Functional Dissection of the Turnip Crinkle Virus Coat Protein Links ATG8 Association to Autophagy‐Associated Antiviral Regulation

ABSTRACT

The coat protein (CP) of turnip crinkle virus (TCV) plays multifaceted roles in viral infection, acting as both an avirulence (Avr) factor recognized by the resistance protein HRT and a viral suppressor of RNA silencing (VSR). However, how these dual activities are coordinated remains unclear. Here, we demonstrate that the position of a fluorescent protein (FP) tag on CP functionally separates its two activities. C‐terminal tagging (CP‐GFP) preserved the ability to trigger HRT‐dependent hypersensitive response (HR) but abolished silencing suppression, whereas N‐terminal tagging (YFP‐CP) retained VSR activity but failed to induce HR. These functional differences correlated with distinct subcellular localizations: Avr activity required both nuclear and cytoplasmic localization, while VSR function was confined to the cytoplasm. We further show that the N‐terminal region of CP associates with autophagy‐related protein 8 (ATG8), linking CP to autophagy‐associated antiviral pathways. Autophagy‐deficient atg5 and atg7 mutants exhibited enhanced susceptibility to TCV, supporting a role for autophagy in antiviral defence during infection. Together, our findings reveal that TCV CP contains spatially and mechanistically separable determinants for Avr and VSR functions and identify CP–ATG8 association as a potential interface between viral infection and host autophagy‐associated regulation.

Turnip crinkle virus coat protein acts as a molecular switch that separates two plant immune responses. It suppresses antiviral RNA silencing while altering hypersensitive cell death through autophagy‐related interactions. This selective modulation allows the virus to balance defence suppression and immune evasion, revealing a strategy used by plant pathogens to optimize infection.

1. Introduction

Plants have evolved diverse defence mechanisms to counteract viral invasion, with RNA silencing serving as a primary antiviral strategy. This conserved eukaryotic gene regulation mechanism not only plays a crucial role in antiviral defence but also regulates various developmental processes (Vaucheret 2006, Ding and Voinnet 2007). Antiviral silencing is triggered by recognition of double‐stranded RNAs (dsRNAs) that arise during viral replication or from intermolecular fold‐back structures within viral genomes. These dsRNAs are cleaved by Dicer‐like ribonucleases to generate 21–24‐nucleotide small interfering RNAs (siRNAs) (Ruiz‐Ferrer and Voinnet 2009). The siRNAs are incorporated into the RNA‐induced silencing complex (RISC), which includes Argonaute (AGO) proteins. Guided by siRNAs, the RISC degrades complementary target mRNAs, effectively silencing viral gene expression.

To counteract RNA silencing, viruses have evolved viral suppressors of RNA silencing (VSRs) that inhibit various steps of this pathway (Ding and Voinnet 2007). More than 50 VSRs have been identified, many of which exhibit multifunctionality in viral replication, assembly and movement. These VSRs primarily suppress silencing through two mechanisms: sequestering small RNA duplexes to prevent AGO loading into the RISC (e.g., potyviral HcPro, beet yellows virus P21, peanut clump virus P15 and turnip crinkle virus [TCV] coat protein [CP]) (Merai et al. 2006) or directly targeting AGO proteins to inhibit RISC activity, as seen with cucumber mosaic virus (CMV) 2b protein, which binds to the PAZ domain of AGO1 (Zhang et al. 2006). Additionally, some VSRs, including sweet potato mild mottle virus P1 and TCV CP, contain glycine/tryptophan repeat motifs that mimic host AGO1‐binding peptides, facilitating their recruitment to the RISC (Azevedo et al. 2010; Giner et al. 2010). These findings highlight the diverse strategies that VSRs employ to suppress RNA silencing and facilitate viral infection.

In addition to RNA silencing, plants employ dominant resistance (R) gene‐mediated defence known as effector‐triggered immunity (ETI), which also targets bacterial and fungal pathogens (Jones and Dangl 2006). ETI is activated when plant R proteins recognize specific pathogen effectors, leading to a hypersensitive response (HR) characterized by localized cell death and activation of defence‐related genes. Examples of virus R genes include N in tobacco for tobacco mosaic virus, Rx1/2 in potato for potato virus X (PVX), and HRT and RCY1 in Arabidopsis thaliana for TCV and CMV, respectively (Cooley et al. 2000; Takahashi et al. 2001; Bendahmane et al. 1999; Whitham et al. 1994). Most R proteins belong to the nucleotide‐binding leucine‐rich repeat family and contain coiled‐coil or Toll/interleukin‐1 receptor domains at their N‐termini (Moffett 2009). These proteins typically recognize effectors through an indirect “guard/decoy” mechanism (Cesari et al. 2013; Dodds and Rathjen 2010).

Emerging evidence implicates autophagy, an evolutionarily conserved intracellular degradation pathway, in plant antiviral defence. While autophagy is best known for maintaining cellular homeostasis under nutrient‐limiting conditions, it has recently been linked with both pro‐ and antiviral roles (Hafren et al. 2018; Clavel et al. 2017). With the identification of core autophagy‐related genes (ATGs) in plants, functional studies have revealed their importance in pathogen responses (Kushwaha et al. 2019). Among them, autophagy‐related protein 8 (ATG8) is a ubiquitin‐like protein that plays a central role in autophagosome formation. Upon lipidation with phosphatidylethanolamine, ATG8 localizes to autophagosomal membranes and acts as a scaffold for selective degradation of cellular or pathogen‐derived cargos, including viral proteins (Yu et al. 2012).

TCV is a small, icosahedral, positive‐sense single‐stranded RNA virus belonging to the Tombusviridae family. TCV can infect a broad range of hosts, including Arabidopsis thaliana (Qu and Morris 2008). TCV CP plays multiple roles in encapsidation, viral movement and systemic spread, and also functions as both an avirulence (Avr) factor and an RNA silencing suppressor (Zhao et al. 2000; Zhu et al. 2013). In the Arabidopsis Dijon ecotype Di‐17, which harbours the R gene HRT, CP triggers HR and activates immune responses. However, most A. thaliana ecotypes lack HRT and are thus susceptible to TCV (Dempsey et al. 1993).

The N‐terminal region of CP is critical for recognition by HRT. Resistance‐breaking TCV mutants (e.g., TCV^D4N and TCV^P5S) carry point mutations at positions 4 (D → N) and 5 (P → S) of CP that allow them to evade HRT‐mediated immunity (Zhao et al. 2000). Despite this knowledge, the molecular mechanisms by which CP is recognized by HRT and activates HR remain poorly understood. CP interacts with host factors such as TCV‐interacting protein, a NAC transcription factor involved in modulating basal resistance (Jeong et al. 2008; Donze et al. 2014). Notably, RNA silencing components are also required for HRT‐mediated resistance, suggesting that crosstalk occurs between RNA silencing and ETI during TCV infection (Zhu et al. 2013). However, the VSR activity of CP is dispensable for HRT‐dependent HR, raising questions about how its Avr and VSR functions are regulated and spatially organized.

In this study, we generated fluorescent protein (FP)‐tagged CP variants with the FP fused to the N‐ or C‐terminus in order to dissect the functional domains of CP. Unexpectedly, C‐terminal tagging (CP‐FP) preserved the ability of CP to activate HRT‐mediated immunity (Avr function) but abolished its VSR activity, whereas N‐terminal tagging (FP‐CP) retained the VSR activity of CP but abrogated its ability to trigger HR. We further show that the N‐terminal region of CP mediates an interaction with ATG8, an autophagy‐related protein, suggesting a potential connection between CP and autophagy‐associated antiviral regulation during viral infection. These findings reveal how the dual functions of TCV CP are functionally separable and indicate that autophagy is a component of antiviral immunity in host plants.

2. Results

2.1. The Position of the Fluorescent Tag Functionally Separates the Avirulence and Silencing Suppression Activities of TCV CP

To dissect how fluorescent tagging affects the biological activities of the TCV CP, we generated two fusion variants in which fluorescent proteins were attached either to the C‐terminus (CP‐GFP) or the N‐terminus (YFP‐CP) of CP. Before characterizing their subcellular localization, we first assessed whether these FP‐tagged forms retained two well‐established CP functions: HRT‐dependent avirulence (Avr) and post‐transcriptional gene silencing suppression (VSR) (Moon et al. 2016; Thomas et al. 2003; Kang et al. 2008; Zhu et al. 2013).

Co‐expression assays in Nicotiana benthamiana revealed functional differences depending on the tag position. Co‐infiltration of HRT with either CP or CP‐GFP triggered a strong HR by 2 days post‐infiltration (dpi), as indicated by visible tissue collapse and enhanced electrolyte leakage. In contrast, YFP‐CP failed to induce cell death under the same conditions (Figure 1a,b). Consistent with HR activation, immunoblot analysis showed that HRT expression selectively reduced the accumulation of CP and CP‐GFP, whereas YFP‐CP levels remained unchanged (Figure 1c). In some samples, an additional lower CP‐reactive band was detected. Because a similar band was observed for wild‐type (wt) CP, this band may reflect partial degradation, processing or altered electrophoretic mobility of CP under the expression conditions used. Thus, C‐terminal tagging preserves the Avr activity of CP, while N‐terminal tagging disrupts it.

FIGURE 1.

CP‐GFP and YFP‐CP exhibit distinct functions as an Avr factor and viral suppressor of RNA silencing (VSR), respectively. (a) Hypersensitive response (HR) induced by co‐expression of coat protein (CP), CP‐GFP or YFP‐CP with HRT in Nicotiana benthamiana leaves. Constructs were transiently expressed via Agrobacterium infiltration. Agrobacteria carrying the empty vector (GV2260; GV) served as a negative control. Leaf images were captured at 2 days post‐infiltration (dpi) under white light (WL) and UV light. (b) Electrolyte leakage assay corresponding to the HR phenotypes in (a). Data represent mean ± standard error from three biological replicates. For each replicate, n = 20 leaf discs (1 cm diameter) per treatment. hpi, hours post‐infiltration. Different letters denote statistically significant differences (p < 0.05, one‐way ANOVA). (c) Immunoblot analysis of TCV CP variants in the samples shown in (a) using an anti‐CP antibody. Relative CP accumulation was quantified using ImageJ and normalized to the RuBisCO band. An additional lower CP‐reactive band was detected in some samples and may reflect partial degradation, processing or altered electrophoretic mobility of CP. Data represent mean ± standard error from independently repeated experiments. (d) Suppression of GFP silencing in transgenic N. benthamiana 16c plants by CP variants. GV2260 (empty vector; GV), CP, CP‐GFP or YFP‐CP was co‐infiltrated with a GFP construct. GFP fluorescence was photographed under UV light at 5 dpi. (e) Immunoblot analysis of GFP accumulation from the silencing suppression assay in (d), using an anti‐GFP antibody. Protein loading was verified by Ponceau S (PS) staining.

We next evaluated VSR activity using the N. benthamiana 16c reporter line, which exhibits robust GFP silencing upon re‐introduction of the GFP gene (Voinnet et al. 1999). In this system, YFP‐CP strongly suppressed GFP silencing, whereas CP‐GFP lacked detectable VSR activity (Figure 1d,e). These reciprocal phenotypes indicate that FP tag position differentially affects the retention of Avr and VSR activities of CP.

To determine whether this orientation effect reflected a general property of N‐ versus C‐terminal fusion, we tested additional constructs (GFP‐CP and CP‐YFP^N). These variants showed the same pattern of functional separation (Figure S1), confirming that the effect is determined by tag orientation rather than fluorophore identity. Because GFP‐CP produced high background fluorescence under the HR‐imaging filter sets, YFP‐CP was used for subsequent experiments. As expected, expression of HRT or CP alone did not induce HR (Figure S1).

To examine whether CP‐GFP‐induced HR restricts viral replication, we used a PVX‐based expression system (Lu et al. 2003, Chapman et al. 1992). PVX expressing CP‐GFP exhibited a strong HR when co‐expressed with HRT, accompanied by a marked reduction in PVX CP accumulation (Figure S2). In contrast, PVX constructs expressing YFP‐CP or GFP did not trigger HR in the presence of HRT and showed no decrease in PVX CP levels, indicating that these variants fail to activate HRT‐mediated antiviral resistance. Notably, all PVX constructs including FP‐tagged CP variants elicited Rx‐dependent HR and the corresponding reduction in PVX CP accumulation, confirming that the Rx‐mediated antiviral pathway remained fully functional in all PVX expression backgrounds.

Collectively, these findings demonstrate that the position of the fluorescent tag differentially affects the Avr and VSR activities of TCV CP, with CP‐GFP preferentially retaining Avr activity and YFP‐CP retaining VSR activity.

2.2. Subcellular Localization Contributes to the Differential Avr and VSR Activities of CP‐GFP and YFP‐CP

Confocal laser scanning microscopy (CLSM) showed that both CP‐GFP and YFP‐CP localized to the nucleus and cytoplasm and, unlike free GFP, displayed punctate fluorescence patterns indicative of compartmentalized accumulation (Figure S3a). Subcellular fractionation of cells expressing untagged CP further confirmed its nuclear presence, demonstrating that this distribution is intrinsic to CP rather than an artefact of fluorescent tagging (Figure S3b).

To investigate whether nuclear localization contributes to the Avr or VSR functions of CP, we redirected CP‐GFP and YFP‐CP to defined subcellular compartments by attaching either a nuclear localization signal (NLS) or a nuclear export signal (NES), with a mutated NLS (nls) serving as a control (Haasen et al. 1999; Wen et al. 1995; Gorlich and Mattaj 1996) (Figure S4; Table S1). CLSM confirmed the expected localization patterns of all constructs (Figure S4). Nuclear and cytoplasmic fractionation followed by immunoblot analysis further validated the altered subcellular distribution of the indicated CP fusion proteins and confirmed their accumulation at detectable levels in the samples used for subsequent functional analyses (Figure 2a).

FIGURE 2.

Subcellular localization contributes to HRT‐mediated hypersensitive response (HR) and viral suppressor of RNA silencing (VSR) function of coat protein (CP). (a) Nuclear and cytoplasmic fractionation and accumulation analysis of CP‐GFP and YFP‐CP variants redirected by nuclear localization signal (NLS), nuclear export signal (NES) or mutant nls sequences. The corresponding confocal laser‐scanning microscopy (CLSM) localization patterns are shown in Figure S4. Nuclear and cytoplasmic fractionation followed by immunoblot analysis was used to biochemically validate the localization patterns and confirm the accumulation of the indicated recombinant proteins. α‐Tubulin and Histone H3 were used as cytoplasmic and nuclear markers, respectively. (b–d) HR phenotypes resulting from co‐expression of HRT with subcellularly redirected CP variants in Nicotiana benthamiana. CP‐GFP was modified with a NLS, NES or nls to control its localization. Agrobacteria carrying the empty vector (GV2260, GV) served as a negative control. (b) Representative images showing HR at 2 days post‐infiltration (dpi). (c) Ion leakage measurements corresponding to HR in (b). Data represent mean ± standard error; n = 20 leaf discs per construct. Statistical differences were assessed by one‐way ANOVA (p < 0.05); different letters indicate significant differences. (d) Immunoblot using an anti‐CP antibody to assess CP accumulation levels. Total protein extracts from the samples in (b) were analysed. The lower CP‐reactive band may reflect partial processing, degradation or altered electrophoretic mobility of the expressed CP fusion protein. (e, f) Silencing suppression assay with YFP‐CP localization variants in N. benthamiana 16c. (e) UV images of infiltrated leaves at 7 dpi showing GFP fluorescence. (f) Immunoblot analysis of GFP accumulation using an anti‐GFP antibody. Two GFP‐reactive bands may reflect simultaneous detection of GFP expressed from the stable 16c transgene and transiently overexpressed GFP, including localization‐dependent GFP forms with different electrophoretic mobility. Equal protein loading was confirmed by Ponceau S (PS) staining.

Co‐expression with HRT showed that CP‐GFP and CP‐nls‐GFP induced visible HR and significant electrolyte leakage, whereas CP‐NLS‐GFP and CP‐NES‐GFP did not (Figure 2b,c). Immunoblot analysis revealed reduced CP accumulation in HRT/CP‐GFP and HRT/CP‐nls‐GFP samples, consistent with HR‐associated protein reduction, while CP levels remained stable in HRT/CP‐NLS‐GFP and HRT/CP‐NES‐GFP. Notably, CP‐NES‐GFP accumulated poorly even in the absence of HRT, suggesting that forced cytoplasmic export destabilizes CP (Figure 2d). These results indicate that proper nucleocytoplasmic distribution of CP is associated with HRT‐dependent HR activation.

In contrast, GFP silencing assays using N. benthamiana 16c plants showed that NES‐YFP‐CP, YFP‐CP and nls‐YFP‐CP efficiently suppressed GFP silencing, whereas NLS‐YFP‐CP did not (Figure 2e,f). In the immunoblot analysis, two GFP‐reactive bands were detected in the silencing suppression assay. Because the anti‐GFP signal in this assay detects the GFP reporter rather than CP fusion proteins, these bands may reflect different GFP species derived from the stable 16c transgene and transiently overexpressed GFP introduced by agroinfiltration. Consistent with this possibility, cytosolic and ER‐associated GFP species can differ in electrophoretic mobility due to compartment‐dependent processing or modification (Lee, Min, et al. 2012). These results suggest that cytoplasmic enrichment favours VSR activity, whereas forced nuclear accumulation reduces silencing suppression activity.

Together, these results indicate that the differential Avr and VSR activities of TCV CP are associated with distinct subcellular localization patterns. CP‐mediated Avr activity is favoured by appropriate nucleocytoplasmic distribution, whereas VSR activity is associated with cytoplasmic enrichment.

2.3. Biochemical Features Distinguishing CP‐GFP and YFP‐CP

Because viral infection reduces AGO1 abundance while elevating miR168 levels (Varallyay et al. 2010), we examined whether CP‐GFP and YFP‐CP differ in biochemical activities associated with RNA silencing suppression. In post‐transcriptional gene silencing (PTGS)‐triggered N. benthamiana 16c plants, transient overexpression of CP or YFP‐CP resulted in reduced AGO1 protein accumulation accompanied by increased miR168 expression, whereas CP‐GFP or YFP expression did not induce AGO1 reduction or miR168 accumulation (Figure 3a). This reciprocal relationship suggests that decreased AGO1 and elevated miR168 correlate with the VSR activity of CP. Consistently, expression of the established silencing suppressor P19 induced miR168 accumulation together with reduced AGO1 abundance (Figure 3a).

FIGURE 3.

Differential RNA silencing‐related biochemical activities of CP‐GFP and YFP‐CP. (a) Effects of coat protein (CP) variants on AGO1 protein abundance and miR168 accumulation in Nicotiana benthamiana 16c plants. AGO1 and GFP protein levels were analysed by immunoblotting following GFP‐silencing induction, and miR168 levels were examined by small RNA blotting using a locked nucleic acid (LNA)‐modified miR168 probe. Loading controls were confirmed by Ponceau S (PS) staining and U6 probing. (b) Electrophoretic mobility shift assay (EMSA) showing dsRNA binding by CP variants. Radiolabelled dsRNA probes were incubated with extracts expressing CP, CP‐GFP or YFP‐CP. CP and YFP‐CP formed specific dsRNA–protein complexes, which were competed by excess unlabelled dsRNA, whereas CP‐GFP showed no detectable binding. (c) Co‐immunoprecipitation of CP variants with DRB4. FLAG‐tagged DRB4 was co‐expressed with CP, CP–GFP or YFP–CP in N. benthamiana. Only YFP‐CP co‐precipitated with DRB4, indicating that DRB4 interaction correlates with the VSR‐active form of CP.

We next tested whether the FP‐tagged CP derivatives retained two biochemical properties previously reported for native CP, namely, dsRNA binding (Merai et al. 2006) and interaction with DRB4 (Zhu et al. 2013). Gel‐shift assays revealed that only CP and YFP‐CP bound radiolabelled dsRNA, and this binding was effectively competed by excess unlabelled dsRNA (Figure 3b). In contrast, CP‐GFP showed no detectable dsRNA‐binding activity. These results indicate that dsRNA binding is associated with the VSR‐active form of CP but is not sufficient to explain Avr activity.

We also examined interactions between the CP derivatives and DRB4, a protein required for RNA silencing suppression. Notably, only YFP‐CP associated with DRB4 (Figure 3c). This finding further supports that DRB4 interaction is specific to the VSR‐active form of CP. The absence of DRB4 association with CP‐GFP is consistent with the loss of VSR‐associated biochemical activities observed for this construct.

2.4. Arabidopsis Plants Expressing FP‐Fused CPs Exhibit Enhanced Susceptibility

To assess the biological significance of the biochemical differences between CP‐GFP and YFP‐CP, we generated A. thaliana Col‐0 transgenic lines overexpressing each construct. Immunoblot analysis confirmed the accumulation of CP‐GFP and YFP‐CP fusion proteins in the corresponding transgenic Arabidopsis lines used for the subsequent analyses (Figure S5). Because Col‐0 lacks the HRT resistance gene, these plants cannot mount HRT‐mediated resistance. YFP‐CP plants displayed narrow and curled leaves, a phenotype previously associated with alterations in RNA silencing‐related pathways (Vaucheret et al. 2004; Gregory et al. 2008), whereas CP‐GFP and YFP plants developed normally (Figure 4a,b). Consistent with the transient expression results in N. benthamiana, miR168 levels were elevated in YFP‐CP plants but not in CP‐GFP or YFP lines (Figure 4c).

FIGURE 4.

Transgenic Arabidopsis plants expressing FP‐fused CPs exhibit increased susceptibility to TCV infection. (a) Growth phenotype of 23‐day‐old Arabidopsis thaliana transgenic lines overexpressing YFP, CP‐GFP or YFP‐CP. (b) Representative images of leaf morphology from transgenic lines in (a), highlighting the curled, narrow leaf phenotype observed in YFP‐CP‐expressing plants. (c) RNA analyses of transgenic plants. Reverse transcription‐PCR showing expression of TCV CP normalized to that of Actin. Small RNA northern blot detecting miR168 using a locked nucleic acid (LNA)‐modified probe. U6 snRNA served as a loading control. (d, e) Virus susceptibility assays. Transgenic plants were inoculated with TCV or buffer (mock). (d) Representative disease symptoms at 21 days post‐inoculation (dpi). The numbers below each image indicate the number of plants showing the same symptom out of the total number of plants treated. M, mild symptoms; S, severe symptoms. (e) Northern blot showing TCV P8/P9 RNA accumulation in inoculated and uninoculated upper leaves. Total RNA loading was visualized by ethidium bromide staining of the agarose gel.

Upon TCV inoculation, both CP‐GFP and YFP‐CP transgenic lines exhibited more severe disease symptoms and accumulated higher viral RNA in systemic tissues compared with YFP control plants (Figure 4d,e). Notably, CP‐GFP and YFP‐CP enhanced TCV susceptibility despite their distinct biochemical properties. In YFP‐CP plants, enhanced susceptibility to TCV was accompanied by elevated miR168 accumulation in the absence of TCV infection (Figure 4c), suggesting that YFP‐CP is associated with altered RNA silencing‐related regulation in planta. In contrast, CP‐GFP also enhanced susceptibility despite lacking the tested biochemical features associated with VSR activity.

Together, these results demonstrate that FP‐tagged CP variants differentially influence plant development and TCV susceptibility, revealing that enhanced susceptibility can occur through distinct functional properties of CP.

2.5. TCV CP Interacts With ATG8 by Its N‐Terminus

Because HRT activation reduced CP and CP‐GFP accumulation, and previous studies suggested a relationship between TCV infection and host autophagy (Shukla et al. 2021), we examined whether CP associates with the autophagy marker ATG8 using CLSM (Ivanov and Harrison 2014).

Co‐expression of GFP and mCherry‐ATG8 produced diffuse cytoplasmic and nuclear fluorescence (Figure 5a). In contrast, YFP‐CP induced punctate structures that partially overlapped with mCherry‐ATG8‐positive structures, whereas CP‐GFP formed prominent punctate structures that showed strong spatial overlap with mCherry‐ATG8‐positive structures. To determine whether CP directly interacts with ATG8, we conducted Co‐IP assays. CP‐GFP co‐precipitated specifically with mCherry‐ATG8, while YFP‐CP and GFP did not exhibit any detectable interaction (Figure 5b). Wild‐type CP also associated with ATG8 (Figure 5c). Reduced detection of CP–ATG8 interaction in the presence of HRT was accompanied by decreased CP accumulation during immune activation.

FIGURE 5.

Co‐localization of FP‐fused coat protein (CP) with ATG8 in Nicotiana benthamiana. (a) Subcellular localization of FP‐tagged CP constructs and mCherry‐ATG8 in N. benthamiana epidermal cells at 2 days post‐infiltration (dpi) observed using confocal laser‐scanning microscopy (CLSM). Images were captured in FITC (GFP/YFP) and TRITC (mCherry) channels. Red arrows indicate CP‐GFP punctate structures showing spatial overlap with mCherry‐ATG8‐positive structures. White arrows indicate putative nuclear regions based on cellular morphology and fluorescence distribution. Scale bars = 10 μm. (b) Co‐immunoprecipitation (Co‐IP) showing the interaction between mCherry‐ATG8 and CP variants in the presence or absence of HRT. FP‐tagged CPs and mCherry‐ATG8 were transiently co‐expressed in N. benthamiana. Proteins were immunoprecipitated with anti‐RFP resin and analysed by immunoblotting with the indicated antibodies at 2 dpi. (c) Co‐IP confirming the interaction of wild‐type (wt) CP with ATG8 in the presence or absence of HRT. Total protein extracts were subjected to RFP‐based pulldown and analysed by immunoblotting.

Additional validation using FLAG‐tagged constructs showed that mCherry‐ATG8 interacted with CP‐FLAG but not with FLAG‐CP, indicating that the N‐terminal region of CP is required for ATG8 binding (Figure S6). These results indicate that TCV CP associates with ATG8 through an N‐terminal region. However, ATG8 co‐expression alone did not substantially alter CP accumulation, suggesting that CP–ATG8 association does not simply reflect ATG8‐mediated CP degradation.

Because CP nucleocytoplasmic distribution influences HRT‐mediated HR (Figure 2), we next examined whether CP localization affects ATG8 association. CP variants engineered for exclusive nuclear (CP‐NLS‐GFP) or cytoplasmic (CP‐NES‐GFP) localization failed to form CP punctate structures overlapping with mCherry‐ATG8, and Co‐IP assays did not detect interaction with ATG8 (Figure S7). These findings indicate that both the CP N‐terminal region and appropriate nucleocytoplasmic distribution are required for detectable CP–ATG8 association.

2.6. Autophagy‐Deficient Mutants Display Enhanced Susceptibility to TCV

To examine the relationship between CP–ATG8 association and HRT‐mediated recognition, we first examined two natural CP mutants, D4N and P5S, which are known to evade HRT detection (Zhao et al. 2000). Consistent with their reported phenotypes, co‐expression of HRT with TCV^D4N or TCV^P5S did not trigger HR, in contrast to wild‐type CP (Figure S8a). Correspondingly, HRT‐dependent reduction in CP abundance was not observed for either mutant (Figure S8b).

We next assessed whether these variants interact with Arabidopsis ATG8a (Shukla et al. 2021). Co‐IP assays revealed detectable interaction only for wild‐type CP (Figure 6a). TCV^P5S levels were strongly reduced upon co‐expression with ATG8, precluding a clear determination of interaction status, whereas TCV^D4N showed no detectable binding. These results indicate that CP determinants required for ATG8 association overlap with or influence determinants involved in HRT‐mediated recognition.

FIGURE 6.

Susceptibility of autophagy‐deficient Arabidopsis mutants to TCV and TCV^D4N. (a) The interaction between mCherry‐ATG8 and D4N or P5S was analysed in Nicotiana benthamiana leaves using a co‐immunoprecipitation (Co‐IP) assay. mCherry‐ATG8 was co‐expressed with coat protein (CP), D4N or P5S, followed by immunoprecipitation with anti‐RFP resin and immunoblotting with indicated antibodies at 2 days post‐inoculation (dpi). (b) Susceptibility of Col‐0, atg5‐1, atg7‐2 and Di‐17 plants to TCV or TCV^D4N infection. Four‐week‐old plants were mechanically inoculated with mock (M), TCV or TCV^D4N, and representative disease symptoms were photographed at 21 dpi. The numbers below each image indicate the number of plants showing the same phenotype out of the total number of plants treated. (c) Reverse transcription‐quantitative PCR analysis of CP transcript levels in uninoculated upper leaves at 5 dpi. Expression levels were normalised to expression of Actin. Data represent mean ± standard deviation from three biological replicates.

Although our initial experiments focused on the interaction of CP with ATG8, we next asked whether canonical autophagy contributes to antiviral defence. We used atg5 and atg7 loss‐of‐function mutants, which disrupt ATG8 lipidation and autophagosome formation and broadly abolish canonical autophagy (Minina et al. 2018). Because the Arabidopsis ATG8 gene family is highly redundant and ATG8 proteins serve multiple binding functions (Shukla et al. 2021; Shpilka et al. 2011), atg5 and atg7 mutants provide a useful genetic approach to assess the contribution of canonical autophagy to antiviral defence.

Compared with wild‐type Col‐0, both atg5 and atg7 mutants exhibited more severe disease symptoms and accumulated higher levels of CP following infection with either TCV or TCV^D4N (Figure 6b,c), supporting a role for autophagy in basal antiviral defence. Infection with the TCV^D4N generally resulted in lower CP accumulation than wild‐type TCV in Col‐0, although TCV^D4N remained fully virulent in Di‐17 plants, consistent with the loss of HRT‐mediated recognition.

Together, these results demonstrate that autophagy deficiency enhances susceptibility to TCV infection and that CP sequence variation influences the relationship between ATG8 association and viral infection outcomes.

3. Discussion

The TCV CP performs two mechanistically distinct functions during infection: activation of HRT‐mediated ETI and suppression of RNA silencing. Our FP‐fusion approach provided an experimental system in which these dual activities could be uncoupled. CP‐GFP retained Avr but not VSR activity, whereas YFP‐CP maintained VSR activity but lost detectable Avr function (Figure 1). These observations suggest that the two activities differ in their sensitivity to terminal FP fusion and are consistent with distinct requirements for CP engagement with host pathways. They support a model in which Avr and VSR activities can be functionally separated (Figure 7), although the fusion experiments alone do not define the structural determinants underlying this separation.

FIGURE 7.

Proposed model for the spatial and functional partitioning of TCV coat protein (CP) activities. (a) Spatial partitioning of CP is associated with functional divergence. CP populations associated with distinct terminal regions show different host‐interaction properties. ATG8‐associated CP requires appropriate nucleocytoplasmic distribution and is linked to autophagy‐associated regulation, whereas DRB4‐associated CP functions predominantly in the cytoplasm and is associated with RNA silencing suppression. (b) Proposed relationship between CP–ATG8 association and HRT‐mediated immunity. The CP N‐terminal region is required for detectable ATG8 association and overlaps with determinants involved in HRT‐mediated immune recognition. N‐terminal CP variants such as D4N fail to associate detectably with ATG8 and evade HRT‐mediated recognition. However, whether CP–ATG8 association directly influences HRT activation, autophagy‐associated defence or viral virulence remains to be determined.

A central finding of this study is that CP associates with ATG8, providing a connection between CP and the host autophagy machinery. Detectable association required the CP N‐terminal region and appropriate nucleocytoplasmic partitioning under the conditions tested (Figures 5, 6 and S6, S7). CP‐GFP also formed punctate structures that partially overlapped with mCherry‐ATG8‐positive structures, consistent with its association with ATG8‐containing compartments. However, neither association nor partial colocalization establishes the functional outcome of this interaction. Autophagic flux, reflecting progression through the autophagic pathway to cargo degradation, was not measured in this study. Consequently, these observations do not establish whether CP enhances or inhibits autophagic flux or whether CP itself undergoes autophagic degradation. Co‐expression of ATG8 alone did not substantially reduce CP accumulation, indicating that ATG8 co‐expression was insufficient to lower steady‐state CP abundance under the conditions tested. This result does not exclude autophagic turnover of CP, because steady‐state protein abundance does not directly measure degradation. We therefore regard the CP–ATG8 association as a potential interface with host autophagy‐related processes whose functional consequences remain unresolved.

The functional diversification of the ATG8 family in plants may provide multiple interfaces through which viral proteins interact with autophagy‐associated pathways (Kellner et al. 2017; Shpilka et al. 2011). In this context, our findings identify a CP sequence‐dependent association with ATG8 but do not establish how this association affects autophagic turnover or antiviral defence.

Our analysis of natural CP variants further implicates the CP N‐terminal region in both ATG8 association and HRT‐mediated immune responses. The D4N and P5S variants, previously shown to evade HRT recognition, failed to trigger HR and did not undergo HRT‐dependent reduction in protein abundance (Figure S8). In Co‐IP assays, ATG8a association was detected for wild‐type CP but not for D4N, whereas P5S showed severely reduced protein accumulation in the presence of ATG8 (Figure 6a). The low abundance of P5S limits interpretation of its Co‐IP result and precludes a firm conclusion about its capacity to associate with ATG8. Although the N‐terminal region is implicated in both ATG8 association and HRT‐mediated recognition, the present data do not establish whether overlapping or distinct amino acid residues underlie these activities. Furthermore, the D4N and P5S substitutions may exert pleiotropic effects on CP properties, including its conformation, stability, localization or interactions with other host factors. These possibilities remain unresolved, and the associated phenotypes cannot therefore be attributed solely to altered ATG8 association. Further residue‐level mapping, together with characterization of variant abundance and other CP properties, would be needed to distinguish the requirements for ATG8 association from those for HRT‐mediated recognition.

Whether ATG8‐associated processes contribute directly to HRT activation also remains unknown. The shared requirement for the CP N‐terminal region does not establish a causal relationship between ATG8 association and HRT‐mediated recognition. Resolving this relationship will require experiments that assess the contribution of ATG8 to HRT‐dependent responses while accounting for potential changes in CP abundance and localization.

To examine the contribution of canonical autophagy to antiviral defence, we analysed atg5 and atg7 mutants, which disrupt ATG8 lipidation and impair canonical autophagy (Minina et al. 2018). These mutants displayed increased susceptibility and elevated CP accumulation following infection with wild‐type TCV and, to a lesser extent, TCV^D4N, supporting a role for canonical autophagy in basal antiviral defence (Figure 6). The effects of autophagy deficiency differed between the two viral genotypes, suggesting that CP sequence variation may influence the infection response to the loss of canonical autophagy. However, this difference does not establish that altered CP–ATG8 association is responsible, because D4N may affect additional CP properties or host interactions. Thus, the genetic evidence for an antiviral role of canonical autophagy does not, by itself, resolve the specific functional contribution of CP–ATG8 association.

The dependence of CP activity on nucleocytoplasmic partitioning further suggests a role for spatially regulated host interactions. CP variants restricted to either the nucleus or cytoplasm failed to associate detectably with ATG8 and did not trigger HR under the conditions tested. These results are consistent with appropriate nucleocytoplasmic distribution being important for both activities. This requirement may reflect access to host factors in different cellular compartments, although the identities of such factors and their relationship to HRT activation remain unknown.

Finally, our findings are consistent with the broader view that plant RNA viruses and host autophagy‐associated pathways are functionally interconnected during infection (Yang and Liu 2022). This study demonstrates that the Avr and VSR activities of TCV CP can be experimentally uncoupled, identifies CP association with ATG8 and provides genetic evidence supporting a contribution of canonical autophagy to antiviral defence. The mechanistic relationships among these observations remain to be established. In particular, neither the residue‐level relationship between ATG8 association and HRT‐mediated recognition nor the effect of CP–ATG8 association on autophagic flux can be resolved from the present data. Together, these findings provide a basis for investigating how CP engages RNA silencing, autophagy‐related processes and HRT‐mediated immunity during infection.

4. Experimental Procedures

4.1. Plasmid Constructs

Plasmids were constructed using standard molecular cloning techniques. The binary vector pBIN19 harbouring cDNAs encoding HRT, TCV CP and the CP mutants D4N and P5S was previously described (Cooley et al. 2000; Zhao et al. 2000). The open reading frames (ORFs) of CP, D4N and P5S were amplified by PCR using primers that introduced XbaI and BamHI restriction sites (primer sequences are provided in Table S1). The resulting PCR products were cloned into the pGEM‐T Easy vector (Promega), digested with XbaI and BamHI and subsequently ligated into the pCamTEVL::smGFP vector (Lee, Kim, et al. 2012).

For FP fusion constructs, the TCV CP ORF was cloned into the pENTR/D‐TOPO vector (Invitrogen) and recombined into pBIN19:YFP:attR using LR Clonase II (Invitrogen), following a previously described protocol (Subramanian et al. 2006). GFP‐CP was generated by removing the YFP‐coding sequence from the YFP‐CP construct via inverse PCR and replacing it with the GFP ORF through T4 DNA ligation following restriction digestion.

To engineer CP constructs with NLS, NES or mutant nls peptides, inverse PCR was performed using Pfu‐X polymerase (SolGent) and pBIN19‐based CP‐GFP and YFP‐CP constructs. The CP‐YFP^N construct was generated by inserting the CP ORF into a bimolecular fluorescence complementation vector as previously described (Walter et al. 2004).

For PVX‐based expression, CP variants or GFP were cloned into the pGR106 vector using AscI and SalI sites (protocol from http://www.plantsci.cam.ac.uk/research/davidbaulcombe/methods/vigs) (Chapman et al. 1992; Lu et al. 2003). CP‐FLAG and FLAG‐CP were generated by PCR amplification of the CP‐coding sequence and recombination into the pDONR207 entry vector using Gateway BP Clonase II (Invitrogen). The resulting entry clones were transferred into pGWB454 (C‐terminal FLAG) or pGWB455 (N‐terminal FLAG) vectors using Gateway LR Clonase II (Invitrogen). All constructs were introduced into Agrobacterium tumefaciens GV2260 or GV3101 by electroporation for use in transient expression assays in N. benthamiana or A. thaliana.

4.2. Plant Growth and Transient Infiltration

Arabidopsis thaliana ecotype Columbia (Col‐0) was used as the wild‐type background in this study. Additional genotypes included the Di‐17 line (provided by Dr. Dan Klessig) and the autophagy‐deficient mutants atg5‐1 (SAIL_129_B07) and atg7‐2 (GABI_655B06), which were obtained from Dr. Kim's laboratory. Homozygous mutant lines were identified and confirmed by genomic DNA‐based PCR (Figure S8c).

For generation of transgenic Arabidopsis lines, the coding sequences of YFP, CP‐GFP and YFP‐CP were cloned into the pCamTEVL binary vector (Moon et al. 2016). These constructs were introduced into A. thaliana via A. tumefaciens ‐mediated transformation using the floral‐dip method (Clough and Bent 1998). Transformants were selected on ½ × Murashige and Skoog agar plates containing 50 mg/L kanamycin. At least 10 independent T₁ lines were selected per construct, and homozygous T₂ and T₃ lines were identified by antibiotic selection and RT‐PCR with CP‐specific primers.

Nicotiana benthamiana plants were grown in a controlled environment chamber at 23°C with a 16 h light/8 h dark photoperiod. For transient expression assays, leaves of 5‐ to 6‐week‐old wild‐type and N. benthamiana 16c plants were used. A. tumefaciens strains harbouring binary vectors were cultured in Luria Bertani (LB) medium supplemented with appropriate antibiotics at 28°C, harvested by centrifugation at 2100 g for 10 min and resuspended in infiltration buffer containing 10 mM MgCl₂, 10 mM MES (pH 5.6) and 200 μM acetosyringone. Bacterial suspensions were adjusted to an OD₆₀₀ of 0.2, incubated at room temperature for 3–4 h and then infiltrated into leaves using a needleless syringe. Transient expression experiments were independently repeated at least three times. GFP fluorescence and cell death phenotypes were monitored using a MultiDoc‐It Digital Imaging System (UVP).

4.3. Ion Leakage Measurements

Electrolyte leakage was measured to assess cell death in N. benthamiana leaves (Moon et al. 2016). Agrobacterium‐infiltrated leaves from 5‐ to 6‐week‐old plants were harvested at 16 h post‐infiltration. A total of 20 leaf discs (1 cm diameter) were excised from the infiltration zone and gently washed in distilled water for 30 min to eliminate ion leakage caused by mechanical damage. The discs were then transferred to Petri dishes containing 20 mL of distilled water and incubated for 6 h in a growth chamber under standard conditions. Electrical conductivity of the bathing solution was measured using a NeoMet EC Meter EC‐470 L (iSTEK). All treatments were performed with three biological replicates, and representative data are shown.

4.4. Immunoblotting

Total proteins were extracted from three infiltrated leaf discs using non‐denaturing or denaturing extraction buffers. The non‐denaturing buffer consisted of 25 mM Tris–HCl (pH 7.5), 1 mM EDTA (pH 7.5), 150 mM NaCl, 5 mM dithiothreitol (DTT), 10% glycerol and a protease inhibitor cocktail (Roche). The denaturing buffer contained 8 M urea, 10 mM Tris–HCl (pH 8.0), 100 mM NaH₂PO₄, 90 mM KOH and protease inhibitors. For AGO1 detection, the following extraction buffer was used: 20 mM Tris–HCl (pH 7.5), 5 mM MgCl₂, 2.5 mM DTT, 300 mM NaCl, 0.1% NP‐40 and protease inhibitors.

Extracted proteins were separated by SDS‐PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Roche) via wet transfer. Immunoblotting was performed using the following primary antibodies: anti‐GFP (Santa Cruz), anti‐TCV CP (Moon et al. 2016), anti‐AGO1 (Agrisera), anti‐mCherry (Bio‐Rad), anti‐ATG8 (Agrisera), anti‐HA (Sigma‐Aldrich) and anti‐FLAG (Cell Signalling Technology). Detection was performed using appropriate horseradish peroxidase‐conjugated secondary antibodies and chemiluminescent substrates.

4.5. TCV Infection and RNA Blot Analysis

Arabidopsis thaliana plants were mechanically inoculated with TCV at the 5‐ to 6‐leaf stage. The viral inoculum was prepared by grinding ~1 g of TCV‐infected N. benthamiana leaf tissue in liquid nitrogen, followed by homogenization in 5 mL of inoculation buffer (20 mM sodium phosphate, pH 7.0). Arabidopsis leaves were dusted with carborundum and gently rubbed with the homogenate to facilitate virus entry. For mock controls, inoculation buffer mixed with tissue from uninfected plants was applied (Li et al. 2016).

To assess TCV RNA accumulation, 10 μg of total RNA from infected leaves was separated by agarose gel electrophoresis under denaturing conditions and transferred to Hybond‐N⁺ membranes (GE Healthcare). Hybridization was carried out with a [³²P]‐labelled probe specific to TCV CP using previously described methods (Thomas et al. 2003). Membranes were washed twice for 5 min and visualized using a BAS‐1800II PhosphorImager (Fujifilm).

For small RNA northern blotting, 15 μg of total RNA was resolved on an 8 M urea/15% polyacrylamide gel, transferred to a Hybond‐N⁺ membrane and hybridized with a locked nucleic acid (LNA)‐modified miR168‐specific probe (Exiqon). A [³²P]‐labelled U6 snRNA probe was used as a loading control (see Table S2).

4.6. Microscopy Imaging

To analyse subcellular localization, leaves of N. benthamiana were co‐infiltrated with FP‐tagged CP constructs and mCherry‐ATG8 or NLS‐RFP. Imaging was performed using a C2 confocal laser scanning microscope (Nikon) with the following filter sets: GFP (λ_ex = 488 nm, λ_em = 505–530 nm) and RFP (λ_ex = 561 nm, λ_em = 570–620 nm). For each construct, three leaves from three individual plants were analysed, and at least three fields of view per leaf were imaged. Data were analysed using NIS‐Elements AR 4.20 software (Nikon).

4.7. Co‐IP

Co‐IP experiments with RFP‐ and FLAG‐fusion proteins were performed according to the manufacturer's protocol using RFP‐Trap (ChromoTek) and Anti‐FLAG M2 Affinity Gel (Sigma‐Aldrich), respectively. Total proteins were extracted from transiently infiltrated N. benthamiana leaves. Immunoprecipitated proteins were analysed by SDS‐PAGE and immunoblotting with appropriate antibodies.

4.8. Statistical Analysis

Statistical analyses were conducted to evaluate differences in ion leakage conductivity. Data were analysed by one‐way ANOVA, and mean comparisons were performed using Tukey's honestly significant difference (HSD) post hoc test. All statistical analyses were carried out with SPSS software (v. 18).

Author Contributions

Sung Hee Jo: investigation, formal analysis, writing – original draft. Ju Yeon Moon: investigation, formal analysis, writing – original draft. Sukyoung Jung: formal analysis, methodology. Gah‐Hyun Lim: formal analysis. Pradeep Kachroo: formal analysis, writing – review and editing. Jeong Mee Park: supervision, formal analysis, writing – original draft, writing – review and editing, conceptualization, investigation, funding acquisition.

Funding

This work was supported by the KRIBB Initiative Program (KGM9942314 and KGM5372322) and the Basic Research Program of the National Research Foundation of Korea (NRF‐2020R1I1A2075335 to JMP), funded by the Korean government (Ministry of Science and ICT).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Functional dissection of CP by addition of truncated YFP to the C‐terminus or GFP to the N‐terminus. (a) Hypersensitive response (HR) phenotypes induced by co‐expression of CP variants with HRT in Nicotiana benthamiana leaves via agroinfiltration. Leaf images were taken at 2 days post‐inoculation (dpi). (b) Electrolyte leakage assay corresponding to HR phenotypes in (a). Data represent mean ± standard error from three biological replicates. Statistical differences evaluated by one‐way ANOVA (p < 0.05). Letters indicate significantly different values. (c) Immunoblot analysis using an anti‐CP antibody to assess the effect of HRT on CP variant accumulation. Total proteins were extracted from leaves shown in (a). (d) Silencing suppression assay in transgenic GFP‐expressing N. benthamiana (16c). CP variants were co‐infiltrated with GFP, and leaf fluorescence was imaged under UV light at 5 dpi to assess suppression of GFP silencing.

Figure S2: PVX expressing TCV CP‐GFP induces HRT‐mediated hypersensitive response (HR) in Nicotiana benthamiana. (a) Schematic diagram of recombinant PVX constructs (PVX:GFP, PVX:CP‐GFP and PVX:YFP‐CP) under the control of the PVX CP promoter. (b) HR triggered by co‐expression of recombinant PVX constructs (PVX:GFP, PVX:CP‐GFP and PVX:YFP‐CP) with Rx or HRT in N. benthamiana leaves. Agrobacterium suspensions carrying the viral constructs and R genes were co‐infiltrated, and HR phenotypes were photographed at 3 days post‐inoculation (dpi) under white light. PVX:GFP was used as a control for viral infection. Rx serves as the cognate R protein for PVX CP. (c) Immunoblot analysis of protein accumulation corresponding to HR phenotypes in (b). Total proteins were extracted at 3 dpi and probed with anti‐TCV CP and anti‐PVX CP antibodies. Equal loading was confirmed by Ponceau S (PS) staining.

Figure S3: CP‐GFP and YFP‐CP localize to both the cytoplasm and nucleus. (a) Confocal laser‐scanning microscopy (CLSM) of CP‐GFP and YFP‐CP in Nicotiana benthamiana epidermal cells at 2 days post‐inoculation (dpi). Fusion proteins were co‐expressed with NLS‐RFP (nuclear marker) and P19 (silencing suppressor). Fluorescence signals were detected using the FITC (GFP/YFP) and TRITC (RFP) channels. Both CP‐GFP and YFP‐CP exhibited diffuse localization in the cytosol and nucleus. Scale bars = 10 μm. (b) Biochemical confirmation of the nuclear and cytoplasmic localization of TCV CP. Wild‐type (wt) TCV CP was transiently expressed in N. benthamiana, followed by nuclear/cytoplasmic fractionation. Protein fractions were analysed by immunoblotting using anti‐α‐tubulin (cytoplasmic marker) and anti‐histone H3 (nuclear marker) antibodies. The presence of CP in both fractions was confirmed by immunoblotting with an anti‐CP antibody.

Figure S4: Nuclear retargeting of CP‐GFP and YFP‐CP by NLS or NES fusion. Confocal laser‐scanning microscopy (CLSM) of CP localization variants in Nicotiana benthamiana leaves at 2 days post‐inoculation (dpi). Constructs were engineered to include a nuclear localization signal (NLS) or nuclear export signal (NES), generating CP‐NLS‐GFP, CP‐NES‐GFP, NLS‐YFP‐CP and NES‐YFP‐CP. Mutant versions of signal peptides (CP‐nls‐GFP and nls‐YFP‐CP) were used as controls. Constructs were co‐expressed with NLS‐RFP (nuclear marker) and P19 (silencing suppressor). Redirection of CP‐GFP and YFP‐CP was confirmed by subcellular localization analysis using CLSM. Scale bar = 10 μm.

Figure S5: Validation of CP‐GFP and YFP‐CP protein accumulation in transgenic Arabidopsis lines. Total proteins extracted from the indicated YFP control, CP‐GFP and YFP‐CP transgenic Arabidopsis lines used in Figure 4 were subjected to immunoblot analysis using anti‐GFP antibodies. Ponceau S (PS) staining was used as a loading control.

Figure S6: CP‐FLAG retains Avr function and interacts with ATG8. (a) Hypersensitive response (HR) phenotypes in Nicotiana benthamiana leaves co‐expressing CP‐FLAG or FLAG‐CP with HRT. Constructs were transiently introduced via Agrobacterium infiltration, and leaf images were captured at 2 days post‐inoculation (dpi) under white light (WL) and UV illumination. (b) Co‐immunoprecipitation (Co‐IP) assay assessing the interaction of CP‐FLAG and FLAG‐CP with mCherry‐ATG8, with or without HRT. Leaf samples were collected at 2 dpi, and total proteins were subjected to immunoprecipitation using anti‐RFP resin, followed by immunoblotting with the indicated antibodies.

Figure S7: Co‐localization and interaction of CP‐GFP variants containing a nuclear localization signal (NLS) or nuclear export signal (NES) with ATG8. (a) Subcellular localization of CP‐GFP variants modified with NLS, NES or mutant nls peptides and co‐expressed with mCherry‐ATG8 in Nicotiana benthamiana leaves at 2 days post‐inoculation (dpi). Confocal laser‐scanning microscopy (CLSM) was used to visualize fluorescence signals. Co‐localization was assessed to determine the spatial overlap of CP variants with the autophagy marker ATG8. Scale bars = 10 μm. (b) Co‐immunoprecipitation (Co‐IP) assay testing the interactions of mCherry‐ATG8 and CP‐GFP localization variants (CP‐nls‐GFP, CP‐NLS‐GFP and CP‐NES‐GFP). Constructs were transiently co‐expressed in N. benthamiana, and total proteins were extracted at 2 dpi. Immunoprecipitation was performed using anti‐RFP resin, and the eluates were analysed by immunoblotting with the indicated antibodies.

Figure S8: Hypersensitive response (HR) induction by natural CP variants D4N and P5S. (a) HR phenotypes triggered by CP variants D4N and P5S compared with wild‐type (wt) CP. Unlike wt CP, neither D4N nor P5S induced HR when co‐expressed with HRT in wt Nicotiana benthamiana at 2 days post‐inoculation (dpi). (b) Immunoblot analysis of CP accumulation for wt and mutant CPs in the presence or absence of HRT at 2 dpi, corresponding to (a). Anti‐CP antibody was used for detection. (c) Genotyping of Arabidopsis thaliana atg5‐1 and atg7‐2 mutants. Genomic DNA from Col‐0, atg5‐1 and atg7‐2 was amplified using LP + RP (wt allele) and BP + RP (T‐DNA insertion allele) primer pairs, and PCR products were resolved by agarose gel electrophoresis to confirm homozygosity.

Table S1: Primer sequences used to construct TCV CP, its mutants (D4N and P5S), and PVX‐based expression vectors.

Table S2: List of synthetic oligonucleotides used for small RNA northern blot and gel mobility shift assays.

Data Availability Statement

All datasets generated for this study are included in the paper/supporting information (Figures [Link], [Link], [Link], [Link], [Link], [Link], [Link], [Link] and Tables S1, S2).