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. 2026 Sep 25;27(9):e70353. doi: 10.1111/mpp.70353

Cucumber Mosaic Virus Promotes Sphingolipid Biosynthesis to Alleviate Host Unfolded Protein Response (UPR)‐Mediated Immunity

Jiayi Yao 1, Shaomin Wu 1, Shushu Gao 1, Zhidan Xiang 1, Qian Wang 1, Fei Ye 1, Qiansheng Liao 1, Zhouhang Gu 1,✉, Zhiyou Du 1,✉
PMCID: PMC13615166  PMID: 42791595

ABSTRACT

RNA viruses usually reprogramme host cellular lipid metabolism to create favourable replication microenvironments, yet the broader biological significance of these lipid changes is largely unknown. To explore the potential roles of virus‐induced lipids, we employed an economically important plant pathogen cucumber mosaic virus (CMV), which has a single‐stranded, positive‐sense, tripartite RNA genome replicating in host tonoplasts. Lipidomic analysis of purified tonoplasts revealed that CMV infection increased the relative accumulation of sphingolipids in tonoplasts. This is consistent with the increased expression levels of these enzymes involved in sphingolipid biosynthesis, particularly 3‐ketosphinganine reductase (TSC10). Virus‐induced silencing of Nb‐TSC10 in Nicotiana benthamiana impaired CMV replication by triggering the unfolded protein response (UPR). Furthermore, we found that salicylic acid (SA) serves as a central regulator of UPR‐mediated antiviral immunity. Notably, a specific sphingolipid, ceramide, was found to alleviate the UPR, suggesting an important role for sphingolipids during CMV infection: CMV subverts SA‐induced immunity by rewiring sphingolipid biosynthesis to inhibit the UPR‐mediated defence. This work advances mechanistic understanding of viral counterdefence strategies through sphingolipid‐mediated modulation of UPR during plant–pathogen interactions.

Keywords: cucumber mosaic virus, resistance, sphingolipid biosynthesis, unfolded protein response (UPR)

Sphingolipid levels are elevated in CMV replication site (the tonoplast). Silencing Nb‐TSC10 activates the host salicylic acid–UPR defence axis, indicating CMV promotes sphingolipid biosynthesis to suppress this antiviral pathway.

graphic file with name MPP-27-e70353-g004.webp

1. Introduction

Lipids are a diverse group of hydrophobic or amphipathic organic molecules, which are categorized into major classes such as phospholipids, sphingolipids and fatty acids. Lipids play essential roles in various cellular processes, including maintaining membrane integrity, generating energy and regulating signalling pathways. Increasing evidence highlights the importance of specific lipids in viral replication (Jiang et al. 2025). For instance, phospholipids, the core components of cellular membranes, such as phosphatidylethanolamines (PE) and phosphatidylcholines (PC), are enriched in viral replication compartments, where they are required for efficient viral replication (Xu and Nagy 2015; Zhang et al. 2016). Viral infections frequently reprogramme host cellular lipid metabolism, resulting in enhanced viral replication in many cases (Zhang et al. 2016; Xu and Nagy 2015; Berger et al. 2011; Nchoutmboube et al. 2013; Hirata et al. 2012; Heaton and Randall 2011; Wang et al. 2024). Except for enhancing viral replication, it is largely unknown whether the alteration of lipids has additional biological significance in the virus–host interaction. Understanding the roles of host lipids in viral infections will not only provide valuable insights into viral pathogenesis but also open avenues for developing novel antiviral strategies targeting lipid–virus interactions.

Sphingolipids are recognized as abundant and essential components of the plasma membrane and other endomembranous compartments of plant cells (Luttgeharm et al. 2016). They are synthesized from ceramide backbones, which contain hydrophobic tails and are typically linked to polar head groups. The ceramide backbone contains a long chain amino alcohol, referred to as a long‐chain base (LCB), which is unique to sphingolipids and linked to a fatty acid via an amide bond. Sphingolipids are required for the assembly of the viral replication complex (VRC) of hepatitis C virus and West Nile virus (Zhang et al. 2019; Strating and van Kuppeveld 2017). Ceramide, as a sphingolipid intermediate, has been characterized as a regulator of plant immunity (Liang et al. 2003; Li, Yin, et al. 2021; Takahashi et al. 2009). The constitution of different ceramide species in cell membranes has distinct biophysical impacts, which are influenced by variations in their acyl chain composition (Pinto et al. 2011). C16‐ceramide (number indicates the carbon length of the acyl chain), mainly generated by ceramide synthase 6 (CerS6), has been implicated in maintaining the integrity of the endoplasmic reticulum (ER) and Golgi membranes. CerS6 inhibition has been shown to activate transcription factor 6 (ATF6)‐induced ER stress, causing cell death in cancer cells (Senkal et al. 2010). These findings imply a potential role for C16‐ceramide in modulating ER stress in humans; however, whether it has a similar function in plants remains unclear.

ER is the essential organelle for protein and lipid synthesis, as well as protein folding and modification. When misfolded or incompletely folded proteins accumulate in the ER lumen, ER stress is triggered, activating the unfolded protein response (UPR) (Lv et al. 2019; Shi et al. 2021). The UPR mitigates ER stress through multiple coordinated mechanisms: (1) upregulation of ER chaperones to facilitate proper protein folding, (2) attenuation of global protein translation, (3) promotion of misfolded protein clearance via ER‐associated degradation, and (4) activation of autophagy pathways (Bao and Howell 2017; Verchot 2016). Plants possess two well‐characterized UPR signalling pathways: the IRE1 (inositol‐requiring enzyme 1) pathway and the bZIP17/28 (basic leucine zipper 17/28) pathway (Howell 2021). IRE1 acts as a stress sensor by catalysing the unconventional splicing of bZIP60 transcripts. The spliced bZIP60 (sbZIP60) regulates the expression of UPR genes in the nucleus (Deng et al. 2011). ER stress also promotes translocation of the membrane‐anchored transcription factors (bZIP17 and bZIP28) from the ER to Golgi membranes, where they are cleaved by Golgi‐resident S1P and S2P proteases, releasing their cytosolic transcription factor domains to induce UPR (Bao and Howell 2017). ER stress is commonly triggered during virus infection due to over‐production and load of viral proteins in the ER, which activates UPR to relieve ER stress (Zhang and Wang 2012; Adhikari et al. 2025). Many plant RNA viruses induce the UPR to maintain ER homeostasis, facilitating viral infection (Hu et al. 2025; Zhang et al. 2022; Li et al. 2020; Li, Zhang, et al. 2021; Li, Xu, et al. 2021), with an exception of sugarcane streak mosaic virus that suppresses host UPR through impeding bZIP60 splicing by viral P1 protein (Zhang et al. 2023).

Cucumber mosaic virus (CMV) is the type member of the genus Cucumovirus in the family Bromoviridae. It has a genome consisting of three (positive‐sense) single‐stranded RNA molecules, designated RNAs 1–3. RNA1 and RNA2 encode the 1a protein with an N‐terminal methyltransferase domain and C‐terminal helicase domain, and the 2a protein (RNA‐dependent RNA polymerase), respectively. Both viral proteins predominantly colocalize to the tonoplast (Cillo et al. 2002), generating CMV‐induced vesicles for viral replication (Hatta and Francki 1981; Wang et al. 2021). RNA2 encodes the 2b protein, which is an RNA silencing suppressor mediating viral pathogenicity (Goto et al. 2007; Guo and Ding 2002; Ding et al. 1994; Duan et al. 2012; Lewsey et al. 2007; Du et al. 2014; Lewsey et al. 2009). In addition, CMV 2b protein primes the induction of salicylic acid (SA) biosynthesis in host plants (Lewsey et al. 2010), but it overcomes SA‐mediated defence against CMV movement and replication in hosts (Ji and Ding 2001; Naylor et al. 1998). CMV infection severely alters the ER morphology and induces the UPR response (Li et al. 2018; Shen et al. 2017). Inspired by the connection between host lipids (specifically ceramide) and ER stress, as mentioned above, we sought to explore the underlying links among virus‐induced host lipids, SA signalling and ER stress‐coupled UPR during CMV infection.

Considering that the tonoplast serves as the site for CMV replication, we first analysed the changes in its lipid composition in response to CMV infection. We found that CMV infection increased the relative amounts of sphingolipids, which is consistent with the upregulated expression of 3‐ketosphinganine reductase (Nb‐TSC10), a key enzyme in sphingolipid metabolism. Furthermore, we demonstrated that both the viral 2b protein and the SA signalling pathway are essential for CMV‐induced UPR activation. Intriguingly, gene silencing of Nb‐TSC10 provoked UPR activation, which subsequently reduced viral replication via the SA–UPR defence axis. To subvert this defence, CMV promotes sphingolipid biosynthesis, either directly or indirectly, to attenuate UPR‐triggered antiviral responses. Collectively, our findings unveil a sophisticated three‐way interplay wherein CMV modulates sphingolipid homeostasis to suppress UPR‐mediated immunity, highlighting a novel viral counterdefence mechanism.

2. Results

2.1. Lipidomic Analysis of CMV‐Infected and Non‐Infected Tonoplasts

Given that CMV replication occurs on the tonoplast membrane, it is conceivable that CMV infection may remodel the membrane lipid composition, as has been reported for other positive‐strand RNA viruses that reprogramme host membranes to facilitate replication. Prior to testing this hypothesis, we first evaluated the feasibility of isolating vacuoles from healthy Nicotiana tabacum leaves using the established protocol for Arabidopsis vacuole isolation (Robert et al. 2007). Proteomic analysis of the isolated tonoplasts showed that abundant unique peptides belonging to tonoplast‐resident proteins were detected, including vacuolar (H+)‐ATPase (Verweij et al. 2008) and tonoplast intrinsic protein 1 (Kim et al. 2006), with a limited number of peptides belonging to other organelle‐membrane‐localized proteins, for example, ammonium transporter in the plasma membrane (Robles‐Martínez et al. 2013) and voltage‐dependent anion channel in the mitochondrial membrane (Bayrhuber et al. 2008) (Table S1). These results indicate that this protocol is practically acceptable for the isolation of vacuoles from Nicotiana leaves.

Then, we isolated the intact vacuoles from the upper systemic leaves of Nicotiana benthamiana plants with CMV infection or buffer‐treated plants (mock) at 7 days post‐inoculation (dpi). The isolated vacuoles were first examined by morphological observation and subsequently subjected to two rounds of ultracentrifugation to enrich the tonoplast fractions (Figure 1A). Reverse transcription (RT)‐PCR was conducted to detect viral RNAs in these tonoplasts. As expected, a DNA fragment corresponding to the 5′ 246‐nt sequence of RNA3 was detected in the CMV‐infected sample, but not in the mock‐treated sample (Figure 1B), suggesting the presence of viral replication complexes in the enriched tonoplasts from CMV‐infected plants. This data encouraged us to measure the lipid composition of both enriched tonoplast samples (mock vs. CMV) using LC–MS. A total of 481 lipids were identified in both mock and CMV‐infected samples, categorized into four lipid groups: fatty acyls, glycerolipids, phospholipids and sphingolipids. Compared with the mock samples, CMV‐infected samples exhibited a significant increase in the content of total sphingolipids, but with the decreased levels of the other lipid groups (Figure 1C,D). Specifically, the accumulation of LCB in CMV‐infected tonoplasts increased to approximately 1.5 times the level in mock tonoplasts, with a 30% increase in their downstream products, ceramides (Figure 1C,D). Additionally, the level of phosphingolipid (phSL) was 2.1‐fold higher than that in the mock‐treated samples (Figure 1C,D). These findings indicate that CMV infection induces the accumulation of sphingolipids in the tonoplast.

FIGURE 1.

FIGURE 1

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Preparation of Nicotiana benthamiana tonoplasts for lipidomic analysis. (A) A flow chart for tonoplast preparation. Protoplasts were prepared from upper systemic leaves of N. benthamiana plants inoculated with CMV or infiltration buffer (mock) at 7 days post‐infiltration by enzymatic digestion of cell walls. Intact vacuoles were released from the protoplasts by osmotic and thermal lysis, and fractionated in a Ficoll density gradient by centrifugation. Isolated vacuoles were verified by visualization under a light microscope. Vacuolar membranes (tonoplasts) were enriched from isolated vacuoles, followed by RNA or lipid extraction. Vacuoles were coloured red by the neutral red dye during isolation. Extracted RNAs were used for detection of viral RNAs, and extracted lipids were used for analysis of lipid composition. White bars represent 50 μm. (B) Molecular detection of CMV RNA3 in enriched tonoplasts. RNAs were isolated from the tonoplasts of mock‐inoculated or CMV‐inoculated samples and used for amplification of a 5′ end fragment of CMV RNA3 by reverse transcription‐PCR. The expected size of the PCR product is 246 bp. (C) Heat map analysis of tonoplast lipids. The tonoplasts were isolated from mock‐ or CMV‐infected N. benthamiana plants at 7 days post‐inoculation. Six biological replicates were used for analysis of mock or CMV samples. Cer, ceramide; DG, diacylglycerol; FA, fatty acids; LCB, long‐chain base; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; MG, monoacylglycerol; OAHFA, (O‐acyl)‐1‐hydroxy fatty acid; PA, phosphatidic acid; PC, phosphatidylcholines; PE, phosphatidylethanolamines; PG, phosphatidylglycerol; phSL, phosphingolipid; PI, phosphatidylinositol; PS, phosphatidylserine; TG, triacylglycerols. (D) Comparative analysis revealed a significant relative accumulation of sphingolipids in CMV‐infected tonoplast relative to mock‐treated controls. Total sphingolipid (SL) content was defined as the sum of LCB, Cer and phSL. Significant differences were determined by t‐test analyses (*p < 0.05, ***p < 0.001, ****p < 0.0001).

2.2. CMV Promotes Plant Sphingolipid Biosynthesis

In sphingolipid de novo synthesis, 3‐ketosphinganine serves as the initial precursor that is subsequently reduced to form other LCB (e.g., sphinganine). These LCB then act as the structural backbone for ceramide biosynthesis through N‐acylation with fatty acids (Figure 2A). Thus, the increased amounts of both LCB and ceramides suggest that CMV promotes plant sphingolipid de novo biosynthesis. To this end, we further analysed the sphingolipid profiles in the mock‐ and CMV‐infected tonoplasts. For LC–MS, it is technically not feasible to distinguish distinct lipid species that belong to the same lipid class and possess an identical mass‐to‐charge ratio (m/z). In this context, while we acknowledge the potential presence of structural isomers, we performed a detailed analysis of C16‐ and C18‐sphinganine, as well as their corresponding dihydroceramides—dhCer(d18:0/15:0) and dhCer(d18:0/16:0)—which were putatively annotated based on their characteristic m/z values. All of these sphingolipid classes were found to be significantly elevated in CMV‐infected samples (Figure 2B).

FIGURE 2.

FIGURE 2

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CMV infection induces upregulation of sphingolipid biosynthesis‐related genes and metabolic intermediates. (A) Schematic representation of the plant sphingolipid biosynthesis pathway. Genes significantly upregulated following CMV infection (in Figure 2C) are highlighted in red. (B) Accumulation of ceramide intermediates with unique m/z in CMV‐infected plants compared to mock‐treated controls. Significant differences were determined by t‐test analyses (**p < 0.01, ****p < 0.0001). (C) Reverse transcription‐quantitative PCR analysis of sphingolipid biosynthesis gene expression in mock‐ and CMV‐infected plants. Total RNA was extracted from systemic leaves at 6 days post‐inoculation (dpi). Significant differences were determined by t‐test analyses (*p < 0.05, **p < 0.01, ***p < 0.001).

In the de novo biosynthesis of sphingolipids, LCB are synthesized via serine palmitoyltransferase‐catalysed condensation of serine and palmitoyl‐CoA, yielding 3‐ketosphinganine as the primary intermediate. Subsequently, 3‐ketosphinganine is reduced by the enzyme 3‐ketosphinganine reductase to yield sphinganine (Figure 2A). As reported in the literature, 3‐ketosphinganine reductase was initially identified in Saccharomyces cerevisiae as the TSC10/YBR265w gene (Beeler et al. 1998). Consequently, this enzyme is designated as TSC10 in the present study. The increasing accumulation of LCB and Cer in CMV‐infected samples prompted us to examine the expression of genes involved in sphingolipid de novo synthesis during CMV infection. As anticipated, a significant upregulation was observed in five of the seven tested genes in the plants infected with CMV as compared to those in the mock controls (Figure 2C). Those upregulated genes participate in nearly all steps in the sphingolipid de novo synthesis pathway (Figure 2A). Notably, two homologues of TSC10 in N. benthamiana, referred as to Nb‐TSC10a and Nb‐TSC10b were upregulated by 2.2‐fold and 4.8‐fold, respectively, in the presence of CMV (Figure 2C). Given markedly stronger CMV‐induced expression, Nb‐TSC10b was selected as the representative allele for subsequent functional characterization of the Nb‐TSC10 gene family.

2.3. CMV‐Induced Nb‐TSC10 Is Coupled With the bZIP60 Branch of UPR Through SA Signalling

SA acts upstream of sphingolipid signalling to stimulate sphingolipid in Arabidopsis thaliana (Shi et al. 2015). Indeed, SA treatment significantly increased the expression of Nb‐TSC10b, as well as the SA‐responsive marker gene Pathogenesis‐related protein 1 (PR1) (Figure 3A). Given that CMV infection is known to induce SA biosynthesis and activate SA signalling (Murphy et al. 2020), our experimental data prompted us to hypothesize that SA may serve as a key regulator in CMV‐mediated Nb‐TSC10b induction. To test this hypothesis, we assessed Nb‐TSC10b expression in wild‐type and NahG transgenic N. benthamiana at 3 dpi. RT‐quantitative PCR (RT‐qPCR) results showed that Nb‐TSC10b transcript levels were comparable in mock‐treated wild‐type and NahG plants, indicating that its basal expression is independent of endogenous SA. However, upon CMV infection, Nb‐TSC10b expression was more strongly induced in wild‐type plants than in NahG plants (Figure 3B). No discernable difference in CMV RNA accumulation was observed in the inoculated leaves between wild‐type and NahG plants (Figure S1A). These results reveal the CMV‐induced expression of Nb‐TSC10b is largely dependent on SA signalling.

FIGURE 3.

FIGURE 3

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CMV induction of both Nb‐TSC10b and Nb‐bZIP60 is dependent on 2b protein and salicylic acid (SA) signalling. (A) Exogenous SA application induces transcriptional activation of Nb‐PR1 and Nb‐TSC10b in Nicotiana benthamiana. Significant differences were determined by t‐test analysis (*p < 0.05, **p < 0.01). (B) Attenuation of CMV‐induced Nb‐TSC10b upregulation in NahG transgenic plants. Significant differences were determined by t‐test analysis (**p < 0.01, n.s.: not significant). (C) The mRNA levels of Nb‐TSC10b in the infiltrated leaves of N. benthamiana plants with virus infection by agrobacteria harbouring infectious clones for CMV, CMV∆2b or CMV∆2b supplied with 2b in trans (CMV∆2b+2b). Significant differences were determined by t‐test analysis (*p < 0.05, **p < 0.01, n.s.: not significant). (D) The relative accumulation of Nb‐bZIP60 in CMV and its derivatives‐infected plants. Significant differences were determined by t‐test analysis (*p < 0.05, **p < 0.01, n.s.: not significant). (E) Both Nb‐bZIP60 and Nb‐PR1 are upregulated by SA treatment. Significant differences were determined by t‐test analysis (*p < 0.05). (F) CMV‐mediated induction of Nb‐bZIP60 is impaired in NahG transgenic N. benthamiana compared to wild‐type controls. Significant differences were determined by the one‐way ANOVA with Tukey test. Data points sharing common lowercase letters above error bars indicate no statistically significant differences (p > 0.05).

Because CMV 2b protein is crucial for priming elicitation of SA biosynthesis (Lewsey et al. 2010), we next examined the responses of Nb‐TSC10b to the infection of a 2b‐deletion mutant of CMV (CMV∆2b) carrying a major deletion in the 2b ORF (Lewsey et al. 2009). Notably, CMV∆2b failed to induce Nb‐TSC10b expression (Figure 3C). However, when the 2b protein was supplied in trans (CMV∆2b + 2b), Nb‐TSC10b expression was significantly induced, although to a lower level to that observed with wild‐type CMV infection (Figure 3C). We further examined the accumulation of CMV RNAs in the infiltrated leaves at 5 dpi. Compared with wild‐type CMV, CMV∆2b had a reduced accumulation of RNA1 and RNA2, and this defect was restored by trans‐expression of 2b protein (CMV∆2b + 2b) (Figure S1B). These data indicate that CMV accumulation has a minimal effect on Nb‐TSC10b expression. Collectively, these data demonstrate SA signalling is important for CMV to induce Nb‐TSC10b expression in a 2b‐dependent manner.

Previous reports have documented that both CMV and SA can induce the UPR in plants (Li et al. 2018; Shen et al. 2017). Interestingly, we found CMV, but not CMVΔ2b, significantly induced a UPR marker, Nb‐bZIP60; the homologue in Arabidopsis, At‐bZIP60, is known to be crucial for UPR activation in response to SA treatment (Moreno et al. 2012). Moreover, provision of the CMV 2b protein in trans restored the ability of CMVΔ2b to induce Nb‐bZIP60 expression (Figure 3D), suggesting that the 2b protein is required for CMV‐induced UPR. Nb‐bZIP60 expression was also induced by SA treatment, along with Nb‐PR1 (Figure 3E). Consistently, the upregulation of Nb‐bZIP60 during CMV infection was compromised in NahG transgenic plants (Figure 3F). Together, these findings establish that both the 2b protein and SA are essential for CMV to activate the UPR through the bZIP60 branch.

Both the 2b protein and SA are required for CMV‐induced Nb‐TSC10 and Nb‐bZIP60, suggesting CMV‐induced Nb‐TSC10 is coupled to the bZIP60 branch of the UPR. To this end, we analysed whether the bZIP60 branch contributes to CMV‐mediated upregulation of Nb‐TSC10b. Interestingly, Nb‐TSC10b expression was further enhanced by CMV infection when Nb‐bZIP60 was silenced (Figure S2). This result indicates that inhibiting the bZIP60 branch of UPR, which exacerbates ER stress, promotes CMV‐induced upregulation of Nb‐TSC10b. It implies that CMV‐mediated upregulation of sphingolipid de novo biosynthesis may function to alleviate UPR through increasing sphingolipids, such as ceramides.

2.4. Ceramide Alleviates CMV‐Induced UPR

To investigate the role of ceramides in the CMV‐induced UPR, candidate Cer molecules needed to be selected for further study. Although total Cer (d34:1), probably including Cer(d14:1/20:0), Cer(d16:1/18:0) and Cer(d18:1/16:0) (commonly referred to as C16‐ceramide), was significantly elevated in CMV‐infected samples, it remains unclear whether C16‐ceramide was specifically increased during CMV infection. Despite this uncertainty, C16‐ceramide was chosen for further study because both its backbone (C18‐sphinganine) and its intermediate product (dhCer(d18:0/16:0)) were significantly increased in CMV‐infected samples (Figure 2B).

Although C16‐ceramide has been reported to induce apoptotic cell death (Aflaki et al. 2012; Stiban and Perera 2015), we found that treatment with 50 μM C16‐ceramide did not induce visible cell death in N. benthamiana leaves, as assessed by trypan blue staining (Figure S3). To determine the potential effect of the C16‐ceramide treatment on plant UPR, Nb‐bZIP60 transcript levels were quantified by RT‐qPCR. Ceramide‐treatment significantly downregulated Nb‐bZIP60 expression (Figure 4A). Similar inhibition was observed following treatment with TUDCA (Figure 4B), a pharmacological small‐molecule chaperone that alleviates ER stress (Hu et al. 2025). These results suggest that C16‐ceramide functions similarly to TUDCA in alleviating UPR. Interestingly, the ceramide treatment moderately inhibited CMV‐induced Nb‐bZIP60 expression, as well as the basal expression of Nb‐bZIP60 in the uninfected leaves (Figure 4A). This suggests that C16‐ceramide can downregulate both basal UPR and CMV‐induced UPR. Thus, it is reasonable to speculate that exogenous C16‐ceramide may benefit CMV accumulation by disturbing UPR‐mediated antiviral immunity. To this end, we treated the CMV‐inoculated leaves with C16‐ceramide or the solvent control, and examined viral accumulation in the upper systemic leaves at 5 dpi. Indeed, the accumulation level of CMV RNA3 was increased in the ceramide‐treated plants compared with the solvent control, although no difference in the accumulation levels of RNA1 and RNA2 was observed between both treatments (Figure 4C). These data suggest that virus‐induced increase of endogenous ceramides facilitate CMV to overcome host UPR‐mediated immunity by alleviating UPR. It also implies that knocking down Nb‐TSC10 will probably hamper CMV accumulation.

FIGURE 4.

FIGURE 4

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Ceramide alleviates the basal and virus‐induced unfolded protein response (UPR). (A) The basal and CMV‐induced Nb‐bZIP60 expressions were attenuated by C16‐ceramide treatment. Significant differences were determined by one‐way ANOVA with Tukey test. Data points sharing different lowercase letters above error bars indicate statistically significant differences (p < 0.05). (B) The expression of Nb‐bZIP60 was downregulated by TUDCA treatment. Significant differences were determined by t‐test analysis (*p < 0.05). (C) Northern blot hybridization analysis of the accumulation level of CMV in the ceramide‐treated or the solvent‐treated plants. CMV RNAs were detected in the upper systemic leaves at 5 days post‐inoculation. Relative accumulation level of viral RNA3 is shown below. Ethidium bromide‐stained ribosomal RNAs were used as a loading control.

2.5. Silencing Nb‐TSC10 Compromises CMV Replication

To explore the potential role of Nb‐TSC10 during CMV infection, we conducted tobacco rattle virus (TRV)‐based virus‐induced gene silencing (VIGS) assays to simultaneously downregulate Nb‐TSC10a/b in N. benthamiana plants. At 7 dpi, plants infected with TRV‐TSC10 exhibited no obvious symptoms, comparable to those inoculated with the TRV‐mCherry control (Figure 5A). As expected, TRV‐TSC10 infection reduced the transcript levels of both Nb‐TSC10 alleles by approximately 80% (Figure 5B). Furthermore, total Cer levels of the tonoplast were significantly reduced in Nb‐TSC10‐silenced plants compared to non‐silenced controls (Figure 5C). When subsequently challenged with CMV, Nb‐TSC10‐silenced plants showed milder viral symptoms than the control plants (Figure 5D), which is consistent with the decreased levels of viral RNAs (Figure 5E). Immunoblot against CMV coat protein (CP) further confirmed this reduction, showing a pattern consistent with the RNA gel blot results (Figure 5F). To assess whether Nb‐TSC10 silencing influences CMV genome replication, we analysed the replication efficiency of CMV RNA3 using a 1a/2a trans‐expression system in Nb‐TSC10‐silenced and control plants. Northern blot results showed the accumulation of RNA3 was decreased at 3 dpi in Nb‐TSC10‐silenced plants (Figure S4). Collectively, these data suggest that downregulation of Nb‐TSC10 expression impairs CMV replication.

FIGURE 5.

FIGURE 5

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Nb‐TSC10 silencing attenuates accumulation of CMV. (A) The phenotype of Nb‐TSC10 knockdown via TRV‐mediated virus‐induced gene silencing (VIGS) at 7 days after inoculation with TRV. (B) Silencing efficiency validation by reverse transcription‐quantitative PCR. Transcript levels of Nb‐TSC10a/b in TRV‐TSC10‐infected plants relative to TRV‐mCherry controls. Significant differences were determined by t‐test analysis (**p < 0.01). (C) Knockdown of Nb‐TSC10 reduced total ceramide (Cer) accumulation in the tonoplast (**p < 0.01). (D) Enhanced CMV symptom progression in Nb‐TSC10‐silenced plants. Representative leaf curling phenotypes at 5 days post‐inoculation (dpi). Red boxes indicate the enlarged part in the upper pictures. (E) Northern blot hybridization analysis of the accumulation level of CMV in the TRV‐mCherry‐ or TRV‐TSC10‐infected plants. CMV RNAs were detected in the upper systemic leaves at 5 dpi. Relative accumulation levels of viral RNAs are shown below. Ethidium bromide‐stained ribosomal RNAs are used as a loading control. (F) Immunoblot analysis of CMV coat protein (CP) in the plants is the same as those in the panel D. Ponceau S‐stained RuBisCO large subunit was used as a loading control.

Given that SA positively regulates Nb‐TSC10b expression in both healthy and CMV‐infected plants (Figure 3A,B), we next investigated whether TSC10 contributes to SA‐mediated resistance against CMV. To this end, we tested CMV infection in NahG transgenic plants, in which Nb‐TSC10 was either pre‐silenced via TRV‐TSC10 or left unsilenced with TRV‐mCherry. Northern blotting analyses showed that the inhibitory effects of silencing Nb‐TSC10 on virus accumulation was alleviated in NahG plants at 4 dpi (Figure S5). These results indicate that Nb‐TSC10 knockdown exerts an additive effect on SA‐induced resistance against CMV. This conclusion is consistent with previous reports that SA primarily restricts the systemic movement of CMV with limited impact on viral replication efficiency (Naylor et al. 1998).

Of note, while exogenous C16‐ceramide suppressed Nb‐bZIP60 expression in healthy plants (Figure 4A), it unexpectedly induced bZIP60 expression in Nb‐TSC10‐silenced plants. In contrast, C18‐ceramide had no significant effect (Figure S6). These findings suggest that C16‐ceramide exerts context‐dependent regulatory effects on the UPR, probably dictated by the host basal sphingolipid homeostasis. We address this phenomenon in the Discussion section.

2.6. Silencing Nb‐TSC10 Impedes CMV Accumulation Through UPR Signalling

Our findings elucidate a potential mechanism by which CMV activates sphingolipid de novo biosynthesis to elevate ceramide levels, thereby counteracting UPR‐mediated immunity. We therefore hypothesised that inhibition of sphingolipid de novo biosynthesis would enhance UPR activation. To test this hypothesis, we analysed Nb‐bZIP60 expression in Nb‐TSC10‐silenced plants. Consistent with the hypothesis, Nb‐bZIP60 was upregulated in Nb‐TSC10‐silenced plants (Figure 6A), implying that Nb‐TSC10 knockdown activates UPR signalling.

FIGURE 6.

FIGURE 6

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Nb‐TSC10 silencing‐induced immunity is dependent on Nb‐bZIP60. (A) Silencing Nb‐TSC10 upregulates Nb‐bZIP60. Significant differences were determined by t‐test analysis (**p < 0.01). (B) Silencing efficiency of TRV‐mediated silencing was measured by reverse transcription‐quantitative PCR. Significant differences were determined by t‐test analysis (**p < 0.01, ***p < 0.001). (C) Symptoms caused by CMV in TRV derivates‐inoculated plants at 5 days post‐inoculation (dpi). Dashed circles indicate the upper systemic leaves, and white lines represent 3 cm. (D) Northern blot analysis of CMV accumulation in silenced and non‐silenced plants. CMV RNAs were detected in the upper systemic leaves at 5 dpi. The relative accumulation of viral RNA3 is shown below. Ethidium bromide‐stained ribosomal RNAs were used as a loading control.

To test whether the reduction in CMV titre mediated by Nb‐TSC10 silencing occurs through the UPR pathway, we investigated the role of Nb‐bZIP60 in Nb‐TSC10‐mediated immunity by simultaneously silencing both genes using TRV‐based VIGS. RT‐qPCR confirmed > 70% knockdown efficiency for both genes (Figure 6B), and no obvious phenotypic abnormalities were caused in the double‐silenced plants, as well as in the Nb‐bZIP60‐silenced plants, at 7 days after TRV inoculation (Figure S7). These plants were subsequently subjected to CMV challenge. At 5 days after CMV inoculation, both Nb‐bZIP60‐ and double‐silenced plants displayed milder leaf curling in the upper systemic leaves than Nb‐TSC10‐silenced plants (Figure 6C). Viral accumulation in the upper systemic leaves of Nb‐bZIP60‐silenced plants was comparable to that in the control plants (TRV‐mCherry) (Figure 6D). Interestingly, reduction of virus accumulation caused by the downregulation of Nb‐TSC10 was restored when Nb‐bZIP60 was silenced together (Figure 6D).

We investigated the involvement of the bZIP17/28 branch in Nb‐TSC10‐mediated resistance. We simultaneously silenced both Nb‐bZIP28 and Nb‐bZIP17 using TRV‐VIGS. Neither the double‐silenced plants nor the triple‐silenced plants exhibited obvious phenotypic abnormalities at 7 days post‐TRV inoculation (Figure 7A). Upon CMV challenge, upper systemic leaves of both Nb‐bZIP17/28‐ and triple‐silenced plants displayed milder leaf curling compared to those of Nb‐TSC10‐silenced plants at 5 dpi (Figure 7B). RT‐qPCR confirmed knockdown efficiencies of > 60% for each target gene (Figure 7C). Notably, viral accumulation in the upper systemic leaves of Nb‐bZIP17/28‐silenced plants was comparable to that in control (TRV‐mCherry) plants. Similar to the phenotype observed upon bZIP60 silencing, the reduction in viral accumulation caused by Nb‐TSC10 downregulation was restored when both Nb‐bZIP28 and Nb‐bZIP17 were silenced (Figure 7D).

FIGURE 7.

FIGURE 7

Open in a new tab

Nb‐TSC10‐related immunity is dependent on Nb‐bZIP17/28. (A) Phenotypes of Nb‐bZIP17/28 knockdown plants at 7 days post‐inoculation (dpi) with TRV, and CMV‐induced symptoms in silenced vs. non‐silenced plants at 5 dpi. Dashed circles denote upper systemic leaves. (B) Silencing efficiency of TRV‐mediated silencing was measured by reverse transcription‐quantitative PCR. Significant differences were determined by t‐test analysis (**p < 0.01, ***p < 0.001, ****p < 0.0001). (C) Northern blot hybridization analysis of the accumulation level of CMV in silenced or non‐silenced plants. CMV RNAs were detected in the upper systemic leaves at 5 dpi. Relative accumulation levels of viral RNA3 are shown below. Ethidium bromide‐stained ribosomal RNAs were used as a loading control.

These data suggest that the Nb‐TSC10‐mediated inhibition of CMV requires both the bZIP60 and the bZIP17/28 branches.

3. Discussion

This study reveals a previously unrecognized counterdefence strategy by which CMV exploits host lipid metabolism to suppress antiviral immunity. Using non‐targeted semiquantitative lipidomic profiling, we showed that CMV infection increases sphingolipid accumulation in the tonoplast, the viral replication site. Genetic and pharmacological analyses demonstrated that increased host sphingolipids de novo biosynthesis, particularly via Nb‐TSC10b, dampens SA–UPR signalling, thereby promoting viral replication. Conversely, silencing Nb‐TSC10b attenuated CMV replication through bZIP60 and bZIP17/28 signalling, unveiling engagement of the UPR regulatory axis in the antiviral defence. These findings establish sphingolipid metabolism as a key regulatory node linking SA signalling and UPR in plant antiviral immunity.

Ceramides are bioactive sphingolipid intermediates with well‐documented roles in cell death and stress signalling (Hannun and Obeid 2008; Wang et al. 2020). In plants, short‐chain ceramides such as C2 and C6 induce cell death (Zhang et al. 2020; Liang et al. 2003; Townley et al. 2005), whereas the roles of long‐chain ceramides (C14–C20) remains unknown. Although C16‐ceramide contributes to apoptosis in human neutrophil, B cell and macrophages (Kroesen et al. 2001; Seumois et al. 2007), our data show that C16‐ceramide inhibited the expression of Nb‐bZIP60 (Figure 4A) without inducing visible cell death in N. benthamiana (Figure S3). This suggests that C16‐ceramide alleviates UPR independently of the cell death‐associated pathway. Notably, despite the long‐held assumptions that exogenous C16‐ceramide cannot penetrate cells due to its hydrophobic, long acyl chain (Fillet et al. 2003), our findings suggest its uptake and functional activity in planta (Figure 4). This aligns with recent work showing that exogenous C16‐ceramide activates jasmonic acid signalling in Arabidopsis (Huang et al. 2021). Intriguingly, C16‐ceramide exhibited distinct roles in the regulation of UPR, probably dependent on host basal sphingolipid homeostasis and, consequently, its UPR status. In healthy leaves, exogenous C16‐ceramide appeared to alleviate UPR (Figure 4A). Conversely, in Nb‐TSC10‐silenced plants where total Cer levels in the tonoplast are reduced (Figure 5C) and the bZIP60 branch of the UPR is activated (Figure 6A), the same treatment exacerbated UPR activation (Figure S6). We speculate that the chronic ER stress caused by Nb‐TSC10 silencing may account for why exogenous C16‐ceramide induced the UPR. Nevertheless, it should be noted that this represents an artificial experimental condition. In the context of CMV infection, we favour the notion that C16‐ceramide alleviates UPR, as evidenced by its reduction of Nb‐bZIP60 (Figure 4A). While this study highlights the specific role of C16‐ceramide in modulating the UPR, we acknowledge that other ceramides could exert analogous effects. Future studies are required to systematically evaluate the functional diversity of other ceramide species.

A striking outcome of our work is the dual role of SA in CMV infection. On one hand, SA is required for CMV‐induced Nb‐TSC10 expression, linking SA signalling to sphingolipid accumulation. On the other, SA activates the UPR pathway, which restricts viral replication under ER stress. Our findings provide novel mechanistic insights into the interplay between CMV infection and host SA signalling pathways. Although SA activates host immune responses that inhibit CMV infection, it enables CMV‐induced sphingolipid production, thereby enabling the virus to subvert UPR‐mediated antiviral defences. In the context of CMV infection, seminal studies demonstrate that SA‐mediated immunity primarily impedes viral systemic movement rather than replication (Ji and Ding 2001; Naylor et al. 1998). Our data further reveal that Nb‐TSC10‐mediated immune response to CMV is orchestrated through an SA‐dependent UPR cascade. It suggests that, at least under silencing Nb‐TSC10‐induced ER‐stress condition, SA could activate the UPR pathway to impair CMV replication. Thus, SA functions both as an antiviral signal and as a proviral factor through its impact on sphingolipid metabolism.

We acknowledge several limitations in the current study. First, the precise molecular mechanism by which CMV induces sphingolipid biosynthesis remains to be fully elucidated. Second, the metabolic fate of exogenously applied C16‐ceramide was not traced in this study; determining its destination would significantly deepen our understanding of how ceramides modulate the host UPR. Finally, our functional analysis relied solely on TRV‐VIGS to knock down Nb‐TSC10. Future studies employing CRISPR/Cas9‐mediated knockout lines would help to validate these findings and, crucially, exclude potential confounding influences associated with the VIGS viral vector itself.

Taken together, our study uncovers a sphingolipid‐mediated viral strategy that counteracts SA‐induced immunity. CMV activates host sphingolipid biosynthesis to relieve ER stress, thereby suppressing UPR‐induced resistance. Although SA confers host resistance against CMV, it paradoxically enhances sphingolipid accumulation in tonoplasts that benefits CMV multiplication. This work advances our mechanistic understanding of virus–host interplay at the interface of lipid metabolism and stress signalling.

4. Experimental Procedures

4.1. Plant Materials and Growth Conditions

Nicotiana tabacum and N. benthamiana plants were cultivated in a plant growth room under constant temperature at 22°C with a 16 h photoperiod.

4.2. Plasmid Construction

For silencing Nb‐TSC10, a 182 bp highly homologous sequence of Nb‐TSC10a/b (SGN number: Niben101Scf11399g00011 and Niben101Scf10363g01002) was amplified from N. benthamiana and cloned into pTRV2 to produce pTRV2‐TSC10. For silencing Nb‐bZIP60, a 300 bp fragment of Nb‐bZIP60 (SGN number: Niben101Scf24096g00018) was amplified and cloned into pTRV2 to generate pTRV2‐bZIP60. For simultaneously silencing both Nb‐TSC10 and Nb‐bZIP60, the same Nb‐bZIP60 fragment was subsequently inserted into pTRV2‐TSC10, generating pTRV2‐TSC10‐bZIP60. For silencing Nb‐bZIP17/28, the construct pTRV2‐bZIP17/28 was generated using primers described previously (Li, Zhang, et al. 2021). The primers used for the fragment amplifications mentioned above were listed in Table S2.

4.3. Protoplast Preparation, Vacuoles Separation, Tonoplast Enrichment

Protoplasts from leaves of N. tabacum or N. benthamiana plants were prepared, which were subsequently subjected to vacuole separation according to the protocol (Robert et al. 2007). The integrity of isolated vacuoles was microscopically inspected. For enrichment of tonoplasts, 2 mL of thawed vacuoles was mixed with 6 mL phosphate‐buffered saline (PBS), followed by ultracentrifugation at 150,000 g for 1 h at 4°C. The membrane pellets were resuspended in 8 mL PBS and centrifuged at 150,000 g for 1 h at 4°C. Finally, the enriched tonoplast fraction was snap frozen in liquid nitrogen and stored at −80°C.

4.4. Lipidomic and Proteomic Analyses of Tonoplasts

The tonoplast fractions prepared from the upper systemic leaves of CMV‐infected or mock‐treated N. benthamiana plants (six independent preparations for each treatment), as well as those from Nb‐TSC10‐silenced or non‐silenced N. benthamiana (five independent preparations for each treatment), were subjected to non‐targeted, semiquantitative lipidomic analysis using LC–MS at Shanghai Luming Biotechnology Co. Ltd. (Shanghai, China). Briefly, the extraction of total lipids from tonoplasts was performed using isopropanol, followed by vacuum dehydration. Total lipids were dissolved in isopropanol/methanol (1:1, v/v) solution, in which the internal standard solution (LPC(12:0), PC(22:0)) was added prior to LC–MS assays. The LC–MS assays were performed using Nexera UPLC and Q Exactive hybrid quadrupole‐Orbitrap mass spectrometer (ThermoFisher Scientific). The total proteins were isolated from the tonoplasts prepared from the leaves of healthy N. tabacum plants. The protein samples were subjected to proteomic analyses using LC–MS/MS served by PTM‐Biolabs Co. Ltd. (Hangzhou, China).

4.5. Detection of Programmed Cell Death

C16‐ceramide‐ or the dissolvent control‐treated leaves were stained with trypan blue as previously reported with minor modifications (Tian et al. 2018; Gu et al. 2017). Briefly, leaves were boiled within trypan blue staining solution (60% ethanol, 10% DL‐lactic acid, 10% glycerol, 10% saturated phenol pH 7.9, 10% water, 0.067% trypan blue [Sangon Biotech]) for 4 min. The decolouration was performed with 2.5 mg/mL chloral hydrate (Sangon Biotech) on a shaker at room temperature for 12 h.

4.6. Agrobacterium‐Mediated Viral Inoculation

All T‐DNA‐containing plasmids were transformed into Agrobacterium tumefaciens GV3101 for agroinfiltration (Duan et al. 2023). Agrobacterial transformants were propagated in Luria Bertani (LB) medium containing 25 μg/mL kanamycin and 25 μg/mL rifampicin. The propagated cells were collected by centrifugation and suspended in the infiltration solution (10 mM 2‐(N‐morpholino) ethanesulphonic acid, 10 mM MgCl2, 200 μM acetosyringone) for 3 h, followed by infiltration into the fifth leaf of 4‐week‐old N. benthamiana plants. To downregulate a specific gene in N. benthamiana plants, TRV variants carrying a fragment of the specific gene were inoculated on the fifth leaves of N. benthamiana plants via agroinfiltration. Seven days later, the sixth leaves (from the bottom of the plant) were inoculated with CMV or CMVΔ2b via agroinfiltration. Viral symptoms were photographed at 4 or 5 days after CMV inoculation unless otherwise stated. The upper systemic leaves were collected for RNA analyses at the same time.

4.7. Chemical Treatment

C16‐ceramide (HY‐100354, MCE) and C18‐ceramide (HY‐100355, MCE) were dissolved in ethanol and diluted to 50 μM using distilled water. The diluted ceramide solution was infiltrated into leaves of N. benthamiana plants. The dissolvent (1% ethanol) was used as the control. To mitigate ER stress, 500 μM tauroursodeoxycholic acid (TUDCA) (HY‐19696, MCE) was infiltrated into leaves of N. benthamiana plants, with a control of 0.5% dimethylsulphoxide (DMSO). Leaf samples were then collected at 12 h post‐treatment. For SA treatment, 5 mM SA (Sangon) was sprayed on the leaves of N. benthamiana, using distilled water as the control. After 24 h, both treated leaves were collected for RNA analysis.

4.8. Protein Extraction and Western Blot Analysis

Proteins were extracted from viral‐inoculated or agroinfiltrated leaves using an SDS extraction buffer (1% SDS, 2% β‐mercaptoethanol in PBS). Subsequently, the total protein was mixed with an equal volume of 2 × loading buffer (4% SDS, 2% β‐mercaptoethanol, 100 mM Tris–HCl pH 6.8, 3% saturated bromophenol blue, 20% glycerol), followed by denaturation at 95°C for 10 min. Immunoblot analysis was performed as previously described (Gao et al. 2018), with the primary mouse monoclonal anti‐CP serum (Yu et al. 2005) and the secondary goat anti‐mouse antibody with conjugation of horseradish peroxidase (SC‐2005, Santa Cruz Biotechnology).

4.9. RNA Extraction, Northern Blot, RT‐qPCR

Total RNA was extracted from 0.1 g leaf tissues using RNA extraction buffer (50 mM sodium acetate pH 5.2, 10 mM EDTA pH 8.0, and 1% SDS). Northern blotting was performed according to our previously report (Qiao et al. 2024). Two micrograms of total RNA was separated in a denatured agarose gel, transferred onto a positively charged nylon membrane, and detected using the DIG‐labelled oligonucleotide probe targeting the conserved sequence in the 3′ untranslated region of CMV genomic RNAs (Du et al. 2014).

Reverse transcription was performed according to the previous reports with minor modifications (Jiao et al. 2024; Zheng, Duan, et al. 2023). Briefly, first‐strand cDNAs were synthesized using FastKing RT Kit (Tiangen) and oligo(dT) primer. For quantitative real‐time PCR, the reaction was performed on ABI QuantStudio 5 with SuperReal PreMix Colour (Tiangen) following the previous reports (Huan et al. 2025; Zhang et al. 2025). The relative quantities were calculated using the 2−ΔΔCt method. The protein phosphatase 2A (SGN number: Niben101Scf09716g01002) was employed as an internal reference gene for normalization in N. benthamiana. The stability of its expression has been confirmed under conditions of CMV infection (Liu et al. 2012) and tunicamycin‐induced ER stress (Ko et al. 2023). Data are presented as the mean ± standard deviation (SD) of at least three independent plants in which three technical replicates were included. The primers used for qPCR were listed in Table [Link], [Link].

4.10. Statistical Analysis

Statistical analyses were performed according to a previous report using GraphPad Prism (v. 6.0.1) (Zheng, Jiao, et al. 2023). An unpaired, two‐tailed t‐test was used for comparisons between two groups; one‐way ANOVA with Tukey's test was used for comparisons among multiple groups. All the data are represented as the mean ± SD.

Author Contributions

Jiayi Yao: investigation, data curation, visualization. Shaomin Wu: investigation. Shushu Gao: investigation. Zhidan Xiang: investigation. Qian Wang: investigation. Fei Ye: formal analysis. Qiansheng Liao: resources. Zhouhang Gu: writing – original draft, writing – review and editing, visualization, supervision, funding acquisition, investigation. Zhiyou Du: writing – review and editing, writing – original draft, funding acquisition, conceptualization, supervision.

Funding

This work was financially supported by the National Natural Science Foundation of China (31600125 to Z.G., 32070154 and 32570174 to Z. D.).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Northern blot analysis of CMV genomic RNAs as showed in Figure 3. (A) Accumulation of CMV RNAs were examined in wild type or NahG lines as shown in (Figure 3B) by northern blot. Total RNAs were extracted from the leaves at 3 days post‐infiltration. (B) Accumulation of CMV RNA in the inoculated leaves of the plants with virus infection as shown in (Figure 3D).

MPP-27-e70353-s008.docx (1MB, docx)

Figure S2: CMV‐induced Nb‐TSC10b is enhanced in Nb‐bZIP60‐silenced Nicotiana benthamiana. Data points sharing different lowercase letters above error bars indicate statistically significant differences (p < 0.05).

MPP-27-e70353-s009.docx (107.2KB, docx)

Figure S3: C16‐ceramide dose not induce a visible cell death phenotype.

MPP-27-e70353-s004.docx (270.1KB, docx)

Figure S4: Knocking down Nb‐TSC10 impairs CMV RNA3 replication in a 1a/2a trans‐expression system.

MPP-27-e70353-s003.docx (125.7KB, docx)

Figure S5: Nb‐TSC10‐silencing‐induced antiviral immunity requires salicylic acid (SA) signalling. (A) CMV‐caused symptoms in Nb‐TSC10‐silenced or non‐silenced transgenic NahG plants at 4 days post‐inoculation (dpi). (B) Silencing efficiency TRV‐mediated silencing of Nb‐TSC10b were measured by reverse transcription‐quantitative PCR. **p < 0.01. (C) Northern blot hybridization analysis of the accumulation level of CMV in the TRV derivates infected wild type or NahG plants. CMV RNAs were detected in the upper systemic leaves at 4 dpi. Relative accumulation level of viral RNA3 was shown below. Ethidium bromide‐stained ribosomal RNAs were used as a loading control.

MPP-27-e70353-s006.docx (359.4KB, docx)

Figure S6: Exogenous C16‐ceramide induced Nb‐bZIP60 under Nb‐TSC10‐silenced background. Statistical differences were determined by t‐test analysis (**p < 0.01, n.s.:not significant).

MPP-27-e70353-s005.docx (56.2KB, docx)

Figure S7: Typic phenotypes of Nb‐TSC10 co‐silencing with Nb‐bZIP60 in Nicotiana benthamiana. White lines represent 3 cm.

MPP-27-e70353-s007.docx (482.8KB, docx)

Table S1: Proteomic analysis of tonoplasts prepared from healthy Nicotiana benthamiana leaves.

MPP-27-e70353-s002.docx (18.6KB, docx)

Table S2: Primers used in this study.

MPP-27-e70353-s001.docx (16.3KB, docx)

Acknowledgements

We are grateful to Professor Jianxiang Wu from Zhejiang University for kindly providing the primary antibody against CMV CP, and Professor Huishan Guo from the Chinese Academy of Sciences for sharing the seeds of NahG transgenic N. benthamiana.

Contributor Information

Zhouhang Gu, Email: hzgu@zstu.edu.cn.

Zhiyou Du, Email: duzy@zstu.edu.cn.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1: Northern blot analysis of CMV genomic RNAs as showed in Figure 3. (A) Accumulation of CMV RNAs were examined in wild type or NahG lines as shown in (Figure 3B) by northern blot. Total RNAs were extracted from the leaves at 3 days post‐infiltration. (B) Accumulation of CMV RNA in the inoculated leaves of the plants with virus infection as shown in (Figure 3D).

MPP-27-e70353-s008.docx (1MB, docx)

Figure S2: CMV‐induced Nb‐TSC10b is enhanced in Nb‐bZIP60‐silenced Nicotiana benthamiana. Data points sharing different lowercase letters above error bars indicate statistically significant differences (p < 0.05).

MPP-27-e70353-s009.docx (107.2KB, docx)

Figure S3: C16‐ceramide dose not induce a visible cell death phenotype.

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Figure S4: Knocking down Nb‐TSC10 impairs CMV RNA3 replication in a 1a/2a trans‐expression system.

MPP-27-e70353-s003.docx (125.7KB, docx)

Figure S5: Nb‐TSC10‐silencing‐induced antiviral immunity requires salicylic acid (SA) signalling. (A) CMV‐caused symptoms in Nb‐TSC10‐silenced or non‐silenced transgenic NahG plants at 4 days post‐inoculation (dpi). (B) Silencing efficiency TRV‐mediated silencing of Nb‐TSC10b were measured by reverse transcription‐quantitative PCR. **p < 0.01. (C) Northern blot hybridization analysis of the accumulation level of CMV in the TRV derivates infected wild type or NahG plants. CMV RNAs were detected in the upper systemic leaves at 4 dpi. Relative accumulation level of viral RNA3 was shown below. Ethidium bromide‐stained ribosomal RNAs were used as a loading control.

MPP-27-e70353-s006.docx (359.4KB, docx)

Figure S6: Exogenous C16‐ceramide induced Nb‐bZIP60 under Nb‐TSC10‐silenced background. Statistical differences were determined by t‐test analysis (**p < 0.01, n.s.:not significant).

MPP-27-e70353-s005.docx (56.2KB, docx)

Figure S7: Typic phenotypes of Nb‐TSC10 co‐silencing with Nb‐bZIP60 in Nicotiana benthamiana. White lines represent 3 cm.

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Table S1: Proteomic analysis of tonoplasts prepared from healthy Nicotiana benthamiana leaves.

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Table S2: Primers used in this study.

MPP-27-e70353-s001.docx (16.3KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from Molecular Plant Pathology are provided here courtesy of Wiley

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