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. 2025 Jul 29;37(8):koaf188. doi: 10.1093/plcell/koaf188

Glutathionylation-mediated degradation of a cap-binding protein enhances Arabidopsis resistance to Plutella xylostella

Ning Lin 1,2,#, Hui Ye 3,4,#, Mengjie Zhao 5,6,#, Xingzhi Chen 7,8, Jing Ma 9,10, Chuanhong Wang 11,12, Tengyue Wang 13,14, Zhen Tao 15,16, Yibing Zhao 17,18, Qingyang Zhang 19,20, Jun Lai 21, Xinqiao Zhang 22,23, Jinghui Dong 24,25, Peijin Li 26,27,28,c,✉,d
PMCID: PMC12366552  PMID: 40729411

Abstract

The lepidopteran insect pest diamondback moth (Plutella xylostella) causes severe yield losses in cruciferous plants worldwide; therefore, there is an urgent need to characterize the genes for resistance to P. xylostella in plants and decipher their mechanisms. We previously demonstrated that inactivating NOVEL CAP-BINDING PROTEIN (NCBP), also known as RESISTANCE TO PLUTELLA XYLOSTELLA (RPX1), enhanced Arabidopsis (Arabidopsis thaliana) resistance to P. xylostella larvae, and the larval infestation caused NCBP degradation. Here, we report that MYB30-INTERACTING WD40 PROTEIN 1 (MIW1), a component of the Cul4-RING ubiquitin ligase complex, interacts with NCBP and causes its degradation through the 26S proteasome pathway. Protein interaction, degradation, and site mutagenesis assays of NCBP indicate that the glutathione transferase GSTF2 also interacts with NCBP and promotes its glutathionylation, ubiquitination, and degradation. GSTF2 and glutathionylation of NCBP enhance the interaction between MIW1 and NCBP. Moreover, consistent with the roles of GSTF2 and MIW1 in P. xylostella resistance, gstf2 and miw1 mutants were sensitive to larval infestation, whereas transgenic Arabidopsis overexpressing GSTF2 and MIW1 were more resistant to the larvae. These findings demonstrate a role for glutathionylation in regulating 26S proteasome-mediated protein degradation in plant resistance to insect pests, thus revealing the functional mechanism of NCBP in this process.

The glutathione S-transferase GSTF2 glutathionylates the cap-binding protein NCBP and promotes its ubiquitination and degradation in Arabidopsis resistance to the insect pest Plutella xylostella.

Introduction

Current management of Plutella xylostella (diamondback moth), one of the most destructive migratory lepidoptera pests of cruciferous plants worldwide, relies heavily on chemical insecticides that are harmful to the environment and human health. Under long-term field application, P. xylostella has evolved increasing resistance to insecticides (Deng et al. 2021; Li et al. 2022; Zhao et al. 2023). It is therefore a priority to mine natural resistance genes and determine their functional mechanisms to facilitate breeding for crop resistance and develop more efficient management approaches.

During co-evolution with insect pests, plants evolved various mechanisms to resist insect infestation, and they can recognize damage from herbivores and generate direct or indirect defensive mechanisms against attack (Croy et al. 2021). Direct defenses involve the physical characteristics of the plant itself, such as villi and silicon structures on the plant surface to reduce insect feeding efficiency (Andama et al. 2020); indirect defenses involve the generation of volatile compounds, which expel insect pests or recruit their natural enemies (Hu et al. 2021). Some volatiles are transmitted to neighboring plants and induce the accumulation of insect-resistant substances (Meents et al. 2019; Jing et al. 2020). Among these volatile compounds, the homoterpene (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) has been widely reported in several species (Lee et al. 2010; Sohrabi et al. 2015; Richter et al. 2016; Liu et al. 2017). Plant hormones such as jasmonic acid (JA) and salicylic acid (SA) play important roles in these processes by activating various regulatory pathways. For example, JA mediates the development of type VI trichomes and trichome terpenoid production to promote insect resistance in tomato (Solanum lycopersicum), and SA functions in resistance to rice fly and pea aphid (Kazan 2018; Hua et al. 2020; Jan et al. 2021).

Glutathione S-transferases (GSTs), which are widespread in most organisms, participate in the metabolic detoxification of exogenous chemicals and play a major role in alleviating oxidative stress. GST genes are induced by various stresses and are involved in the tolerance of various biotic and abiotic stresses (Sappl et al. 2009; Ma et al. 2018), playing overlapping and functionally diverse roles in plants. For instance, GST genes in cotton (Gossypium hirsutum) are induced or repressed in response to salt stress (Dong et al. 2016), and GST genes in fig (Ficus carica) are involved in anthocyanin biosynthesis in the fruit epidermis (Vaish et al. 2018). The class phi glutathione transferases GSTF2, GSTF8, GSTF10, and GSTF11 function as SA-binding proteins, as revealed using photoaffinity labeling and surface isoelectric resonance-based techniques, suggesting that they might function in SA signaling pathways related to plant defense (Gleason et al. 2011; Tian et al. 2012), although this has not been experimentally validated. Apart from their well-documented function in glutathione (GSH) homeostasis, GSTs may also participate in the modification of proteins, especially those related to redox and defense control (Michelet et al. 2005); the stability of glutathionylated protein is altered, but the underlying mechanism is unclear.

We recently reported that RESISTANCE TO PLUTELLA XYLOSTELLA (RPX1), also known as NOVEL CAP-BINDING PROTEIN (NCBP, AT5G18110), plays an important role in resistance to P. xylostella. Inactivation of NCBP leads to the accumulation of the terpene DMNT, which kills insects by disrupting the peritrophic matrix structure in the midgut (Chen et al. 2021, 2023); notably, NCBP can be degraded by P. xylostella infestation, which might provide a reversible strategy for resisting pests. However, the mechanism underlying NCBP degradation remains to be addressed. Here, we performed protein immunoprecipitation assays coupled with mass spectrometry (IP-MS) analysis of NCBP-GFP in NCBPpro:NCBP-GFP ncbp and identified two NCBP-interacting proteins: MIW1 (MYB30-INTERACTING WD40 PROTEIN 1) and GSTF2. GSTF2 glutathionylated NCBP at specific cysteine sites, promoting its degradation in a 26S proteasome–dependent pathway via MIW1. Our findings establish the role of a GSTF2-NCBP-MIW1 regulatory module in Arabidopsis resistance to P. xylostella and demonstrate a mechanism linking protein glutathionylation and degradation.

Results

NCBP interacts with MIW1, a component of the Cul4-RING E3 ubiquitin ligase complex

We have previously reported that the Arabidopsis ncbp mutant exhibits enhanced resistance to P. xylostella and that NCBP is degraded in response to P. xylostella infestation and mechanical wounding (Chen et al. 2023). To investigate the mechanism underlying NCBP degradation, we identified MIW1 (AT1G52730), a component of the CUL4-RING E3 ubiquitin ligase complex (Zheng et al. 2012; Zhan et al. 2023), as a potential NCBP-interacting protein. This interaction was revealed through immunoprecipitation followed by mass spectrometry (IP-MS) of NCBPpro:NCBP-GFP ncbp transgenic seedlings (Supplementary Table S1). To validate the interaction between NCBP and MIW1, we performed yeast two-hybrid (Y2H) assays using NCBP fused to the GAL4 DNA-binding domain (BD-NCBP) and MIW1 fused to the GAL4 activation domain (AD-MIW1). Only yeast cells co-expressing BD-NCBP and AD-MIW1 grew on synthetic dropout medium lacking Trp, Leu, Ade, and His (SD -Trp/-Leu/-Ade/-His), confirming their interaction in yeast (Fig. 1A). Subsequently, we performed bimolecular fluorescence complementation (BiFC) assays by co-transfecting Arabidopsis protoplasts with plasmids encoding MIW1 fused to the N-terminal region of yellow fluorescent protein, YFP (MIW1-YNE) and NCBP fused to the C-terminal region of YFP (NCBP-YCE). The BiFC results revealed positive interaction signals, further supporting the association between MIW1 and NCBP (Fig. 1B). To further corroborate these findings, we employed a split-luciferase (Split-LUC) system in Nicotiana benthamiana cells. The assay yielded similar positive results, confirming the interaction between MIW1 and NCBP in vivo (Fig. 1C). We then performed a co-immunoprecipitation (Co-IP) assay using Arabidopsis seedlings expressing MIW1-MYC and NCBP-FLAG, derived from a cross between transgenic MIW1pro:MIW1-MYC and 35Spro:NCBP-FLAG lines. The Co-IP experiment demonstrated that MIW1-MYC could be successfully pulled down by NCBP-FLAG, further validating their interaction (Fig. 1D). Additionally, recombinant NCBP and MIW1 were expressed and purified from Escherichia coli for in vitro pull-down assays. The results showed direct binding between NCBP and MIW1, reinforcing the evidence for their physical interaction (Fig. 1E). In addition, in Arabidopsis protoplast cells, MIW1-RFP and NCBP-GFP proteins could co-localize as evidenced by the overlapping of fluorescence signals (Fig. 1F). Together, these results provide robust evidence that MIW1 interacts with NCBP, suggesting a potential role in the regulation of NCBP stability and function in the context of plant defense mechanisms.

Figure 1.

Figure 1.

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NCBP interacts with MIW1 in vitro and in vivo. A) NCBP and MIW1 interacts in yeast (Saccharomyces cerevisiae) cells. The interaction of T and P53 was used as a positive control. pGBKT7 (empty bait vector, BD) with MIW1 and pGADT7 (empty prey vector, AD) with NCBP were used as negative controls. B and C) Bimolecular fluorescence complementation (BiFC) (B) and luciferase (LUC) complementation imaging (LCI) (C) assays show that NCBP interacts with MIW1 in Arabidopsis protoplasts cells (B) and Nicotiana benthamiana cells (C). D) Co-immunoprecipitation (Co-IP) assay showing the association of NCBP with MIW1 in stable transgenic Arabidopsis lines co-expressing NCBP-FLAG and MIW1-MYC. E) In vitro pull-down assay confirming direct interaction between NCBP and MIW1. MIW1 was fused to MBP-His and NCBP to GST. MBP-His was used as a negative control. M: protein marker. F) Subcellular localization analysis showing co-localization of MIW1-RFP and NCBP-GFP in Arabidopsis protoplasts. Scale bars, 5 μm.

MIW1 contributes to Arabidopsis resistance against P. xylostella in a dependence on NCBP

To investigate the role of MIW1 in response to P. xylostella, we first examined its transcriptional dynamics in wild-type Col-0 following larval infestation. MIW1 was significantly induced at 12 h post-infestation and remained moderately elevated at 24 h (Supplementary Fig. S1A). MIW1 expression was also rapidly induced by mechanical wounding, with a time-dependent increase in protein accumulation (Supplementary Fig. S1, B and C). A T-DNA insertion mutant miw1 (SALK_130999C) exhibited markedly reduced MIW1 transcript levels, as confirmed by RT-qPCR (Supplementary Fig. S1D, Supplementary Table S2). To explore MIW1 function, we generated transgenic lines overexpressing MIW1 under the CaMV 35S promoter (35Spro:MIW1) (Supplementary Fig. S1E). In no-choice feeding assays, P. xylostella larvae fed on miw1 mutants showed significantly greater weight gain, survival, and pupation rates compared to those fed on Col-0, whereas the 35Spro:MIW1 lines had the opposite phenotype (Fig. 2, A to C). In two-choice preference assays, P. xylostella larvae preferred miw1, but not 35Spro:MIW1, to Col-0 (Fig. 2D). To investigate the molecular basis of this resistance, we analyzed the expression of PENTACYCLIC TRITERPENE SYNTHASE 1 (PEN1), a gene upregulated in ncbp mutants, which encodes a key DMNT biosynthetic enzyme (Chen et al. 2023). PEN1 transcript levels were significantly downregulated in miw1 and upregulated in 35Spro:MIW1 plants compared to Col-0 (Fig. 2E). Consistently, solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS) revealed reduced DMNT levels in miw1 and elevated levels in 35Spro:MIW1 (Fig. 2F). To investigate the genetic relationship between NCBP and MIW1, miw1 was crossed with ncbp to obtain the miw1 ncbp double mutant, which was tested for P. xylostella infestation resistance along with Col-0, ncbp and miw1. In no-choice feeding assays, analysis of P. xylostella weight, survival and pupation rates showed that miw1 ncbp had a similar negative effect on P. xylostella as ncbp (Fig. 3, A to C). In preference assays, P. xylostella larvae preferred miw1, but not ncbp and miw1 ncbp compared to Col-0 (Fig. 3D). Moreover, PEN1 expression in miw1 ncbp was more similar to ncbp and much higher than in miw1 (Fig. 3E). Overall, these findings suggest that MIW1-mediated resistance to P. xylostella is dependent on functional NCBP, acting within the same genetic pathway to regulate downstream defense responses.

Figure 2.

Figure 2.

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MIW1 contributes to Arabidopsis resistance against P. xylosella. A to D) Effects of MIW1 on P. xylostella performance: larval weight (A), survival rate (B), pupation rate (C), and preference (D) on Col-0, miw1 mutant, and 35Spro:MIW1 overexpression lines. E) Relative expression of PEN1 in Col-0, miw1 and 35Spro:MIW1 plants. F) DMNT content in Col-0, miw1, and 35Spro:MIW1 plants, as determined by SPME-GC-MS. Data are presented as means ± SE (n = 3). In (A, B, and D), different letters represent significant differences that were determined by two-way ANOVA followed by Tukey's multiple comparison test (P < 0.05). In (C, E, and F), one-way ANOVA with Tukey's test was used (P < 0.05); different letters indicate statistically significant differences.

Figure 3.

Figure 3.

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MIW1 confers resistance to P. xylostella in the dependence on NCBP. A to D) Comparison of larval weight (A), survival rate (B), pupation rate (C) and preference (D) between P. xylostella larvae fed on Col-0, miw1, ncbp, and miw1 ncbp seedlings. E) Relative expression levels of PEN1 in Col-0, miw1, ncbp and miw1 ncbp. Data are presented as the mean ± SE (n = 6 in A, B, and C), (n = 3 in D and E). In (A, B, and D), different letters represent significant differences determined by two-way ANOVA followed by Tukey's multiple comparison test (P < 0.05). In (C and E), significance was determined by one-way ANOVA with Tukey's multiple comparison test (P < 0.05). Different letters indicate statistically significant differences.

MIW1 reduces the stability of NCBP

MIW1 is a component of the Cul4 E3 ligase protein complex; we hypothesized that MIW1 may regulate the stability of NCBP. We transiently transformed N. benthamiana leaves with Agrobacterium cells containing the NCBP-LUC construct with either empty vector or MIW1 overexpression driven by the CaMV 35S promoter (35Spro:MIW1) for luciferase imaging. The LUC signals from leaves transiently transformed with NCBP-LUC and 35Spro:MIW1 showed significantly reduced luminescence compared to those co-expressing NCBP-LUC with the empty vector (Fig. 4, A and B). Consistently, luciferase enzyme activity was significantly higher in miw1 protoplasts than in Col-0 (Fig. 4C). To confirm this effect, we co-transformed Col-0 protoplasts with NCBP-GFP and either the empty vector or 35Spro:MIW1. The fluorescent NCBP-GFP signal and protein abundance were significantly weaker than in protoplasts co-transformed with NCBP-GFP and 35Spro:MIW1 vs. NCBP-GFP with empty vector (Supplementary Fig. S2, A to C). Insect infestation causes mechanical damage in plants, and our previous study found that wounding treatment–induced NCBP degradation similar to the finding in P. xylostella infestation (Chen et al. 2023). We therefore examined NCBP degradation in NCBPpro:NCBP-GFP, NCBPpro:NCBP-GFP miw1, and NCBPpro:NCBP-GFP 35SproMIW1-MYC without or after 1 h of wounding treatment, and found that NCBP protein degradation was attenuated in NCBPpro:NCBP-GFP miw1 and accelerated in NCBPpro:NCBP-GFP 35SproMIW1-MYC (Fig. 4D). To test whether this degradation is dependent on the 26S proteasome, we treated seedlings with 50 μM MG132 (Z–Leu–Leu–Leu–al), a proteasome inhibitor. MG132 effectively blocked NCBP degradation (Fig. 4E), supporting a proteasome-mediated mechanism. Furthermore, ubiquitination of NCBP increased upon wounding, reinforcing the conclusion that NCBP was degraded via the ubiquitin–26S proteasome pathway (Fig. 4F). To further examine if MIW1 degrades NCBP via the 26S proteasome, we assessed NCBP ubiquitination levels in NCBPpro:NCBP-GFP, NCBPpro:NCBP-GFP miw1 and NCBPpro:NCBP-GFP 35Spro:MIW1-MYC seedlings following treatment with 50 μM MG132. Immunoprecipitation and immunoblot analysis revealed that NCBP ubiquitination was reduced in the miw1 background, whereas it was markedly enhanced in the 35Spro:MIW1 overexpression line (Fig. 4G). These results indicate that MIW1 is a pivotal regulatory factor involved in NCBP ubiquitination and degradation. In addition, we analyzed whether NCBP expression was affected by MIW1 and found that NCBP expression level was upregulated in miw1 (Supplementary Fig. S3), suggesting that MIW1 regulates NCBP in multiple ways.

Figure 4.

Figure 4.

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MIW1 reduces NCBP stability. A and B) NCBP-LUC signals are reduced by MIW1 overexpression. The results of quantitative analysis are shown in (B) (n = 8). EV: empty vector. C) NCBP-LUC activity is higher in miw1 protoplast cells than in Col-0 (n = 4). D) Wounding-induced NCBP degradation was attenuated in NCBPpro:NCBP-GFP miw1 and enhanced in NCBPpro:NCBP-GFP 35Spro:MIW1-MYC compared with NCBPpro:NCBP-GFP. E) Wound-induced NCBP degradation was inhibited by 50 μM MG132 treatment. F) Wounding treatment induced the ubiquitination level of NCBP in NCBPpro:NCBP-GFP. G) Ubiquitination of NCBP-GFP was reduced in NCBPpro:NCBP-GFP miw1 and enhanced in NCBPpro:NCBP-GFP 35Spro:MIW1-MYC compared with NCBPpro:NCBP-GFP. In (B and C), box plot: lower vertical bar, sample minimum; lower box, lower quartile; middle line, median; upper box, upper quartile; upper vertical bar, sample maximum, data are shown as the mean ± SE, significant differences are indicated by asterisks (**P < 0.01, ns, not significant; Student's t-test).

GSTF2 interacts with NCBP and is involved Arabidopsis resistance to P. xylostella

To investigate the regulatory mechanisms underlying NCBP degradation, we identified and focused on a glutathione transferase protein, GSTF2 (AT4G02520), a known salicylic acid-binding protein (SABP) (Gleason et al. 2011; Tian et al. 2012), from the IP-MS data using NCBPpro:NCBP-GFP transgenic plants. This interaction was further supported by a reciprocal IP-MS analysis using the GSTF2pro:GSTF2-FLAG line (Supplementary Table S1). To validate their interactions, we performed the Y2H assay with GSTF2 and NCBP fused to the GAL4 DNA-binding domains, respectively. Only yeast cells co-expressing both proteins grew on synthetic dropout medium, confirming a physical interaction in yeast (Fig. 5A). In LCI assays, N. benthamiana leaves co-infiltrated with NCBP-nLUC (NCBP fused to the N-terminal half of LUC) and cLUC-GSTF2 (GSTF2 fused to the C-terminal half of LUC) exhibited strong luminescence, indicating reconstitution of luciferase due to protein interaction (Fig. 5B). Similarly, BiFC assays using Arabidopsis protoplasts co-transformed with GSTF2-YNE and NCBP-YCE confirmed in vivo interaction (Fig. 5C). Further in vivo Co-IP assay were performed using Arabidopsis seedlings containing GSTF2-MYC and NCBP-FLAG generated from a cross between transgenic GSTF2pro:GSTF2-MYC and 35Spro:NCBP-FLAG seedlings, which showed that GSTF2-MYC could be pulled down by NCBP-FLAG (Fig. 5D). We finally expressed and purified NCBP and GSTF2 using an Escherichia coli protein expression system for in vitro pull-down assay, finding that these two proteins directly interacted (Fig. 5E). In agree with their interaction, confocal microscopic imaging showed that GSTF2-RFP and NCBP-GFP co-localized in Arabidopsis protoplast cells (Fig. 5F).

Figure 5.

Figure 5.

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GSTF2 interacts with NCBP and is involved in Arabidopsis resistance to P. xylostella. A) NCBP interacts with GSTF2 in yeast cells. The interaction of T and P53 was used as a positive control. pGBKT7 (empty bait vector, BD) with NCBP and pGADT7 (empty prey vector, AD) with GSTF2 were used as negative controls. B and C) Luciferase (LUC) complementation imaging (LCI) (B) and bimolecular fluorescence complementation (BiFC) (C) show that NCBP interacts with GSTF2 in N. benthamiana leaves (B) and Arabidopsis protoplast cells (C). D) Co-immunoprecipitation (Co-IP) assays show NCBP interacts with GSTF2 in vivo. E) Protein pull-down assays show that NCBP interacts with GSTF2 in vitro. F) GSTF2-RFP and NCBP-GFP co-localize in Arabidopsis protoplast cells. Scale bars: 5 μm. G to J) Comparison of larval weight (G), survival (H), pupation (I) and preference (J) between P. xylostella larvae fed on Col-0, gstf2, and 35Spro:GSTF2 transgenic seedlings. K) Comparison of PEN1 expression analysis in Col-0, gstf2 and 35Spro:GSTF2 plants. L) DMNT content in Col-0, gstf2, 35Spro:GSTF2 plants. DMNT: (3E)-4,8-dimethyl-1,3,7-nonatriene. In (G to L), data are shown as the mean ± SE (n = 3). In (G to J), 15 P. xylostella larvae were used for each replicate. In (G, H, and J), different letters represent significant differences determined by two-way ANOVA with Tukey's multiple comparison test (P < 0.05). In (I, K, and L), different letters represent significant differences determined by one-way ANOVA with Tukey's multiple comparison test (P < 0.05).

Then, we set up experiments to investigate the biological role GSTF2 P. xylostella resistance. Apart from ordering a gstf2 (a T-DNA insertion mutant: SALK_030186C, Supplementary Fig. S4A) mutant, we also made a construct harboring GSTF2 driven by the CaMV 35S promoter (35Spro:GSTF2) to generate GSTF2 overexpression transgenic lines (Supplementary Fig. S4B) for bioassays with P. xylostella larvae. After a longer feeding time in no-choice assays, larvae fed on gstf2 were heavier than those on Col-0 (Fig. 5G), and the larvae fed on gstf2 showed higher survival and pupation rates than those fed on Col-0 (Fig. 5, H and I), Conversely, the 35Spro:GSTF2 lines had the opposite effects (Fig. 5, G to I), suggesting that GSTF2 represses larval growth. In preference assays, the larvae preferred gstf2, but not 35Spro:GSTF2 compared to Col-0 (Fig. 5J). Furthermore, we hypothesized that GSTF2 expression might respond to larval infestation and sampled P. xylostella-infested Col-0 at 0, 6, 12, and 24 h after infestation for assessing GSTF2 expression: GSTF2 was significantly upregulated after 12–24 h (Supplementary Fig. S4C). Similarly, GSTF2 expression was wound-inducible and showed a corresponding temporal accumulation of its protein products (Supplementary Fig. S4, D and E). Meanwhile, we also analyzed whether NCBP expression was regulated by GSTF2 and found that NCBP expression was unchanged in gstf2 mutants, indicating that GSTF2 does not regulate NCBP at the transcriptional level (Supplementary Fig. S5). Expression analysis of PEN1, a gene involved in DMNT biosynthesis, showed downregulation in gstf2 and upregulation in 35Spro:GSTF2 plants (Fig. 5K). Consistently, DMNT accumulation followed the same trend (Fig. 5L). These findings suggest that GSTF2 contributes to resistance against P. xylostella by promoting PEN1-mediated DMNT production.

GSTF2 affects P. xylostella larvae dependent on NCBP

We crossed gstf2 with ncbp and used the gstf2 ncbp double mutants for insect resistance assays. Analysis of P. xylostella weight, survival, pupation, and preference rates indicated that gstf2 ncbp negatively affected P. xylostella at a level similar to ncbp (Fig. 6, A to D). Both ncbp and gstf2 ncbp significantly inhibited P. xylostella growth, whereas larvae fed on gstf2 were bigger than those fed on these lines or Col-0 (Fig. 6E). Comparative analysis of PEN1 expression in gstf2, ncbp, and gstf2 ncbp seedlings together with Col-0 showed that the PEN1 expression in gstf2 ncbp was more similar to ncbp but much higher than in gstf2 (Fig. 6F). These results suggest that GSTF2 represses P. xylostella larvae indispensable to NCBP.

Figure 6.

Figure 6.

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GSTF2 confers Arabidopsis resistance to P. xylostella in the dependence on NCBP. A to D) Comparison of larval weight (A), survival rate (B), pupation rate (C), and preference (D) between P. xylostella larvae fed on Col-0, gstf2, ncbp, and gstf2 ncbp plants. E) Representative images of P. xylostella larvae fed on Col-0, ncbp, gstf2, and gstf2 ncbp. Images were digitally extracted for comparison. F) Relative PEN1 expression analysis in Col-0, gstf2, ncbp, and gstf2 ncbp. In (A, B, C, D, and F), data are shown as the mean ± SE (n = 3). In (A, B, and D), different letters represent significant differences determined by two-way ANOVA with Tukey's multiple comparison test (P < 0.05). In (C and F), different letters represent significant differences determined by one-way ANOVA with Tukey's multiple comparison test (P < 0.05).

To check whether GSTF2 influences NCBP stability, we introduced Agrobacterium cells containing the 35Spro:NCBP-LUC construct with either the empty vector or the 35Spro:GSTF2 vector into N. benthamiana leaves via infiltration to examine the effect of GSTF2 on NCBP based on LUC activity. The LUC signals from 35Spro:NCBP-LUC and 35Spro:GSTF2 were significantly weaker than that of 35Spro:NCBP-LUC with empty vector (Fig. 7, A and B). Meanwhile, 35Spro:NCBP-LUC plasmid was transformed into Col-0 and gstf2 protoplasts, which showed that the NCBP-LUC activity of gstf2 was significantly higher than in Col-0 (Fig. 7C). We also co-transformed Col-0 protoplasts with NCBP-GFP and either empty vector or 35Spro:GSTF2. The fluorescent NCBP-GFP signal and protein abundance were significantly less in protoplasts co-transformed with NCBP-GFP and 35Spro: GSTF2 vs. NCBP-GFP with empty vector (Supplementary Fig. S6, A to C). We then transformed NCBP-GFP plasmid into miw1 protoplasts with 35Spro:GSTF2 or the corresponding empty vector. It turned out that in miw1 background, the NCBP-GFP fluorescent signal and protein abundance were not significantly different between the combination with empty vector and 35Spro:GSTF2 (Supplementary Fig. S6, D to F). These results suggest that MIW1 is required for GSTF2 to inhibit the abundance of NCBP-GFP protein.

Figure 7.

Figure 7.

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GSTF2 and wounding promote the interaction of NCBP with MIW1, thereby affecting its stability. A and B) Overexpressing GSTF2 represses NCBP-LUC signals in N. benthamiana leaves. Quantitative analysis of the LUC images is shown in (B) (n = 7). EV: empty vector. C) Weaker NCBP-LUC signals are present in Col-0 than in gstf2 protoplast cells (n = 4). D) Wounding treatments induce NCBP degradation, which is attenuated in the NCBPpro:NCBP-GFP gstf2. E) Luciferase (LUC) complementation imaging (LCI) assays demonstrate that GSTF2 promotes the interaction of NCBP with MIW1 in N. benthamiana leaves. F) Co-immunoprecipitation (Co-IP) assays indicate that GSTF2 promotes the interaction of NCBP and MIW1. G and H) Bimolecular fluorescence complementation (BiFC) assays demonstrates that the interaction of NCBP and MIW1 was weaker in gstf2 protoplasts than that in Col-0 (n = 207). Quantitative analysis of the YFP fluorescence is shown in (H). I) Co-immunoprecipitation (Co-IP) assays in stable transgenic Arabidopsis lines show that wounding enhances the interaction between NCBP and MIW1. In (H), box plot: lower vertical bar, sample minimum; lower box, lower quartile; middle line, median; upper box, upper quartile; upper vertical bar, sample maximum. In (B and C), data are shown as the mean ± SE. In (B, C, and H), significant differences are indicated by asterisks (**P < 0.01, ***P < 0.001; Student's t-test).

To obtain more in vivo evidences, we crossed NCBPpro:NCBP-GFP transgenic Arabidopsis to gstf2 and used the hybridization seedlings for NCBP abundance analysis. After 1-hour wounding treatment, NCBP level in NCBPpro:NCBP-GFP gstf2 was much less affected, in contrast to the finding in NCBPpro:NCBP-GFP (Fig. 7D). We reasoned that GSTF2 may regulate NCBP by affecting MIW1 interactions with NCBP. To test this hypothesis, we co-infiltrated N. benthamiana leaves with Agrobacterium cells containing MIW1-cLUC and NCBP-nLUC with empty vector or 35Spro:GSTF2, the imaging results showed that the LUC signals were significantly stronger in the leaves co-infiltrated with 35Spro:GSTF2 vs. the empty vector (Fig. 7E). We also co-injected Agrobacterium cells containing 35Spro:MIW1-MYC and 35Spro:NCBP-GFP as well as empty vector or 35Spro:GSTF2-FLAG with a FLAG overexpression tag into N. benthamiana leaves and performed Co-IP assay, finding that GSTF2 enhanced the interaction between NCBP and MIW1 (Fig. 7F). Moreover, consistently, the BiFC assays using MIW1-YNE and NCBP-YCE plasmids showed that the interaction signals of YFP in gstf2 protoplasts were significantly weaker than in those in Col-0, suggesting that inactivation of GSTF2 attenuated the interaction between NCBP and MIW1 (Fig. 7, G and H). Consistent with these observations, wounding of transgenic seedlings expressing NCBPpro:NCBP-GFP 35Spro:MIW1-MYC enhanced the interaction between NCBP and MIW1 (Fig. 7I). Together, these results suggest that GSTF2, in conjunction with wounding signals, promotes the destabilization of NCBP by facilitating its interaction with MIW1.

Glutathionylation of NCBP by GSTF2 affects its stability and confers resistance to P. xylostella

Since GSTF2 is a glutathione transferase and the above results indicate it interacts with NCBP and negatively affects its stability, we hypothesized that GSTF2 might modify NCBP through glutathionylation. To assess this, glutathionylated peptides were identified in raw IP-MS data from NCBPpro:NCBP-GFP transgenic Arabidopsis seedlings. As expected, NCBP contained several glutathionylated peptides (Supplementary Fig. S7, A and B). In silico prediction using the pCysMod platform (http://pcysmod.omicsbio.info/index.php) further identified cysteine 117 (C117) of NCBP as a candidate glutathionylation site (false positive rate < 5%) (Supplementary Fig. S7C). To validate this modification in vivo, NCBPpro:NCBP-GFP seedlings were subjected to wounding for 1 h, followed by immunoblotting with an anti-GSH antibody. Wounding significantly enhanced NCBP glutathionylation and corresponded with decreased NCBP-GFP protein levels (Fig. 8A). However, these effects were abolished in the gstf2 background, indicating that GSTF2 is required for NCBP glutathionylation and its subsequent degradation (Fig. 8A). Transgenic plants of 35Spro:NCBPC117A-GFP were generated to verify the effect of the mutation C117 to Alanine (A) on the glutathionylation modification of NCBP by immunoblotting analysis (Li et al. 2021). The western blotting assays using an anti-GSH antibody showed that glutathionylation on NCBP in 35Spro:NCBP-GFP increased upon 1-hour wounding treatment, which is companied with the reduction of NCBP level, whereas in contrast, the glutathionylation and protein level of NCBP did not show much changes in 35Spro:NCBPC117A-GFP (Fig. 8B). In protein ubiquitination assays, we found the ubiquitination levels of NCBP were decreased in both NCBP-GFP gstf2 and NCBPC117A-GFP compared with those in NCBP-GFP, suggesting GSTF2 and the amino acid C117 are important for NCBP ubiquitination (Fig. 8C). Moreover, BiFC and Co-IP assays showed that the NCBPC117A-MIW1 interaction was significantly reduced compared to the NCBP-MIW1 interaction (Fig. 8, D to F). These results indicate that the glutathionylation modification of NCBP is important for its stability.

Figure 8.

Figure 8.

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Glutathionylation modification on NCBP affects its stability. A) Wounding treatments induce NCBP degradation and GSH modification, which is attenuated in the NCBPpro: NCBP-GFP gstf2 background. B) Wounding-induced degradation and GSH modification of NCBP are attenuated by the C117A mutation of NCBP. ACTIN was used as a control. C) Ubiquitination levels of NCBP in 35Spro:NCBP-GFP gstf2 and 35Spro:NCBPC117A-GFP seedlings were attenuated compared with those in 35Spro:NCBP-GFP. Col-0 was used as a control for immunoblotting analysis of NCBP ubiquitination. D and E) The C117A mutation of NCBP attenuates the interaction between NCBP and MIW1 in bimolecular fluorescence complementation (BiFC) assays. The results of quantitative analysis are shown in (E) (n = 314). F) Co-immunoprecipitation (Co-IP) assays show that the C117A mutation of NCBP attenuates the interaction between NCBP and MIW1. In (E), significant differences are indicated by asterisks (***P < 0.001; Student's t-test).

In bioassays, feeding and preference assays were carried out to examine the biological role of the C117A mutation in NCBP. In no-choice feeding assays, the larvae fed on 35Spro:NCBPC117A-GFP showed higher weight, survival and pupation rates than those on 35Spro:NCBP-GFP (Fig. 9, A to C). Following two hours of larval pretreatment, P. xylostella tended to select transgenic 35Spro:NCBPC117A-GFP over 35Spro:NCBP-GFP plants (Fig. 9, D to F). These results indicate that C117A of NCBP is important for Arabidopsis resistance to P. xylostella larvae.

Figure 9.

Figure 9.

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C117A mutation of NCBP affects Arabidopsis resistance to P. xylostella larvae. A to C) Comparison of weight (A), survival rate (B), and pupation rate (C) of larvae fed on 35Spro:NCBP-GFP and 35Spro:NCBPC117A-GFP transgenic Arabidopsis seedlings that had been pre-infested with P. xylostella larvae. D to F) Comparison of preference of P. xylostella larvae between 35Spro:NCBP-GFP and 35Spro:NCBPC117A-GFP seedlings that had been pre-infested with P. xylostella larvae. In (A to F), the data are shown as the mean ± SE (n = 3). In (A, B, D, E, and F), different letters represent significant differences determined by two-way ANOVA with Tukey's multiple comparison test (P < 0.05). In (C), different letters represent significant differences determined by one-way ANOVA with Tukey's multiple comparison test (P < 0.05).

Discussion

Protein glutathionylation exists in various organisms, but the role and mechanism of this modification on the target proteins remains unclear in plants. Through the analysis of NCBP complex in Arabidopsis, we report that GSTF2 glutathionylates NCBP, which in turn promotes its interaction with MIW1, leading to NCBP degradation and resistance to P. xylostella larvae.

We previously reported that the Arabidopsis ncbp mutant has improved resistance to P. xylostella and that NCBP is degraded by P. xylostella infestation and wounding treatment (Chen et al. 2023). The degradation character of NCBP is important for its role in P. xylostella resistance, as NCBP inactivation activates PEN1 expression, leading to DMNT accumulation. This compound kills P. xylostella larvae by destroying the peritrophic matrix structure in the midgut (Chen et al. 2021, 2023). Here, we further report that NCBP degradation is established through ubiquitination mediated by the 26S proteasome pathway. In Y2H, BiFC, pull-down, and Co-IP assays, NCBP physically interacted with MIW1, a component of the Cul4-RING complex linked to the 26S protein degradation system, in vivo and in vitro (Fig. 1). In agreement, NCBP was more highly ubiquitinated upon wounding treatment, and the ubiquitination level in miw1 and MIW1 overexpression background was reduced and upregulated, respectively (Fig. 4, F and G), which is consistent with protein degradation via the 26S proteasome. In miw1, NCBP degradation upon wounding was slower than in Col-0 control, and degradation was inhibited by MG132 treatments (Fig. 4, D and E). MIW1 overexpression reduced the abundance of NCBP (Fig. 4, A, B and D; Supplementary Fig. S2), and the downstream regulatory target of NCBP, PEN1 expression, were upregulated in 35Spro:MIW1 and downregulated in miw1 (Fig. 2E), and meanwhile the effective compound DMNT changed consistently in these plant materials (Fig. 2F). As found for the influence of the ncbp mutant on the resistance in the P. xylostella (Chen et al. 2023), MIW1 overexpression decreased and knockout of MIW1 increased the weight, survival, pupation and preference rates of P. xylostella larvae compared to those of larvae fed on Col-0 (Fig. 2, A to D), and further genetic analysis using miw1 ncbp double mutants showed that MIW1 functions in the dependence on NCBP (Fig. 3, A to D). Notably, MIW1 expression was transiently induced by P. xylostella infestation (Supplementary Fig. S1A), and this gene downregulates NCBP expression (Supplementary Fig. S3), which provides multiple layers of regulation to inactivate NCBP, thereby enhancing P. xylostella resistance in Arabidopsis.

GSH is a tripeptide with the sequence γ-Glu–Cys–Gly. Apart from its role in detoxification through binding to toxic compounds, GSH in mammalian cells functions in S-glutathionylation of target proteins, which is accomplished by various types of GSTFs (Michelet et al. 2005). This post-translational modification involves the reversible addition of a proximal donor of GSH to thiolate anions of cysteines in target proteins (Grek et al. 2013), most of which function in redox-mediated abiotic stress responses (Cha et al. 2017). However, up to date in plants, the mechanism by which glutathionylation regulates the target proteins in plants is unclear. Here, we dissected the role of GSTF2 in glutathionylation modification and P. xylostella resistance in Arabidopsis. A series of experiments, including Y2H, BiFC, pull-down, Co-IP, and luciferase complementation assays, indicated that GSTF2 interacts with NCBP (Fig. 5, A to E). GSTF2 reduces the abundance of NCBP (Fig. 7, A and B; Supplementary Fig. S6, A to C). More in-depth, we demonstrated that GSTF2 regulates NCBP via glutathionylation and 26S proteasome-mediated degradation (Fig. 8, A to C); Knockdown of GSTF2 or mutation of the C117 cysteine modification site increased the stability of NCBP in plants (Figs. 7D and 8, A and B). This process depends on MIW1, as in the absence of MIW1, GSTF2 did not effectively destabilize NCBP (Supplementary Fig. S6, D to F), and more importantly, GSTF2 enhances MIW1–NCBP interaction and thus accelerates the ubiquitination-mediated degradation of NCBP (Figs. 7, E to H and 8C). GSTF2 has been proposed to function as SA-binding proteins (Tian et al. 2012); therefore, we hypothesize that GSTF2 may play a link between SA and insect resistance, and it will be interesting to check the relationship between SA signaling pathways and GSTF2-NCBP regulatory module and whether SA has roles in regulating NCBP stability and DMNT accumulation in the future.

Wounding treatment–induced NCBP degradation is associated with enhanced GSH modification of this protein, when the glutathionylation site of NCBP (C117) was mutated to A, GSH modification did not increase under the treatment (Fig. 8B). Consistent with the finding that NCBPC117A became stable upon wounding (Fig. 8B), GSH modification affected NCBP-MIW1 interaction as well as the associated ubiquitination status of NCBP (Fig. 8, C to F), which were supported by bioassays that the glutathionylation site of NCBP (C117) is important in Arabidopsis resistance to P. xylostella (Fig. 9). These results elucidate that NCBP glutathionylation plays an important role in regulating NCBP stability and Arabidopsis resistance to P. xylostella. How the glutathionylation modification on NCBP affects the interaction between NCBP and MIW1 remains to be addressed. In mammalian cells, it has been proposed that the glutathionylation of cysteine residues can result in the unfolding of the α-helical lid structure (Yang et al. 2020). Thus, it is possible that the cysteine glutathionylation in NCBP influences NCBP's interaction with MIW1 by altering its protein structure.

Overall, we showed that GSTF2 mediates NCBP glutathionylation and causes its degradation by enhancing its interaction with MIW1 (Fig. 10). The discovery of this regulatory module expands our understanding of the roles of NCBP and glutathionylation in plant resistance to P. xylostella, providing a theoretical basis for insect resistance breeding.

Figure 10.

Figure 10.

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A working model illustrating that GSTF2 glutathionylates NCBP and promotes its degradation via MIW1 and 26S proteasome, to confer resistance to P. xylostella larvae. Under wounding conditions by P. xylostella, GSTF2 glutathionylates NCBP (shown with S-Glutathionylation, SSG), which promotes the interaction of NCBP with MIW1 and causes to higher level of ubiquitination and degradation NCBP, leading to DMNT accumulation and resistance to P. xylostella larvae. DMNT: (3E)-4,8-dimethyl-1,3,7-nonatriene.

Materials and methods

Plant materials and growth conditions

The Arabidopsis gstf2, ncbp, and miw1 mutants were ordered from the Arashare website (Zheng et al. 2012). Seeds were sterilized with 5% NaClO solution, washed three to five times with sterile water, and transferred to 1/2 MS growth medium. After 3 days of cold treatment in the dark at 4 °C, the seeds were transferred to a growth chamber and incubated at 22 °C under long-day conditions (16 h light/8 h dark). At the four-leaf stage, the seedlings were transplanted into soil for further growth and experiments (Wang et al. 2020).

IP-MS analysis methods

We used NCBPpro:NCBP-GFP ncbp transgenic Arabidopsis seedlings for NCBP-GFP protein extraction. NCBP-GFP protein sample separated by SDS-PAGE, SDS-PAGE gel pieces were destained and then incubated with trypsin (Promega, 10 ng/μL) overnight at 37 °C. Peptides were extracted by incubation with 5% TFA for 1 h, followed by the addition of 2.5% TFA/50% acetonitrile for 1 h at 37 °C. The combined supernatants were dried in a SpeedVac concentrator (Thermo Fisher) for MS analysis. Samples were analyzed by nanoLC-MS/MS using an rapid separation liquid chromatography system interfaced with a Q Exactive instrument (Thermo Fisher, San Jose, CA). Mass spectrometry data were acquired using a data-dependent acquisition procedure with a cyclic series of a full scan acquired with a resolution of 120,000 followed by MS/MS scans (30% collision energy in the high energy collision dissociation cell) with a resolution of 30,000 for the 20 most intense ions, with a dynamic exclusion duration of 20 s. The LC-MS/MS peak list files from each experiment were searched against the corresponding TAIR10 database. The assay methods were performed as described previously (Wang et al. 2020).

Wounding treatment

Holes were punched in the leaves of seedlings at the four-leaf stage with a sterile 1-mL syringe to mimic P. xylostella gnawing; all the leaves of every seedling were wounded, and one hole was made on each leaf (Xu et al. 2024). After further growth for different lengths of time, the seedlings were harvested and frozen in liquid nitrogen for subsequent experiments.

Yeast two-hybridization (Y2H) assay

The Y2H assay was performed as previously described (Wang et al. 2023a). The coding regions of NCBP, GSTF2, and MIW1 were amplified using high-fidelity PCR enzymes, sequenced, and ligated to the pEasy-T3 vector to generate the pGBKT7 and pGADT7 vectors, respectively, using the T4 ligation method (Kulich et al. 2018). After co-transforming different plasmids into yeast cells, the cells were cultured in solid synthetic dropout medium without leucine and tryptophan (Sd –Leu, –Trp) at 28 °C for 3 days. Single colonies were picked and cultured in leucine- and tryptophan-free growth medium at 28 °C overnight. The yeast cells were precipitated and washed three times with liquid SD medium minus leucine, tryptophan, adenine, and histidine (Sd –Leu, –Trp, –Ade, –His). The concentrations of the yeast cells were measured with a spectrophotometer, and the cells were diluted in Sd medium and spread on solid Sd –Leu, –Trp, –Ade, –His growth medium. After an additional 3 to 5 days of culture, the yeast colonies were photographed and analyzed.

Luciferase complementation imaging (LCI) and bimolecular fluorescence complementation (BiFC) assay

For LCI analysis, the coding regions of NCBP, GSTF2, and MIW1 were amplified, sequenced, cloned into the empty vectors JW771 and JW772 containing the C-terminal region of LUC (cLUC) and the N-terminal region of LUC (nLUC), respectively, and transformed into Agrobacterium cells. Agrobacterium cells containing different combinations of constructs were infiltrated into N. benthamiana leaves. The infiltrated N. benthamiana plants were kept in the dark for 24 h and transferred to a greenhouse for further growth. The leaves were sprayed with luciferin substrate (5 mg/mL), and the LUC signals from the leaves were detected using a Tanon 5,200 chemiluminescent imaging system (Tanon, Shanghai, China) (Gou et al. 2011; Shan et al. 2014). For BiFC, the coding regions of NCBP, GSTF2, and MIW1 were amplified and cloned into the pYNE and pYCE vectors. Different combinations of plasmids were co-transformed into Col-0 protoplasts using standard methods. Following 1 day of incubation in the dark, fluorescent signals from YFP were captured and analyzed under a confocal laser-scanning microscope (Zeiss LSM 800, Jena, Germany; Laser excitation: 488 nm, collection bandwidth: 500 to 530 nm, digital gain: 1.0) (Walter et al. 2004).

Pull-down and co-immunoprecipitation assays

Pull-down and co-immunoprecipitation (Co-IP) assays were performed as described previously (Liang et al. 2018). The coding regions of NCBP, GSTF2, and MIW1 were amplified, sequenced, and cloned into the PET28a-MBP vector in-frame with His or GST tags using the T4 ligation method. The correct clones were transformed into E. coli BL21 (DE3) cells using the E. coli protein expression system (CloneTec, Beijing, China, cat: 635607). The purified proteins were mixed and incubated in a solution of 50 mm Tris-HCl of pH 8.0, 150 mm NaCl, 0.1% (v/v) IGEPAL, 2.5 mm EDTA of pH 8.0, 10% (v/v) glycerol, 10 mm β-mercaptoethanol, 1 mm phenylmethanesulfonyl fluoride (PMSF), 10 μM leupeptin, and 1× Roche protease inhibitor cocktail tablets (Roche) overnight at 4 °C. The samples were collected using magnetic beads attached with specific antibodies, washed, separated by SDS-PAGE, and detected by immunoblotting with anti-His (EnoGene, E12-004-3, dilution: 1/2,000) or anti-GST (Abmart, AB_2864360, dilution: 1/5,000) antibodies.

For the Co-IP assays, the coding region of NCBP was cloned into pGreenII-0179 fused with a GFP tag and the PLGNL-FLAG vector. The coding region of GSTF2 was cloned into pCAMBIA1307 fused with the MYC tag and the PLGNL-FLAG vector. The coding region of MIW1 was cloned into the pCAMBIA1307 vector (Neefjes et al. 2021). The vectors were verified by PCR, introduced into Agrobacterium GV3101 cells and transformed into Col-0 to obtain transgenic lines. Transgenic lines harboring different constructs were crossed to obtain double transgenic lines. Total protein was extracted from the samples, and the supernatants were collected and incubated with magnetic beads with specific antibodies at 4 °C for 4 h. The beads were washed five times with protein extraction buffer, separated by SDS-PAGE, and subjected to immunoblot analysis with anti-GFP (Roche, 11814460001, dilution: 1/1,000) and anti-MYC (Abways, AB0001, dilution: 1/2,000) and anti-Flag (MBL, M185, dilution: 1/10,000) antibodies.

Preference and no-choice feeding assays using P. xylostella larvae

The P. xylostella eggs used in this study were purchased from Henan Jiyuan Biological Company (Cat: HJB004). Preference tests were conducted using 3-week-old Arabidopsis seedlings with 15 fourth-instar P. xylostella larvae for each assay. The non-choice feeding assays were carried out using second instar P. xylostella larvae. The larval phenotypes were analyzed quantitatively at regular time intervals (Chen et al. 2021).

Analysis of protein stability in N. benthamiana leaves based on florescence

To investigate whether GSTF2 is involved in NCBP protein degradation, we infiltrated 35Spro:NCBP-LUC with empty or 35Spro:GSTF2 vectors into N. benthamiana leaves. The leaves were incubated in the dark for 24 h, transferred to the light, and grown for an additional 24 h. The fluorescent signals from NCBP-LUC were analyzed using a fluorescence analysis platform (Tanon 5200). For quantitative LUC activity assays, equal amounts of infiltrated leaves were subjected to LUC enzyme activity assays using a LUC enzyme activity kit; Renilla-LUC fluorescence was used as an internal control for Agrobacterium infiltration (Neefjes et al. 2021).

In vivo protein degradation assay

Fourteen-day-old seedlings after wounding treatment were frozen in liquid nitrogen immediately, ground to a powder and extracted with TAP protein extraction buffer (50 mm Tris-HCl of pH 8.0, 150 mm NaCl, 0.1% [v/v] IGEPAL, 2.5 mm EDTA of pH 8.0, 10% [v/v] glycerol, 10 mm β-mercaptoethanol, 1 mm PMSF and 1× Roche protease inhibitor cocktail tablets [Roche]) at a ratio of 1 mL buffer per gram of ground plant tissue. Following centrifugation at 4 °C to remove cellular debris, the supernatant was collected for protein stability analysis. The protein was collected using magnetic beads with specific antibodies and separated by SDS-PAGE. Protein abundance was analyzed by immunoblotting with anti-GFP (Roche, 11814460001, dilution: 1/1,000) and anti-MYC (Abways, AB0001, dilution: 1/2,000) and anti-Flag (MBL, M185, dilution: 1/10,000) and anti-ACTIN (Abcam, ab197345, dilution: 1/5,000) antibodies (Wang et al. 2020).

Western blotting analysis of protein ubiquitination and glutathionylation

14-day-old seedlings were grounded in liquid nitrogen and extracted using protein extraction buffer (50 mm Tris-HCl f pH 8.0, 150 mm NaCl, 0.1% [v/v] IGEPAL, 2.5 mm EDTA of pH 8.0, 10% [v/v] glycerol, 10 mm β-mercaptoethanol, 1 mm PMSF and 1× Roche protease inhibitor cocktail tablets [Roche]) at a ratio of 1 mL buffer per gram materials. The supernatants were collected and incubated with magnetic beads with specific antibodies at 4 °C for 4 h. The beads were washed five times with protein extraction buffer and eluted with loading buffer. Seedlings and protoplasts treated with 50 μM MG132 (Wang et al. 2023b; Luo et al. 2024). SDS-PAGE was used to separate the proteins, and the target proteins were detected by immunoblotting with anti-GSH (Abcam, ab19534, dilution: 1/10,000) (Li et al. 2021), or anti-Ubiquitin [Ubi-1] (Abcam, ab7254, dilution: 1/2,000) antibodies (Wang et al. 2020). The intensity of immunoblotting bands was quantified using Image J software.

GC-MS assay of DMNT content

Three-week-old Arabidopsis seedlings were selected for the GC-MS assays coupled with solid-phase microextraction (SPME) (Sigma-Aldrich, 57328-U). The samples were lyophilized and weighed to approximately 100 mg, then placed in an adsorbent bottle (2 cm in diameter, 5 cm in height) and stabilized in a water bath at 40 °C for 10 min. Then the extraction needle was inserted into the bottle to absorb the volatile compounds for 30 min. The samples were analyzed using Agilent 5977B GC/MSD. The GC oven temperature was initially maintained at 40 °C for 1 min and then increased to 220 °C at a rate of 5 °C/min, held for 38 min, and finally increased to 240 °C at a rate of 5 °C/min and held for 6 min.

Data analysis

The SPSS 16.0 package (http://www.spss.com) was used for data analysis. Independent Student's t-test, one-way, and two-way analysis of variance (ANOVA) were performed using default settings without data transformation. The detailed statistical results are shown in Supplementary Data Set 1.

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers. The Accession numbers based on The Arabidopsis Information Resource (https://www.arabidopsis.org) for all genes examined in this study are NCBP (AT5G18110), MIW1 (AT1G52730), UBC (At1g50490), and GSTF2 (AT4G02520).

Supplementary Material

koaf188_Supplementary_Data
koaf188_supplementary_data.zip (10.8MB, zip)

Acknowledgments

We are grateful to Dr. Minglei Yang (University of Science and Technology of China) for insightful discussions, and to Dr. Nipapan Kanjana (Anhui Agricultural University) for her meticulous language editing, which greatly improved the clarity of the manuscript.

Contributor Information

Ning Lin, The National Engineering Laboratory of Crop Stress Resistance, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China; Center for Crop Pest Detection and Control, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.

Hui Ye, The National Engineering Laboratory of Crop Stress Resistance, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China; Center for Crop Pest Detection and Control, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.

Mengjie Zhao, The National Engineering Laboratory of Crop Stress Resistance, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China; Center for Crop Pest Detection and Control, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.

Xingzhi Chen, The National Engineering Laboratory of Crop Stress Resistance, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China; Center for Crop Pest Detection and Control, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.

Jing Ma, The National Engineering Laboratory of Crop Stress Resistance, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China; Center for Crop Pest Detection and Control, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.

Chuanhong Wang, The National Engineering Laboratory of Crop Stress Resistance, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China; Center for Crop Pest Detection and Control, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.

Tengyue Wang, The National Engineering Laboratory of Crop Stress Resistance, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China; Center for Crop Pest Detection and Control, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.

Zhen Tao, The National Engineering Laboratory of Crop Stress Resistance, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China; Center for Crop Pest Detection and Control, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.

Yibing Zhao, The National Engineering Laboratory of Crop Stress Resistance, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China; Center for Crop Pest Detection and Control, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.

Qingyang Zhang, The National Engineering Laboratory of Crop Stress Resistance, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China; Center for Crop Pest Detection and Control, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.

Jun Lai, The Laboratory of Systematic & Evolutionary Botany and Biodiversity, College of Life Sciences, Zhejiang University, Hangzhou 310058, China.

Xinqiao Zhang, The National Engineering Laboratory of Crop Stress Resistance, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China; Center for Crop Pest Detection and Control, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.

Jinghui Dong, The National Engineering Laboratory of Crop Stress Resistance, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China; Center for Crop Pest Detection and Control, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.

Peijin Li, The National Engineering Laboratory of Crop Stress Resistance, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China; Center for Crop Pest Detection and Control, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China; The Anhui Province Key Laboratory of Resource Insect Biology and Innovative Utilization, School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.

Author contributions

P.L., N.L., and H.Y. conceived the project. N.L., M.Z., J.M., and Z.T. conducted the experiments. X.C., C.W., and T.W analyzed the data. Y.Z., Q.Z., J.L., X.Z., and J.D. helped in the experiments. P.L. and N.L. wrote and revised the manuscript.

Supplementary data

The following materials are available in the online version of this article.

Supplementary Figure S1.  MIW1 expression and protein abundance analysis (supports Fig. 2).

Supplementary Figure S2. MIW1 negatively affects NCBP-GFP abundance in Arabidopsis protoplast cells (supports Fig. 4).

Supplementary Figure S3.  NCBP expression level is higher in miw1 than that in Col-0 (supports Fig. 4).

Supplementary Figure S4.  GSTF2 expression and protein abundance analysis. (supports Fig. 5).

Supplementary Figure S5.  NCBP expression is not influenced by gstf2 compared with Col-0 (supports Fig. 7).

Supplementary Figure S6. GSTF2 negatively affects the abundance of NCBP-GFP via a process dependent on MIW1 (supports Fig. 7).

Supplementary Figure S7. Analysis of glutathionylation sites in NCBP protein (supports Fig. 8).

Supplementary Table S1. List of NCBP-GFP and GSTF2-FLAG mass spectrometric data.

Supplementary Table S2. List of the primers used in this study.

Supplementary Data Set 1. Statistical data.

Funding

This work was supported by the National Natural Science Foundation of China (32171954), the National Key Research and Development Program of China (2022YFD1201803-2), and the Anhui Provincial Major Science and Technology Project (202203a06020005).

Data availability

All data generated and analyzed in this study are available within the article and its Supplementary Information files. The data are available from the corresponding author upon reasonable request.

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

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Associated Data

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

Supplementary Materials

koaf188_Supplementary_Data
koaf188_supplementary_data.zip (10.8MB, zip)

Data Availability Statement

All data generated and analyzed in this study are available within the article and its Supplementary Information files. The data are available from the corresponding author upon reasonable request.

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