S: PbeZFP3-GFP were only discovered from the nucleus, whereas with the 35S: GFP were in all cellular space, confirming that PbeZFP3 is a nuclear protein (Figure 1D). The above results indicate that PbeZFP3 is a typical transcription factor that belongs to the C2H2 family.

2.2. The Expression of PbeZFP3 Respond to Multiple Stress-Related Signals

To further investigate probable signals regulating the expression of PbeZFP3, we predicted the cis-elements in it promote the region via the online database Plant CARE. The results demonstrated that the promoter region of PbeZFP3 contains various stress-related cis-elements, such as G-box, TCA-element, and ABRE, which respond to salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), and oxidative stress signals, respectively (Figure 2A). Expression pattern analysis of PbeZFP3 in 20% VpM-infected “Duli-G03” suspension cells revealed that it was significantly upregulated at three time points (Figure 2B). Furthermore, we examined the response pattern of PbeZFP3 in “Duli-G03” to exogenous ABA, SA, and JA signals. Compared to the control, the expression of PbeZFP3 was significantly activated in cells treated with exogenous ABA, SA, and JA (Figure 2C–E). For instance, under ABA treatment, the expression continued to increase up to 48 h, while SA and JA treatments induced significant responses at 12 h and 3 h, respectively. These findings collectively demonstrate that the expression of PbeZFP3 could be induced by VpM and multiple stress-related signals.

2.3. PbeZFP3 Positively Regulate Valsa Canker Resistance of Pear Fruits

To address the mechanism of the influence on Valsa canker resistance, we transiently overexpressed PbeZFP3 in fruits of both “Huangguan” pear and “Fuji” apple (Figure 3). The evaluation of the disease lesions showed that the lesion diameters in both pear and apple fruits infiltrated with PbeZFP3 were significantly smaller than that with vector controls (Figure 3A,B), suggesting positive roles in fruit resistance against Valsa canker. As symptoms progressively developed at the inoculation sites, we further measured lesion diameter in each treatment. The data demonstrated that the overexpression of PbeZFP3 significantly reduced the lesion diameters caused by Vp in both “Huangguan” pear and “Fuji” apple fruits (Figure 3E,F). qRT-PCR analysis revealed that the expression level of PbeZFP3 at the injection sites was significantly upregulated compared with the empty vector controls (Figure 3C,D). These findings indicate that the overexpression of PbeZFP3 positively regulates resistance to Vp in both pear and apple fruits.

2.4. Overexpression PbeZFP3 Elevated Valsa Canker Resistance of “Duli-G03” Suspension Cells

Because of the positive roles of Valsa canker resistance of apple and pear fruits, we speculate that PbeZFP3 also regulates the resistance of other tissues. Therefore, we further transformed PbeZFP3 into “Duli-G03” suspension cells and generated three transgenic cell lines, PbeZFP3-OE1, PbeZFP3-OE2, and PbeZFP3-OE4. The incubation of the cells with the Vp isolate showed that the growth of the pathogen was obviously inhibited compared to the wild-type (WT) cells. Further MTT staining revealed higher cell viability of the transgenic cell lines than that of WT (Figure 4A). The overexpression of PbeZFP3 in transgenic cell lines was further validated by the qRT-PCR assay (Figure 4B). At 72 h post-inoculation, the colony diameter was recorded as 27 mm on the WT, whereas it only varied from 12.5 to 14 mm on the overexpression cell lines (Figure 4C). Taken together, these results suggested that the overexpression of PbeZFP3 greatly increased the Valsa canker resistance of pear “Duli-G03” cells.

2.5. Overexpression of PbeZFP3 Enhanced Tolerance of Suspension Cells Against VpM

Among three transgenic cell lines, PbeZFP3-OE4 displayed the highest expression level of the target gene and Valsa canker resistance. We subsequently detected the influence of PbeZFP3 on cellular tolerance to Valsa pyri metabolism (VpM). Under the VpM exposure, the cell viability of PbeZFP3-OE4 was significantly higher than that of WT, especially under high concentration (20%) (Figure 5A,B). Meanwhile, ROS fluorescences were significantly increased in PbeZFP3-OE4 compared to WT at the early stages of VpM exposure (1–3 h) (Figure 5C,D). These results collectively indicate that the overexpression of PbeZFP3 significantly enhances the tolerance of “Duli-G03” suspension cells to VpM.

2.6. PbeZFP3 Initiated Immune Responses

To elucidate the immune pathways regulated by PbeZFP3, we analyzed the expression patterns of 12 immune-related genes in both wild-type (WT) and PbeZFP3-overexpressing (PbeZFP3-OE4) cell lines following treatment with 20% VpM (Figure 6). Compared to the WT, the overexpression of PbeZFP3 promotes the upregulation of several genes during VpM exposure, including the SA-related genes PbePR1 and PbePR5, and the JA-related genes PbePR1b and PbePDF1.2. Among these, the SA-related gene PbePR5 was induced 8.25-fold relative to the control at 1 h post-treatment. Taken together, these results indicate that PbeZFP3 activates multiple plant immune signaling pathways, thereby enhancing resistance to Valsa canker.

2.7. PbeZFP3 Contribute to the Resistance of Suspension Cells Against Botrytis Cinerea but Not Colletotrichum Fructicola

Due to the positive regulation of PbeZFP3 on Valsa canker resistance, we further explored its roles on host resistance against other important pathogenic fungal pathogens, Colletotrichum fructicola and Botrytis cinerea (Figure 7). The results exhibit that the overexpression dramatically reduced the colonial expansion of B. cinerea, but not C. fructicola (Figure 7A). The lesion diameters of B. cinerea were recorded as 13.2 mm, 15.8 mm and 12.7 mm on three overexpression lines, which is significantly smaller than that on WT (29.2 mm) (Figure 7B). On the other hand, compared to the WT, the colonial size of C. fructicola obviously enlarged in overexpressed cells (Figure 7C). Collectively, these findings demonstrate that PbeZFP3 also enhances the resistance of “Duli-G03” cells against infection by B. cinerea, but might act as a negative regulator to C. fructicola.

3. Discussion

Transcription factors (TFs) regulate the spatiotemporal expression of target genes and act as essential regulatory factors in the plant lifecycle [27,28]. In this study, we identified that PbeZFP3, a C2H2 member in Pyrus betulifolia, positively regulates the resistance against Valsa canker and infection by B. cinerea, but acts as a negative mediator for C. fructicola. The overexpression of PbeZFP3 activates the expression of JA- and SA-related genes during cell responds to Valsa canker signals.

Zinc finger proteins (ZFPs) are among the largest transcription factor families in plants, and members of the C2H2 type play crucial roles in regulating responses to biotic and abiotic stresses [17,29]. C2H2 zinc finger proteins primarily rely on their zinc finger structural motifs to bind enhancer sequences, thereby regulating gene expression, cell differentiation, embryonic development, and enhancing plant stress tolerance [30]. Early research demonstrated that the CCCH-type zinc finger protein GhZFP1 from cotton interacts with stress-related proteins GZIRD21A, effectively improving salt stress tolerance and resistance to fungal infection in transgenic tobacco [31]. In Arabidopsis, the overexpression of the C2H2 member PUX2 enhances resistance to powdery mildew by modulating salicylic acid (SA) signaling [24]. The TFAIII-type transcription factor OsZFPH, involved in signaling pathway regulation, confers resistance to Xanthomonas oryzae pv. oryzae in rice [32]. In this study, stable overexpression of PbeZFP3 in suspension-cultured cells of “Duli-G03” further confirmed that this gene enhances resistance to Valsa canker by inhibiting pathogen growth and maintaining cell viability. Meanwhile, PbeZFP3 not only enhances the resistance of “Duli-G03” cells to Botrytis cinerea infection but may also act as a negative regulator against Colletotrichum fructicola. Botrytis cinerea is a necrotrophic pathogen, against which plant defense primarily relies on the jasmonic acid (JA) signaling pathway [33,34]. In contrast, Colletotrichum fructicola is a hemibiotrophic pathogen, with early plant defense against it mainly dependent on the salicylic acid (SA) signaling pathway [35,36,37]. The JA pathway and pathogenesis-related (PR) proteins activated by PbeZFP3 overexpression can effectively curb the invasion of B. cinerea. C. fructicola, however, employs a stealth-invasion strategy. The overexpression of PbeZFP3 may impair the defensive function of effector-triggered immunity (ETI) components, such as PbeEDS1, while concurrently and prematurely hyperactivating the broad-spectrum defense responses mediated by both SA and JA signaling pathways [38,39]. This imbalanced immune activation creates more favorable conditions for the successful colonization of C. fructicola, which is adept at establishing early latent infections. These hypotheses regarding the potential roles of ETI and PbeEDS1 await further verification by more direct experiments in future studies.

Plant innate immunity relies on two interconnected pathways: pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) [38,39]. Reactive oxygen species (ROS) play a crucial role in plant immunity, but excessive accumulation can lead to oxidative damage [40]. PbeZFP3-overexpressing cells effectively regulated ROS bursts at early stress stages (1–3 h), suggesting that this gene may mitigate oxidative damage by enhancing antioxidant capacity. This observation aligns with previous reports indicating that “C2H2-type zinc finger proteins can improve stress tolerance by modulating ROS homeostasis” [41]. Salicylic acid (SA) and jasmonic acid (JA), two well-characterized defense hormones, play key roles in inducing local defense responses and systemic resistance [42]. Generally, the JA pathway mainly mediates plant defense against necrotrophic pathogens, while the SA pathway is primarily associated with resistance against biotrophic pathogens [43]. The interaction between SA and JA pathways can be antagonistic, synergistic, or neutral [44,45]. A recent Arabidopsis genome-wide transcriptome study revealed widespread synergistic interactions between JA and SA [46]. In this study, upon VpM treatment, the expression of SA pathway marker genes (PbePR1, PbePR5) and JA pathway marker genes (PbePR1b, PbePDF1.2) was significantly upregulated in PbeZFP3-overexpressing cells. Therefore, we emphasize the important role of PbeZFP3 in the JA and SA signaling pathways in conferring resistance to pear Valsa canker and propose that the disease resistance mediated by PbeZFP3 may depend on the activation of the JA and SA pathways. However, the specific regulatory mechanisms of these two signaling pathways require further investigation.

4. Materials and Methods

4.1. Plant Materials and Pathogen Isolation

Suspension cells of the pear rootstock variety “Duli-G03” (Pyrus betulifolia) were induced from leaves of tissue-cultured plantlets and cultured at 25 °C in darkness with shaking at 100 rpm [47]. A pathogenic Valsa pyri (isolate Vp-007), Botrytis cinerea and Colletotrichum fructicola were cultured on potato dextrose agar (PDA) medium at 25 °C darkness [48,49]. To obtain the metabolites of Vp (VpM), 10 disks of 5 mm diameter of the activated PDA cultures were transferred into 100 mL of potato dextrose broth (PDB) liquid medium and cultured at 25 °C in darkness and for 3 d statically. The samples were centrifuged at 5500 g for 8 min, and the supernatant was filtered with a 13 mm PES syringe with a 0.22 μm filter and counter ring to obtain the metabolites.

4.2. Bioinformatic Analysis of PbeZFP3

The coding sequence (CDS) and protein sequence of PbeZFP3 were retrieved from the Genome Database for Rosaceae (GDR, https://www.rosaceae.org accessed on 12 November 2025). Genomic information was obtained by searching the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/ accessed on 12 November 2025) and The Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org accessed on 12 November 2025). Full-length amino acid alignments of PbeZFP3 with orthologues from multiple species were performed using DNAMAN 9.0. A neighbor-joining phylogenetic tree based on the protein sequences of PbeZFP3 and its homologues was constructed with MEGA 11. Protein domains were predicted with the SMART online resource (http://smart.embl-heidelberg.de/ accessed on 17 November 2025) using an E-value ≤ 1 × 10^−5^. Putative cis-acting elements in the PbeZFP3 promoter were identified with the Plant CARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ accessed on 17 November 2025) and visualized with TBtools-II v2.420.

4.3. Subcellular Localization of PbeZFP3

The CDS of PbeZFP3 was cloned into pCAMBIA1300-GFP, and the recombinant plasmid 35S: PbeZFP3-GFP was introduced into Agrobacterium GV3101 [50]. 35S: PbeZFP3-GFP or empty vector was transformed into GV3101 and incubated to an OD600 value of 1.0. Agrobacterium was resuspended in MES buffer and kept at 4 °C for 4 h. The suspension was injected into the leaves of Nicotiana benthamiana. After culturing at 25 °C for 48 h, GFP fluorescence signals at the injection site were detected by confocal microscopy (FV1200, Olympus LS, Manufacturer: Carl Zeiss; City: Jena; Country: Germany).

4.4. Transient Expression of PbeZFP3 in Pear Fruits

Agrobacterium strains carrying the target gene and the empty vector (control) were activated, expanded to OD_600_ = 0.6–1.0, harvested by centrifugation, and resuspended in MES-KOH buffer. After 4 h at 4 °C, 0.2 mL of the suspension was infiltrated into each pear and apple fruit. The fruits were incubated at 25 °C for 3 days and then used for pathogen inoculation and qPCR analysis of target gene expression at the infiltration site. A mycelial plug of Valsa pyri (Vp) was placed on the agroinfiltrated area, and lesion diameters were measured at 36, 48, 60, 72 and 84 h post-inoculation. The experiment was conducted with five biological replicates.

4.5. Stable Overexpression and Functional Verification of PbeZFP3

Agrobacterium GV3101 carrying 35S-PbeZFP3 was transformed into “Duli-G03” suspension cells by using Agrobacterium-mediated transformation. The overexpression cell lines were screened by PCR detection and qRT-PCR assay. PbeZFP3 overexpression cells and WT cells were cultured in MS solid medium and inoculated with Vp. The change in lesion diameter was measured with Vernier calipers. PbeZFP3-overexpressing cells were infected with VpM at a concentration of 20%. For the control, WT cells were treated in the same conditions as above. All samples were harvested at 0, 1, 3 and 6 h, respectively, and the cell activity was determined by the methyl thiazolyl tetrazolium (MTT) method [51]. For ROS measurement, all samples were treated with the above method and were stained with reactive oxygen-specific fluorescent dye H2DCFDA (10 µM). Quantitative analysis of ROS was performed by enzyme labeling apparatus (TECAN, Spark, Switzerland) at excitation wavelength at 488 nm and emission wavelength at 525 nm. All assays were biologically and technology replicated five times.

4.6. Gene Expression Assays

The expressional patterns were detected by using data extraction from released RNAseq data and quantitative real-time PCR (qRT-PCR) assay. For RNAseq data, the suspension cells of “Duli-G03” were treated with VpM for 1, 3 and 6 h [52]. The log2-fold-change values of the target gene were extracted as expressional levels. To perform the qRT-PCR assay, total RNA was extracted using the RNA out kit (160906-50, Tiandz, Beijing). All primers of target genes and acting genes were designed on the online software Primer 3.0 (http://primer3.ut.ee/ accessed on 12 August 2025) (Table S1). cDNA synthesis and qRT-PCR amplification were conducted using a Prime Script RT reagent Kit with gDNA Eraser (RR047, TaKaRa, Dalian) and SYBR Green Pro Taq HS premix qPCR kit (AG11718, TaKaRa, Dalian). The fluorescence curve and melting curve were analyzed, and the relative gene expression was calculated by the 2^−ΔΔCT^ method. The above assays were technologically and biologically repeated at least three times.

4.7. Statistical Analysis

The basic data were statistically analyzed in Microsoft Excel 2016 and visualized with Origin 2022, and differences between means were analyzed using Student’s t-test (* p < 0.05; ** p < 0.01).

5. Conclusions

Taken together, PbeZFP3, a member of the C2H2-type zinc finger gene family, positively enhances resistance to Valsa canker in both “Duli-G03” cells and pear/apple fruits. It also augments the resistance of “Duli-G03” cells to pear B. cinerea. Signaling pathways related to ROS, SA, and JA are likely involved in the PbeZFP3-mediated immune response. Furthermore, expression analysis indicates that JA and SA signaling play crucial roles in initiating Valsa canker resistance triggered by PbeZFP3. This study provides a theoretical basis for disease-resistant breeding in pear trees.