Nuclear coat protein drives a multifaceted tolerance program during Turnip crinkle virus infection

TL;DR

This paper shows how a plant virus uses a protein in the cell nucleus to control infection and prevent host damage.

Contribution

The study reveals a new role for viral coat protein in activating host tolerance mechanisms during infection.

Findings

Nuclear localization of TCV coat protein is essential for host reprogramming and virus accumulation.

Loss of ROS or autophagy leads to severe tissue damage and similar transcriptional responses.

Nuclear CP both accelerates symptoms and prevents cell death through ROS and autophagy.

Abstract

Plant viruses must replicate yet maintain host tissue viability to sustain infection and transmission. We show that Turnip crinkle virus (TCV) uses nuclear localization of its coat protein (CP) to coordinate this balance. A nuclear-export mutant (TCVNES) retained core CP functions but causd attenuated symptoms and accumulated ~10-fold less than wild-type TCV. Transcriptomics and functional assays demonstrated that nuclear CP is required for large-scale host reprogramming, including SA accumulation and RBOH-dependent reactive oxygen species (ROS) production that promote TCV accumulation, as well as autophagy induction. Genetic loss of NADPH oxidase-derived ROS (rbohD rbohF) or autophagy (atg5-1) triggered rapid tissue collapse, necrosis, and convergent collapse-associated transcriptional responses following TCV infection. Nuclear CP consistently accelerated symptom onset in all backgrounds and exacerbated cell death and necrosis in rbohD rbohF and atg5-1. Nuclear CP is therefore responsible for early symptom development yet also required to activate ROS- and autophagy-dependent tolerance pathways.

Figures

Transmission electron microscopy (TEM) was used to visualize wild-type TCV and TCVNES virion formation. Sap from systemically infected A. thaliana Col-0 tissue (14 dpi) was imaged by TEM. Raw images were processed in ImageJ/Fiji. Red arrows denote virions.

CPNES retains VSR activity. a, VSR activity was visualized in 16c N. benthamiana leaves following agroinfiltration of CP constructs with a GFP expression construct. The p14 VSR from Pothos latent virus was included as a positive control. b, GFP transcripts from infiltrated spots (Panel a) were quantitated using RT-qPCR. *p<0.05; p<0.01; p<0.0001 one-way ANOVA with Tukey’s multiple comparisons test. Points represent biological replicates from two independent experiments (n=6 for all conditions). For all bar graphs, bars indicate the mean and error bars indicate the standard deviation.

atg5-1 plants exhibit impaired autophagic flux. Lysates from mature rosette leaves were subjected to western blotting using anti-NBR1 antibody. The intact >100 kDa NBR1 protein is shown. Impaired autophagy in atg5-1 plants results in elevated NBR1 levels. RuBisCO was stained with Ponceau S and shown as a loading control.

rbohD rbohF and atg5-1 plants exhibit increased symptom severity and necrosis. Representative plants from genotypes used in this study and their Trainable Weka Segmentation (TWS) profiles are shown. TWS profiles were generated using a custom classifier in ImageJ/Fiji. Only TCV-infected rbohD rbohF and agt5-1 show evident necrosis at 14 dpi.

Exogenous SA rescues TCV accumulation in sid2-2, but reduces TCVNES accumulation. For exogenous SA treatments, plants were sub-irrigated with 500 μM SA at 24 h post-infection, then foliar-sprayed with 500 μM SA every 48 h thereafter. Systemic leaves were harvested at 14 dpi for RT-qPCR measuring TCV. Data represents at least two independent experiments for each genotype; the five median values are plotted (Two-way ANOVA; *p<0.05 p<0.01 ns not significant). Bar height represents mean and error bars represent standard deviation.

The number of shared DEGs between rbohD rbohF and atg5-1 plants is increased during TCV infection, but not during TCVNES infection. Venn diagram of all DEGs identified in rbohD rbohF (teal circles) vs atg5-1 (orange circles). Total numbers of genes in each category are shown along with percent overlap between samples (hypergeometric significance test).

Full text

Plants experience large fitness costs during viral infection, yet disease severity frequently does not correlate with plant virus accumulation.^1–3^ This disconnect highlights the importance of tolerance, the capacity to maintain tissue function and limit damage despite infection, as distinct from resistance, which restricts pathogen growth.^4^ Tolerance is especially consequential for plant viruses because the majority rely on insect vectors for transmission between hosts,^5^ underscoring the importance of sustained viral replication in infected tissues to ensure acquisition and subsequent spread by vectors. However, host programs that preserve tissue integrity during ongoing viral replication, and how viral factors engage or subvert these programs, remain poorly defined.

When pathogen defense and stress programs are not properly restrained, they can become self-damaging and drive tissue collapse.^6,7^ Salicylic acid (SA) is a central defense hormone that restricts many pathogens but can also trigger growth arrest and cell death, when not properly buffered.^8,9^ Reactive oxygen species (ROS) production by NADPH oxidases (RBOHs) can coordinate defense responses,^10,11^ whereas dysregulated ROS contributes to tissue damage.^12^ Autophagy is a conserved degradation pathway, that supports cellular homeostasis under stress and shapes immunity by clearing damaged components and remodeling metabolic resources.^13^ During viral infection, autophagy can impose antiviral pressure,^14–16^ yet its broader role in sustaining tissue function and preventing collapse during prolonged infection is less clear.

Turnip crinkle virus (TCV, Tombusviridae) provides a tractable system to dissect how a single viral protein influences host transcriptional state and disease outcome. While plant virus coat proteins are typically viewed as structural components, many also “moonlight” as regulators of host responses and symptom development.^17–19^ Likewise, TCV coat protein (CP) carries functions beyond virion assembly and RNA silencing suppression, including modulating host RNA turnover through interactions with XRN4^20^ and promoting ubiquitin-dependent depletion of DCP1,^21^ activities thought to be predominantly cytoplasmic. In contrast, the best-defined nuclear consequence of CP is host recognition and hypersensitive response (HR) activation.^22^ Whether nuclear CP also has pro-viral roles that reprogram host responses remains unknown.

To address this question, we combined a CP nuclear export mutant (TCV^NES^), functional assays, and transcriptomics to demonstrate that nuclear CP is required for maximal virus accumulation, SA/ROS accumulation, and activation of autophagy. We show that host tolerance depends on pathways engaged during nuclear CP-competent infection since ROS-deficient rbohD rbohF and autophagy-deficient atg5-1 plants undergo rapid tissue collapse and necrosis. Together, these findings show that nuclear CP drives a tolerance-associated host state that sustains long-term viral accumulation.

We first confirmed prior transient-expression results in Nicotiana benthamiana^22^ by detecting CP in both the soluble and chromatin-associated (insoluble) fractions of nuclei fractionated from TCV-infected Col-0 Arabidopsis (Fig. 1a). To test whether this nuclear pool is functionally important, we appended a nuclear-export signal to CP (CP^NES^), which excluded CP from nuclei following over-expression in N. benthamiana (Fig. 1b–c). We introduced the same modification into full-length TCV and compared with wild-type, TCV^NES^ produced milder symptoms and a ~10-fold reduction in viral accumulation at 14 dpi (Fig. 1d–f). Disease severity (0–5 scale), quantified using automated rosette image segmentation and a trained classifier that scores size, chlorosis/yellowing, and necrosis, was significantly higher for TCV than TCV^NES^ (Fig. 1e). Control assays showed that CP^NES^ assembled virions and retained RNA silencing-suppressor activity (Extended Data Fig. 1,2), arguing that the phenotype reflects loss of nuclear CP rather than defects in known CP functions.^23,24^ These results identify nuclear CP as a virulence determinant and motivated RNA sequencing (RNA-seq) to define host program(s) it engages. At 14 dpi, principal component analysis (PCA) separated mock, TCV, and TCV^NES^ into three distinct clusters. TCV^NES^ samples remained closer to mock than wild-type TCV along PC1, consistent with a weaker global transcriptional response (Fig. 1g). Consistent with this, genes elevated only in wild-type TCV were enriched for response to SA, oxidative stress, and autophagy, while genes reduced only in wild type were primarily associated with photosynthesis (Fig. 1h, Supplementary Tables 1–2). Because these signatures were prominent only in nuclear CP-competent infection, we asked whether wild-type TCV, but not TCV^NES^, measurably induces SA accumulation, oxidative stress, and autophagic flux.

Using an Acinetobacter sp. ADP1-derived salicylate biosensor^25^ we detected a robust increase in free SA in wild-type TCV-infected Col-0, whereas mock- and TCV^NES^-infected plants remained near background (Fig. 2a). Consistent with SA induction arising from de novo synthesis in response to TCV, SA remained at baseline in sid2-2 plants^26^ (defective in pathogen-inducible SA production via ICS1/SID2) across all conditions (Fig. 2a). We next assessed oxidative stress using 3,3′-diaminobenzidine (DAB) staining to visualize H_2_O_2_ accumulation. Wild-type TCV infection produced strong DAB signal in Col-0, whereas mock and TCV^NES^ plants showed minimal staining (Fig. 2b–c), indicating that the ROS burst is associated with infection by nuclear CP-competent virus. To identify the NADPH oxidase driving this response, we examined rbohD, rbohF, and rbohD rbohF mutants.^27,28^ DAB staining was retained in rbohF, significantly decreased in rbohD, and absent in the rbohD rbohF double mutant (Fig. 2b–c), implicating RBOHD as the predominant NADPH oxidase responsible for the TCVinduced ROS burst. Finally, we tested whether autophagy is activated preferentially by wild-type TCV. In p35S:B2-GFP/pUbi10:mCherry-ATG8A reporter plants,^29^ wild-type TCV increased ATG8A-labelled autophagosome abundance at 7 dpi (but not 3 dpi), whereas TCV^NES^ remained at background (Fig. 2d). This timing was supported by immunoblot-based flux assays, where free mCherry release from mCherry-ATG8A increased specifically in wild-type TCV samples at 7 dpi (Fig. 2e–f). Together, these assays validate the RNA-seq predictions, demonstrating that wild-type TCV, but not TCV^NES^, induces SA accumulation, an RBOHD-dependent oxidative burst, and increased autophagic flux.

Our results linking nuclear CP to SA-, ROS-, and autophagy-associated programs prompted us to test whether these responses simply mark host defense, or are instead co-opted by TCV to sustain robust accumulation via increased host tolerance. To address this genetically and transcriptome-wide, we profiled infection outcomes and gene expression across mutants that disrupt each arm of this response. At 14 dpi, we collected mock-, TCV-, and TCV^NES^-inoculated tissues from Col-0, sid2-2, rbohD, rbohF, rbohD rbohF, and autophagy-deficient atg5-1 plants^30^ (Fig. 3a). We validated atg5-1 by NBR1 immunoblotting, where NBR1 accumulates in atg5-1 but is largely depleted in Col-0 due to autophagic turnover^31^ (Extended Data Fig. 3). Following infection of the above plants, disease severity was measured using our automated segmentation-based scoring pipeline. Phenotypically, rbohD rbohF plants exhibited the most extreme loss of tolerance, with severe necrosis and maximal disease scores, followed by atg5-1, which also showed substantial necrosis and elevated disease relative to Col-0 (Fig. 3b–c; see Extended Data Fig. 4 for magnified images). In contrast, Col-0, sid2-2, rbohD, and rbohF displayed comparatively tolerant phenotypes: visible disease and growth suppression, but limited yellowing and absent necrosis (Fig. 3b–c, Extended Data Fig. 4). Virus accumulation did not track directly with symptom severity. Viral RNA levels were largely similar between Col-0, rbohD, and rbohF, while atg5-1 showed a modest increase (Fig. 3d), consistent with the notion that autophagy exerts antiviral pressure while simultaneously supporting virus accumulation.^32^ By contrast, the nearly lethal rbohD rbohF double mutant had significantly reduced RNA accumulation (Fig. 3d), possibly due to reduced tissue longevity and/or failure to sustain productive infection as tolerance collapses. Notably, sid2-2 also accumulated less virus than Col-0 despite SA’s well-established role in conferring resistance against TCV through the HR response,^33^ suggesting that SA may actually benefit TCV. In support of this, exogenous SA applied beginning 24 h after inoculation restored TCV accumulation in sid2-2, whereas it further reduced TCV^NES^ levels (Extended Data Fig. 5), consistent with previous findings of nuclear CP counteracting SA-dependent basal antiviral immunity.^34^

Building on our 14-day infections, which suggested that tolerance was lost in the rbohD rbohF double mutant and reduced in atg5-1, we repeated these assays and extended the time course to 21 and 28 dpi to test whether tolerance fully breaks down and culminates in plant death. By 21 dpi, TCV-infected rbohD rbohF and atg5-1 plants were fully necrotic, exhibiting maximum disease scores (Fig. 3e–f). Notably, loss of nuclear CP (TCV^NES^) delayed symptom development by 7 days in both backgrounds, shifting death to ~21 dpi in rbohD rbohF and ~28 dpi in atg5-1 (Fig. 3e–f). Interestingly, symptom severity in TCV-infected Col-0 remained stable over time, whereas disease scores in TCV^NES^-infected plants increased significantly by 28 dpi (Fig. 3e–f). Taken together, this data suggests that nuclear CP strikes a balance by accelerating symptom development while also engaging NADPH oxidase–derived ROS and autophagy, which restrain runaway necrosis. When these tolerance mechanisms are removed, TCV infection rapidly progresses to tissue collapse and death. Consistent with this, even TCV^NES^ infections ultimately caused plant death in rbohD rbohF and atg5-1 plants, likely reflecting cumulative stress from ongoing viral replication over time.

To define the transcriptional basis of tolerance loss during TCV infection, we performed RNA-seq on tolerant genotypes (Col-0, sid2-2, rbohD, and rbohF) and on the collapse-prone rbohD rbohF and atg5-1 mutants at 14 dpi, when the induction of TCV-associated collapse is phenotypically evident. We then asked whether rbohD rbohF and atg5-1 share a common transcriptional signature associated with rapid necrosis and death. We observed three PCA-defined clusters that closely mirrored infection outcomes (Fig. 4a, Supplementary Tables 3–4): (i) an “uninfected-like” cluster containing all mock samples together with TCV^NES^-infected rbohD rbohF plants, consistent with their near-absent disease scores (Fig. 3c) and low viral RNA accumulation at 14 dpi (Fig. 3d); (ii) a broad “tolerant” cluster comprising Col_-0, sid2-_2, rbohD, and rbohF, in which both wild-type TCV and TCV^NES^-infected samples largely grouped, and (iii) a distinct “loss-of-tolerance” cluster containing TCV-infected rbohD rbohF and atg5-1, the two genotypes displaying near-lethal disease that clustered together despite being deficient in different host pathways.

Next, we compared TCV- or TCV^NES^-infected plants of each genotype against infected Col_-0, distinguishing infection-driven (TCV-specific) DEGs from genotype-associated DEGs evident in mock comparisons (Fig. 4b–c, Supplementary Tables 5–7). sid2-2 and the rbohD and rbohF single mutants exhibited few infection-specific DEGs, regardless of virus, relative to Col-_0, with most differences attributable to genotype-associated shifts detected in uninfected controls (Fig. 4b–c). This was expected as sid2-2 and the rbohD and rbohF single mutants clustered closely with Col-0 in the PCA space (Fig. 4a), consistent with a Col-0–like transcriptional response in these backgrounds. In contrast, TCV infection in rbohD rbohF and atg5-1 triggered thousands of DEGs not observed in Col-0, whereas these large-scale transcriptional changes were largely absent during TCV^NES^ infection (Fig. 4b–c). In uninfected rbohD rbohF and atg5-1 plants, there were few overlapping DEGs relative to Col-0 (Extended Data Fig. 6), but TCV infection elicited over 1,700 shared DEGs between these genotypes, compared with <50 DEGs in TCV^NES^-infected plants (Fig. 4d). KEGG enrichment analyses of the DEGs shared between rbohD rbohF and atg5-1 revealed a collapse-associated program, with up-regulated genes linked to fatty acid degradation and down-regulated genes linked to photosynthesis, metabolic pathways, and glycolysis/gluconeogenesis (Fig. 4e, Supplementary Table 8). We hypothesize that coordinated loss of the above core cellular functions likely underlies the tissue collapse/necrosis phenotype.

In summary, our results establish nuclear CP as a viral factor that engages host tolerance pathways during TCV infection (see Fig. 4f for model). The shared collapse-associated transcriptional state in rbohD rbohF and atg5-1 highlights a convergent route to lethal disease when buffering capacity is lost, marked by broad repression of photosynthesis and other core metabolic functions. We speculate that TCV ultimately benefits from increased host tolerance since virus transmission depends on insect vectors, which typically feed on living tissue. Thus, by preserving host tissue while sustaining high viral loads, TCV (and other vector-transmitted plant viruses) may enhance the duration and efficiency of vector-mediated transmission. More broadly, these findings indicate that although CP can elicit HR-mediated resistance in some ecotypes, in this host context it accumulates in the nucleus and instead engages host tolerance programs. This shift from resistance to tolerance preserves host viability while still supporting viral fitness, highlighting nuclear-localized viral proteins as potent modulators of disease outcomes.

Methods

Plants and Growth conditions

All Arabidopsis thaliana lines used in this study are Col-0 background and were obtained from the Arabidopsis Biological Resource Center (ABRC, The Ohio State University, Columbus, Ohio): Col-0 (CS70000), sid2-2 (CS16438), rbohD (CS9555), rbohF (CS9557), rbohD rbohF (CS68522), and atg5-1 (CS39993). All plants (including N. benthamiana LAB strain) were grown and maintained in plant growth chambers (Caron Scientific, Marietta, Ohio) with the following conditions: 12/12 hr light/dark, LED lights set at 200 μmol m^−2^s^−1^ intensity, 25°C, and 65% humidity.

Construction of CP expression vectors and TCVNES

TCV isolate M (GenBank: PX830476) was used for this study. A CP entry vector for gateway cloning (pTWIST:CP) was synthesized by Twist Bioscience (San Francisco, CA). Site-directed mutagenesis (SDM) was performed using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA) following the manufacturers protocol to append the pyruvate kinase NES to the C-terminus of CP (pTWIST:CP^NES^). CP or CP^NES^ were then cloned into the destination vectors pGWB552 (Addgene #74882) or pGWB418 (Addgene #74812), generously gifted by Tsuyoshi Nakagawa,^35^ using the LR Clonase II enzyme per manufacturers protocol (Invitrogen, Waltham, MA) to produce N-terminal GFP-tagged or Myc-tagged CP constructs, respectively. Full-length TCV (isolate TCV-M) has been previously described^36^ and was used as a template for SDM to append the pyruvate kinase NES to the C-terminus of CP in full-length TCV to create TCV^NES^. Resulting clones were sequenced using whole plasmid sequencing (Plasmidsaurus) to verify cloning accuracy. SDM primers are listed in Supplementary Table 9.

TCV infections and nuclei isolation

Four-week-old A. thaliana plants were selected and grouped for experiments based on size. Full-length TCV plasmids were linearized with SmaI overnight at 25°C. TCV RNAs were synthesized using HiScribe^®^ T7 Quick High Yield RNA Synthesis Kit (New England Biolabs) per the manufacturers protocol. Silicon carbide powder was applied to two rosette leaves per plant and 1 μg RNA or 5 μL H_2_O (mock-infected plants) was gently rubbed on selected leaves. For exogenous SA treatments, plants were sub-irrigated with 500 μM SA at 24 hours post-infection and subsequently sprayed with 500 μM SA every 48 hours. Systemic leaves were harvested for downstream analysis at 14-days post-infection (dpi) unless otherwise noted. Nuclei were isolated from 200 mg of systemically infected A. thaliana rosette leaves using the Minute^™^ Plant Cytosolic and Nuclear Protein Isolation Kit (Invent Biotechnologies Inc., Plymouth, Minnesota) per the manufacturer’s protocol. Nuclei were separated into soluble (nucleoplasm) and insoluble (chromatin) fractions as previously described.^37^ Fractions were subjected to ethanol precipitation overnight to concentrate the DNA for visualization with ethidium bromide.

Western blotting

Systemically-infected A. thaliana leaves, agroinfiltrated N. benthamiana leaves, or isolated nuclei were stored at −80°C prior to processing. Samples were pulverized with liquid nitrogen, resuspended in 1X TBS + 3% β-mercaptoethanol supplemented with protease inhibitor cocktail (Thermo Scientific, Waltham, Massachusetts), mixed with Laemmli buffer and boiled at 95°C for 5-10 min. Proteins were separated using SDS-PAGE and transferred to nitrocellulose membrane using the Bio-Rad Trans-Blot Semi-Dry Transfer system. Membranes were stained with Ponceau S (0.2% w/v) to detect the RuBisCO large subunit for loading controls. Following blocking in 1X TBS + 0.1% Tween 20, 3% bovine serum albumin, primary antibodies (1:10,000 dilution) were added and incubated at 4°C overnight. TCV CP was detected using antigen-purified rabbit polyclonal anti-CP generated by Sino Biological (Paoli, Pennsylvania). Other primary antibodies used in this study include anti-RFP (Invitrogen, Cat#:PA1–986), anti-GFP (Invitrogen, Cat#:PA1–980A), anti-NBR1 (Agrisera, Sweden, Cat#:AS14 2805), and anti-Myc (Invitrogen, Cat#:PA1–981). Following secondary antibody incubation (Thermo Scientific Pierce Goat Anti-Rabbit IgG Peroxidase Conjugated at 1:10,000 dilution), proteins were detected using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) per manufacturer’s protocol. Blots were imaged using an Azure 600 Imaging System (Azure Biosystems, Dublin, CA).

Agroinfiltrations

Agrobacterium cultures (strain C58C1) transformed with expression plasmids were passaged in Luria Broth (LB) twice in 48 hours with appropriate antibiotics. 24 hours prior to agroinfiltration, 20 μM acetosyringone was added to the passaged cultures. Infiltration solutions were created by pelleting agrobacterium cultures and resuspending them in agroinfiltration buffer (10 mM MgCl_2_, 10 mM MES-K [pH 5.6], and 100 μM acetosyringone) to the desired OD_600_. The RNA silencing suppressor p14 from Pothos latent virus^38^ was included in all infiltration solutions at a final OD_600_ of 0.2, unless otherwise specified, to increase transient gene expression. All other constructs were used at a final OD_600_ of 0.2. Infiltration solutions were incubated at room temperature for 1–2 hours before infiltration and leaves were harvested 2–3 days following infiltration for downstream analyses.

Quantification of disease severity

Plant disease severity was quantified from RGB images using a threshold-based workflow in ImageJ/Fiji (TWS pipeline) coupled to supervised pixel classification. Briefly, whole plants were segmented and analyzed using a trained classifier to assign each plant pixel to one of three tissue classes: healthy (green), yellowing (chlorotic), or necrotic. To quantify growth effects independently of tissue quality, plant size was expressed as a size ratio relative to mock controls within each genotype (infected plant total area divided by the mean total area of mock plants of the same genotype). To summarize symptom severity as a single continuous score, we calculated a disease score index from the segmented fractions and size ratio. For each plant, a damage term was computed as a weighted sum of yellowing and necrosis fractions, with necrosis weighted more strongly than yellowing. A stunting term was computed from the reduction in plant size relative to mock $[eqn]$ , and was capped to prevent extreme size differences from dominating the score. The final score was scaled to an approximately 0–5 range and capped at 5:

$$[eqn]$$

where $[eqn]$ and $[eqn]$ are the yellowing and necrotic tissue fractions, respectively. The size weight $[eqn]$ was set to 0.8 for wild-type TCV infections and 0.2 for TCV^NES^ infections to reflect that symptoms in TCV^NES^ infections were primarily captured by visible tissue damage rather than stunting in this dataset. Per-plant scores were calculated from the segmented outputs, and genotype-level disease severity was summarized as the mean disease score across plants within each condition.

RNA extraction and RT-qPCR

Systemically infected leaves were harvested and stored at −80°C prior to processing. Samples were pulverized in liquid nitrogen, resuspended in TRIzol (Thermo Scientific), and RNA was extracted following the manufacturers protocol. After extraction, an RQ1 DNase digest was performed (Promega, Madison, Wisconsin) according to the manufacturers protocol to remove residual DNA. One-step reverse-transcription quantitative PCR (RT-qPCR) was performed using approximately 20 ng of purified RNA and the SYBR Green-based Luna^®^ Universal One-Step RT-qPCR kit (New England BioLabs) per manufacturers protocol with reaction volumes reduced to 10 μL. Samples were analyzed using a BioRad CFX Connect Real-Time PCR detection system with CFX Maestro software. Primer sequences used in this study are available in Supplementary Table 9.

RNA-seq and computational analyses

Poly(A)-enriched RNA-seq libraries (Fig. 1) were prepared from DNase-treated total leaf RNA and sequenced as paired-end reads (Illumina NovaSeq X, University of Missouri Genomics Technology Core; ~50 million PE100 reads per sample) Raw reads were subjected to quality control and adapter/quality trimming with fastp^39^ (v0.24.0) prior to quantification, and only filtered, high-quality reads were used for Salmon-based quantification.^40^ Gene-level quantification was performed with Salmon in quasi-mapping mode against the A. thaliana TAIR10 transcriptome, and transcript per million (TPM) values as well as raw and normalized count matrices were exported for statistical analyses. Gene-level differential expression was carried out within the RNAseqChef framework^41^ using DESeq2^42^ for pairwise comparisons and EBSeq^43^ for three-condition comparisons, after filtering lowly expressed transcripts and applying Benjamini-Hochberg correction for multiple testing. Functional enrichment analysis was performed using g:Profiler^44^ (g:GOSt), querying Gene Ontology (GO) terms.

Alternatively, Poly(A)-enriched RNA-seq libraries (Fig. 4) were sequenced as 3′-tag single-end libraries on an Illumina platform (Plasmidsaurus; ~20 million raw reads and ~10 million deduplicated 3′ end counting reads per sample) following quality filtering as described above. Filtered reads were aligned to the A. thaliana TAIR10 reference genome using STAR^45^ (v2.7.11). PCR and optical duplicates were eliminated via UMI-guided deduplication using UMIcollapse^46^ (v1.1.0). Gene-level expression was quantified with featureCounts^47^ (Subread v2.1.1). Raw count matrices were analyzed in iDEP^48^ (v2.01) for PCA and differential expression testing using DESeq2. Functional enrichment was performed with g:Profiler, as described above.

Free salicylic acid quantification

Acinetobacter ADPWH_lux^25^ was a generous gift from Dr. Denise Pallett (UK Centre for Ecology and Hydrology) and free SA was quantified as previously described.^49^ Reactions were carried out in a 96-well plate and luminescence was detected using the BioTek Synergy Neo2 hybrid multimode microplate reader (Agilent Technologies, Santa Clara, CA) and the BioTek Gen6 1.04 software. For each experiment, a standard curve was generated using sid2-2 lysates spiked with known amounts of SA (0, 10, 25, 50, 100, and 200 ng). Luminescence measurements were imported into GraphPad Prism, where a standard curve was fitted and used to interpolate experimental sample concentrations of free SA.

ROS quantification

Selected leaves were incubated in DAB stain (0.1% 3,3’-Diaminobenzidine, 10 mM Na_2_HPO_4_, and 0.05% Tween-20) at room temperature overnight, protected from light. Leaves were placed in DAB clearing solution (3:1:1 ethanol:glacial acetic acid:glycerol) with gentle heating until chlorophyll was fully leached from leaves. Cleared leaves were imaged with an Azure 600 Imaging System (Azure Biosystems). Raw images were converted to RGB images and processed using the ImageJ Color Deconvolution Tool with the “H DAB” stain selected. Threshold adjustments were performed on Color 2 to 1.25% to eliminate background signal. Leaves were selected with the ROI tool and the Analyze -> Measurements function was then used to measure the area of each leaf and calculate the mean gray value. DAB intensity was calculated as: mean gray value/area. rbohD rbohF mock leaves were used for normalization for all conditions to calculate relative DAB intensity.

Autophagosome Quantification and flux analyses

Transgenic Arabidopsis p35S:B2-GFP/pUbi10:mCherry-ATG8A and pUbi10:mCherry-ATG8A were generous gifts from Dr. Marion Clavel (Max Planck Institute of Molecular Plant Physiology). For autophagosome counts and flux analyses, at 3- and 7- dpi, systemically infected p35S:B2-GFP/pUbi10:mCherry-ATG8A plants were harvested five to six hours after the daylight cycle commenced. For autophagosome visualization, fresh roots were rinsed, wet-mounted, and imaged using a 40X objective from a Nikon Ti2 confocal microscope with Nikon Elements software. Raw images and the ImageJ “Analyze Particle” function were used to count the number of autophagosomes within the field with a size threshold of <15 μm^2^ to eliminate counting large diffuse regions of mCherry expression. Autophagosome counts/mm^2^ of TCV- and TCV^NES^-infected samples were normalized to mock-infected plants from the same experiment. For flux assays, systemically infected leaves were harvested at −80°C until processing for western blot as previously described using anti-RFP (1:10,000) as the primary antibody. Densitometry measurements of intact and degraded mCherry-ATG8A were used to calculate autophagic flux (free mCherry/mCherry-ATG8A). Flux from TCV- and TCV^NES^-infected samples were normalized to mock-infected plants from the same experiment.

Transmission electron microscopy (TEM)

Sap was harvested from systemically infected A. thaliana Col-0 tissue at 14 dpi, placed on a 300 mesh formvar-coated copper grid, and stained with 2% (w/v) uranyl acetate. Images were obtained using a Philips CM12 TEM at an accelerating voltage of 80 kV at the University of Missouri-Kansas City School of Dentistry Electron Microscope Core Facility. Raw images were processed using ImageJ.

VSR assay

16c N. benthamiana plants,^50^ expressing a GFP transgene, were agroinfiltrated with either GFP alone, or alongside the p14 VSR,^38^ Myc:CP, or Myc:CP^NES^. At 5 dpi, whole leaves were imaged for GFP fluorescence using an Azure 600 Imaging System (Azure Biosystems). Infiltrated spots were harvested for western blotting or RNA purification and RT-qPCR using primers targeting GFP or ubiquitin (reference gene).

Statistics and reproducibility

GraphPad Prism 10.6.1 software was used for plotting and statistical analyses. Information on biological replicates, numbers of independent experiments, and statistical analyses are provided in the figure and extended data figure legends. Mathematical modeling and formula development for disease scoring, along with language refinement, were performed with the assistance of large-language models.

Extended Data

Transmission electron microscopy (TEM) was used to visualize wild-type TCV and TCVNES virion formation. Sap from systemically infected A. thaliana Col-0 tissue (14 dpi) was imaged by TEM. Raw images were processed in ImageJ/Fiji. Red arrows denote virions.

*CPNES retains VSR activity. a, VSR activity was visualized in 16c N. benthamiana leaves following agroinfiltration of CP constructs with a GFP expression construct. The p14 VSR from Pothos latent virus was included as a positive control. b, GFP transcripts from infiltrated spots (Panel a) were quantitated using RT-qPCR. *p<0.05; **p<0.01; ***p<0.0001 one-way ANOVA with Tukey’s multiple comparisons test. Points represent biological replicates from two independent experiments (n=6 for all conditions). For all bar graphs, bars indicate the mean and error bars indicate the standard deviation.

atg5-1 plants exhibit impaired autophagic flux. Lysates from mature rosette leaves were subjected to western blotting using anti-NBR1 antibody. The intact >100 kDa NBR1 protein is shown. Impaired autophagy in atg5-1 plants results in elevated NBR1 levels. RuBisCO was stained with Ponceau S and shown as a loading control.

rbohD rbohF and atg5-1 plants exhibit increased symptom severity and necrosis. Representative plants from genotypes used in this study and their Trainable Weka Segmentation (TWS) profiles are shown. TWS profiles were generated using a custom classifier in ImageJ/Fiji. Only TCV-infected rbohD rbohF and agt5-1 show evident necrosis at 14 dpi.

*Exogenous SA rescues TCV accumulation in sid2-2, but reduces TCVNES accumulation. For exogenous SA treatments, plants were sub-irrigated with 500 μM SA at 24 h post-infection, then foliar-sprayed with 500 μM SA every 48 h thereafter. Systemic leaves were harvested at 14 dpi for RT-qPCR measuring TCV. Data represents at least two independent experiments for each genotype; the five median values are plotted (Two-way ANOVA; *p<0.05 *p<0.01 ns not significant). Bar height represents mean and error bars represent standard deviation.

The number of shared DEGs between rbohD rbohF and atg5-1 plants is increased during TCV infection, but not during TCVNES infection. Venn diagram of all DEGs identified in rbohD rbohF (teal circles) vs atg5-1 (orange circles). Total numbers of genes in each category are shown along with percent overlap between samples (hypergeometric significance test).

Supplementary Material

Supplementary Table 1 EBSeq output for three-condition RNA-seq analysis in Col-0, including differential expression results for TCV vs. mock and TCV vs. TCV^NES^ comparisons.

Supplementary Table 2 Filtered lists of TCV-specific DEGs, along with g:Profiler GO:Biological Process enrichment results for each gene set (up and down).

Supplementary Table 3 Table contains DESeq2 size factor-normalized counts (mean expression values) for all genes across all experimental conditions (Fig. 3).

Supplementary Table 4 DESeq2 outputs for all comparisons in Figure 3, including genotype vs Col-0 comparisons under Mock, TCV, and TCV^NES^ conditions. For every gene, log2 fold-change and adjusted p-values are reported for each comparison.

Supplementary Table 5 Table lists genotype-associated DEGs identified by comparing mock (uninfected) samples from each genotype to Col-0 mock controls. Table includes log2 fold-change and adjusted p-values for all DEGs.

Supplementary Table 6 Table lists TCV-responsive DEGs identified by comparing each genotype’s TCV-infected samples to Col-0 TCV controls, then removing genotype-associated DEGs.

Supplementary Table 7 Table includes DEGs that are TCV-specific and also significant in the TCV^NES^ comparisons (each genotype vs Col-0 TCV^NES^). Genes are retained only if they show the same direction of change (up or down) in both TCV and TCV^NES^.

Supplementary Table 8 Table lists shared TCV-specific genes between atg5-1 and rbohD rbohF TCV infections. Shared genes were tested by Welch’s t-test for atg5-1 and rbohD rbohF versus a pooled baseline (Col-0, sid2-2, rbohD, and rbohF). KEGG pathway enrichment for the significant shared gene set was performed using g:Profiler.

Supplementary Table 9 DNA sequences of site-directed mutagenesis and RT-qPCR primers used in this study.

This is a list of supplementary files associated with this preprint. Click to download.

• SupplementaryTable7.xlsx
• SupplementaryTable2.xlsx
• SupplementaryTable9.docx
• SupplementaryTable8.xlsx
• SupplementaryTable5.xlsx
• SupplementaryTable1.xlsx
• SupplementaryTable4.xlsx
• SupplementaryTable3.xlsx