PpTOR, a Major Factor Associated with Phytophthora parasitica Virulence, Serves as a Candidate RNAi Target for Disease Control

Highlights

What are the main findings?

• Our study systematically investigated the role of the target of rapamycin (TOR) in the growth and pathogenicity of Phytophthora parasitica, a devastating oomycete pathogen, and revealed that PpTOR acts as a major regulator of P. parasitica pathogenicity by affecting the transcription of numerous virulence-related genes, especially key effector genes belonging to the PpRxLR and PpCRN families.
• Functional characterization of PpRxLR3, a PpTOR-related effector, revealed that it promotes P. parasitica infection by modulating plant jasmonic acid biosynthesis and signaling and the plant immune response.
• Host-induced gene silencing (HIGS) of PpTOR confers strong resistance to P. parasitica in Nicotiana benthamiana, which is associated with downregulated expression of PpTOR and its downstream effector genes in P. parasitica.

What is the implication of the main findings?

• The findings identify PpTOR as a promising target for P. parasitica disease management.

Abstract

Phytophthora parasitica is a devastating oomycete pathogen that causes significant crop losses worldwide. Identifying master regulators of its virulence is crucial for the development of novel control strategies. Here, we demonstrate that the conserved eukaryotic kinase TOR (target of rapamycin) is essential for both growth and pathogenicity in P. parasitica. Transcriptomic analysis revealed that PpTOR inhibition broadly reprograms the transcriptome of P. parasitica, notably leading to the downregulation of numerous PpRxLR and PpCRN effector genes. Among these genes, the overexpression of PpRxLR3 increased plant susceptibility to P. parasitica by affecting jasmonic acid biosynthesis and signaling. On the basis of the crucial role of PpTOR, we evaluated its potential as a target for intervention. Host-induced gene silencing (HIGS) of PpTOR in Nicotiana benthamiana conferred strong resistance to P. parasitica, which was associated with the downregulation of the expression of PpTOR and key effector genes during P. parasitica infection. Furthermore, small RNA sequencing confirmed the production of PpTOR-specific siRNAs in HIGS plants. Exogenous application of synthetic siRNAs targeting PpTOR effectively reduced P. parasitica virulence. Our findings establish PpTOR as a global regulator of pathogenicity and validate PpTOR as a promising target for RNA-based disease control strategies.

1. Introduction

Phytophthora parasitica is a devastating oomycete pathogen with a broad geographical distribution and host range and poses a persistent threat to global agriculture. It has been successfully isolated from numerous economically important crops, trees, and medicinal plants, as well as from soil and aquatic environments, demonstrating its remarkable adaptability [1]. Renowned for its high level of pathogenicity, which is similar to that of other oomycete pathogens, such as P. infestans and P. sojae. P. parasitica is capable of causing root and crown rot in more than 250 genera of herbaceous and perennial plant species, as well as on the leaves and fruits of these plants, posing a serious threat to global agriculture and ecosystems [1].

Currently, chemical control remains the primary strategy for managing Phytophthora pathogens because of its immediate effectiveness and operational convenience, particularly in outbreak scenarios [2,3]. However, the frequent application of chemical pesticides not only prompts the gradual development of drug resistance in Phytophthora, thereby weakening the effectiveness of the prevention and control of Phytophthora but also raises serious environmental and health concerns, including soil and water pollution and potential threats to human health through food chain accumulation [3,4]. Therefore, revealing the molecular mechanisms underlying the interaction between P. parasitica and its host plants is particularly important, as it can help us explore innovative, environmentally friendly, and sustainable disease prevention and control strategies.

In the context of the ongoing coevolutionary competition between plants and pathogens, plants have evolved a highly efficient immune system to defend against pathogenic invasions. When plants are subjected to pathogenic stress, the pattern recognition receptors (PRRs) of plant cells can recognize pathogen-associated molecular patterns (PAMPs) from pathogens and quickly activate PAMP-triggered immunity (PTI) to confer resistance to pathogen invasion [5]. In response, pathogens have evolved countermeasures to overcome the PTI response in plants. Pathogens employ various strategies to suppress the PTI response of plants, the most crucial of which is the secretion of effector proteins by pathogens to infiltrate and dismantle the host immune barrier [6]. Effector proteins accomplish their goals by disrupting cellular gene expression and metabolism to suppress host defenses [7]. Among the most studied cytoplasmic effector subclasses, the RxLR and Crinkler (CRN) families, characterized by conserved N-terminal motifs (RxLR-EER and LXLFLAK, respectively), play important roles in pathogen-infecting plants. For example, the RxLR effector AVR3a^EM from P. infestans can prevent host–cell death by interacting with and stabilizing the E3 ligase CMPG1 during the biotrophic phase of infection [8]. The Plasmopara viticola RxLR effector interacts with and stabilizes oxygen-evolving enhancer 2 (VpOEE2 or VpPsbP) to reduce H₂O₂ accumulation, thereby promoting disease in Vitis piasezkii [9]. Although P. parasitica is known to be a potent pathogen, the functional repertoire of its effectors remains largely unexplored. Huang et al. reported that the RxLR effector PpE4 promotes the infection of P. parasitica and triggers cell death in the host [10]. Maximo et al. reported that two CRN effectors, PpCRN7 and PpCRN20, increase the susceptibility of Nicotiana benthamiana to P. parasitica by modulating plant cell death [11]. This limited knowledge highlights a significant gap in understanding the effector arsenal of P. parasitica. In plant defense responses, the apoplast serves as a crucial site for resistance to pathogen invasion, and plants secrete digestive enzymes, including PR-1 and apoplastic proteases, into the apoplast [12]. Beyond intracellular sabotage, pathogens must also contend with apoplastic defenses. Consequently, pathogens secrete apoplastic effectors that act as inhibitors of these host enzymes, thereby aiding pathogen infection and colonization. Notably, the atypical RxLR effector PsAvh181 from P. sojae interacts with the host Gm SNAP-1 protein to disrupt the vesicle trafficking of defense-related proteins, effectively blocking their secretion into the apoplast [13]. Whether P. parasitica employs similar sophisticated strategies to neutralize apoplastic defenses is an open question.

Beyond effector secretion, pathogens employ an arsenal of enzymes to breach the physical and chemical barriers of host plants [14,15]. For example, pathogens secrete cutinases to degrade the protective cuticle layer of plants, creating ports of entry for invasion [16,17]. Similarly, cell wall-degrading enzymes (CWDEs) are crucial for tissue maceration and nutrient release. For instance, P. parasitica produces endo-β-1,4-xylanases to break down hemicellulose, facilitating penetration and spread within host tissues [18]. Additionally, Zhang et al. reported that the P. parasitica cysteine proteases PpCys44 and PpCys45 can promote pathogen virulence and trigger host–cell death at the late stages of infection [19]. Collectively, these results underscore the multifaceted nature of plant–pathogen interactions, and P. parasitica may have evolved a complex repertoire of tools—from apoplastic and cytoplasmic effectors—to suppress host immunity across different spatial and temporal contexts.

The coordinated expression of this diverse virulence arsenal strongly suggests the existence of a central regulatory hub within the pathogen. The target of rapamycin (TOR), a highly conserved atypical serine/threonine protein kinase, represents a prime candidate for such a role. TOR functions as a master integrator of environmental, nutritional, and hormonal signals, directly phosphorylating downstream substrates to govern cell growth, proliferation, and metabolism [20]. Since its initial discovery in yeast, the structure and function of TOR have been gradually revealed. The TOR protein encompasses five highly conserved domains: HEAT repeat, FAT, FRB, kinase, and FATC. Among these domains, the kinase domain is the most conserved and is responsible for its serine/threonine kinase activity [21]. In mammals and yeast, it typically operates within two distinct multiprotein complexes, TOR complex 1 (TORC1) and TOR complex 2 (TORC2). The core components of TORC1 include TOR, RAPTOR, and LST8, whereas TORC2 comprises TOR, RICTOR, and LST8 [20]. Among the two complexes, TORC1 is uniquely and acutely sensitive to inhibition by the macrolide antibiotic rapamycin. Rapamycin, which was originally isolated from Streptomyces hygroscopicus on Easter Island, exerts its inhibitory effect by binding to the cellular protein FKBP12 (FK506-binding protein of 12 kDa) to form a rapamycin-FKBP12-TOR ternary complex via the FRB domain of TOR [20]. This specific pharmacological property has made rapamycin an indispensable tool for dissecting TOR functions. Moreover, owing to functional differences in plant FKBP12, rapamycin selectively inhibits pathogen TOR signaling without affecting the plant host. This specificity makes it an ideal tool for dissecting the pathogen-specific role of TOR during plant–pathogen interactions [22,23].

Recent studies on plant pathogenic fungi and oomycetes have revealed that TOR is also a crucial regulator of pathogenicity, with its inhibition consistently impairing hyphal growth, development, and virulence [23,24,25,26,27,28,29]. P. parasitica is a destructive and widely distributed pathogen, yet the specific functions and mechanistic basis of TOR in this economically important oomycete remain largely unknown. In this study, we employed a multifaceted approach combining rapamycin treatment, phenotypic assays, transcriptomics, and RNA-based silencing strategies to investigate the potential function of PpTOR in P. parasitica. We demonstrate that PpTOR is essential for hyphal growth, development, and pathogenicity in P. parasitica. Transcriptomic analysis revealed that inhibition of PpTOR expression led to a broad downregulation of the expression of virulence-associated genes, most strikingly those encoding RxLR and CRN effector genes. The functional characterization of a key effector, PpRxLR3, confirmed its role in suppressing host resistance by attenuating jasmonic acid (JA) biosynthesis and signaling in plants. Furthermore, we generated PpTOR RNA interference-treated plants that were highly resistant to P. parasitica. In addition, synthetic siRNAs targeting PpTOR effectively reduced the pathogenicity of P. parasitica. Our findings establish PpTOR as a crucial regulator of pathogenicity in P. parasitica by affecting the expression of a suite of effector proteins and validate it as a promising target for disease control strategies.

2. Materials and Methods

2.1. Plant Material and Pathogen Strains

The N. benthamiana, Nicotiana tabacum cv. Honghua Dajinyuan (HD) and transgenic plants were cultivated in a greenhouse at 22 °C under a 16 h light/8 h dark photoperiod with the light intensity maintained at 10,000 lux. The P. parasitica strain WL1-7 was routinely preserved and subcultured on oatmeal agar (OMA) medium in constant darkness at 25 °C for all subsequent experimental analyses.

2.2. P. parasitica Inoculation Experiments

Plant infection assays were performed using seedlings at defined developmental stages, with tobacco at the six true-leaf stage and N. benthamiana at the seven true-leaf stage. The detached leaves or seedling stem bases were inoculated with uniform P. parasitica mycelial plugs and then incubated in the dark at 30 °C with 100% relative humidity. Disease phenotypes were photographed and quantified at designated time points post-inoculation.

2.3. Transcriptome and Small RNA Sequencing

To prepare P. parasitica samples for transcriptome sequencing, root extract was prepared by grinding 5 g of fresh N. benthamiana roots in liquid nitrogen and homogenizing them in 50 mL of phosphate buffer (pH 7.2) to simulate host-derived infection signals. Approximately 0.1 g of fresh P. parasitica mycelium was incubated in root extract medium supplemented with 10 μM rapamycin (Sigma-Aldrich, St. Louis, MO, USA) or an equivalent volume of DMSO (solvent control; Sigma-Aldrich, St. Louis, MO, USA) for 6 h at 25 °C. Treated mycelia were collected by centrifugation, snap-frozen in liquid nitrogen, and stored at −80 °C for total RNA extraction. Three biological replicates were generated for transcriptome sequencing.

To prepare plant samples for transcriptome sequencing, six true-leaf-stage HD and PpRxLR3-OE whole seedlings were harvested, flash-frozen in liquid nitrogen, and preserved at −80 °C for RNA isolation and transcriptome sequencing, with three independent biological replicates.

Total RNA library construction and paired-end Illumina sequencing were completed by Novogene Bioinformatics Technology (Beijing, China). Raw sequencing reads underwent strict quality filtering to remove adapters, ambiguous N bases and low-quality reads. Each library generated approximately 6 Gb of clean PE150 reads, with Q20 > 98% and Q30 > 95%. Clean reads were mapped to the P. parasitica reference genome (http://protists.ensembl.org/Phytophthora_parasitica_gca_000509525/, accessed on 12 March 2026) and the N. tabacum reference genome (http://lifenglab.hzau.edu.cn/Nicomics/, accessed on 5 April 2026) using TopHat2 software (version 2.0.10) [30], yielding overall unique mapping rates greater than 85% (P._parasitica) and 90% (N. tabacum), respectively. The fragments per kilobase of transcript per million mapped reads (FPKM) of each gene were calculated on the basis of the length of the gene and the read count mapped to this gene. The R package edgeR (version 4.2.1) was applied to the raw read counts to identify differentially expressed genes (DEGs) [31].

DEG screening thresholds were set as |log₂ (fold change)| ≥ 1 and an adjusted false discovery rate (FDR) < 0.05 for the P. parasitica transcriptome data or an adjusted p-value (Padj) < 0.05 for the RxLR3-OE plant transcriptome data.

For small RNA sequencing and data processing, whole six-leaf-stage WT and PpTOR-RNAi N. benthamiana seedlings were collected for small RNA (sRNA) library construction and sequencing at Novogene Bioinformatics Technology (Beijing, China). After quality filtering, clean reads ranging from 18 to 36 nt were retained and aligned to the conserved target fragment sequence of PpTOR1/2. The number of mapped sRNA reads targeting PpTOR was counted to quantify transgene-derived silencing small RNAs.

2.4. Vector Construction and Plant Transformation

To generate overexpression vectors, full-length coding sequences (CDSs) of the PpRxLR and NtJAZ genes were amplified from P. parasitica cDNA and tobacco leaf cDNA, respectively. Signal peptide coding regions at the N-terminus of the PpRxLR genes were truncated before primer design. Purified PCR amplicons were inserted into the entry vector P35S-8GWN to construct donor plasmids. Gateway LR recombination reactions were performed between the donor vectors and the KANA30 destination vector to generate 35S-driven effector overexpression constructs (p35S-RxLR/JAZ-KANA303). The recombinant plasmids were subsequently transformed into Agrobacterium tumefaciens strain GV3101 for subsequent transient and stable transformation experiments.

To generate the PpTOR1/2 RNAi hairpin vector, a 300 bp fragment specific to PpTOR1 and PpTOR2 was amplified and cloned in sense and antisense orientations, separated by the IN4 intron spacer, into the p35S-IN4-8GWN entry vector as previously described [22]. The complete hairpin expression cassette was recombined into KANA303 via a Gateway LR reaction to produce the p35S-PpTOR1/2-RNAi-KANA303 silencing vector.

Finally, all the KANA303 destination vectors were transferred into Agrobacterium strain GV3101 to generate transgenic N. benthamiana and N. tabacum plants using the Agrobacterium-mediated transformation method, following previously described protocols [32]. All the primers used for gene cloning are listed in Table S11.

2.5. Agrobacterium-Mediated Transient Expression of RxLR Genes in N. benthamiana

Agrobacterium carrying the PpRxLR overexpression construct or an empty vector (EV) control was cultured overnight in liquid LB medium supplemented with 50 mg/L rifampicin (Solarbio, Beijing, China) and 50 mg/L kanamycin (Solarbio, Beijing, China). Agrobacterium cells were then collected by centrifugation and resuspended in infiltration buffer (10 mM MES (Solarbio, Beijing, China), 10 mM MgCl₂ (Solarbio, Beijing, China), 200 μM acetosyringone (Solarbio, Beijing, China), and 5 g/L glucose (Solarbio, Beijing, China)). The Agrobacterium suspension was infiltrated into the abaxial side of fully expanded N. benthamiana leaves following standard agroinfiltration protocols [33]. At 36 h after infiltration, the infiltrated leaves were used for subsequent experiments. Infiltrated leaf tissues were incubated for 36 h under normal growth conditions before P. parasitica inoculation or molecular detection.

2.6. Jasmonic Acid (JA) Measurement in PpRxLR3-OE and Control HD Plants

Seedlings grown in the greenhouse at the six-leaf stage were inoculated with P. parasitica for 48 h. JA was extracted from tobacco, and the total JA content in the plants was quantified using a Plant JA ELISA test kit (MEIMIAN, Yancheng, China) following the manufacturer’s instructions.

2.7. RNA Extraction and Quantitative Real-Time PCR (RT–qPCR)

Total plant RNA was extracted using the RNA PrepPure Plant Plus Kit (Tiangen Biotech, Beijing, China), while fungal RNA from P. parasitica mycelia was isolated with the HiPure Fungal RNA Mini Kit (Magen, Guangzhou, China) following the manufacturer’s protocols. cDNA was reverse transcribed using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). RT–qPCR was performed using TB Green Premix Ex Taq II (TaKaRa, Dalian, China), and the parameters were as follows: 94 °C for 5 min, followed by 40 cycles of 94 °C for 15 s and 60 °C for 30 s each, and a dissociation stage of 95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s. All reactions were performed with 3 biological replicates. The plant internal reference gene GAPDH and the P. parasitica reference gene UBC were used to calculate the relative transcript abundance via the 2^−ΔΔCt method in plants and P. parasitica, respectively. The primers used for RT–qPCR are listed in Table S11.

To assess the relative P. parasitica biomass, approximately 100 mg of inoculated plant tissue was collected for genomic DNA extraction. P. parasitica DNA levels were quantified via RT–qPCR targeting P. parasitica reference genes, with identical thermal cycling parameters as RT–qPCR, to calculate the relative pathogen biomass within plant tissues.

3. Results

3.1. Identification and Phylogenetic Analysis of PpTORs

To investigate the function of TOR in P. parasitica, we first identified its homologs. Using TOR protein sequences from Arabidopsis, yeast and humans, we performed a BLAST (version 2.17.0) search against the P. parasitica genome database (http://protists.ensembl.org/Phytophthora_parasitica_gca_000509525/, accessed on 2 March 2026). Two TOR homologs were identified and named PpTOR1 (L914_08005) and PpTOR2 (L914_16893). Bioinformatic analysis revealed that the full-length PpTOR1 and PpTOR2 genes are 11.6 kb and 8.16 kb long, respectively, and contain no introns (Figure S1A). Their full-length coding sequences are 9748 bp and 7970 bp long, encoding 3242 and 2653 amino acid residues with predicted molecular masses of approximately 355 kDa and 296 kDa, respectively. Domain analysis revealed that both PpTOR1 and PpTOR2 contain the typical FAT, FRB, and FATC domains and a highly conserved kinase domain (Figure S1B,C). Phylogenetic analysis revealed that PpTORs are more closely related to AtTOR from Arabidopsis than to TOR proteins from other organisms (Figure S1B,C). Furthermore, TOR proteins are evolutionarily conserved across the oomycete genus (Figure S2, Table S1). Previous studies have shown that TOR recruits shared and distinct core components to form TORC1 and TORC2 complexes. Homologs of the TORC1 core components (RAPTOR and LST8) and the TORC2 core components (RICTOR and LST8) were also identified in the P. parasitica genome (Table S2). These results suggest that the TOR pathway is evolutionarily conserved in P. parasitica.

3.2. PpTOR Affects the Growth and Pathogenicity of P. parasitica

To characterize the biological function of PpTOR, the growth and development of P. parasitica under TOR inhibition were evaluated by using rapamycin treatment. The results showed that rapamycin significantly inhibited the vegetative growth of P. parasitica in a dose-dependent manner. Moreover, rapamycin-exposed hyphae displayed distinct morphological abnormalities, including excessive branching, hyphal distortion, and irregular cellular swelling (Figure 1A–C). Collectively, these results demonstrate that PpTOR plays a critical role in the hyphal growth of P. parasitica. We further investigated the role of PpTOR in regulating the pathogenicity of P. parasitica. The data revealed that both the lesion area and pathogen biomass significantly decreased with increasing rapamycin concentration, suggesting that PpTOR inhibition significantly attenuated P. parasitica infection in N. benthamiana (Figure 1D,E). Notably, PpTOR2 was significantly upregulated during early host infection, whereas the abundance of the PpTOR1 transcript remained relatively stable across all the sampled time points. This divergent expression pattern strongly implies functional specialization between the two PpTOR paralogs.

To further elucidate the function of PpTOR, transcriptome sequencing of P. parasitica mycelia treated with rapamycin under conditions simulating plant infection (using tobacco root extract) was performed (Figure 2A). Transcriptome data revealed that inhibition of PpTOR resulted in the differential expression of 1618 genes, comprising 974 upregulated genes and 644 downregulated genes (Figure 2B, Table S3). Gene Ontology (GO) enrichment analysis indicated that these differentially expressed genes (DEGs) were significantly enriched in various functional categories, including metabolic processes, cellular biosynthetic processes, the cell cycle, and cell development (Figure S3, Table S4). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis further revealed that the upregulated and downregulated DEGs were annotated to 74 and 79 pathways, respectively (Table S5). Among these functions, TOR-related conserved functions such as ribosome biogenesis, amino acid metabolism, autophagy, and the MAPK signaling pathway were significantly enriched (Figure 2C,D; Table S5), suggesting that PpTOR performs evolutionarily conserved core regulatory functions in P. parasitica.

Oomycetes secrete a large array of effector proteins, including RxLR and CRN effectors, into host plants to facilitate infection [34]. Therefore, we focused on analyzing the changes in the expression of RxLR and CRN effector genes, as well as other previously reported virulence-related factors in P. parasitica (such as the cysteine protease PpCys44/45 and the hydrolase PpXYN1/2), under PpTOR inhibition [10,11,18,19]. The expression of numerous effector proteins and virulence-related genes was significantly altered upon rapamycin treatment (Figure S4). RT–qPCR assays further validated the transcriptional changes in key virulence genes. Most RxLR/CRN effector and hydrolase genes were repressed by rapamycin-mediated TOR inhibition, whereas cysteine protease genes were upregulated (Figure 3A). In addition, the expression of these virulence genes was rapidly activated in response to host root-derived signals, supporting their crucial roles during infection (Figure 3B).

Five differentially expressed genes, PpRxLR genes (named PpRxLR1-5), were selected to investigate whether the PpTOR-regulated PpRxLR effectors directly participate in pathogenicity. RT–qPCR assays revealed that the expression of these genes significantly increased during the early stages of P. parasitica infection (Figure 3C). Additionally, transient overexpression of these effectors led to larger lesion areas, suggesting that these effectors increased plant susceptibility to P. parasitica. Among them, PpRxLR3 exhibited the most pronounced virulence-promoting effect (Figure 3D,E). Collectively, these results indicate that PpTOR acts as an upstream regulator of P. parasitica pathogenicity by governing the expression of a suite of effector genes, with PpRxLR3 representing a key downstream virulence determinant.

3.3. PpRxLR3 Contributes to P. parasitica Infection by Attenuating JA Biosynthesis and Signaling in Plants

To characterize the biological function of PpRxLR3, PpRxLR3-overexpressing tobacco plants (PpRxLR3-OE1 and PpRxLR3-OE2) whose transgene expression levels were confirmed to be high by RT–qPCR were generated (Figure 4A). Compared with those on wild-type (HD) leaves, lesions on leaves from PpRxLR3-OE plants were significantly greater after inoculation with P. parasitica (Figure 4B,C). Consistent with these findings, whole-plant inoculation assays also revealed that compared with HD plants, PpRxLR3-OE plants developed more severe disease symptoms, including rapid leaf wilting and a black shank phenotype (Figure 4D). The disease incidence rate and index were consistently greater in the transgenic plants than in the WT plants (Figure 4E). These results demonstrate that PpRxLR3 functions as a virulence effector that increases plant susceptibility to P. parasitica.

To elucidate the molecular mechanisms underlying PpRxLR3-mediated susceptibility, we performed transcriptome sequencing on PpRxLR3-OE and HD plants. A total of 3644 differentially expressed genes (DEGs) were identified, with 2076 upregulated and 1568 downregulated genes in the transgenic plants (Figure S5, Table S6). Functional enrichment analysis indicated that these DEGs were significantly overrepresented in categories related to plant–pathogen interactions and immunity, including systemic acquired resistance (SAR), the MAPK signaling pathway, cell wall organization/biogenesis, photosynthesis, and multiple hormone signaling pathways (Figures S6–S8, Table S7). This global transcriptional reprogramming suggests that PpRxLR3 perturbs multiple defense and signaling networks in the host.

In plant–pathogen interactions, multiple phytohormones participate in the regulation of pathogen resistance in plants [35,36]. Among them, jasmonic acid (JA)-mediated signaling is important in plant immune responses against pathogens [37,38]. Our transcriptomic analysis revealed that numerous genes involved in JA biosynthesis (e.g., LOX, AOS, AOC, and OPR3) and signaling (e.g., COI1, JAZ, and MYC2) were differentially expressed in PpRxLR3-OE plants even before infection (Figure 5A, Table S8). More importantly, while P. parasitica infection promoted the expression of these JA-related genes in HD plants, this induction was markedly attenuated or reversed in PpRxLR3-OE plants (Figure 5B). To determine whether these transcriptional changes corresponded to physiological alterations, we quantified JA levels in PpRxLR3-OE plants. Consistent with the gene expression data, the JA content was significantly lower in the PpRxLR3-OE plants than in the HD plants (Figure 5C). Similarly, the expression of genes involved in JA signal transduction and response was also significantly suppressed in the OE plants after P. parasitica infection (Figure 5D). Collectively, these results indicate that PpRxLR3 overexpression is associated with the suppression of both JA biosynthesis and signaling.

To functionally validate the importance of JA signaling in plant resistance to P. parasitica, we performed two complementary experiments. First, exogenous application of methyl jasmonate (MeJA), a bioactive JA derivative, significantly increased plant resistance to P. parasitica, reducing disease severity (Figure 6A,B). Second, since JAZ proteins are negative regulators of JA signaling, we generated JAZ-overexpressing plants (Figure S9). Conversely, overexpression of JAZ (which represses JA responses) significantly compromised plant resistance, leading to increased susceptibility (Figure 6C,D). These results suggest that JA signaling plays a crucial role in regulating plant resistance to P. parasitica.

In addition to jasmonic acid (JA) signaling, plant innate immunity (PTI) and salicylic acid (SA) signaling play central roles in plant defense against pathogen invasion [36]. Our transcriptomic analysis revealed differential expression of key genes in these pathways (Figures S6–S8). Further validation by quantitative PCR confirmed that these genes are strongly upregulated during infection (Figure S8C) [40,41,42,43,44]. In contrast, this induction was significantly suppressed in PpRxLR3-overexpressing (OE) plants (Figure S8C). In addition to disrupting immune signaling, PpRxLR3 also targets fundamental aspects of host physiology. Transcriptomic profiling revealed substantial dysregulation of numerous photosynthesis-related genes in PpRxLR3-OE plants (Figure S8, Table S9). Compared with the wild-type controls, these plants consistently exhibited growth retardation and a significant reduction in leaf photosynthetic capacity (Figure S10). In summary, PpTOR is essential for the pathogenicity of P. parasitica. It functions as a crucial regulator of P. parasitica virulence by modulating the expression of a suite of effector proteins, including PpRxLR3, which act in concert to dismantle multiple layers of host defense.

3.4. Silencing of PpTOR Promotes Plant Resistance to P. parasitica

Having established PpTOR as a master regulator of growth and pathogenicity in P. parasitica, we explored its potential as a target for disease control using host-induced gene silencing (HIGS). HIGS is a promising strategy wherein the host plant expresses double-stranded RNA (dsRNA) homologous to essential pathogen genes, leading to their silencing and consequent reduction in disease [45,46,47]. We generated stable N. benthamiana lines expressing dsRNA targeting PpTOR (PpTOR-RNAi plants) (Figure S11). In detached leaf assays, compared with wild-type (WT) control plants, PpTOR-RNAi plants developed significantly smaller lesions after inoculation with P. parasitica (Figure 7A,B). Whole-plant inoculation at the stem base further confirmed enhanced resistance. When the WT plants exhibited severe wilting within 3 days of incubation, the PpTOR-RNAi plants presented minimal disease symptoms under the same conditions (Figure 7C). Consequently, both the disease incidence rate and the disease index were significantly lower in the PpTOR-RNAi plants (Figure 7D). In addition, we confirmed the efficacy of HIGS. Compared with those in P. parasitica-infected WT plants, the transcript levels of both PpTOR1 and PpTOR2 were drastically reduced in pathogen-colonizing PpTOR-RNAi plants, with a silencing efficiency of more than 90% (Figure 7E). Consistently, the expression of pathogenicity-related genes in P. parasitica was also markedly suppressed (Figure 7F).

To verify the silencing signal, we performed sRNA sequencing (sRNA-Seq) on PpTOR-RNAi plants. The data revealed abundant production of PpTOR-specific small interfering RNAs (siRNAs) in the transgenic plants compared with the WT controls (Table S11). Furthermore, we investigated whether P. parasitica could take up sRNAs from the environment. Fluorescence labeling experiments revealed that P. parasitica mycelia efficiently internalized exogenous siRNAs, suggesting that direct application of synthetic siRNAs could serve as a nontransgenic strategy to silence PpTOR (Figure S12). Additionally, we artificially synthesized four siRNA sequences that can specifically target PpTOR1 and PpTOR2 and reported that treatment with PpTOR2-specific dsRNA significantly impaired pathogen virulence, whereas treatment with PpTOR1-specific siRNA had a more modest effect. The most pronounced reduction in disease was observed when a combination of PpTOR1- and PpTOR2-specific siRNAs was applied (Figure 8A–D). Corroborating these findings, the expression of virulence-associated effector genes in the treated pathogen was most strongly suppressed under combined siRNA treatment (Figure 8E).

In summary, our results demonstrate that silencing PpTOR through either HIGS or exogenous siRNA application effectively reduces the pathogenicity of P. parasitica by dampening the expression of its downstream virulence program. This finding establishes TOR not only as a critical virulence regulator but also as a promising target for developing RNA-based control strategies against oomycete diseases.

4. Discussion

P. parasitica is a devastating oomycete pathogen that causes severe root and crown rot diseases worldwide [1]. Effective control remains challenging because of its rapid infection cycle and limited management options [48]. In this study, the conserved kinase PpTOR was established as an essential regulator of P. parasitica growth and pathogenicity. We demonstrate that PpTOR coordinates the expression of a suite of virulence factors, including RxLR and CRN effectors, and that one key downstream effector, PpRxLR3, promotes infection by broadly suppressing plant resistance-related signaling. Furthermore, HIGS- and sRNA-mediated silencing of PpTOR proved to be effective strategies for enhancing plant resistance, highlighting its potential as a novel target for disease control.

TOR kinase is a well-conserved integrator of nutrient and environmental cues that modulates the growth and pathogenicity of pathogens [23,24,25,26,27,28,29]. In this study, our findings extend its pivotal role to pathogenic oomycetes. The inhibition of PpTOR by rapamycin severely impaired the mycelial growth and pathogenicity of P. parasitica, which is consistent with the findings of previous reports on other pathogens [23,24]. Transcriptomic profiling further revealed that PpTOR inhibition dysregulated a vast network of genes involved in core cellular processes such as ribosome biogenesis, amino acid metabolism, and autophagy. More significantly, we identified a pronounced suppression of genes encoding key virulence determinants, including RxLR and CRN effectors. This positions PpTOR not only as a growth regulator but also as a critical signaling nexus that integrates upstream cues to modulate downstream virulence programs. Under favorable conditions, active PpTOR signaling may promote the growth of P. parasitica and the production of the effector arsenal necessary for host invasion.

Recent studies in fungal pathogens have illuminated the expanded role of TOR beyond growth regulation. For instance, in Fusarium oxysporum, FoTOR not only promotes ribosome biogenesis via FoSIK1 but also directly regulates the expression of numerous cell wall-degrading enzymes (CWDEs), which are crucial for breaching the plant cell wall barrier during early infection [24,49]. These findings highlight the unique function of TOR in pathogenicity. Our findings in the oomycete Phytophthora parasitica align with this paradigm, as PpTOR inhibition led to the coordinated downregulation of a suite of CWDEs and key effector genes (e.g., PpRxLR3). Furthermore, evidence from F. oxysporum indicates that TOR can affect virulence through downstream transcription factors, such as the bZIP protein MeaB [26]. This convergence of evidence—from fungal systems to our oomycete model—highlights a paradigm in which TOR acts as a switch, synchronizing the pathogen’s biosynthetic capacity (e.g., ribosome biogenesis) with the timed expression of invasion machinery (e.g., CWDEs and effectors) through the modulation of key transcription factors.

Given that TOR inhibition broadly suppresses pathogen growth and metabolism, a potential confounding factor is that the observed repression of virulence genes might result from nonspecific secondary transcriptional remodeling triggered by growth arrest rather than direct PpTOR-dependent regulation. To rule out this possibility, we performed rigorous cross-validation using two independent gene-specific silencing systems, namely, transgenic host-induced gene silencing (HIGS) and exogenous spray-induced gene silencing (SIGS). Both RNAi approaches specifically reduced PpTOR transcript abundance without causing global metabolic repression or obvious growth inhibition during early pathogen colonization. Importantly, both genetic silencing systems recapitulated the specific repression of RxLR/CRN virulence effectors observed in rapamycin-treated mycelia. The high consistency between chemical inhibition and gene-specific silencing strongly indicates that the altered virulence transcriptome is a direct outcome of impaired PpTOR signaling rather than a general stress response secondary to growth suppression.

We further excluded growth-associated confounding effects through time-course expression analysis of virulence genes in response to host-derived signals. RxLR and CRN effector genes were rapidly and specifically induced within 3–6 h following exposure to host root cues, which occurred long before any detectable alteration in mycelial growth or biomass accumulation. These rapid induction kinetics, together with the independent expression pattern of effector genes relative to general growth-related transcripts, further demonstrate that TOR signaling directly modulates pathogen virulence programs in response to host signals. Therefore, PpTOR may govern infection-related effector expression in a growth-independent manner, confirming its specific and central role in coordinating oomycete pathogenicity. However, the specific identity of the downstream factors of PpTOR that orchestrate this virulence-related transcriptional reprogramming during P. parasitica infection remains a pivotal and unresolved question and represents a key frontier for future research.

Notably, unlike most fungi that harbor only a single TOR copy, the oomycete P. parasitica possesses two distinct TOR paralogs, designated PpTOR1 and PpTOR2. Our transcriptional and functional assays revealed clear functional divergence between these two isoforms. PpTOR2 expression sharply increased during the early stage of host infection, and compared with suppression of PpTOR1, siRNA-mediated silencing of PpTOR2 had a more potent inhibitory effect on pathogen virulence. These findings may indicate that PpTOR2 serves as an infection-specific TOR isoform that activates the full virulence repertoire of P. parasitica, including RxLR/CRLR effector expression and cell wall-degrading enzyme activity. In contrast, PpTOR1 maintains basal TOR signaling to support routine vegetative growth under saprophytic conditions and contributes minimally to pathogenicity. The retention of dual TOR paralogs in oomycetes may represent an adaptive evolutionary strategy enabling the independent regulation of saprophytic growth and parasitic infection. Further genetic knockout or knockdown assays in P. parasitica will help fully elucidate the distinct downstream regulatory networks of the two TOR isoforms.

In Phytophthora species, RxLR effector proteins contribute to infection by suppressing host plant resistance [34]. Our integrated approach identified PpRxLR3 as a key effector downstream of PpTOR. PpRxLR3 overexpression in plants increased susceptibility, and transcriptomic profiling of these plants revealed a systematic dampening of defense responses, particularly within the JA pathway—a well-established pillar of plant disease resistance [50,51,52]. Pathogens can increase their virulence by interfering with JA biosynthesis or JA signal transduction [53,54,55]. Within the Phytophthora genus, pathogens frequently employ effector proteins to suppress JA biosynthesis or disrupt JA-mediated signaling, thereby facilitating host colonization [56,57]. Specifically, our data demonstrate that PpRxLR3 suppresses the transcription of genes involved in JA biosynthesis and signaling. Functional validation confirmed that an active JA pathway enhances resistance to P. parasitica, whereas its inhibition (via JAZ overexpression) increases susceptibility. Taken together, these findings position JA signaling as a critical host target compromised by Phytophthora genus pathogens to facilitate infection and underscore its potential value as a target for breeding resistance against Phytophthora genus pathogens. Notably, the endogenous JA content in this study was quantified using an ELISA-based method. Although this approach is widely applicable for relative hormone quantification in plant immune studies, it has lower detection specificity and resolution than the gold-standard LC–MS/MS platform does. Future studies will adopt LC–MS/MS-based hormone profiling to achieve more accurate and high-confidence quantification of hormone accumulation (e.g., that of salicylic acid) and further validate the hormone-suppressive effect of PpRxLR3.

In addition to JA signaling, SA represents a central hormone in plant defense against pathogens [58]. PpRxLR3 significantly attenuated the infection-triggered induction of the SA receptor gene NPR1 and the marker gene PR1, effectively disabling this major defense branch. During the initial stages of plant infection, PTI is rapidly activated to protect plants from pathogenic invasion. In response, pathogens deliver a large number of effector proteins into plant cells to disrupt PTI and facilitate invasion and colonization [5]. PpRxLR3 also abolished the pathogen-induced expression of core PTI components, such as the coreceptor NtBAK1, its negative regulator NtBIR2 and SAR-related genes, suggesting that PpRxLR3 likely blocks the early activation of PTI signaling cascades, including the MAPK pathway and SAR. Furthermore, in addition to directly suppressing the defense response in plants, the manipulation of host physiology by PpRxLR3 likely involves fundamental processes such as photosynthesis. Overall, this multipronged attack on diverse defense-related pathways and host physiology highlights a sophisticated mechanism through which P. parasitica dismantles host defenses. By simultaneously targeting these interconnected networks, PpRxLR3 ensures the comprehensive suppression of defense signaling, preventing the host from mounting an effective counterresponse and facilitating successful colonization.

A limitation of the current functional characterization of PpRxLR3 is the absence of pathogen-intrinsic gene deletion data, which are restricted by the low genetic tractability of P. parasitica. Despite the lack of loss-of-function mutants, multiple layers of gain-of-function evidence sufficiently support the virulent role of PpRxLR3. This evidence includes transient agroinfiltration assays and stable overexpression in two independent tobacco varieties, together with its infection-induced and TOR-dependent expression profile during host colonization. Collectively, these results strongly demonstrate that PpRxLR3 acts as a core virulence effector of P. parasitica to inhibit host immune defenses. Future studies should perform gene knockout assays of PpRxLR3 to verify its indispensable function during infection with an authentic pathogen. Further investigation into the molecular mechanisms by which PpRxLR3 suppresses plant resistance and identification of its host-interacting targets will advance our mechanistic understanding of oomycete virulence strategies and provide novel insights for the development of precise disease control approaches.

Given the central role of PpTOR in P. parasitica, we evaluated PpTOR as a target for HIGS, a promising RNAi-based strategy for disease control [45,59]. Our PpTOR-RNAi plants exhibited strong resistance, accompanied by the silencing of both PpTOR1/2 and key downstream virulence genes such as PpCRN7, PpXYN1, and PpRxLR3. This successfully translates our mechanistic understanding into a proof-of-concept application. In a previous study, Zhang et al. reported that the production of dsRNA in Arabidopsis did not effectively silence the expression of target genes of P. parasitica [60]. However, HIGS has been used to suppress the occurrence of several other oomycete diseases. Govindarajulu et al. reported that the HIGS method could effectively control B. lactucae, a biotrophic oomycete that induces downy mildew disease in lettuce [61]. In Phytophthora, the HIGS method could also confer resistance to P. capsici and P. infestans in N. benthamiana and potato by targeting different target genes [32,62]. Notably, a recent study revealed that Phytophthora could inhibit the biogenesis of pentatricopeptide-repeat protein (PPR)-derived small interfering RNAs (siRNAs), which are located in extracellular vesicles and silence target genes in Phytophthora, through the effector protein PSR2 in Arabidopsis, suggesting that pathogens may regulate the production of siRNA in Arabidopsis through the secretion of effector proteins [63]. There may be complex and different mechanisms for dsRNA or siRNA exchange between different hosts and pathogens, and the mechanism by which pathogens take up dsRNA or siRNA molecules from plant cells remains unclear.

In addition to HIGS, spray-induced gene silencing (SIGS) has emerged as a rapid and effective method for disease prevention and control in plants [64,65]. SIGS can constrain pathogen infection through the use of double-stranded RNA (dsRNA) or single-stranded RNA (sRNA) to directly suppress the transcription levels of pathogen virulence-related genes [65,66]. Interestingly, a recent study revealed that Phytophthora capsici can effectively take up environmental small interfering RNAs (siRNAs) and that SIGS can be used to silence the expression of P. capsici virulence-related genes to restrict the infection of P. capsici in tobacco [67]. Our results also demonstrate that P. parasitica can take up exogenous dsRNA, making the SIGS method viable for controlling P. parasitica by silencing PpTOR. By designing specific siRNAs against PpTOR1/2 and employing efficient delivery systems, it may be possible to develop a nontransgenic, environmentally friendly control method. Furthermore, given the highly conserved functional role of TOR across plant pathogens, targeting TOR via SIGS represents a promising strategy to effectively control multiple oomycete diseases by directly impairing the pathogen at its core regulatory node, as demonstrated in this study. Even though some pathogens have difficulty taking up siRNA from the environment, Wang et al. reported that nanocarriers enable more efficient delivery of siRNA into P. infestans [68,69] for the management of potato late blight. These findings indicate that the SIGS method can be utilized to inhibit the expression of pathogen TOR genes, thereby effectively controlling diseases caused by various oomycete pathogens in a nontransgenic and environmentally friendly manner. Our synthetic siRNA assays establish a proof-of-concept RNA-based control strategy against P. parasitica. Quantitative comparison of TOR transcript knockdown efficiency further explains the differential disease-suppressive potency between single siRNA treatments and combined siRNA cocktails. Despite these promising results, the translational application of laboratory-based SIGS technology in commercial agricultural practices requires further evaluation of key environmental parameters. These key factors include siRNA persistence on leaf surfaces, resistance to rain wash-off, tolerance to UV degradation, and systemic absorption efficiency in mature crop tissues. Nanoparticle delivery systems have been experimentally validated to optimize SIGS performance in Phytophthora pathosystems and can substantially increase siRNA stability and pathogen uptake under field conditions. Therefore, nanoparticle-assisted delivery represents a promising strategy for future translational research to advance the field application of RNA-mediated disease control approaches.

Fungicide application has emerged as the primary method for preventing and controlling P. parasitica because of its high efficiency and convenience. However, issues related to environmental pollution and pathogen resistance to fungicides caused by long-term application cannot be ignored. In this study, we provide compelling evidence that PpTOR is indispensable for the pathogenicity of P. parasitica and demonstrate that TOR suppression can effectively mitigate P. parasitica-induced disease under laboratory conditions. Moreover, considering that P. parasitica shares numerous biological characteristics with most Phytophthora pathogens, the TOR gene not only plays a pivotal role in P. parasitica but is also highly conserved in other Phytophthora pathogens and even broader oomycete genera, suggesting that TOR may have similar functional mechanisms and regulatory pathways in these organisms. These findings provide a theoretical foundation for developing broad-spectrum and efficient control strategies against oomycete diseases by targeting TOR.