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. 2024 Nov 25;25(11):e70034. doi: 10.1111/mpp.70034

Cucumber Green Mottle Mosaic Virus Coat Protein Hijacks Mitochondrial ATPδ to Promote Viral Infection

Xue Yang 1,2,✉, Xing‐Lin Jiang 1, Han Fu 1, Lian‐Wei Yu 1, Niu Ai 1, Ya‐Juan Shi 1, Yu‐Wen Lu 2, Zi‐Hao Xia 3, Hong‐Lian Li 1,4, Yan Shi 1,✉
PMCID: PMC11588859  PMID: 39587446

ABSTRACT

The production and scavenging of reactive oxygen species (ROS) are critical for plants to adapt to biotic and abiotic stresses. In this study, we investigated the interaction between the coat protein (CP) of cucumber green mottle mosaic virus (CGMMV) and ATP synthase subunit δ (ATPδ) in mitochondria. Silencing of ATPδ by tobacco rattle virus‐based virus‐induced gene silencing impeded CGMMV accumulation in Nicotiana benthamiana leaves. Both the overexpression of ATPδ in transgenic plants and transient expression promoted CGMMV infection. Nitro blue tetrazolium (NBT) and 3,3′‐diaminobenzidine (DAB) staining revealed that ATPδ inhibited O2 − production but not H2O2 production. The treatment of CGMMV‐infected leaves with the ROS inhibitor diphenylene iodonium (DPI) induced a ROS burst that inhibited CGMMV infection. Reverse transcription‐quantitative PCR and superoxide dismutase (SOD) activity assays showed that ATPδ, CGMMV infection, and CP expression specifically induced NbFeSOD3/4 expression and SOD activity, and silencing NbFeSOD3/4 inhibited CGMMV infection. We speculate that CGMMV CP interacts with ATPδ and hijacks it, thereby enhancing O2 − quenching by upregulating NbFeSOD expression and, in turn, SOD activity.

Keywords: ATP synthase subunit δ, cucumber green mottle mosaic virus superoxide, superoxide dismutase

Cucumber green mottle mosaic virus uses the viral coat protein to hijack ATPδ, which in the absence of virus regulates the expression of NbFeSODs to induce superoxide dismutase activity and thus O2 − quenching.

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1. Introduction

Reactive oxygen species (ROS) have both benefits and drawbacks for basic biological processes and stress responses in living organisms. In plants, both abiotic and biotic stresses lead to ROS production in several different subcellular compartments (Mittler 2017). In plant apoplasts, ROS are produced by NADPH oxidase, which generates superoxide (O2 −) (Smirnoff and Arnaud 2019). Mitochondria are another important source of ROS, and mitochondrial ROS (mROS) are involved in plant defence and immunity as well as cell signalling (Wang, Xu, et al. 2022; Huang et al. 2016). The site of mROS production is the electron transport chain (ETC), which drives ATP production in the mitochondrial respiratory chain (Vercellino and Sazanov 2022). When mitochondria are under abiotic or biotic stress, mROS levels increase (Wang, Xu, et al. 2022). The mROS burst has also been linked to programmed cell death and plant immunity during pathogen infection (Wang, Xu, et al. 2022; Huang et al. 2016). A previous study showed that mROS accumulation is induced in maize leaves infected with sugarcane mosaic virus (SCMV) (Jiang et al. 2023). The loss of functionality of RTP7, an RNA processing factor of the ETC complex I subunit, leads to an increased mROS burst, thereby enhancing plant immunity to broad‐spectrum pathogens (Yang, Zhao, et al. 2022). However, although the assembly and overall function of mitochondria are highly conserved in eukaryotes, the mitochondrial processes responsible for plant defences and immunity remain poorly understood.

Mitochondrial ATP synthase produces energy for plant cells, whereas the availability of chloroplast‐derived ATP is limited by chloroplast energy dissipation systems and the capacity of chloroplast ATP export shuttles. Given these limitations, substantial mitochondrial ATP synthesis is required to fulfil cytosolic ATP requirements (Shameer, Ratcliffe, and Sweetlove 2019). Most of the required energy is obtained from the ETC and mitochondrial ATP synthase (Yang, Xue, et al. 2022). The mitochondrial ETC generates a transmembrane proton gradient across the inner mitochondrial membrane, with the flow of protons toward the mitochondrial matrix, leading to the synthesis of ATP mediated by F0F1‐ATP synthases (Zancani et al. 2020). The subunit composition and assembly pathway of plant mitochondrial ATP synthase are mostly conserved across species (Röhricht, Schwartzmann, and Meyer 2021). The F0F1‐ATP synthase complex consists of a water‐soluble, catalytic F1 head and a membrane‐embedded F0 subcomplex. The subunits of mitochondrial ATP synthase are involved in biotic and abiotic stress in plants. For the F1 component of mitochondrial ATP synthase, the polypeptide subunit composition is α3β3γδε (Bason et al. 2015). In mitochondria, the F1 complex of ATP synthase (Gellért et al. 2018) and the ATP synthase OSCP subunit (He et al. 2023) are involved in plant responses to virus infection. In maize, reduced F0F1‐ATP synthase activity triggers a ROS burst and the premature programmed cell death of tapetal cells (Yang, Xue, et al. 2022). Plant responses to abiotic stresses such as salt excess, drought, and low temperatures include the induction of ATP6 expression (Zhang, Liu, and Takano 2008) and the oxidative burst‐mediated degradation of subunits α, β, and δ of mitochondrial ATP synthase (Sweetlove et al. 2002). In wheat, aluminium stress induces α‐ and β‐subunit expression (Hamilton, Good, and Taylor 2001).

The nuclear‐encoded δ subunit of F0F1‐ATP synthase (ATPδ) acts as a linker between F0 and F1 (Gibbons et al. 2000). In Arabidopsis thaliana , the δ subunit is represented by a single gene (At5g47030) (Geisler et al. 2012). In cotton, GhATPδ1 determines the final fibre length (Gibbons et al. 2000). In A. thaliana , ATPδ is highly expressed in pollen, ovules, and floral primordia, which are tissues with high energy requirements (Geisler et al. 2012). Knockdown of the δ subunit in the mitochondrial ATP synthase of Arabidopsis results in greater sensitivity to heat stress (Liu et al. 2021). However, the function of ATPδ in viral infection is currently unknown.

Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus. It consists of a single‐stranded, positive‐sense genomic RNA of approximately 6.4 kb (Ugaki et al. 1991). In a previous study, we showed that interaction between CGMMV coat protein (CP) and movement protein (MP) suppresses host antiviral defences to facilitate systemic virus infection (Shi et al. 2023). In the present study, we used a yeast two‐hybrid (Y2H) screen of an Nicotiana benthamiana cDNA library to identify plant proteins including ATPδ in mitochondria that interact with CGMMV CP. The silencing of ATPδ impeded CGMMV accumulation in N. benthamiana, whereas the overexpression of ATPδ enhanced virus accumulation in leaves. ATPδ specifically inhibited O2 − accumulation but not H2O2 accumulation, suggesting its involvement in regulating ROS signalling by specifically activating NbFeSOD3/4 and thus superoxide dismutase (SOD) activity.

2. Results

2.1. Mitochondrial ATPδ Interacts With CGMMV CP

A Y2H assay was performed to screen a N. benthamiana cDNA library to identify plant proteins that interact with CGMMV CP (Table S2). Under stringent selective conditions, mitochondrial ATPδ (Niben101Scf08179g01015.1) was identified as a candidate interacting protein and selected for further investigation. The complete open reading frame of ATPδ was amplified from N. benthamiana cDNA and cloned into pGEM‐T for sequencing and further analysis. ATPδ was fused to the N‐terminus of green fluorescent protein (GFP) and transiently expressed in N. benthamiana leaves for examination by confocal microscopy. The mitochondrial localization of ATPδ was confirmed by the detection of ATPδ–GFP in N. benthamiana leaves using the mitochondrial marker MitoTracker Red (Figure 1a). The localization of CGMMV CP in the cytoplasm and nucleus was shown in our previous research (Shi et al. 2023). The interaction of ATPδ with CP was confirmed by bimolecular fluorescence complementation (BiFC) experiments, by the transient expression of paired proteins in N. benthamiana leaves. ATPδ and CP were fused at the C‐terminus of either the amino (n) domain or the carboxy (c) domain of yellow fluorescent protein (YFP) to generate the constructs ATPδ–cYFP, ATPδ–nYFP, CP–cYFP, and CP–nYFP. Co‐expression of ATPδ–cYFP and CP–nYFP or of ATPδ–nYFP and CP–cYFP resulted in YFP fluorescence signals in the agroinfiltrated cells at 72 h post‐infiltration (hpi), but there were no detectable signals in the negative control combinations ATPδ‐cYFP and nYFP, ATPδ‐nYFP and cYFP, CP‐cYFP and nYFP or CP‐nYFP and cYFP (Figure 1b, bottom panel). These results were consistent with the interaction of ATPδ and CP. YFP‐fused ATPδ and CP fluorescent granules mostly showed aggregation in the cytoplasm and nucleus with a small portion (19%) in the mitochondria. These demonstrated that CGMMV CP alters the localization of a portion of ATPδ.

FIGURE 1.

FIGURE 1

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Mitochondrial ATP synthase subunit δ (ATPδ) interacts with cucumber green mottle mosaic virus (CGMMV) coat protein (CP). (a) Subcellular localization of ATPδ–green fluorescent protein (GFP) in agroinfiltrated Nicotiana benthamiana leaves and using the fluorescence marker MitoTracker Red. Scale bars = 20 μm. (b) Interaction between ATPδ‐nYFP and CP‐cYFP/ ATPδ‐nYFP and CP‐cYFP revealed by bimolecular fluorescence complementation in agroinfiltrated N. benthamiana leaves and using the fluorescence marker MitoTracker Red. Co‐expressing ATPδ‐nYFP /cYFP, ATPδ‐cYFP /nYFP, nYFP/CP‐cYFP, and cYFP/CP‐nYFP were used as negative controls. Scale bars = 20 μm. (c) Interaction between ATPδ–GFP and CP–Myc verified by co‐immunoprecipitation. Input proteins were detected by western blotting with anti‐GFP and anti‐Myc antibodies. IP denotes the immunoprecipitated fraction probed with anti‐Myc antibodies, and the ATPδ–GFP is marked by an asterisk.

A co‐immunoprecipitation (Co‐IP) assay was performed to further explore the interaction between ATPδ and CP. Plasmids encoding ATPδ–GFP and CP–Myc were transiently expressed in N. benthamiana, using a C‐terminal Myc‐tagged truncated β‐glucuronidase (GUS) fragment (1–166 amino acids; GUS‐Myc) as a negative control. Myc–TRAP_M beads were used to purified Myc‐tagged proteins and any interacting partners that had accumulated in the plants. Although all proteins were expressed in the plants (Figure 1c, Input), ATPδ–GFP was co‐precipitated only in the presence of CP–Myc, not when expressed together with GUS‐Myc (Figure 1c, IP).

2.2. Silencing of ATPδ Impedes CGMMV Infection

The involvement of ATPδ in CGMMV infection was investigated using tobacco rattle virus‐induced gene silencing (TRV VIGS) to silence ATPδ in N. benthamiana plants prior to their inoculation with CGMMV. At 10 days post‐inoculation (dpi), reverse transcription‐quantitative PCR (RT‐qPCR) showed that the transcript levels of ATPδ in TRV:ATPδ‐silenced plants were only 5% of that in TRV:00‐treated plants (Figure 2a). ATPδ‐silenced plants (TRV:ATPδ) were shorter and exhibited more severe chlorosis and necrosis symptoms than TRV:00‐treated plants (Figure 2b). At 10 dpi, TRV:ATPδ and TRV:00 inoculated plants were infected with CGMMV. At 7 dpi, ATPδ‐silenced plants (TRV:ATPδ) were shorter and smaller than the TRV:ATPδ plants without CGMMV infection (Figure 2b). All TRV:00 plants showed signs of systemic CGMMV infection at 7 dpi, whereas the systemic leaves (uppermost leaves) of TRV:ATPδ plants did not. Neither curling nor mosaic symptoms were observed in the uppermost leaves of TRV:ATPδ plants. However, after CGMMV infection, the first leaf above the inoculated leaves exhibited chlorosis symptoms, and severe necrosis was observed in the inoculated leaves (Figure 2c). Western blotting and RT‐qPCR showed that CGMMV CP mRNA and protein expression were significantly inhibited in the systemic leaves (uppermost leaves) of TRV:ATPδ plants (Figure 2d,g). CGMMV CP mRNA expression in the first leaves above inoculated leaves and inoculated leaves of TRV:ATPδ plants was also significantly downregulated compared with TRV:00 leaves (Figure 2e,f). These results suggested that the silencing of ATPδ inhibits systemic infection by CGMMV in N. benthamiana.

FIGURE 2.

FIGURE 2

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Silencing of ATPδ impedes CGMMV infection. (a) The silencing efficiency of ATPδ was detected by reverse transcription‐quantitative PCR. The experiment was repeated three times. (b) Symptoms caused by TRV‐mediated virus‐induced gene silencing with TRV:ATPδ following CGMMV infection or mock treatment as observed at 17 days post‐inoculation (dpi). (c) Systemic infection by CGMMV at 7 dpi in plants pretreated with TRV:00 or TRV: ATPδ. Top panel, Whole plants; second panel, systemic leaves (uppermost leaves) of CGMMV‐infected plants; third panel, first leaves above the inoculated leaves; bottom panel, inoculated leaves. The accumulation of CGMMV coat protein (CP) in (d) systemic leaves (uppermost leaves), (e) the first leaves above the inoculated leaves, and (f) the inoculated leaves was detected by reverse transcription‐quantitative PCR. (g) CGMMV CP accumulation in systemic leaves (uppermost leaves) was assayed by western blotting using CGMMV CP antibody. Every treatment had four biological repeats. Every lane was an individual plant. Bars represent the standard errors of the means. A two‐sample unequal variance directional t test was used to test the significance of the difference (**p < 0.01).

2.3. Overexpression of ATPδ Promotes CGMMV Infection

A construct consisting of the full‐length ATPδ gene driven by the cauliflower mosaic virus (CaMV) 35S promoter in the binary vector pFGC5941 (Cao et al. 2023) was used to obtain stable transgenic lines of N. benthamiana overexpressing ATPδ through Agrobacterium‐mediated transformation. Stable transgenic lines with upregulated expression of ATPδ (OE ATPδ1–5) were analysed using PCR (Figure 3a). The primers were designed based on TMVΩ‐ATPδ sequence, which has a translational enhancer from the tobacco mosaic virus 5′‐leader sequence in the pFGC5941 vector (Table S1). The level of ATPδ mRNA was increased in overexpression (OE) ATPδ1–5 transgenic lines with RT‐qPCR (Figure 3b). OE ATPδ1–5 developed normally, with no obviously altered phenotypes. The fourth to sixth leaves of wild‐type (WT) and OE ATPδ1–5 plants were then inoculated with CGMMV. At 6 dpi, curling and mosaic symptoms were more severe in the systemic leaves of OE ATPδ1–5 plants than in WT plants (Figure 3c). Western blotting and RT‐qPCR showed greater accumulation of CGMMV CP protein and mRNA in the inoculated leaves of OE ATPδ1–5 plants than in WT plants (Figure 3d,e). ATPδ–GFP was then transiently expressed in leaves that were also infected with CGMMV, with transiently expressed free GFP and CGMMV used as controls in a separate area of the same leaves. At 2 dpi, CGMMV accumulation was assessed by western blotting, which showed higher levels of CGMMV coat protein (CP) in plants transiently expressing ATPδ–GFP than in those transiently expressing free GFP (Figure 3f,g). These results suggested that the overexpression of ATPδ promotes CGMMV infection.

FIGURE 3.

FIGURE 3

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Overexpression of ATPδ promotes CGMMV infection. ATPδ expression levels in transgenic and wild‐type plants detected by PCR (a) and reverse transcription‐quantitative PCR (RT‐qPCR) (b). (c) Total plants and systemic leaves of overexpression (OE) ATPδ1–5 and wild‐type (WT) plants infected with CGMMV as observed at 6 days post‐inoculation (dpi). The accumulation of CGMMV coat protein (CP) and mRNA in OE ATPδ1–5 and wild‐type (WT) systemic leaves was analysed by western blotting (d) with an anti‐CGMMV CP antibody and by RT‐qPCR (e). (f, g) ATPδ–GFP and CGMMV were co‐inoculated in the same leaves, using free GFP and CGMMV as controls. CGMMV CP and ATPδ–GFP accumulation in the same leaf (at 2 dpi) as detected by western blotting. The accumulation of CP and ATPδ–GFP was detected using anti‐CGMMV CP and anti‐GFP antibodies, respectively; the levels of each relative to co‐expressed CGMMV and GFP were calculated using ImageJ. Bars represent the standard errors of the means. A two‐sample unequal variance directional t test was used to test the significance of the difference (**p < 0.01).

2.4. ATPδ Inhibits ROS Accumulation to Promote CGMMV Infection

Previous studies demonstrated that the mitochondrial gene atp6c confers male sterility in CMS‐C maize by inhibiting ATP6 interactions with ATP8 and ATP9, thereby reducing the quantity and activity of assembled F0F1‐ATP synthase and triggering a ROS burst (Yang, Xue, et al. 2022). In our study, ATPδ‐silenced leaves exhibited severe necrosis (Figure 2b). These observations suggested that ATPδ may be involved in the regulation of ROS production. This hypothesis was tested by examining H2O2 and O2 − levels in TRV:ATPδ and TRV:00 plants using 3,3′‐diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) staining. The accumulation of O2 − was significantly higher in the leaves of TRV:ATPδ than in those of TRV:00 plants, whereas there were no changes in H2O2 accumulation (Figure 4a). In OE ATPδ transgenic plants, ATPδ overexpression reduced the content of O2 −, but not that of H2O2 (Figure 4b). These results implied that ATPδ specifically inhibits O2 −.

FIGURE 4.

FIGURE 4

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ATPδ inhibits reactive oxygen species (ROS) accumulation to promote CGMMV infection. Leaves were stained with nitro blue tetrazolium (NBT) or 3,3′‐diaminobenzidine (DAB). Accumulation of H2O2 (upper panels, DAB staining) and O2 − (lower panels, NBT staining) in (a) TRV:ATPδ and TRV:00 leaves, (b) Overexpressing (OE) ATPδ1–5 and wild‐type (WT) leaves, and (c) CGMMV‐infected and WT plant leaves. (d, e) CGMMV coat protein (CP) accumulation in diphenylene iodonium (DPI)‐treated leaves following CGMMV infection as observed 24 h post‐inoculation (hpi) and detected by western blotting. Dimethyl sulphoxide (DMSO, 1%) was used in the control mock treatment (CK) in the same leaves. CP accumulation was normalised to RuBisCO levels, and its expression was calculated relative to the mock treatment. The experiments were repeated four times with similar results. Relative protein levels were calculated using ImageJ. Bars represent the standard errors of the means. A two‐sample unequal variance directional t test was used to test the significance of the difference (**p < 0.01).

Infection by multiple pathogens, including fungi, bacteria, viruses, and oomycetes, triggers plant immunity and ROS accumulation (Wang, Pruitt, et al. 2022). Although the high toxicity of ROS kills infecting pathogens, excessive ROS may also harm the plants (Afzal et al. 2014). Therefore, we investigated the relationship between CGMMV infection and ROS in N. benthamiana. At 12 dpi with CGMMV, curling and mosaic symptoms were observed in systemic leaves (Figure 4c). ROS accumulation in CGMMV‐infected plants was monitored by DAB and NBT staining, which showed higher levels of H2O2 and O2 − in CGMMV‐infected plants than in WT plants (Figure 4c). In leaves treated with the ROS inhibitor diphenylene iodonium (DPI) for 3 h with 1% dimethyl sulphoxide (DMSO) used as controls in a separate area of the same leaves and then inoculated with CGMMV for 24 h, CGMMV accumulation was significantly increased during the early stage of CGMMV infection (Figure 4d,e). These results demonstrated that ROS inhibits early‐stage CGMMV infection.

2.5. ATPδ Enhances O2 − Quenching by Increasing Superoxide Dismutase Activity

Superoxide dismutases (SODs) are a family of enzymes that protect cells by acting as ROS scavengers through catalysing the conversion of O2 − to O2 and H2O2 (Alscher, Erturk, and Heath 2002). Four NbFeSODs, four NbCuZnSODs, and four NbMnSODs were identified in the N. benthamiana v1.0.1 database (https://solgenomics.net/). Their transcript levels in OE ATPδ 1–5 and ATPδ‐silenced plants were then examined. Interestingly, NbFeSOD1/2 and NbFeSOD3/4 mRNA levels were reduced in ATPδ‐silenced leaves compared with non‐silenced leaves (Figure 5a), whereas the mRNA level of NbFeSOD3/4 was significantly increased in OE ATPδ plants (Figure 5b). Decreased transcript levels of NbCuZnSODs and NbMnSODs were detected both in ATPδ‐silenced and in OE ATPδ plants (Figure 5a,b). By contrast, there were no changes in the transcript levels of other antioxidant metabolic enzymes in OE ATPδ plants, including catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutathione S‐transferase (GST), and alternative oxidase (AOX) (Zhu et al. 2015; Noctor and Foyer 1998) (Figure 5c). Assays of SOD activity in OE ATPδ transgenic plants showed significantly higher levels than in WT plants (Figure 5d). Together, we speculated that ATPδ specifically regulates NbFeSOD3/4 expression to increase SOD activity, thereby enhancing O2 − quenching.

FIGURE 5.

FIGURE 5

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ATPδ enhances O2 − quenching by increasing superoxide dismutase (SOD) activity. The transcript levels of NbFeSOD1/2, NbFeSOD3/4, NbCuZnSOD1/2, NbCuZnSOD3/4, NbMnSOD1, NbMnSOD2, NbMnSOD3, and NbMnSOD4 in ATPδ‐silenced (a) and overexpressing (OE) ATPδ (b) plants as detected by reverse transcription‐quantitative PCR (RT‐qPCR). (c) Transcript levels of other reactive oxygen species‐scavenging enzymes, including NbAPXs, NbCATs, NbGPXs, NbGSTs, NbAOXa, and NbAOXb, in OE ATPδ1–5 and wild‐type (WT) plants. SOD activity in OE ATPδ1–5 and WT leaves (d), CGMMV‐infected and negative control (CK) plants (e), and CP‐Myc and GUS‐Myc expressed leaves (f). The transcript level of NbFeSOD3/4 during CGMMV infection (g) and coat protein (CP) expression (h). (i) The silencing efficiency of NbFeSOD3/4 was detected by RT‐qPCR. The experiment was repeated three times. (j) Symptoms caused by TRV‐mediated virus‐induced gene silencing with TRV:NbFeSOD3/4 following CGMMV infection or mock treatment as observed at 17 days post‐inoculation (dpi). CGMMV CP accumulation in systemic leaves (uppermost leaves) was assayed by RT‐qPCR (k) and western blotting (l). Every treatment had four biological repeats. Every lane was an individual plant. Expression levels relative to mock transfection were calculated using ImageJ. Bars represent the standard errors of the means. A two‐sample unequal variance directional t test was used to test the significance of the difference (*p < 0.05, **p < 0.01).

To further investigate the relationship between CGMMV infection and NbFeSOD3/4 in viral infection, we measured the SOD activity and the transcript level of NbFeSOD3/4 during CGMMV infection or CP expression. The results showed that SOD activity and the transcript level of NbFeSOD3/4 were significantly increased during CGMMV infection or CP expression (Figure 5e–h). We used TRV VIGS to silence NbFeSOD3/4 (Figure 5i). As expected, NbFeSOD3/4‐silenced plants developed less severe viral symptoms in upper leaves than TRV:00‐treated plants (Figure 5j). In agreement with this observation, lower levels of viral RNAs and proteins were found in NbFeSOD3/4‐silenced plants than in control plants at 7 dpi (Figure 5k,l). These results demonstrated that CGMMV infection and CP expression could induce the accumulation of NbFeSOD3/4 and the activity of SOD, and silencing NbFeSOD3/4 inhibited the sensitivity of plants to CGMMV infection.

3. Discussion

3.1. CGMMV CP Hijacks Mitochondrial ATPδ to Facilitate Viral Infection

Viral infection usually triggers plant immune responses and causes ROS bursts. However, ROS either resist or facilitate viral infection. For instance, ROS can have a negative impact on the accumulation of citrus tristeza virus (Sun and Folimonova 2019) and citrus leprosis virus C (Arena et al. 2020). In addition, ROS can enhance the accumulation of several viruses, such as barley yellow dwarf virus, bamboo mosaic virus, plum pox virus, and red clover necrotic mosaic virus (Díaz‐Vivancos et al. 2008; Hyodo et al. 2017; Lin et al. 2022; Tian et al. 2024). In our study, we used DPI (a NADPH oxidase inhibitor) to inhibit the accumulation of ROS after the inoculation with CGMMV, demonstrating that ROS can hinder CGMMV infection.

In infected cells, viruses use mitochondria (DeBlasio et al. 2018; Kopek et al. 2007) to promote their replication, as described for positive‐sense RNA viruses, using membranous structures derived from mitochondria (Xu, Huang, and Nagy 2012; Lee et al. 2022). In this study, the mitochondrial protein ATPδ was shown to be targeted by CGMMV CP (Figure 1), presumably to promote viral infection in host cells. Previous studies have reported that certain mitochondrial ATP synthases are involved in viral infection in plants (He et al. 2023; Gellért et al. 2018). For example, the capsid protein of a mutant strain of cucumber mosaic virus (CMV‐R3E79R) was shown to strongly interact with the F1 complex of ATP synthase in mitochondria and chloroplasts. CMV‐R3E79R causes necrosis in systemic leaves by lethally blocking the rotation of the ATP synthase F1 motor complex, leading to cell apoptosis and finally plant death (Gellért et al. 2018). The OSCP subunit of apple mitochondrial ATP synthase was shown to facilitate apple necrotic mosaic virus (ApMV) infection (He et al. 2023). In our study, CGMMV CP interacted with mitochondrial ATPδ and hijacked a portion of ATPδ, preventing its localization to the mitochondria (Figure 1). ATPδ supported viral infection (Figures 2 and 3), whereas silencing ATPδ caused dwarfing and leaf chlorosis followed by necrosis of the leaves and almost complete immunity to viral infection (Figure 2). Therefore, silencing ATPδ may facilitate the ROS burst, thereby inhibiting viral infection (Figure 4). DAB and NBT staining results suggested that ATPδ specifically regulates O2 − accumulation without involving H2O2 (Figure 4). Together, these results indicated that CGMMV uses CP to hijack ATPδ and inhibit ROS accumulation to promote viral infection.

3.2. ATPδ Regulates NbFeSODs Expression to Enhance O2 − Quenching

Superoxide (O2 −) is highly reactive and unstable and is dismutated either spontaneously or, more rapidly, by the action of SOD, which catalyses its conversion to H2O2 (Waszczak, Carmody, and Kangasjärvi 2018). SODs play an important role in the balance between oxidation and oxidation resistance. Based on their metal co‐factors, plant SODs are classified into three groups, MnSODs, FeSODs and CuZnSODs, which are found at different sites of O2 − production (Alscher, Erturk, and Heath 2002). Although FeSODs are typically located in chloroplasts (Myouga et al. 2008), a cytosolic subfamily of FeSODs has been found in soybean and cowpea tissues (Asensio et al. 2012). MnSODs (Alscher, Erturk, and Heath 2002) are located in the mitochondria and peroxisomes, and CuZnSODs (Lightfoot, McGrann, and Able 2017) are located in chloroplasts, the cytosol, and the nucleus. In our study, the overexpression of ATPδ induced SOD activity (Figure 5c), but amongst the different types of SODs detected in N. benthamiana, only NbFeSOD3/4 expression was decreased in ATPδ‐silenced plants and increased in OE ATPδ plants (Figure 5a,b). The expression levels of NbMnSODs and NbCuZnSODs were downregulated independent of ATPδ. Thus, ATPδ was shown to specifically regulate the expression of NbFeSODs, but not NbMnSODs or NbCuZnSODs. Therefore, ATPδ may be involved in maintaining ROS levels across different compartments. The expression of other ROS‐scavenging enzymes did not change in OE ATPδ plants. We speculated that ATPδ upregulates the expression of NbFeSODs to induce SOD, thereby enhancing O2 − quenching. Simultaneously, CGMMV infection and CP expression also trigger this process that induces the accumulation of NbFeSOD3/4 and the activity of SOD. Silencing NbFeSOD3/4 inhibited CGMMV infection. We suppose that CGMMV CP interacts with ATPδ and alters the localization of ATPδ to induce the transcription of NbFeSOD3/4 and the activity of SOD to quench ROS. In addition, in the previous study, CGMMV CP could interact with MP, but the role of MP in this process requires further research to confirm.

In conclusion, CGMMV uses CP to hijack ATPδ, which in the absence of virus regulates the expression of NbFeSODs to induce SOD activity and thus O2 − quenching (Figure 6).

FIGURE 6.

FIGURE 6

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A proposed working model. CGMMV uses coat protein (CP) to hijack ATPδ to facilitate CGMMV infection, by specifically regulating the expression of NbFeSOD3/4 to induce superoxide dismutase (SOD) activity, which in turn quenches O2 − and thereby reduces reactive oxygen species (ROS) accumulation to promote CGMMV infection.

4. Experimental Procedures

4.1. Plant Materials and Virus Inoculation

Nicotiana benthamiana WT plants were grown in pots in a growth room at 25°C with 60% humidity under a 16‐h/8‐h light/dark photoperiod. CGMMV was obtained from a previously published source and maintained in our laboratory (Shi et al. 2023). Four to six leaves of wild‐type and OE ATPδ transgenic N. benthamiana were inoculated mechanically with the virus according to the classical method. For agroinfiltration, Agrobacterium tumefaciens GV3101 carrying individual infectious viral clones was suspended in infiltration buffer (10 mM MgCl2, 10 mM MES, 200 μM acetosyringone, pH 5.6) to an optical density at 600 nm (OD600) of 1, incubated at room temperature for 2–4 h, and then infiltrated into N. benthamiana leaves using 1‐mL needleless syringes.

4.2. Agrobacterium Infiltration

For A. tumefaciens ‐mediated transient expression, strain GV3101 containing the expression vector was grown overnight at 28°C, pelleted, resuspended in infiltration buffer, and incubated at room temperature for 4 h. N. benthamiana leaves were infiltrated with A. tumefaciens cultures that had reached an OD600 = 0.5; at 48 h post‐inoculation (hpi), the leaves were detached for further analysis. For co‐infiltrations, equal volumes of individual Agrobacterium cultures were mixed.

4.3. Total RNA Extraction and RNA Analysis

Total RNA was isolated from leaves using TRIzol reagent (TaKaRa) following the manufacturer's instructions and then treated with RNase‐free DNase I. First‐strand cDNA was synthesised using 1 μg of total RNA (per 20 μL reaction), random primers, and M‐MLV reverse transcriptase. Tenfold‐diluted cDNA products and the SYBR Green master mix (Vazyme) were used for qPCR, performed on an ABI real‐time PCR system (Applied Biosystems). The NbUBC gene (GenBank accession no. AB026056.1) was used as the internal control. Relative gene expression levels were calculated using the 2−ΔΔCt method.

4.4. Generation of Transgenic N. benthamiana Plants

Overexpression ATPδ transgenic N. benthamiana plants were generated through Agrobacterium‐mediated transformation by the vector pFGC5941. The plasmids were transformed into A. tumefaciens GV3101. The transformation of the leaf disc was performed to generate the transgenic N. benthamiana plants. We obtained five OE ATPδ lines. Then transgenic plants were transferred to soil and screened by PCR and RT‐qPCR, while we obtained T2 stable transgenic lines OE ATPδ 1–5. The primers were designed based on the TMVΩ‐ATPδ sequence, which has a translational enhancer from the tobacco mosaic virus 5′‐leader sequence in pFGC5941 vector.

4.5. Western Blotting

Plant tissues were extracted from 0.1 g in 400 μL lysis buffer (100 mM Tris–HCl pH 8.8, 60% SDS, 2% β‐mercaptoethanol). The total proteins were separated by SDS‐PAGE on 12% gels and detected with primary antibodies anti‐GFP and anti‐Myc with 1:5000 dilution (Abmart), anti‐CGMMV CP with 1:5000 dilution (prepared in our laboratory), and secondary antibodies with 1:10,000 dilution (Sigma‐Aldrich). The proteins were then visualized using the EasySee western blot kit (TransGen Biotech) and imaged on a chemiluminescence apparatus (Imager 680; Amersham Biosciences). Coomassie brilliant blue staining of the large subunit of RuBisCO was conducted as the loading control. Expression levels were calculated using ImageJ (National Institutes of Health).

4.6. ROS Inhibitor Treatment

The ROS inhibitor DPI (Sigma‐Aldrich) was used to prevent the ROS burst (Yang, Zhao, et al. 2022). DMSO (1%) was used as the control. Equal volumes of 1% DMSO and 10 μM DPI were infiltrated into a separate area of the same leaves. At 3 hpi, the leaves were inoculated with CGMMV, and virus accumulation at 24 hpi was determined by western blotting.

4.7. Confocal Laser Scanning Microscopy

A Nikon Eclipse Ti2 confocal laser scanning microscope was used to examine the subcellular localization of ATPδ–GFP and BiFC of ATPδ and CP at excitation and emission wavelengths of 488 and 490–542 nm, respectively. Mitochondria were labelled using 100 nM MitoTracker Red CM‐H2XRos (Invitrogen) at excitation and emission wavelengths of 561 and 588–648 nm, respectively.

4.8. VIGS

The TRV vectors pYL196 (TRV1) and pTRV (TRV2) were kindly provided by Dr. Yule Liu, Tsinghua University (Beijing, China) (Liu, Schiff, and Dinesh‐Kumar 2002). Host genes were silenced by expression of their partial sequences in pTRV. ATPδ was silenced using a 300‐bp fragment of ATPδ inserted into the pTRV vector, using empty pTRV vector as the control (TRV:00). The primers used for the various constructions are listed in Table S1. Silenced and control plants at 10 dpi were used for further analysis.

4.9. DAB Staining

Leaves were soaked in DAB solution (2 mg/mL, pH 3.8) in the dark at 25°C for 12 h and then transferred to boiling 95% ethanol to remove chlorophyll. After cooling, the leaves were placed in 75% ethanol for 2–3 h.

4.10. NBT Staining

Leaves were soaked in NBT solution (1 mg/mL) in the dark at 25°C for 12 h and then transferred to boiling 95% ethanol to remove chlorophyll. After cooling, the leaves were placed in 75% ethanol for 2–3 h.

4.11. SOD Activity Assays

SOD activity was measured using a kit (Beijing Solarbio Science & Technology Co. Ltd). A crude enzyme extract was prepared by mixing 0.04 g of tissue with 0.6 mL of 0.5 M phosphate buffer (pH 7.8). The supernatant, containing crude enzyme, was obtained by centrifugation at 8000 g for 10 min at 4°C. The reaction was initiated by illuminating the reaction mixture for 20 min to photochemically induce the production of superoxide, which then reacted with NBT. The absorbance of formazan, the product of NBT reduction, was recorded at 560 nm. One unit of SOD activity was defined as the amount of enzyme that caused 50% of the maximum inhibition of NBT reduction. These experiments were repeated three times.

4.12. Quantification and Statistical Analysis

Data are reported as means ± SD, calculated using GraphPad Prism software (GraphPad Software Inc.). The statistical significance of RT‐qPCR results was evaluated using Student's t test (*p < 0.05, **p < 0.01).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1. Primers used in the study.

MPP-25-e70034-s001.docx (21.1KB, docx)

Table S2. The candidate proteins by yeast two‐hybrid screen.

MPP-25-e70034-s002.docx (15.1KB, docx)

Acknowledgements

This work was funded by the National Nature Science Foundation of China (3210170372), State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro‐products (2021DG700024‐KF202321).

Funding: This work was supported by National Natural Science Foundation of China (3210170372) and State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro‐products (2021DG700024‐KF202321).

Xue Yang and Xing‐Lin Jiang contributed equally to this work.

Contributor Information

Xue Yang, Email: yangxuepphappy@126.com.

Yan Shi, Email: shiyan@henau.edu.cn.

Data Availability Statement

The data that support the finding 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

Table S1. Primers used in the study.

MPP-25-e70034-s001.docx (21.1KB, docx)

Table S2. The candidate proteins by yeast two‐hybrid screen.

MPP-25-e70034-s002.docx (15.1KB, docx)

Data Availability Statement

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

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