Mild to Virulent: Coat Protein Mutations Restore Mosaic Symptom Induction in a Korean PepMV Isolate

Abstract

Pepino mosaic virus (PepMV) is a significant threat to global tomato production, with symptom severity varying widely among strains and often leading to significant economic losses. Despite extensive studies on aggressive variants, the molecular determinants of mild symptomatology in field isolates, particularly from Korea, remain underexplored. In this study, we characterized a mildly infecting PepMV isolate from asymptomatic tomato plants during a field survey in Jeonju, South Korea. The full-length genome sequence and phylogenetic analysis classified it as a CH2 strain. A full-length cDNA infectious clone of this isolate was constructed and confirmed to induce no mosaic symptoms in tomato plants. To identify symptom determinants, targeted mutagenesis was performed in the coat protein (CP) open reading frame. Substitution mutations at CP position 236 or combined 6/155 substitutions converted the mild isolate into a severe variant, inducing strong mosaic symptoms and significantly higher viral accumulation (up to tenfold). These results demonstrated that specific CP residues act as key regulators of symptom severity in PepMV CH2 strains and provide defined severe mutants as useful tools for screening resistance in tomatoes. Although the mechanism underlying symptom modulation remains unclear, this work advanced our understanding of molecular differences between mild and severe strains and supported targeted strategies for managing this economically important virus.

1. Introduction

Potexvirus is the largest and most diverse genus in the family Alphaflexiviridae, comprising 52 recognized species that infect a wide range of economically important crops [1,2,3]. Some members of this genus have recently emerged as significant pathogens, attracting considerable attention in plant virology, with pepino mosaic virus (PepMV) serving as a representative example. PepMV causes highly variable disease phenotypes, ranging from latent infections to severe mosaic and growth suppression [4]. This striking variability in symptom severity poses challenges for disease diagnosis, epidemiological monitoring, and resistance breeding, yet the viral determinants underlying mild versus aggressive PepMV infections remain incompletely understood.

PepMV was first observed in pepino (Solanum muricatum) plants in Peru in the mid-1970s and was characterized in the early 1980s [5]. This virus has spread rapidly since its emergence in tomato (Solanum lycopersicum) in Europe in the late 1990s and is now prevalent across major tomato-producing regions worldwide [6,7,8]. Although PepMV can negatively affect fruit quality and marketability, its impact on yield and symptom severity varies considerably among regions and production systems. In the United Kingdom, glasshouse trials reported substantial downgrading of marketable Class I fruit (6.5–38%) without consistent reductions in total yield, whereas several European countries observed modest overall yield losses (~4%) and reduction in marketable yield (~14%) associated with aggressive isolates [7,9,10]. Due to its potential threat, PepMV has been classified as a dangerous pathogen and has been included in the European and Mediterranean Plant Protection Organization (EPPO) alert lists since 2009 [11]. Such inconsistencies suggest that differences in viral genetic composition, rather than PepMV presence alone, play a central role in disease outcomes. Among the five recognized PepMV genetic strains (EU, CH2, US1/CH1, LP, and PES), isolates differ markedly in their ability to induce symptoms, further highlighting the need to define strain- and mutation-specific pathogenicity determinants [12].

The PepMV genome encodes five major open reading frames, including the CP, which plays critical roles in virion assembly, virus movement, and suppression of host RNA silencing [13,14]. Importantly, accumulating evidence indicates that specific amino acid substitutions in the CP can dramatically alter symptom severity, including the induction of yellow mosaic or necrosis, particularly in EU and CH2 strain isolates. However, the contribution of CP mutations to symptom modulation in naturally occurring mild PepMV isolates, especially those from under-characterized regions, remains unclear.

Despite the increasing global importance of PepMV, its molecular and biological characteristics in Korea remain poorly defined. To date, only a single study has reported PepMV in Korean tomato production, reporting limited disease incidence and moderate symptoms [15]. Comprehensive genomic characterization of Korean PepMV isolates and experimental validation of their pathogenic potential have not been performed. This lack of knowledge hinders accurate risk assessment and the development of effective disease management and resistance screening strategies in Korean tomato production systems.

In this study, we addressed these gaps by characterizing a mild PepMVisolate collected from asymptomatic tomato plants during a 2024 field survey in Jeonju, South Korea. We aimed to (i) determine the genomic and phylogenetic properties of this isolate, (ii) experimentally assess its pathogenicity using an infectious cDNA clone, and (iii) identify CP mutations responsible for the transition from mild to severe disease phenotypes through targeted mutagenesis. By defining key molecular determinants of symptom severity, this work provides the first comprehensive functional characterization of PepMV in Korea and establishes genetically defined viral tools for resistance screening and future studies of PepMV pathogenicity.

2. Materials and Methods

2.1. PepMV Isolation

To monitor the presence of emerging and re-emerging plant viruses, a field survey was conducted in Jeonju, South Korea, in July 2024. 50 tomato samples were collected from a field, including five asymptomatic plants located adjacent to the symptomatic yellow tomatoes. Total RNA was extracted from five asymptomatic samples using the RNeasy Plant Mini Kit (Qiagen Hilden, Germany) according to the manufacturer’s instructions. The extracted RNA was used as a template for one-step reverse transcription polymerase chain reaction (RT-PCR) to detect PepMV, employing the specific primers (PepMV-det-F/PepMV-det-R) listed in Table S1. RT-PCR was performed using SuPrimeScript RT-PCR Premix (2×) in a 20 μL reaction mixture containing 1 μL of total RNA, 1 μL each of forward and reverse primers (10 μM), 10 μL of RT master mix, and 7 μL of nuclease-free water. Thermal cycling conditions consisted of reverse transcription at 50 °C for 30 min, initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 1 min, with a final extension at 72 °C for 2 min. The PCR products were analyzed by electrophoresis on a 1% agarose gel, and the identity of PepMV was confirmed by sequencing of the amplicons (Macrogen, Seoul, Korea).

2.2. Virus Amplification, Cloning, and Sequencing

To determine the complete genome sequence and phylogenetic placement of the PepMV isolate, viral RNA was reverse-transcribed, amplified, cloned, and analyzed using comparative sequence approaches. Following the detection of PepMV in five asymptomatic samples, one representative PepMV-positive sample was selected for all subsequent experiments. Total RNA was extracted, and first-strand cDNA was synthesized using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). According to the manufacturer’s instructions, 1 µg of total RNA was mixed with 1 µL of oligodT primer (10 µM) and nuclease-free water to a final volume of 13 µL. The mixture was incubated at 65 °C for 5 min and then immediately placed on ice for at least 1 min. Next, the following components were added to bring the total reaction volume to 20 µL: 4 µL of 5× SuperScript IV Reaction Buffer, 1 µL of 100 mM DTT, 1 µL of 10 mM dNTP mix, and 1 µL of SuperScript IV Reverse Transcriptase. Reverse transcription was carried out at 50 °C for 10 min, followed by enzyme inactivation at 80 °C for 10 min. To generate the full-length sequence of the detected PepMV isolate, two overlapping amplicons were designed based on the closely related PepMV-Korean reference genome (GenBank accession No. LC656469). Primer pairs PepMV-F1-F/R and PepMV-F2-F/R were used to produce fragments with a 20-nt overlap and terminal homology to the binary vector pJL89 for subsequent seamless assembly. Amplification was performed in 50 μL reactions containing 2 μL cDNA template, 0.4 μM each primer, and 25 μL PrimeSTAR Max DNA Polymerase Premix Ver. 2 (Takara Bio, Kusatsu, Japan), in nuclease-free water. Cycling parameters were: 98 °C for 3 min; 30 cycles of 98 °C for 10 s, 58 °C for 5 s, and 72 °C for 16 s; followed by a final extension at 72 °C for 2 min. The PCR products were purified using the AccuPrep PCR/Gel Purification Kit (Bioneer, Daejeon, Republic of Korea) and initially cloned into pJET1.2/blunt using the CloneJET PCR Cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA). Recombinant plasmids were introduced into Escherichia coli DH5α competent cells by heat-shock transformation. Positive colonies were identified by PCR screening and were sequenced. The full-length genomic sequences were deposited in the GenBank^® (www.ncbi.nlm.nih.gov/genbank/, accessed on 23 July 2025) under accession number PV927494.1 [16].

The complete genome sequence of the PepMV mild isolate was aligned with 29 different full-length PepMV genomes available in GenBank using the MUSCLE algorithm implemented in MEGA11 [17]. The accession numbers of all sequences used are listed in Table S2. A phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replications, employing the Tamura-Nei substitution model within MEGA11. The tree was rooted using Tomato mottle mosaic virus (ToMMV) as the outgroup.

Pairwise nucleotide sequence identities were calculated and visualized using the Sequence Demarcation Tool (SDT) v1.3 with the MUSCLE alignment as the input, generating a color-coded identity matrix to facilitate direct comparison among isolates [18].

2.3. Infectious Clone Construction and Virus Inoculation

To generate an infectious cDNA clone of PepMV, two overlapping PCR fragments covering the complete genome of the PepMV-CT isolate (GenBank: PV927494) were cloned into the linearized binary vector pJL89 (Addgene, Cambridge, MA, USA) by Gibson Assembly using Gibson Assembly^® Master Mix (New England Biolabs, Hitchin, UK) following a previously reported method [19,20]. The assembly reaction was performed in a total volume of 20 μL, containing 3 μL of linearized pJL89 vector (95 ng/μL), 4 μL of gel-purified fragment 1 (70 ng/μL), 3 μL of fragment 2 (95 ng/μL), and 10 μL of 2× Gibson Assembly master mix. The reaction was incubated at 50 °C for 30 min. The assembled product was transformed into E. coli DH5α competent cells via heat shock. Positive clones were screened by colony PCR with fragment-specific primers, and plasmids from the selected colonies were purified and fully sequenced to confirm the integrity of the construction. Verified full-length clones were then introduced into Agrobacterium tumefaciens strain GV3101 using the freeze–thaw method for subsequent agro-inoculation studies.

The infectivity of the PepMV full-length clone was evaluated by agro-inoculation to Nicotiana benthamiana. Agrobacterium harboring the infectious clone was grown overnight in 20 mL LB medium supplemented with kanamycin (50 µg/mL), rifampicin (50 µg/mL), and gentamicin (50 µg/mL) at 28 °C with shaking until OD₆₀₀ reached 1.0. Cells were pelleted by centrifugation, resuspended in infiltration buffer (10 mM MgCl₂, 10 mM MES pH 5.6, 200 µM acetosyringone), and incubated at room temperature for 2–4 h to induce virulence. Agroinfiltration of N. benthamiana leaves were performed using a needleless syringe. Systemic infection was monitored based on the appearance of symptoms and confirmed by RT-PCR at 14 days post-inoculation (dpi) with specific primers (PepMV-det-F/PepMV-det-R).

Viral inoculations were performed on S. lycopersicum (tomato cv. Moneymaker) to assess the infectivity of the different mutants. Three-week-old plants were inoculated for infectivity assays by pinpricking the main apical shoot. A set of commercial tomato cultivars was also tested, including four widely available cultivars: Redeubel, Noranjangwon, Yeonghwabangul, and Yegwang (abbreviated as cv. 1 to cv. 4) from Daenong Seed Company (Jeonnam, Korea), and eight cultivars carrying Ty resistance genes, Bacchus, Ty-Senseukwi, Mini-maru, Benekia 220, Ty-Kiseumi, Ty-Jangsu, Ty-Wineo, and Titichal (abbreviated as Rcv. 1 to Rcv. 7) from the Nongwoo Bio Company (Seoul, Korea). These cultivars were used to evaluate the infectivity of the mutated PepMV clone and to conduct preliminary screening to test whether resistance genes are effective against other viruses that similarly confer resistance to PepMV, using the same inoculation procedure as for Moneymaker cv.

2.4. Site-Directed Mutagenesis

To identify coat protein (CP) amino acid residues associated with PepMV symptom severity, site-directed mutagenesis was performed on the CP coding region of the mild PepMV infectious clone. Site-directed mutants of the PepMV CP were generated using the Q5^® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. Based on amino acid differences identified through multiple sequence alignment between the PepMV isolate characterized in this study and two reference isolates—the aggressive CH2-type PepMV-KLP2 (OR733204) and the EU-type PepMV-H30 (OR733205) from a previous report [21]—eight single-point mutants were designed and generated using specific primer sets (Table S3). Mutations were introduced by replacing the corresponding codons at defined positions in the PepMV genome. The resulting amino acid substitutions were CP^S5P (serine to proline; UCU → CCU), CP^A6T (alanine to threonine; GCA → ACC), CP^T10P (threonine to proline; ACA → CCG), CP^A13V (alanine to valine; GCA → GTG), CP^S94F (serine to phenylalanine; UCU → UUU), CP^M98I (methionine to isoleucine; AUG → AUU), CP^E155K (glutamic acid to lysine; GAA → AAA), and CP^E236K (glutamic acid to lysine; GAA → AAA). In addition, a series of double mutants combining the substitution at position 155 with each of the other mutations was constructed and designated CP^E155K/S5P, CP^E155K/A6T, CP^E155K/T10P, CP^E155K/A13V, CP^E155K/S94F, and CP^E155K/M98I. All mutant sequences were verified by sequencing and subsequently transformed into A. tumefaciens to generate infectious clones using the same agro-inoculation methodology as previously described.

2.5. Quantitative Viral Accumulation

To quantify PepMV accumulation in systemically infected tomato tissues, viral RNA levels were measured by one-step reverse transcription quantitative PCR (RT-qPCR). Briefly, total RNA was isolated from systemic leaves at 12 dpi, and RNA concentration and purity were determined using an Epoch Microplate Spectrophotometer (BioTek, Winooski, VT, USA). RNA samples were adjusted to 50 ng/µL with nuclease-free water. RT-qPCR reactions were performed in a total volume of 20 μL using AccuPower^® GreenStar™ RT-qPCR Master Mix (Bioneer, Daejeon, Korea) and contained 2 μL of RNA template, 10 pmol of TGB1 gene-specific primer (Table S1), 10 μL of master mix, and 6 μL of NFW. Thermal cycling conditions were as follows: reverse transcription at 50 °C for 15 min, initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 15 s, and extension at 72 °C for 20 s. Elongation factor 1-alpha (EF1α) served as an internal reference gene, and all reactions were performed in triplicate. Relative viral accumulation was calculated using the 2^−ΔΔCt method [22], and statistical significance was determined by Student’s t-test using GraphPad Prism 8.0.2 (GraphPad Software, Boston, MA, USA).

2.6. Molecular Docking Analysis of PepMV CP and Host Factors

To examine the interaction between the CPs of wild-type and mutant strains and the host factor, molecular docking was performed using a modified protocol adapted from a previously reported method [23]. Initially, 3D models of the host proteins (HSP70, SIOSCA4, SISTU38) and the virus CP were generated via homology modeling with the I-TASSER server (https://zhanggroup.org/I-TASSER/, accessed on 27 September 2025) and the SWISSMODEL server (http://swissmodel.expasy.org, accessed on 27 September 2025). Moreover, the 3D structure model was assessed using the Ramachandran score in GalaxyWEB (https://galaxy.seoklab.org/index.html, accessed on 27 September 2025) to identify low-favorable models, and the low-quality models were improved using these web-based services. Then, molecular docking using the HADDOCK web server (https://rascar.science.uu.nl/haddock2.4/, accessed on 27 September 2025) was performed with the scoring function using 400 iterations and 10,000 pre-placements [24]. The best docking result was chosen based on the Z-score and docking quality. Thus, the PRODIGY web server (http://rascar.science.uu.nl/prodigy, accessed on 27 September 2025) was used to predict protein-protein binding energy. The interacted residues of complex protein were analyzed using DIMPLOT in LigPlot + v.2.2.4 [25].

3. Results

3.1. New Mild PepMV Isolated in Korea

PepMV was detected from asymptomatic tomato plants collected during a field survey in Jeonju, South Korea. RT-PCR detection yielded the expected amplicon (Figure S1), and sequence analysis revealed 99% nucleotide identity with a previously reported Korean isolate (LC656469). The complete genome sequence of the PepMV CT isolate (GenBank accession No. PV927494) is 6419 nucleotides in length, and its ORF composition and genomic organization are conserved relative to those of other PepMV isolates infecting tomatoes.

The complete genome sequence of the identified PepMV isolate was aligned with multiple representative full-length PepMV genome sequences retrieved from GenBank, covering the five established strains (EU, CH2, LP, PES, and US1). The isolate identified in this study was designated PepMV-CT. A phylogenetic tree was constructed using the neighbor-joining method. As shown in Figure 1, the CT isolate clustered most closely with a previously reported Korean isolate (LC656469) and groups within the CH2 strain, forming a distinct clade separate from other strains. Consistent with the phylogenetic analysis, the SDT results revealed high nucleotide identity between the CT isolate and the other CH2 isolates. The CT isolate shared more than 98% identity with the Korean isolate LC656469 and approximately 94% identity with the CH2 isolates from Chile (DQ000985), Spain (OR733204), Poland (JX417070, HQ650559), Belgium (JN835466), Germany (MK133092), and Switzerland (MF422613). In contrast, isolates representing other PepMV strains exhibited less than 83% nucleotide identity with the CT isolates.

3.2. Symptom Expression in Tomato Infected with PepMV cDNA Infectious Clone and Mutant Derivatives

A full-length infectious cDNA clone of PepMV was generated by assembling two overlapping genomic fragments into the binary vector pJL89 using Gibson assembly (Figure 2a). The resulting plasmid was introduced into A. tumefaciens strain GV3101 and the recombinant cells were agroinfiltrated into N. benthamiana leaves. At 7 dpi, inoculated plants exhibited early symptoms, such as mild blistering and slight leaf distortion. At 14 dpi, typical PepMV symptoms, including pronounced leaf distortion, puckering, blistering, and mild chlorosis, were clearly observed (Figure 2b). RT-PCR confirmed systemic PepMV infection in all inoculated plants (Figure 2c). These results demonstrated the successful construction and infectivity of the full-length cDNA clone of the mild Korean PepMV isolate.

The infectivity of PepMV infectious clone was also evaluated in tomato plants (cv. Moneymaker). Although RT-PCR confirmed successful viral infection, no visible symptoms developed, consistent with the mild phenotype of this isolate (Figure 3, wild-type only).

Previous studies have demonstrated that specific amino acid substitutions in the CP ORF are associated with bright yellow mosaic induction in PepMV, particularly K236 in EU isolates and K155 in aggressive CH2 isolates [21]. These residues have been identified as the key determinants of symptom severity. Based on these findings, we introduced site-directed mutations at positions 155 and 236—substituting the native glutamic acid (E) residue in the mild PepMV-CT isolate, to generate mutant infectious clones and assess their role in symptom development. Each mutation was introduced individually into the PepMV-CT genome and inoculated into N. benthamiana and tomato plants. In N. benthamiana, infection with either the mutant or wild-type PepMV clone resulted in disease symptoms, and no significant differences were observed (Figure S2). Both CP^E155K and CP^E236K induced mosaic symptoms in tomatoes, in contrast to the asymptomatic phenotype caused by the wild-type clone (Figure 3a,

Table 1. Summary of the infectivity of wild-type and CP mutant PepMV infectious clones in tomato.

Infectious Clone	Infectivity
	PCR	Symptom Description
WT	15/15	No symptoms
CPS5P	15/15	No symptoms
CPA6T	15/15	No symptoms
CPT10P	15/15	No symptoms
CPA13V	15/15	No symptoms
CPS94F	15/15	No symptoms
CPM98I	15/15	No symptoms
CPE155K	15/15	Mild leaf yellowing mosaic
CPE236K	15/15	Severe yellowing mosaic on leaves
CPE155K/S5P	15/15	Mild leaf yellowing mosaic
CPE155K/A6T	15/15	Severe yellowing mosaic on leaves
CPE155K/T10P	15/15	Mild leaf yellowing mosaic
CPE155K/A13V	15/15	Mild leaf yellowing mosaic
CPE155K/S94F	15/15	Mild leaf yellowing mosaic
CPE155K/M98I	15/15	Mild leaf yellowing mosaic

), and RT-PCR confirmed systemic infection in all plants (Figure 3b). Among the two mutants, CP^E236K induced the most severe mosaic symptoms, appearing as early as 9 dpi, and spreading rapidly across the entire leaf surface after 3 weeks post-inoculation (wpi). CP^E155K caused mild yellow spotting starting at 16 dpi, which did not spread extensively and led to mild mosaic symptoms. These results indicate that residue 236 plays a stronger role than residue 155 in determining the severity of mosaic symptoms. Quantitative RT-PCR analysis of viral accumulation correlated with symptom intensity, showing that CP^E236K accumulated at the highest levels, followed by CP^E155K, and then the wild-type isolate.

Sequence comparison with the severe CH2 isolate, PepMV-KLP2, revealed seven amino acid differences. Based on these results, we generated six additional single mutants (positions 5, 6, 10, 13, 94, and 98) and corresponding double mutants combined at 155K. Infectivity assays showed that only the double mutant CP^E155K/A6T produced severe mosaic symptoms, whereas the other single or double mutants did not increase symptom severity (Figure 4a, Table 1). The RT-PCR and relative accumulation data supported these observations, with higher viral accumulation observed in the CP^E155K/A6T mutant than in the CP^E155K single mutant (Figure 4a,c). To confirm the persistence of the introduced mutations, viral progeny from symptomatic plants inoculated with mutant clones were sequenced. All analyzed samples retained the targeted mutations, demonstrating genetic stability of the mutant constructs during infection. These results indicate that, in the mild Korean isolate, residue 155 alone is insufficient and that both Thr6 and Lys155 are required to induce severe yellow mosaic symptoms.

Based on previous reports demonstrating interactions between PepMV CP and specific host proteins, three host factors (HSP70, SIOSCA4, SISTU38) were selected for in silico docking analysis [14,26,27]. Docking analysis (Table S4) revealed notable differences in host-binding patterns across CP variants, suggesting a potential mechanism underlying the differential symptom expression observed in our study. The CP^E155K and CP^E236K mutations against HSP70 enhanced complex formation, indicated by higher HADDOCK scores and binding energies, while the CP^E155K/A6T mutation weakened the interaction. In contrast, the highest binding affinity to SlOSCA4.1, a key regulator of Ca²⁺ homeostasis, was observed for the CP^E236K and CP^E155K/A6T variants, indicating that residue 236 is critical for maintaining this interaction. SISTU38 showed the greatest affinity for the wild-type capsid protein, whereas all evaluated mutations reduced binding strength. SISTU38 demonstrated a strong interaction with the CP of PepMV and facilitates viral infection. These data indicate that capsid mutations specifically alter host interaction characteristics instead of universally improving or diminishing host binding. While additional functional validation is required, the present study provides direct evidence linking specific CP mutations to the transition from mild to aggressive symptomatology in Korean PepMV isolates, laying an important groundwork for future investigations of this virus in Korea.

3.3. Evaluation of Tomato Cultivar Responses to the Severe PepMV CP Mutant

To preliminarily evaluate the pathogenicity of the severe PepMV mutant and screen for potential resistance, we examined several commercial tomato cultivars available in the Korean market, including four standard cultivars and eight cultivars containing Ty or Tm resistance genes. The CP^E236K mutant was used as the inoculum. All tested cultivars became infected and developed mosaic symptoms (Figure 5). Several cultivars—including the common cv.2 ‘Noranjangwon’ (Daenong), Rcv.4 ‘Benekia 220’, Rcv.5 ‘Ty-Kiseum’, and Rcv.7 ‘Ty-Wineo’ (Nongwoo) showed milder mosaic symptoms compared with the other tested cultivars, suggesting partial tolerance to PepMV. However, all cultivars were infected, with a 100% infection rate. These results confirm that resistance genes are effective against other viruses, such as Ty (TYLCV resistance) and Tm (Tobamovirus resistance), but do not protect PepMV. These findings further highlight the strong pathogenicity of the CP^E236K mutant, and the potential risk posed by PepMV if it acquires an aggressive phenotype comparable to natural severe isolates.

4. Discussion

PepMV is widely considered a re-emerging plant virus that has transitioned from being a minor pathogen to a globally significant threat to greenhouse tomato production, despite having been first identified several decades ago [7,10,28]. Numerous studies have documented its pathogenicity, particularly the wide variation in symptom severity associated with the infection. Among the five recognized PepMV strains, CH2 and EU strains are the most prevalent in several regions, especially across Europe, including Spain, Poland, and Belgium [11,29,30,31]. Notably, considerable variability exists within these strains, with some isolates inducing only mild symptoms while others cause markedly aggressive disease. Only a few PepMV isolates have been identified in South Korea. Since 2021, a single official report has documented the presence of PepMV strains belonging to CH2 and US1 [15]. In this study, we identified a mild PepMV isolate from an asymptomatic tomato plant during a field survey, in contrast to the virus-like symptomatic plants previously reported in Korea. Phylogenetic analysis revealed that our PepMV isolate was most closely related to earlier Korean isolates classified as the CH2 strain, further highlighting the genetic diversity of this strain. To better characterize and understand PepMV in Korea, we constructed an infectious clone using Gibson assembly. Infectious clones serve as essential tools on precisely manipulating viral genomes and studying viral pathogenicity, gene function, and host–virus interactions under controlled conditions. Upon inoculation, the clone successfully infected N. benthamiana and induced typical disease symptoms, confirming its functionality. However, in the primary host tomato, the clone did not induce visible symptoms, which was consistent with the mild nature of this isolate.

Symptom development in PepMV-infected tomato plants is influenced by multiple factors, including viral genotype, tomato cultivar, and environmental conditions [4]. Therefore, understanding the genetic determinants driving aggressive symptom expression is essential for predicting and mitigating destructive outbreaks. Previous studies have demonstrated that mutations in several viral genes, including TGB3 and CP, modulate symptom severity, with CP mutations being the most extensively investigated [4,32,33]. Among the CH2 isolates, two point- mutations in the PepMV CP gene, E155K and D166G, have been associated with interveinal leaf yellowing, and these substitutions have been identified in multiple yellowing-inducing CH2 isolates. In contrast, for isolates within the EU strain, a single mutation in the CP gene, E236K, has been reported to be the major determinant of symptom expression [21]. To validate these observations and obtain highly aggressive isolates for further studies, we generated a series of CP point mutations using the sequence of an aggressive CH2 isolate as a reference. Seven different mutations were introduced, including a substitution at position 236 to mimic the key CP mutation (CP^E236K) characteristic of EU strains, although this mutation has never been naturally reported in CH2 isolates. Infectivity assays demonstrated that E236K is not only a major determinant of mosaic symptoms in EU strains, but also dramatically enhances symptom severity when introduced into the CH2 background. CH2 clones carrying the E236K substitution (CP^E236K) induced severe mosaic and yellowing symptoms in tomatoes, compared to the mild phenotype of the Korean wild-type isolate. The CP^E155K mutant also induced mosaic symptoms, although the onset and severity were noticeably weaker and slower than those induced by CP^E236K. These findings indicate that residue 155 contributes to pathogenicity; however, its effects alone are insufficient to generate aggressive symptoms. Therefore, we hypothesized that the amino acid at position 155 might require additional cooperative mutations to induce severe yellow mosaic symptoms. To test this hypothesis, we generated double mutants by substituting residue 155 with six other point mutations. Among these, the double mutant affecting residues 6 and 155 (CP^E155K/A6T) caused markedly stronger yellowing symptoms, nearly comparable to those of the aggressive CP^E236K mutant, which was correlated with higher viral accumulation. These results provide direct evidence that a combination of mutations at residues 6 and 155 plays a crucial role in determining the virulence of PepMV in the CH2 genotype. Previous studies have proposed that the E236K substitution in EU isolates alters CP properties and host-interaction interfaces, virion localization. Based on these findings, we performed in silico analyses of the interactions between the CP of the mild wild-type PepMV isolate and its aggressive mutants and various host proteins known to interact with PepMV. In silico docking analysis (Table S4) revealed notable differences in host-binding patterns across CP variants, suggesting a potential mechanism underlying the differential symptom expression observed in our study. Docking analysis between the CP of wild-type and mutant viruses with known host factors suggests that these mutations may alter interactions with host defense components, potentially affecting recognition and symptom development. These findings support the possibility that structural changes in CP contribute to immune evasion, complementing the phenotypic differences observed between wild-type and mutant derivatives. Further experimental validation will be necessary to confirm these host–virus interaction effects in vivo. The observed effects of single and combined CP mutations suggest that PepMV coat protein evolution is driven by incremental, cooperative amino acid changes rather than single deterministic substitutions. While certain residues, such as position 236, act as major virulence determinants across genetic backgrounds, other residues (positions 6 and 155) appear to function as modulators whose effects depend on epistatic interactions. This pattern is consistent with stepwise adaptive evolution of the CP to optimize host interactions while maintaining structural integrity. Overall, our results demonstrate that a circulating PepMV isolate in Korea is phenotypically mild but genetically poised to transition to aggressiveness through a limited number of coat protein mutations. This finding highlights that symptom severity in PepMV is not fixed at the strain level but can shift rapidly through specific evolutionary changes in key viral proteins.

Viruses generally exhibit high mutation rates, that can lead to substantial genetic variability and rapid evolutionary dynamics in their populations. Although many mutations are detrimental, this intrinsic mutational capacity provides the plasticity necessary for viruses to adapt quickly to new environmental conditions [34,35]. Successive epidemic outbreaks of PepMV have been linked to the emergence of different strains with varying levels of adaptation to host plants [4,13]. Therefore, in addition to the molecular characterization of our PepMV isolate, we assessed its ability to infect a broad panel of tomato cultivars commonly available in the Korean market to evaluate the potential risks should an outbreak occur. In addition, we included cultivars carrying resistance genes effective against viruses from other families, such as Ty genes (against Begomoviruses) and Tm genes (against Tobamoviruses) [36,37,38], to determine whether such resistance mechanisms also provide cross-protection against PepMV. Using the most aggressive clone generated (CP^E236K), we inoculated various tomato cultivars and found that most were susceptible to infection. Although a few cultivars exhibited partial tolerance, showing only milder symptoms, none displayed complete resistance, indicating that the currently available Korean commercial cultivars do not provide sufficient protection against PepMV. Therefore, the additional screening of a wider range of cultivars is necessary. Nevertheless, this preliminary evaluation is valuable because it demonstrates the infectivity of a novel CP mutation not naturally reported in CH2 isolates and provides a foundation for future resistance breeding. Although the CP mutations analyzed in this study were artificially introduced, they highlight the potential for similar mutations to arise under natural conditions. Such changes could alter symptom expression and host interactions, potentially impacting disease severity or spread. Monitoring the emergence of novel CP variants in field and greenhouse populations would be important to anticipate and manage possible outbreaks. These results can also inform broader disease management strategies, such as deploying tolerant cultivars, implementing cultural practices to reduce virus spread, and monitoring for early detection in greenhouses. From an epidemiological perspective, our findings suggest that mild PepMV isolates may act as hidden reservoirs for the emergence of more aggressive variants, as similar transitions from mild to severe phenotypes have been observed in Europe. The recurrence of comparable virulence-associated CP mutations across geographically distinct regions indicates shared selective pressures under intensive greenhouse production systems, underscoring the global risk posed by PepMV evolution. While this study contributes meaningful insights, some limitations remain, and more work will be necessary to corroborate and expand on the present findings. Collectively, our results indicate that PepMV constitutes a persistent and evolving threat to tomato production in Korea. The rapid transition from mild to highly aggressive phenotypes underscores the urgent need for strengthened surveillance, symptom-independent diagnostic approaches, and the accelerated development of resistant tomato cultivars. Proactive and adaptive plant protection strategies will be essential to reduce the risk of future outbreaks driven by continued viral evolution.

5. Conclusions

In this study, we identified a new PepMV isolate belonging to the CH2 strain in Korea that induced only mild symptoms in tomatoes following agro-inoculation with its full-length cDNA infectious clone. By introducing specific amino acid substitutions based on previously reported aggressive isolates, we demonstrated that these mutations can convert a mild CH2 isolate into a severe variant, producing markedly stronger mosaic symptoms. In parallel, we conducted preliminary resistance screening using commercial tomato cultivars available in the Korean market; however, no cultivars with strong resistance were identified. This is the first in-depth characterization of a Korean PepMV isolate in relation to symptom induction and virulence determinants. Moreover, the infectious clone and its derived mutants provide valuable tools for future resistance screening and understanding PepMV pathogenicity in Korea. However, we acknowledge that this study is limited by the number of PepMV isolates analyzed and the experimental conditions used. Validation with larger isolate collections and additional functional assays, along with future mechanistic studies of host–virus interactions, will be required to fully elucidate the determinants of PepMV virulence and symptom severity.