Truncated CMV2b^N43 enhances virus-induced gene silencing in pepper by retaining systemic but not local silencing suppression

Abstract

See More

Pepper is an economically important crop. Owing to its recalcitrance to genetic transformation, virus-induced gene silencing (VIGS) is currently the major technique available for validating gene function in pepper. However, the low efficiency and difficulty of silencing genes in reproductive organs remain major challenges in pepper VIGS studies. To address these limitations, we developed an optimized VIGS system by structure-guided truncation of the Cucumber mosaic virus 2b (C2b) silencing suppressor. A silencing suppression assay revealed that the C2b^N43 mutant retained systemic silencing suppression while abrogated local silencing suppression activity in systemic leaves. The engineered TRV-C2b^N43 system significantly enhanced VIGS efficacy in pepper, providing a powerful tool for functional genomics studies in pepper. By leveraging transcriptomic profiles, we identified CaAN2, an anther-specific MYB transcription factor, whose suppression via TRV-C2b^N43 perturbation resulted in coordinated downregulation of structural genes in anthocyanin biosynthesis pathway and abolished anthocyanin accumulation in anthers establishing its essential regulatory role in pigmentation. This study validated and provided mechanistic insight for a further optimized VIGS system in pepper.

Introduction

RNA silencing is a small-RNA-mediated gene silencing mechanism present in most eukaryotes, wherein small interfering RNAs (siRNAs) or microRNAs (miRNAs) 20 to 25 nt length form an RNA-induced silencing complex (RISC) with the Agonaute (AGO) protein and mediate sequence-specific gene silencing via transcription inhibition, mRNA degradation or translational repression [1,2,3,4]. RNA silencing has long been recognized as a natural antiviral defence mechanism. Viral replication inside host cells leads to the formation of viral siRNAs, which form RISCs to silence viral gene expression [5, 6]. VIGS not only targets viral RNAs but also represses homologous plant genes, both transgenic and endogenous [7,8,9,10]. Thus, viruses could be developed as reverse genetics tools to knock down cellular gene expression by inserting cellular gene fragments into the virus genome [11,12,13]. Tobacco rattle virus (TRV) was first developed as a VIGS vector for silencing endogenous genes in Nicotiana benthamiana and Solanum lycopersicum, in which the wild-type virus induces very mild symptoms, making it suited for plant gene functional analysis [9, 12,13,14]. In the past 20 years, TRV has been widely used in many plant species, including both dicots and monocots, with variable efficiency [9, 15, 16].

To counteract RNA silencing, viruses encode viral suppressors of RNA silencing (VSRs) [17, 18]. VSRs target key components of the plant RNA silencing pathway using diverse molecular strategies, including disrupting RISC assembly, inhibiting viral RNA cleavage, and suppressing secondary siRNA biogenesis, thereby facilitating viral accumulation and spread [19, 20]. Systemic silencing, where mobile silencing signals propagate through the phloem and confer antiviral immunity in distal tissues, was previously discovered [21, 22]. VSRs deficiency compromises both intracellular viral replication and cell-to-cell/systemic movement, underscoring the critical role of VSRs in suppressing this long-range defence [19]. The C2b protein exhibits dual-suppression activity by binding both long and short dsRNAs to inhibit plant RNA silencing [23,24,25]. Critically, the capacity of C2b to disrupt secondary siRNA amplification provides a mechanistic basis for its essential role in promoting systemic viral spread [26,27,28]. Notably, while the systemic suppression of VSRs enhances spread, their local suppression may paradoxically reduce the efficacy of gene silencing in initially infected tissues [29,30,31]. Previous studies have highlighted that the VSRs exhibit distinct, separable inhibitory functions. Notably, P19 protein demonstrates two independent activities: siRNA binding and regulation of the miR168-mediated AGO1 expression pathway [32, 33]. Importantly, disruption of P19’s siRNA-binding ability does not impair its capacity to modulate miR168 and AGO1 expression [34]. Similarly, analysis of C2b deletion mutants has revealed that its ability to suppress AGO1 activity via direct interaction is independent of its dsRNA-binding function [35]. These findings underscore that the multiple inhibitory functions of VSRs can be effectively separated, allowing the generation of truncated VSR mutants with single functional activity. On the basis of previous findings showing that C2b incorporation enhances TRV-mediated VIGS efficiency in pepper [36], we reason that the decoupling of C2b’s dual activities by mutagenesis could further improve VIGS efficiency: retaining systemic suppression to promote the dissemination of TRV vectors while abolishing local silencing suppression to potentiate the efficacy of silencing in systemically infected tissues. This functional segregation strategy represents a viable approach to increase VIGS efficacy across phylogenetically diverse nonmodel crop species.

The pepper anther colouration is a critical phenotypic trait, manifests as predominant purple pigmentation in most cultivars through anthocyanin accumulation. This distinctive attribute has consequently been established as the premier phenotypic marker for assessing VIGS efficiency specifically in reproductive organs [37]. In the rpf1 mutant of pepper, a single nucleotide polymorphism (G to A) causes premature termination of the CabHLH gene, resulting in yellow anthers. RNA-sequencing analysis revealed significant downregulation of key flavonoid biosynthetic genes, including DFR, ANS, and RT, in the rpf1 mutant. Concurrently, the expression of DTX35, a gene associated with pollen fertility and flavonoid transport, had reduced expression levels. This transcriptional suppression correlates with the observed loss of anther pigmentation and compromised male fertility in the mutant [37]. While MYB-family transcription factors are well-established regulators of anthocyanin biosynthesis across plant species, their specific involvement in modulating anthocyanin accumulation in pepper anthers remains to be fully elucidated [38,39,40].

In this study, we conducted structure-guided mutagenesis to generate truncation mutants of the C2b protein to systematically investigate how domain-specific modifications affect its dual suppressive effects on local and systemic RNA silencing, as well as its effects on TRV-mediated VIGS efficiency. This rational mutagenesis strategy aimed to decouple the local and systemic silencing suppression activity of C2b, thereby identifying potential variants capable of enhancing TRV-based VIGS performance in pepper plants. Intriguingly, both the C2b^N43 and C2b^C79 truncation variants displayed compromised local RNA silencing suppression activity, but exhibited increased systemic VIGS efficiency when the marker gene CaPDS was targeted. This functional preservation facilitated long-distance movement of the recombinant TRV vectors through phloem-mediated transport pathways and significantly improved the target gene silencing efficacy. Through this structure-function dissection, we demonstrated that the selective disruption of local silencing suppression, coupled with the maintenance of systemic suppression activity, provides a viable strategy for optimizing viral vectors for VIGS. Our findings demonstrate that targeted modifications of the C2b domain can significantly enhance TRV-mediated VIGS in pepper, suggesting a practical strategy for gene function studies in other crop species.

Materials and methods

Plant material cultivation

N. benthamiana and C. annuum seedlings (L265) were grown in greenhouse under long-day conditions (light/dark: 16 h/8 h) at a temperature of 25 °C before inoculation. All the post inoculation plants were grown under long-day conditions at a temperature of 20 °C.

Construction of vectors

For pH7lic4.1-based vectors, full-length C2b and truncated variants (C2b^N43, C2b^N69 and C2b^C79), were amplified by PCR and cloned into the pH7lic4.1 expression vector. These constructs are driven by the CaMV 35 S promoter and fused with a C-terminal 3×Flag tag for protein detection. For generation of TRV-based vectors, full-length C2b and truncated variants, were PCR-amplified and fused at the 5-terminus with the subgenomic RNA promoter form Pea Early Browning Virus (PEBV). These fragments were subsequently cloned into the pTRV2-lic vector to generate recombinant plasmids pTRV2-C2b, pTRV2-C2b^N43, pTRV2-C2b^N69 and pTRV2-C2b^C79. For CaPDS silencing vector construction, a 368-bp fragment of the CaPDS gene (CA03g36860) was amplified by PCR using cDNA derived from pepper and inserted into the respective base vectors containing various viral suppressors of RNA silencing (VSRs) to generate silencing constructs. For CaAN2 silencing, a 250-bp CaAN2 fragment was cloned into the pTRV2-C2b^N43 vector to create the pTRV2-C2b^N43-CaAN2 construct. The primers are listed in the Table S1.

Imaging of plants and flowers

GFP fluorescence signals were visualized with a hand-held ultraviolet meter (ZF-5, Shanghai Jiapeng Technology Co., Ltd). Images of plant and flowers were taken using a camera (D7500, Nikon corp., Minato, Japan).

Quantitative real time PCR (qRT-PCR) analysis

RT-qPCR was performed as previously described [33]. Total RNA was extracted from C. annuum tissue with Trizol (ET101-01, Transgen Biotech, Beijing, China). First-strand cDNA was synthesized using 2 µg total RNA with random primer. RT-qPCR was performed using the ChamQ SYBR qPCR Master Mix (Q311-02, Vazyme, Nanjing, China) in a 10 µL volume (5 µL 2× ChamQ SYBR qPCR Green Master Mix, 1.0 µL of primers, 1.0 µL of cDNA, and 3 µL of distilled, deionized water). Relative gene expression values were calculated using the 2^−ΔΔCt method. The pepper GAPDH gene (CA03g24310) was used as an internal reference gene. At least three biological replicates were included for each treatment, and each replicate was from independent sampling.

Western blot analysis

Protein extraction and western blot was performed as previously described [33]. Protein was extracted from leaves of individual plant of N. benthamiana with 2 x sample buffer (50 mM Tris-HCl (pH 7.4), 100 mM KCl, 2.5 mM MgCl₂, 0.1% NP-40, 2× complete protease inhibitor cocktail). Protein extracts were separated on 10% SDS-PAGE gels and transferred to a polyvinylidene fluoride (PVDF) membrane blocked in 5% nonfat milk for 1 h at room temperature. Primary Anti-GFP monoclonal antibody (HT801-01, Transgen Biotech, Beijing, China) at a dilution of 1:5000 and secondary Goat Anti-Rabbit IgG (H + L), HRP Conjugate (HS101-01, Transgen Biotech, Beijing, China) at a dilution of 1:10000 were used for detection of GFP and GFP-fusion proteins.

Anthocyanin extraction and quantitation

The anthocyanin extraction protocol was adapted from previously reported methods with minor modifications [41]. Briefly, frozen pepper anthers were ground to a fine powder in liquid nitrogen and homogenized with five volumes (based on fresh weight) of ice-cold extraction buffer (45% methanol and 5% acetic acid, v/v). The homogenate was subjected to two sequential centrifugation steps (12,000 × g, 5 min, room temperature), with the supernatant collected after each centrifugation. Anthocyanin content was quantified spectrophotometrically using the formula:

$$\begin{array}{*{20}c} {{\text{Anthocyanin}}\,{\text{content}}} {\left( {{\text{Abs}}_{{{\text{53}}0}} /{\text{ g}}~{\text{FW}}} \right)} \\ \end{array} = \begin{array}{*{20}c} {\left[ \begin{gathered} {\text{A}}_{{{\text{53}}0}} \hfill - {\text{ }}\left( {0.{\text{25 }} \times {\text{ A}}_{{{\text{657}}}} } \right) \hfill \\\end{gathered} \right]} { \times {\text{ 5}}} \\ \end{array} {\text{ }} $$

where A_530​ and A₆₅₇​ represent absorbance values at 530 nm and 657 nm, respectively, to correct for background interference from chlorophyll degradation products.

Results

C2b^N43 and C2b^C79 mutations abolish local silencing suppression capacity

The 2b protein encoded by CMV (Q strain) consists of 100 amino acid residues, featuring two α-helical domains (residues 8–35 and 41–58) and dual nuclear localization signal (NLS) motifs at its N-terminus. To dissect the functional segregation between local and systemic silencing suppression activities in C2b, we engineered three truncation variants through structure-guided domain analysis: C2b^N43 retains both NLS motifs and the first α-helical domain; C2b^N69 encompasses both NLS motifs and complete helical domains; and C2b^C79 contains two NLS motifs, the intact second α-helix, and C-terminal regions (Fig. 1A). Transient expression assays via Agrobacterium-mediated infiltration in N. benthamiana leaves confirmed successful protein accumulation for all truncation mutants through western blotting (Fig. 1B). The functional characterization of local silencing suppression was performed through coinfiltration assays with a GFP reporter. Three days postinfiltration, fluorescence imaging revealed marked GFP signal enhancement in leaves coinfiltration with full-length C2b or C2b^N69, whereas C2b^N43 and C2b^C79 presented attenuated fluorescence levels similar to the empty vector control (Fig. 1C). Western blot analysis of GFP accumulation patterns corroborated these phenotypic observations (Fig. 1D). These findings demonstrate that the C2b^N43 and C2b^C79 mutants exhibit impaired local silencing suppression activity.

Fig. 1

Loss of local silencing suppression in C2b^N43 and C2b^C79 mutants. A The full-length C2b protein is depicted along with their respective domains, including Helix 1, Helix 2 and two NLSs. The truncated variants are shown below the full-length protein, indicating the positions of the truncations and the corresponding amino acid positions at the ends of the variants. The green boxes indicate the NLS1 and NLS2 motifs. B Western blot analysis of transiently expressed truncated C2b proteins. Total protein extracts from agroinfiltrated leaves were probed with anti-flag antibodies to detect the C2b proteins and anti-actin antibodies as a loading control. C Coinfiltration with GFP was used to assess the local silencing suppression capacity of C2b variants. UV indicates that the leaf photographs were captured under ultraviolet light illumination. D Western blot analysis of GFP expression in N. benthamiana leaves agroinfiltrated with different VSRs. The upper panel shows the detection of GFP, and the lower panel shows the detection of actin as an internal control. The numbers above the blots represent the relative expression levels of GFP normalized to actin

C2b^N43 retains systemic silencing suppression activity

To determine whether the C2b^N43 and C2b^C79 mutants retain systemic silencing inhibition capacity, we leveraged the established 16c system, in which GFP overexpression induces GFP-siRNA production and subsequent systemic silencing. This experimental framework enabled targeted evaluation of systemic signal suppression capabilities (Fig. 2A). As C2b^N43 and C2b^C79 lose their local silencing suppression activity, they may not accumulate to a sufficient level for systemic silencing suppression. Interestingly, the potyvirus VSR, HcPro was reported to efficiently suppress local silencing but not systemic silencing in grafting using transgenic tobacco plants [42, 43]. To test whether HcPro maintains these properties in a transient assay, Agrobacterium strains carrying either 35 S::GFP or HcPro constructs were mixed at varying ratios and infiltrated onto 16c plant leaves. At a HcPro: GFP ratio of 0.1:0.5 (OD600), 66% of the infiltrated plants exhibited systemic GFP silencing, whereas complete suppression occurred at a ratio of 0.3:0.5 (OD600) (Fig. 2B,C). Subsequent coexpression experiments with C2b^N43 and C2b^C79 at a HcPro: GFP: C2b^N43/C2b^C79 ratio of 0.1:0.5:0.5 (D600) demonstrated that C2b^N43 significantly suppressed systemic silencing, with only 26% of the plants showing systemic GFP silencing. This finding contrasts with the 69% silencing observed in C2b^C79-expressing plants and 74% in empty vector controls (Fig. 2D). RT-qPCR quantification revealed significantly lower GFP mRNA levels in silenced tissues than in nonsilenced tissues (Fig. 2E). These findings demonstrate that the truncated C2b^N43 protein retains systemic silencing suppression activity.

Fig. 2

Validation of systemic silencing suppression retention in C2b^N43 and C2b^C79. A Phenotypic comparison of GFP expression in 16c plants showing non-systemic and systemic GFP silencing. The left panel shows a plant with non-systemic GFP silencing. The right panel shows a plant exhibiting systemic GFP silencing, with yellow arrows highlighting the regions affected by systemic silencing. The scale bar represents 1 cm. B Phenotypic comparison of GFP expression in 16c leaves following GFP overexpression alone versus coexpression with HcPro. The scale bar represents 1 cm. C Systemic silencing efficiency of GFP in response to increasing concentrations of HcPro. The percentage of systemic silencing (y-axis) is shown for different HcPro/GFP ratio (mean ± SD, n = 3). D Systemic silencing efficiency in plants infiltrated with C2b^N43, C2b^C79 and EV. Efficiency was quantified as the percentage of silenced plants relative to total plants (mean ± SD, n = 3). Statistical significance (one-way ANOVA with Tukey’s test) is denoted by different lowercase letters. E RT‒qPCR analysis of GFP mRNA expression levels in systemically silenced versus non-silenced leaves of 16c (mean ± SD, n = 3). Statistical significance (Student’s t test; ** p < 0.01) is denoted by asterisks

Differential enhancement of VIGS efficiency by truncated VSR variants in pepper

To investigate whether C2b truncation mutants could enhance TRV-mediated gene silencing efficiency, we engineered TRV-based vectors expressing full-length C2b or its truncated variants. The recombinant vectors were constructed by cloning each VSR sequence into the pTRV2 backbone, followed by insertion of a CaPDS gene fragment to simultaneously evaluate VSR functionality and quantify CaPDS silencing efficiency through the photobleaching phenotype. Agrobacterium strains harbouring these pTRV2 constructs were coinfiltrated with pTRV1-carrying Agrobacterium into pepper cotyledons. At the systemic level, leaves of all TRV2-VSR infected plants presented confluent CaPDS silencing phenotype (Fig. 3A). TRV-specific RT‒PCR confirmed the presence of TRV in all inoculated plants. No amplification was detected in the uninfected controls (Fig. 3B). RT‒PCR analysis of silencing-affected leaf tissues revealed significant reductions in CaPDS transcript levels across TRV-infected plants compared with those in uninfected controls (Fig. 3C). Quantitative analysis of CaPDS silencing efficiency revealed that the control group (without C2b) exhibited a silencing efficiency of 70%, with considerable variation among biological replicates. Strikingly, the C2b^N43 and C2b^C79 expressing constructs achieved significantly higher silencing efficiencies of 93.33% and 91.67%, respectively, outperforming both the full-length C2b and C2b^N66 (Fig. 3D). These results demonstrate that structural modulation of VSRs differentially regulates TRV-mediated VIGS efficacy, with the C2b^N43 truncation conferring optimal enhancement under our experimental conditions.

Fig. 3

Phenotypic and molecular analysis of TRV-mediated VIGS targeting CaPDS in pepper using different VSRs. A Phenotypic analysis of TRV-mediated VIGS targeting the CaPDS gene in pepper plants using different VSRs. Images were taken 14 days post-inoculation and demonstrate the effects of different VSRs on the efficiency of gene silencing, as indicated by the photobleaching phenotype resulting from CaPDS gene silencing. The scale bar represents 1 cm. B RT-PCR analysis of TRV-coat protein expression in different experimental groups. The presence of TRV-coat protein transcript was detected using RT‒PCR, with Actin serving as an internal control to ensure equal RNA loading. C The relative expression levels of CaPDS were analyzed by RT‒PCR. The relative expression levels of CaPDS are presented in the bar graph, with the y-axis representing the relative expression level. D VIGS efficiency of TRV vectors expressing different VSRs. The graph shows the efficiency of CaPDS silencing mediated by TRV vectors carrying various VSRs (mean ± SD, n = 3). Statistical significance (one-way ANOVA with Tukey’s test) is denoted by different lowercase letters. E Plant height statistics of pepper following TRV inoculation carrying distinct VSRs (n = 12) at 28 dpi. Control groups comprised TRV-free plants and plants inoculated with TRV without VSR. Statistical significance (one-way ANOVA with Tukey’s test) is denoted by asterisks (** p < 0.01, ns represents no significant difference). F ROS detection in systemic leaves of TRV-infected and uninfected pepper at 28 dpi. ROS accumulation was analyzed by DAB staining. G Histograms quantify relative DAB-staining intensity (gray values) from F (n = 12). Statistical significance (one-way ANOVA with Tukey’s test) is denoted by asterisks (ns represents no significant difference)

Symptom severity following viral infection represents a critical determinant of VIGS utility. To assess whether TRV expressing C2b^N43 exacerbates infection symptoms, we quantified plant height in Capsicum annuum inoculated with unmodified TRV or TRV expressing full-length C2b or C2b^N43. Compared with the mock-inoculated controls, all the virus-inoculated groups presented significant height reductions; however, the stunting severity of the plants infected with C2b^N43-TRV was comparable to that of those receiving C2b-TRV or unmodified TRV (Fig. 3E). Further analysis of hydrogen peroxide accumulation via DAB staining in apical systemic leaves at 28 dpi revealed no significant differences between mock-inoculated plants and TRV-infected plants harbor different suppressors (Fig. 3F, G). These findings demonstrate that compared with conventional TRV vectors, C2b^N43 expression does not intensify infection symptoms.

CaAN2 regulates anthocyanin accumulation in pepper anthers

Anthocyanin accumulation in pepper anthers not only serves as a protective mechanism against UV-induced damage but also functions as a critical phenotypic marker for assessing the purity of F1 hybrids through visible colour based screening. To assess the gene-silencing efficiency of C2b^N43-incorporated TRV vectors during reproductive development in pepper and the genetic basis of anthocyanin accumulation in pepper anthers, we investigated the function of MYB family genes specifically expressed in pepper anthers using the C2b^N43-incorporated TRV vector. Database mining from PepperHub identified Capana10g001433 as displaying stage-correlated expression with anther pigmentation dynamics (Fig. 4A). Capana10g001433 was exclusively expressed during the F4-F6 developmental stages, which coincided with the observed anthocyanin accumulation initiation at the F5 stage, whereas it remained undetectable in the F1, F2, and F7‒F10 stages (Fig. 4A). Sequence alignment analysis against the NCBI (www.ncbi.nlm.nih.gov) database revealed that Capana10g001433 encodes a canonical R2R3-MYB transcription factor, characterized by conserved DNA-binding domains. Phylogenetic reconstruction using orthologous sequences from nine angiosperm species (Capsicum annuum, Solanum lycopersicum, Solanum tuberosum, Solanum pennellii, Nicotiana benthamiana, Arabidopsis thaliana, Petunia riograndensis, Lycium ruthenicum, and Nicotiana. attenuata) placed the R2R3-MYB within a conserved Solanaceae clade alongside potato and tomato homologues, named Anthocyanin2 (AN2) or MYB113 (Figure S1A) [44, 45]. Comparative sequence analysis revealed pronounced conservation in R2R3 DNA-binding domains, suggesting evolutionary preservation of functional specificity (Figure S1B).

Fig. 4

Silencing of CaAN2 disrupts anthocyanin biosynthesis in pepper anthers. A Tissue-specific expression profiling of CaAN2 across 10 floral developmental stages (F1–F10). Heatmap analysis (top) reveals tissue-specific CaAN2 expression patterns. Color intensity in the heatmap reflects log2-transformed expression levels (red: high; blue: low). Corresponding floral images (bottom) document progressive anther color changes. The scale bar represents 5 mm–1 cm. B Tissue-specific expression of CaAN2 in floral organs (sepal, petal, anther, and carpel) by RT‒qPCR (n = 3, mean ± SD). C Anther phenotypes of wild-type (WT) and CaAN2-silenced plants via VIGS. The scale bar represents 1 cm. D RT‒qPCR validation of CaAN2 suppression in silenced anthers (n = 3, mean ± SD). Statistical significance (Student’s t test; ** p < 0.01) is denoted by asterisks. E Anthocyanin content in control and CaAN2-silenced anthers (n = 3, mean ± SD). Statistical significance (Student’s t test; ** p < 0.01) is denoted by asterisks. F Schematic of key structural genes and metabolites in the plant anthocyanin biosynthesis pathway, and heatmap analysis of gene expression profiles before and after CaAN2 silencing. The heatmap was generated from RT‒qPCR data, with red and blue gradients indicating high and low relative expression levels, respectively

Spatiotemporal expression profiling via RT‒qPCR revealed strong anther-specific expression of CaAN2 at the F5 stage, with no detectable transcript accumulation in sepals, petals, or carpels (Fig. 4B). Functional validation through optimized virus-induced gene silencing was performed with pTRV2-C2b^N43 vectors containing a unique CaAN2 fragment. Cotyledonary infiltration of 14-day-old seedlings with Agrobacterium (OD600 = 0.004) carrying recombinant vectors resulted in complete anthocyanin ablation, resulting in yellow–green anthers in contrast to the purple anthers of the wild-type controls (Fig. 4C). Quantitative analysis confirmed 91% suppression of CaAN2 expression in the silenced plants (Fig. 4D). Anthocyanin extraction and quantification were performed for both CaAN2-silenced and control anthers. Spectrophotometric analysis revealed a striking reduction in anthocyanin content, with control samples exhibiting 20 mg/g anthocyanin, whereas CaAN2-silenced anthers presented no detectable accumulation (Fig. 4E). To investigate the regulatory role of CaAN2 in anthocyanin biosynthesis, we quantified the transcript levels of 10 core structural genes within the pathway using RT‒qPCR analysis. RT‒qPCR revealed coordinated downregulation of the structural genes CaPAL, CaC4H, Ca4CL, CaCHS, CaF3’5 H, CaDFR, CaANS, and CaUFGT, whereas CaCHI and CaF3H expression remained unaffected, demonstrating that CaAN2 plays a critical regulatory role in anthocyanin biosynthesis in pepper anthers (Fig. 4F, Figure S2A‒J). These findings establish CaAN2 as the principal regulator of floral anthocyanin production in pepper and validate C2b^N43-modified TRV vectors as effective tools for functional studies in reproductive tissues. The identified molecular marker system improves the technical capacity for hybrid purity assessment in pepper breeding programs.

Discussion

VSRs counteract host RNA silencing mechanisms to facilitate viral immune evasion, primarily through two functional components: local silencing suppression (blocking siRNA biogenesis/function in initially infected cells) and systemic silencing suppression (inhibiting the cell-to-cell or long-distance spread of silencing signals) [19, 20]. For example, Citrus tristeza virus (CTV) P23 exhibits potent local silencing suppression, whereas CTV CP specifically suppresses systemic silencing [46,47,48]. In contrast, multiple VSRs such as C2b and TBSV p19 possess dual activities [49, 50]. Notably, the reduced VSR activity of TAV 2b was critical for achieving efficient VIGS in turnip vein-clearing virus-based vectors [29]. Consistently, TBSV p19 was shown to suppress GFP-targeted VIGS when expressed in homologous TBSV vectors, whereas p19-deficient TBSV enabled robust GFP silencing [30, 31]. The heterologous VSRs turnip mosaic virus p38 and tobacco mosaic virus 126-kDa protein inhibited TRV-mediated VIGS of the endogenous PDS gene [51]. Our data revealed that C2b^N69 retained local silencing suppression activity (Fig. 1C, D), whereas C2b^N43 abolished local silencing suppression but maintained systemic silencing suppression capacity (Fig. 2D). This enables efficient systemic infection by TRV. At the systemic level in leaves, C2b^N43 fails to block the siRNA-mediated cleavage of both TRV and the target gene, ultimately resulting in more efficient and thorough target gene silencing (Fig. 5). Previous studies demonstrated that the N-terminal truncation mutant (residues 1–37) of the 2b protein from CMV (SD strain), designated C2b (1–37), loses PTGS suppression activity and lacks binding capacity for 21-nt siRNA but retains affinity for the longer 24-nt siRNAs and 55-nt dsRNAs [52, 53]. Given that long siRNAs serve as critical components of systemic silencing signals [54], our findings signals that 24-nt siRNAs or long dsRNAs may function as systemic signals in antiviral RNA silencing. Furthermore, the compact size of the inserted C2b^N43 fragment may increase TRV genomic stability and facilitate efficient systemic spread. It should be noted that the VIGS system developed in this study and its functional validation have so far been conducted exclusively in pepper, and its generalizability to other plant species has not yet been tested. Nevertheless, this domain separation strategy, which involves enhancing systemic spread from local silencing suppression, provides a promising framework for improving VIGS efficiency in other virus‒host systems, particularly those currently suboptimal for cross-species applications.

Fig. 5

Schematic representation of C2b^N43-mediated enhancement of VIGS efficiency in pepper. Compared to full-length C2b, the C2b^N43 truncation retains systemic silencing suppression but lacks local silencing suppression activity. In inoculated leaves, both proteins facilitate systemic spread of TRV through suppression of mobile silencing signals. Crucially, in systemic leaves, absence of local suppression in C2b^N43 enables unimpeded RISC-mediated cleavage of target transcripts, resulting in enhanced VIGS efficacy

Anthocyanin accumulation in anthers is a key agronomic trait in cultivated pepper, with the purple phenotype being a hallmark of most cultivars [37]. Previous studies have identified CaAN2 as a regulator of anthocyanin biosynthesis in pepper cotyledons [55]. However, the tissue-specific expression pattern of CaAN2 in anthers and its functional role in establishing purple pigmentation remain uncharacterized. Here, our spatiotemporal expression profiling revealed a strong positive correlation between CaAN2 transcript levels and anthocyanin accumulation during critical stages of anther development (Fig. 4A). Using an optimized TRV-C2b^N43-based VIGS system, we functionally validated CaAN2 by demonstrating that its silencing in anthers completely abolished anthocyanin synthesis, resulting in the loss of the purple phenotype (Fig. 4C). Intriguingly, prior work on the rpf1 mutant in pepper indicated that a mutation in the CabHLH gene significantly reduced in flavonoid content in anthers and impaired pollen fertility [37]. Given the conserved regulatory role of the MYB-bHLH-WD40 ternary complex in plant anthocyanin biosynthesis, our findings raise an important question: does CaAN2 coordinately regulate both the biosynthesis of anthocyanin and other flavonoid in pepper anthers through interactions with CabHLH? Addressing this question will advance our understanding of the functional interplay between anthocyanin accumulation and pollen development.

Conclusions

We pioneered a structure-guided decoupling strategy for viral silencing suppressors, engineering C2b^N43 to enable tissue-specific VIGS enhancement in pepper achieving 93.33% CaPDS silencing efficiency and establishing CaAN2-regulated anther pigmentation as a morphological marker for VIGS validation in reproductive tissues. This optimized TRV platform provides a universally applicable framework for efficient gene function validation in recalcitrant species, advancing functional genomics across plant systems.