Proteome and Ubiquitinome Analyses Reveal the Involvement of Ubiquitination in Resistance to Maize Lethal Necrosis

ABSTRACT

The co‐infection of maize chlorotic mottle virus (MCMV) and sugarcane mosaic virus (SCMV) causes maize lethal necrosis (MLN), which seriously affects the yield and quality of maize. Ubiquitination is one of the most important protein post‐translational modifications. However, the role of ubiquitination modification in regulating maize resistance to viral infection remains largely unknown. In this study, we found that the ubiquitination levels in SCMV‐ and/or MCMV‐infected maize plants were higher than that in the non‐infected maize plants. Ubiquitinome and proteome analyses of the above maize plants revealed that most down‐regulated differentially accumulated proteins that possessed up‐regulated lysine ubiquitination sites were mainly involved in photosynthesis, fructose and mannose metabolism, and glyoxylate and dicarboxylate metabolism. Functional analyses of three DAPs involved in glyoxylate metabolism demonstrated that silencing ZmGOX1 facilitated SCMV and MCMV single and co‐infection, while knockdown of ZmHPR1 or ZmHPR2 suppressed viral infections. Moreover, overexpression of ZmGOX1 and its mutants at Kub sites enhanced maize resistance to SCMV infection. We also found that exogenous application of sodium sulphide could up‐regulate the expression of ZmGOX1 and effectively inhibit viral infections. These findings provide novel insights into the roles of ubiquitination in the regulation of maize resistance to viral infection.

This study analysed the proteome and ubiquitinome of SCMV‐ and/or MCMV‐infected maize plants and demonstrated the role of ZmGOX1 in the maize response to viral infections.

1. Introduction

Protein post‐translational modifications (PTMs) are important regulatory mechanisms to enrich the structure, function and subcellular localisation of proteins by adding different groups to the amino acid chain (Withers and Dong 2017). Ubiquitination, one of the most common PTMs, plays vital roles in the regulation of plant growth and development, and in response to various stresses by selective degradation of proteins (Xu and Xue 2019). Ubiquitin‐activating enzymes (E1), ubiquitin‐conjugating enzymes (E2) and ubiquitin‐ligase enzymes (E3) are key enzymes involved in the ubiquitin‐26S proteasome degradation pathway (Ozkaynak et al. 1987). Ubiquitin is activated and conjugated to the target proteins by a series of E1, E2 and E3 activities, and the target protein is selectively degraded by the 26S proteasome (Zientara‐Rytter and Sirko 2016).

The ubiquitin‐26S proteasome pathway is one of the main pathways involved in specific degradation of target proteins in vivo, and its importance in regulating various plant life activities has been widely reported (Millar et al. 2019). In the process of plant immunity against viral infection, the host can specifically degrade key proteins encoded by the virus through the ubiquitin‐26S proteasome pathway to resist virus infection (Langin et al. 2023). In tobacco, NtRFP1, a RING E3 ligase, can interact with βC1 encoded by tomato yellow leaf curl China betasatellite (TYLCCNB) to promote its ubiquitylation for proteasome‐mediated degradation, thus inhibiting viral infection (Shen et al. 2016). The RING type E3 NbubE3R1 interacts with the replicase of bamboo mosaic virus (BaMV) to inhibit virus replication in Nicotiana benthamiana (Chen et al. 2019). In addition, some studies have also shown that viruses can destroy the ubiquitin‐26S proteasome pathway of plants, and even use the ubiquitination pathway to promote their own infection (Dubiella and Serrano 2021). The RNA silencing suppressor P22 protein encoded by tomato chlorosis virus (ToCV) disrupts the stability of NbSCF^TIR1 through interaction with the C‐terminal of NbSKP1, thus affecting auxin signal transduction and promoting ToCV infection (Liu, Wang, et al. 2021). Cotton leaf curl Multan virus (CLCuMuV) βC1 interacts with NbSKP1 in N. benthamiana, disrupting the interaction between NbSKP1s and NbCUL1 and interfering with the biosynthesis of jasmonic acid (JA) and gibberellin, thereby promoting CLCuMuV infection (Jia et al. 2016). These results indicate the importance of ubiquitination modification in plant–virus interaction.

The K‐ε‐GG antibody can be used to enrich ubiquitinated proteins after trypsin digestion by recognising the ubiquitin remnant motif Lys‐e‐Gly‐Gly (di‐Gly‐Lys) (Xu et al. 2010). Ubiquitinome analysis using the K‐ε‐GG antibody is a novel approach that has been widely used in research on the changes of protein ubiquitination during plant growth and stress response (Porras‐Yakushi and Hess 2014). The results of dynamic ubiquitinome analyses show that protein ubiquitination regulates the process of rice seed germination, and differentially ubiquitinated proteins are involved in protein processing, DNA and RNA processing/regulation, signalling and transport (He et al. 2020). Integrated proteome and ubiquitinome analysis revealed that half of 12 up‐regulated pathogenesis‐related 10 (PR10) proteins show reduced ubiquitination levels in Botrytis cinerea‐infected rose plants, suggesting that ubiquitination is involved in the regulation of rose resistance (Li et al. 2023). A total of 224 up‐regulated and 155 down‐regulated ubiquitinated lysine (Kub) sites have been identified during rice stripe virus (RSV) infection of N. benthamiana; the proteins with up‐regulated Kub sites are significantly enriched in the ribosome (Liu et al. 2022).

Maize chlorotic mottle virus (MCMV) belongs to the genus Machlomovirus in the family Tombusviridae (Bockelman et al. 1982). Maize lethal necrosis (MLN) is caused by co‐infection of maize chlorotic mottle virus (MCMV) with one of several viruses from the Potyviridae, such as sugarcane mosaic virus (SCMV) (Redinbaugh and Stewart 2018). Synergistic infection of SCMV and MCMV increases MCMV titre and MCMV‐derived small interfering RNAs (siRNAs), resulting in chlorosis and mottling of leaves, necrosis of leaf tips and even death of the whole plant, which causes serious economic losses in maize production (Jiao et al. 2022; Xia et al. 2016). Although previous studies have explored the maize responses to SCMV, MCMV and synergistic infection at transcriptional, post‐transcriptional and post‐translational levels (Chen et al. 2017; Dang et al. 2019; da Silva et al. 2020; Gao et al. 2023; Hao et al. 2023; Wu et al. 2013; Xia et al. 2016, 2018, 2019), how viral infection affects protein ubiquitination of maize is still elusive.

In this study, we obtained quantitative ubiquitinome and proteome profiles of SCMV and MCMV single‐ and co‐infected (S+M) maize leaves by 4D‐label‐free quantification (LFQ) technology and a K‐ε‐GG‐based qualitative method. Bioinformatics analyses elucidated the potential functions and associations of differentially accumulated proteins (DAPs) and whether these proteins possessed differentially ubiquitinated sites (DUSs) in response to different viral infections in maize. Moreover, parallel reaction monitoring (PRM) was performed to further verify several DAPs with DUSs. We then analysed the antiviral roles of three key genes involved in the glyoxylate metabolism pathway, showing differential expression and protein ubiquitination levels in maize through cucumber mosaic virus (CMV)‐based virus‐induced gene silencing (VIGS) assays. Virus‐mediated protein overexpression (VOX) using an SCMV infectious clone further elucidated the role of ubiquitination modification in regulating the anti‐SCMV activity of glycolate oxidase 1 (ZmGOX1). In addition, the effects of exogenous spraying sodium sulphide (Na₂S) on the accumulation of ZmGOX1 and viral infections in maize were clarified. Our study contributes to understanding the roles of ubiquitination in the maize antiviral process and provides candidate genes for resistance breeding.

2. Results

2.1. The Ubiquitin‐Proteasome System Is Involved in Maize Response to Viral Infections

Ubiquitination is one of the classic PTMs that regulates plant stress resistance by targeting proteins for degradation (Xu and Xue 2019). In our previous transcriptome data (Hao et al. 2023), we found that the expression levels of most ubiquitination pathway‐related genes were significantly up‐regulated in maize plants infected with SCMV, MCMV and S+M (Figure S1a), suggesting that ubiquitination modification is involved in antiviral responses in maize. To test this hypothesis, we inoculated maize with phosphate‐buffered saline (PBS), SCMV, MCMV or S+M at the three‐leaf stage (Figure 1a). We found that the accumulation of SCMV RNAs was similar in S+M‐ and SCMV‐infected maize plants, while the accumulation of MCMV genomic RNAs in S+M‐infected maize plants was significantly higher than that in MCMV‐infected maize plants at 9 days post‐inoculation (dpi) (Figure 1b,c). Total protein was extracted from the first systemically infected maize leaves of the four different inoculation groups at 9 dpi for western blot analysis using an anti‐Ub antibody, with proteasome inhibitor MG132‐treated healthy maize leaves as a control. The results showed that many proteins were ubiquitinated in maize leaves and were mainly distributed in the size range of 25–250 kDa (Figure 1d). Interestingly, the ubiquitination levels of total proteins in virus‐infected maize plants were significantly higher than that in the non‐infected maize plants, especially in the S+M group, which showed similar results to the MG132‐treated healthy maize plants (Figure 1d). To further investigate whether the ubiquitin‐proteasome system plays a role in the antiviral response of maize, MG132 was used for treatment of maize leaves (Figure 1e). At 7 dpi, the leaves of MG132‐treated maize plants showed more severe viral symptoms than those of control plants (Figure 1e). Reverse transcription‐quantitative PCR (RT‐qPCR) results showed that the accumulation of SCMV RNAs was up‐regulated by about 1.5–1.7‐fold in MG132‐treated SCMV and S+M plants, and the accumulation of MCMV RNAs was up‐regulated by about 12–16‐fold in MG132‐treated MCMV and S+M plants (Figure 1f,g). The results of western blotting were consistent with that of RT‐qPCR (Figure 1h,i).

FIGURE 1.

The ubiquitin‐proteasome system participates in maize antiviral defence. (a) The symptoms of whole maize and the first systemically infected leaves in four groups at 9 days post‐inoculation (dpi). PBS, phosphate‐buffered saline; S, sugarcane mosaic virus (SCMV); M, maize chlorotic mottle virus (MCMV); S+M, SCMV and MCMV co‐infection. Bars, 5 cm. (b) Relative accumulation of SCMV RNAs in S and S+M plants determined by reverse transcription‐quantitative PCR (RT‐qPCR). (c) Relative accumulation of MCMV RNAs in M and S+M groups determined by RT‐qPCR. (d) The ubiquitination levels of total proteins in maize determined by western blotting. Healthy maize samples sprayed with MG132 once 24 h before harvest were used as a positive control. (e) The symptoms of the first systemically infected maize leaves infected with SCMV, MCMV and S+M after MG132 treatment. DMSO, dimethyl sulphoxide. (f) Relative RNA accumulation of SCMV RNAs in S and S+M groups after MG132 treatment. (g) Relative RNA accumulation of MCMV RNAs in M and S+M groups after MG132 treatment. (h) The accumulation of SCMV coat protein (CP) in the first systemically infected maize leaves treated with MG132. (i) The accumulations of MCMV CP in the first systemically infected maize leaves treated with MG132. Asterisks indicate significant difference between treatments, determined by the two‐tailed t test (*p < 0.05, **p < 0.01, ***p < 0.001). The relative densities of each band detected in western blotting were analysed by ImageJ software. CBB, Coomassie brilliant blue staining of loading control.

2.2. Impacts of Virus Infections on the Global Proteome Level in Maize

To explore the effects of SCMV, MCMV and S+M infection on the expression levels of maize proteins, we performed 4D label‐free quantitative proteomic analysis of the first systemically infected maize leaves under four different treatments (Figure 2a). The results showed that 31,610 unique peptides were obtained from 364,519 matched spectra in this database (1,432,935 total spectra) and resulted in 4115 comparable proteins (Table S2). About 79% of identified proteins had > 10% sequence coverage, and over 80% of them matched at least two peptides (Table S3). Pearson's correlation coefficients of three replications from 12 samples were between 0.89 and 0.98, which indicated that the correlation between each group of biological replicates was high (Figure 2b). A total of 2593 DAPs were identified, of which 787, 1372, 1768, 1490 and 1047 DAPs were found in S versus PBS, M versus PBS, S+M versus PBS, S+M versus S and S+M versus M, respectively (Figure 2c). The heat map showed the expression patterns of all DAPs in S+M versus PBS (Figure 2d,e). Most of the DAPs identified in S+M versus PBS were also differentially expressed in S versus PBS and/or M versus PBS (Figure 2d,e). Furthermore, we identified 178 DAPs that were up‐regulated only in S+M versus PBS, which were mainly enriched in proteasome and oxidative phosphorylation pathways (Table S4). A total of 308 DAPs that were down‐regulated only in S+M versus PBS were identified, which were mainly involved in porphyrin metabolism and glycolysis/gluconeogenesis pathways (Table S4).

FIGURE 2.

Statistical analyses of differently accumulated proteins (DAPs) in sugarcane mosaic virus (SCMV)‐, maize chlorotic mottle virus (MCMV)‐ and SCMV + MCMC (S+M)‐infected maize plants. (a) The systematic workflow of proteome and ubiquitinome analyses. (b) Correlation heatmap of three biological replicates in four groups. The closer Pearson's correlation coefficient is to 1, the stronger the correlation between the two duplicate samples is. (c) The number of DAPs in five different comparisons. (d) The expression pattern of up‐regulated DAPs in S+M versus phosphate‐buffered saline (PBS). (e) The expression pattern of down‐regulated DAPs in S+M versus PBS.

Based on these differences, Gene Ontology (GO) analysis of DAPs was performed (Figure S2a). The co‐infection of SCMV and MCMV resulted in MLN, and the accumulation of MCMV and MCMV‐derived siRNAs in S+M group was increased compared with that in M group (Xia et al. 2016). Therefore, for host plants, the DAPs in S+M versus M were hypothesised to be closely related to MLN caused by co‐infection. In S+M versus M, the up‐regulated DAPs were involved in ‘polysome binding’, ‘mRNA binding’ and ‘proteasome assembly’, while the down‐regulated DAPs were mainly involved in ‘starch metabolic process’ and ‘chlorophyII binding’ (Figure S2a). In addition, we performed KEGG enrichment analysis of DAPs (Figure S2b). In S+M versus M, the up‐regulated DAPs were mainly in ‘proteasome’ and ‘spliceosome’, while the down‐regulated DAPs were mainly in ‘photosynthesis’, ‘carbon fixation in photosynthetic organisms’ and ‘carotenoid biosynthesis’ (Figure S2b). Classification of subcellular location revealed that DAPs were mainly localised in chloroplast and cytoplasm in the five different comparisons (Figure S2c).

2.3. Impacts of Virus Infections on the Ubiquitinome Level in Maize

To characterise the protein ubiquitination events of SCMV‐, MCMV‐ and co‐infected maize plants, we performed LC–MS/MS with a 4D‐lLFQ approach to generate ubiquitinome datasets. A total of 9662 lysine ubiquitination (Kub) sites on 3667 proteins were identified, of which 3681 Kub sites on 1158 proteins were quantified (Table S5). To further elucidate underlying function of these proteins with DUSs from different treatments, we performed GO and KEGG enrichment analyses (Figure 3 and Table S6). Notably, we found that the proteins that possessed DUSs in S+M versus M were mainly involved in molecular function (MF) term, including ‘glutathione transferase activity’, ‘lyase activity’ and ‘oxidoreductase activity’ (Figure 3a). The results of KEGG enrichment analyses showed that ‘carbon fixation in photosynthetic organisms’, ‘glyoxylate and dicarboxylate metabolism’ and ‘fatty acid degradation’ were the top three pathways in S+M versus M (Figure 3b and Table S6). Subcellular localisation prediction showed that these proteins with DUSs were mainly distributed in the chloroplast and cytoplasm in the five different comparisons (Figure 3c and Table S6).

FIGURE 3.

Functional categorisations of the proteins that possessed differentially ubiquitinated sites (DUSs) in five comparisons. (a) GO analyses of the proteins with DUSs. (b) KEGG enrichment analyses of the proteins with DUSs. (c) Classification of subcellular location of the proteins with DUSs.

2.4. Crosstalk Between the Global Proteome and Ubiquitinome

To further clarify the effects of virus infections on maize proteins, we conducted an integrative analysis of proteome and ubiquitinome data. A total of 1765 identified proteins were found in both the proteome and ubiquitinome libraries (Figure 4a). These proteins are presented according to their expression patterns (Figure 4b and Figure S3).

FIGURE 4.

Crosstalk analysis between global proteome and ubiquitinome. (a) Venn diagrams of total identified proteins in proteome and ubiquitinome. (b) Scatter plot of the differentially accumulated proteins (DAPs) in the proteome and ubiquitinome data based on their fold change values in S+M versus M. The coloured dots indicate proteins with fold change values of either > 1.5 or < 0.67 in DAPs and differentially ubiquitinated sites (DUSs). Ps, proteins; Us, ubiquitinated sites. The blue dotted line represents the trend line. (c) GO and KEGG analysis of DAPs between proteome and ubiquitinome in S+M versus M. (d) The association between enriched KEGG pathways in S+M versus M. Dots showing DAPs of different KEGG pathways detected in proteomics, and the dot colours indicate fold change values. Asterisks indicate the DUSs; red asterisks represent up‐regulated DUSs, blue asterisks represent down‐regulated DUSs, yellow asterisks represent both up‐regulated and down‐regulated DUSs.

The results of GO analyses showed that the down‐regulated DAPs with up‐regulated DUSs in five different comparisons were mainly involved in ‘photosynthesis, light harvesting’, ‘chloroplast’ and ‘chlorophyII binding’ (Figure 4c and Figure S4). KEGG pathway clustering analyses revealed that these DAPs were mainly enriched in ‘photosynthesis’, ‘photosynthesis‐antenna proteins’ and ‘carbon fixation in photosynthetic organisms’ in S versus PBS, M versus PBS and S+M versus S (Figure S4). In S+M versus PBS, the DAPs were significantly enriched into more KEGG pathways, including ‘glycolysis/gluconeogenesis’, ‘alanine, aspartate and glutamate metabolism’ and ‘fructose and mannose metabolism’ (Figure S4). In S+M versus M, ‘photosynthesis’ and ‘glyoxylate and dicarboxylate metabolism’ were the two main KEGG enrichment pathways (Figure 4c).

2.5. Integrative Proteome and Ubiquitinome Analysis Revealed the Different Expression Patterns of Multiple Pathway‐Related Maize Proteins in Response to Viral Infections

2.5.1. Photosynthesis

Virus infection often disrupts the photosynthesis of host plants and affects their growth and metabolism (Zhao et al. 2016). In S versus PBS, M versus PBS and S+M versus PBS comparisons, a total of 7, 12 and 15 DAPs, respectively, involved in the photosystem I (PSI) and II (PSII) assemblies were down‐regulated, and most of them had up‐regulated DUSs (Figure S5a–c; Table S7). One down‐regulated F‐type ATP synthase (ZmATPF1E) in S versus PBS, and four down‐regulated F‐type ATP synthases (ZmATPF1A, ZmATPF1B, ZmATPF1E and ZmATPF1G) in both M versus PBS and S+M versus PBS were identified, all of which had up‐regulated DUSs (Figure S5a–c; Table S7). Moreover, in S+M versus PBS, we also identified one ferredoxin‐NADP reductase (ZmFNR) that was down‐regulated with up‐regulated DUSs (Figure S5c; Table S7). In S+M versus M, we found that 12 DAPs involved in PSI and PSII, two cytochrome b6 (ZmPetC and ZmPetB) and two ZmATPFs (ZmATPF1A and ZmATPF1B) were down‐regulated, all of which had up‐regulated DUSs (Figure 4d; Table S7).

2.5.2. Fructose and Mannose Metabolism

In S versus PBS, five DAPs involved in fructose and mannose metabolism were up‐regulated, including GDP‐L‐fucose synthase (ZmGFS), fructokinase 2 (ZmFRK2), triosephosphate isomerase 2 (ZmTPI2) and two fructose‐bisphosphate aldolases (ZmFBA1 and ZmFBA4) (Figure S5a; Table S7). Among them, ZmGFS had down‐regulated DUSs, while ZmFRK2 and ZmFBA1 had up‐regulated DUSs (Figure S5a; Table S7). In addition, the other two ZmFBAs (ZmFBA2 and ZmFBA3) were down‐regulated and had up‐regulated DUSs (Figure S5a; Table S7). In M versus PBS, we found that the protein levels of one ZmGFS, three ZmFRKs (ZmFRK1, ZmFRK2 and ZmFRK3), one sorbitol dehydrogenase (ZmSDH), one ZmTPI2, one hexokinase 2 (ZmHK2) and one ZmFBA1 were up‐regulated (Figure S5b; Table S7). Among them, ZmGFS had down‐regulated DUSs, ZmFRKs, ZmSDH and ZmHK2 possessed up‐regulated Kub sites, while ZmFBA1 possessed both up‐regulated and down‐regulated Kub sites (Figure S5b; Table S7). We also found that fructose‐2,6‐biphosphatase 1 (ZmFBPase1) and the other two ZmFBAs (ZmFBA2 and ZmFBA3) were down‐regulated and had up‐regulated DUSs (Figure S5b; Table S7). In S+M versus PBS, eight down‐regulated DAPs were identified, including one ZmFBPase1, one ZmTPI1, one ZmHK6, three ZmFBAs (ZmFBA2, ZmFBA3 and ZmFBA5) and two fructose‐1,6‐bisphosphatases (ZmFBP1 and ZmFBP3), and eight up‐regulated DAPs were identified, including one ZmGFS, three ZmFRKs (ZmFRK1, ZmFRK2 and ZmFRK3), one ZmTPI2, one ZmHK2 and two ZmFBAs (ZmFBA1 and ZmFBA4) (Figure S5c; Table S7). All of these proteins had up‐regulated DUSs except ZmGFS (Figure S5c; Table S7).

2.5.3. Glyoxylate and Dicarboxylate Metabolism

We found that ribulose‐bisphosphate carboxylase small chain 1 (ZmRbcS1) was down‐regulated, while acetyl‐CoA acetyltransferase (ZmACAT) and acetyl‐CoA synthetase (ZmACSS) were up‐regulated in S versus PBS and M versus PBS (Figure S5a,b; Table S7). In S+M versus PBS, ZmGOX1, alanine‐glyoxylate transaminase (ZmAGT), ZmRbcS1, ribulose‐bisphosphate carboxylase large chain (ZmRbcL) and glutamine synthetase (ZmGLUL) were down‐regulated (Figure S5c; Table S7). It is worth noting that most of the glyoxylate and dicarboxylate metabolism‐related proteins had up‐regulated DUSs (Figure 5d and Figure S5; Table S7).

FIGURE 5.

Functional analyses of three genes involved in glyoxylate metabolism through cucumber mosaic virus (CMV)‐based virus‐induced gene silencing assays. (a) Disease symptoms of the first systemically infected leaves on different gene‐silenced maize plants after maize chlorotic mottle virus (MCMV; M), sugarcane mosaic virus (SCMV; S) and SCMV + MCMV (S+M) infection. (b) The accumulation of SCMV and MCMV genomic RNAs determined by reverse transcription‐quantitative PCR (RT‐qPCR) in different gene‐silenced maize plants. (c) The accumulation of SCMV and MCMV coat protein (CP) in the first systemically infected maize leaves determined by western blot assays in different gene‐silenced maize plants. (d) Silencing efficiencies of target genes determined through RT‐qPCR. Asterisks indicate statistical difference between treatments, determined by the two‐tailed t test (*p < 0.05, **p < 0.01, ***p < 0.001). The relative density of each band was analysed by the ImageJ software. CBB, Coomassie brilliant blue staining of loading control.

2.6. Validation of Ubiquitinome and Proteome Data by PRM Analysis

In this study, six proteins, including hydroxypyruvate reductase 2 (ZmHPR2, A0A1D6KCW2), ZmGOX1 (A0A3L6E0R4), ZmHPR1 (B4FLP0), photosystem I P700 chlorophyll a apoprotein A1 (ZmPsaA, P04966), ZmFRK2 (Q6XZ78) and ZmFBA3 (B4FTI5), were selected for PRM analysis. We successfully measured their protein levels under four different treatments, and the expression trends were basically consistent with the results of the proteome (Figure S7a, Table S8). With regards to the Kub sites, we detected K15, K133, K136, K151 and K215 of ZmGOX1, K565 and K708 of ZmPsaA and K61 of ZmFRK2 (Figure S7b, Table S8), but failed to detect the Kub sites on ZmHPR2, ZmHPR1 and ZmFBA3, which were related to the strict signal cut‐off.

2.7. Roles of Three Key Genes Related to Glyoxylate Metabolism Pathway in Maize Antiviral Responses

In this study, proteins with DUSs that were related to the glyoxylate and dicarboxylate metabolism pathway were mainly enriched in S+M versus M (Figure 4c), which may be closely related to MLN caused by synergistic infection. Therefore, we constructed an expression pattern diagram of proteins involved in the glyoxylate metabolism pathway (Figure S6). We found that the protein level of ZmGOX1 (A0A3L6E0R4), which possessed up‐regulated DUSs in five different comparisons (Figure S6), was down‐regulated after virus infection. The protein level of ZmHPR2 (A0A1D6KCW2) was up‐regulated after virus infection, which possessed up‐regulated DUSs in S+M versus M (Figure S6). The protein level of ZmHPR1 (B4FLP0) had an up‐regulation trend after virus infection, and possessed up‐regulated DUSs in M versus PBS, S+M versus PBS, S+M versus S and S+M versus. M (Figure S6). To further examine the role of glyoxylate metabolism pathway‐related genes in maize antiviral responses, we selected these three genes for functional verification through CMV‐based VIGS assays. We found that ZmGOX1‐silenced plants showed more severe symptoms in SCMV and MCMV single‐ and co‐infection, while ZmHPR1‐ and ZmHPR2‐silenced plants exhibited milder symptoms (Figure 5a). The accumulation of SCMV and MCMV genomic RNAs and CP was consistent with the symptom severity determined by RT‐qPCR and western blot assays, respectively (Figure 5b,c). The silencing efficiencies of these three genes were between 58% and 34% determined by RT‐qPCR (Figure 5d).

2.8. The Ubiquitination Modification of ZmGOX1 Affected Maize Resistance to SCMV Infection

To investigate whether ZmGOX1 was degraded through the 26S proteasome pathway, we examined the effect of MG132 on its accumulation. The results showed that the ZmGOX1 protein level was increased after treatment with MG132 (Figure 6a). Combining the results of ubiquitinome and PRM analysis, we mutated two Kub sites on ZmGOX1, K136 and K361, separately or simultaneously to arginine (R) to produce mutants ZmGOX1^K136R, ZmGOX1^K361R and ZmGOX1^RR. The results of western blot assays revealed that the protein levels of these three mutants with lower ubiquitination levels were higher than that of ZmGOX1, especially ZmGOX1^RR (Figure 6b,c). To further elucidate the role of ZmGOX1 ubiquitination modification in the regulation of maize resistance to SCMV infection, the pSCMV‐3×Flag‐ZmGOX1, pSCMV‐3×Flag‐ZmGOX1^K136R, pSCMV‐3×Flag‐ZmGOX1^K361R and pSCMV‐3×Flag‐ZmGOX1^RR vectors were constructed (Figure 6d). As shown in Figure 6e, the maize plants infected with SCMV‐3×Flag‐ZmGOX1 and its mutants displayed milder mosaic symptoms compared to that inoculated with SCMV‐GFP (Figure 6e). The accumulation of SCMV RNAs and CP was also down‐regulated in maize plants infected with SCMV‐3×Flag‐ZmGOX1 and its mutants (Figure 6f,g). Notably, the accumulation of SCMV RNAs and CP in maize plants infected with SCMV‐3×Flag‐ZmGOX1^K136R, SCMV‐3×Flag‐ZmGOX1^K361R or SCMV‐3×Flag‐ZmGOX1^RR was lower than that in plants infected with SCMV‐3×Flag‐ZmGOX1 (Figure 6f,g).

FIGURE 6.

The ubiquitination of ZmGOX1 affected maize resistance to sugarcane mosaic virus (SCMV) infection. (a) The protein level of ZmGOX1 was increased by MG132 treatment in Nicotiana benthamiana. (b) The protein levels of ZmGOX1 and its mutants at lysine ubiquitination (Kub) sites in N. benthamiana at 3 days post‐agroinfiltration (dpi). (c) The ubiquitination levels of ZmGOX1 and its mutants at Kub sites were detected through immunoblotting with an anti‐Ub antibody. (d) Schematic organisation of the recombinant SCMV‐related constructs. (e) Viral symptoms on the first systemically infected leaves of maize plants infected with SCMV‐GFP and its derivate viruses. (f) The accumulation of SCMV RNA in the first systemically infected leaves of maize plants infected with SCMV‐GFP and its derivate viruses determined by reverse transcription‐quantitative PCR. (g) The accumulations of SCMV coat protein (CP) in the first systemically infected leaves of maize plants infected with SCMV‐GFP and its derivate viruses determined by western blotting. Asterisks indicate statistical difference between treatments, determined by the two‐tailed t test (**p < 0.01, ***p < 0.001). The relative density of each band was analysed by the ImageJ software. CBB, Coomassie brilliant blue staining of loading control.

2.9. Na₂S Treatment Enhanced Maize Resistance to Virus Infections

It has been reported that exogenous application of Na₂S can promote the plant photorespiration rate by increasing the expression level of GOX1 to improve its activity (Xu and Wang 2003). To further explore the roles of ZmGOX1 in resistance to virus infections, the assays of Na₂S treatment were performed and the expressions of ZmGOX1 were detected in PBS‐inoculated maize plants at 24 h, 72 h and 120 h after Na₂S treatment. The results revealed that the expression levels of ZmGOX1 were indeed induced after Na₂S treatment, especially at 72 h, which were up‐regulated 2.7‐fold and 4.6‐fold in the maize plants sprayed with 1 mM and 2 mM Na₂S, respectively (Figure S8). In addition, we found that exogenous Na₂S treatment significantly inhibited viral infection in SCMV‐ and/or MCMV‐inoculated maize plants (Figure 7a). The first systemically infected leaves of Na₂S‐treated maize plants showed milder viral symptoms than that of the control plants (Figure 7a). The RT‐qPCR results showed that the accumulations of SCMV RNAs were down‐regulated by about 34%–70% in Na₂S‐treated SCMV and S+M plants, and the accumulation of MCMV RNA was down‐regulated by about 34%–58% in Na₂S‐treated MCMV and S+M groups (Figure 7b). The results of western blot assays were consistent with those of RT‐qPCR (Figure 7c). Meanwhile, the expression level of ZmGOX1 in virus‐infected maize leaves was up‐regulated after Na₂S treatment (Figure 7d).

FIGURE 7.

Effects of Na₂S treatment on antiviral responses of maize. (a) Viral symptoms on the first systemically infected leaves of maize plants under Na₂S treatment. S, sugarcane mosaic virus (SCMV); M, maize chlorotic mottle virus (MCMV); S + M, SCMV + MCMV. (b) The accumulation of viral genomic RNAs in the first systemically infected leaves of maize plants under Na₂S treatment determined by reverse transcription‐quantitative PCR (RT‐qPCR). (c) The accumulation of viral coat protein (CP) in the first systemically infected leaves of maize plants under Na₂S treatment determined by Western blotting. (d) The expression level of ZmGOX1 gene in the first systemically infected leaves of maize plants under Na₂S treatment determined by RT‐qPCR. Lowercase letters indicate significant difference between treatments. The statistical significance was determined using one‐way analysis of variance followed by Duncan's multiple comparison test (p < 0.05). The relative density of each band was analysed by the ImageJ software. CBB, Coomassie brilliant blue staining of loading control.

3. Discussion

With the continuous development of sequencing technology, a growing body of evidence suggests that PTMs play important roles in altering protein structure and function, and regulating immune response (Vu et al. 2018). Ethylene regulates the petal senescence of petunia corollas by affecting the ubiquitination level of protein in the hormone signal transduction pathway and endoplasmic reticulum‐associated degradation (Guo et al. 2017). The ubiquitinome analysis proved that nitrogen regulates the secondary metabolism of tea plants by affecting the ubiquitination levels of the hub enzymes in theanine and flavonoid biosynthesis (Wang et al. 2021). At present, there are only a few reports on the plant ubiquitinome in response to virus infection. The co‐infection of SCMV and MCMV causes MLN in maize, which leads to leaf yellowing and necrosis followed by serious economic losses (Johnmark et al. 2022). In our previous transcriptome data we reported that the expression levels of most ubiquitination pathway‐related genes were up‐regulated in virus‐infected maize plants (Figure S1a). In this study, we found that the proteins in SCMV‐ and/or MCMV‐infected maize plants exhibited higher levels of ubiquitination than that in mock‐inoculated plants, especially in co‐infected maize plants (Figure 1d). We also found that SCMV infection up‐regulated 26S proteasome activity, whereas neither MCMV infection nor SCMV+MCMV co‐infection significantly affected 26S proteasome activity (Figure S1c). Additionally, the integrated analysis of transcriptomic and proteomic data indicated that the accumulation of most proteasome subunit genes and proteins was up‐regulated after viral infection, especially in co‐infected maize plants (Figure S1a,b). Moreover, we found that some of these proteins possessed up‐regulated DUSs (Figure S1b). These results collectively suggested that maize plants may maintain protein homeostasis in response to viral infection by enhancing proteasome assembly or activity, and viruses could promote the degradation of these proteins to facilitate infection through ubiquitination modification. However, the relationship between the accumulation of proteasome subunit genes/proteins and the activity of 26S proteasome still requires further study. Furthermore, when the proteasome function is impaired, these down‐regulated DAPs may be degraded through other pathways, such as the autophagy‐lysosome pathway and/or the unfolded protein response. In this study, we found that the transcript levels of autophagy‐related 18 (ZmATG18), ZmATG4 and ZmATG3 were significantly up‐regulated in S+M infected maize plants (Figure S9a). The accumulation of ZmATG8 was up‐regulated in SCMV‐infected maize plants while down‐regulated in MCMV‐infected maize plants (Figure S9a). Based on proteomic data, we found that ZmATG7 was significantly up‐regulated in S+M infected maize plants (Figure S9b). These results indicated that after viral infection, autophagy is involved in the regulation of protein degradation, although the specific mechanism of action needs to be further explored.

Chloroplasts are the key organelles for plant photosynthesis and are targets of plant viruses for viral pathogenesis or propagation (Zhao et al. 2016). Chloroplasts are closely involved in plant immunity (Liu et al. 2024). Previous studies have shown that after MCMV infection, the photochemical efficiency and the electron transfer rate of maize leaves were significantly reduced, and the expression levels of 14 photosynthesis‐related proteins were decreased (Dang et al. 2019). The accumulation of seven photosynthesis‐related proteins decreased in maize after SCMV infection (Chen et al. 2017). Consistent with previous reports, the results of proteome and ubiquitinome analyses showed that the expression levels of most of the photosynthesis‐related proteins were decreased and up‐ubiquitinated in maize after SCMV, MCMV and S+M infection, especially after S+M infection (Figure 5d and Figure S5). In addition to providing energy and metabolic products for plants, photosynthesis can also regulate gene expression and signal transduction, enhancing the disease resistance of plants (Lu and Yao 2018). For example, silencing chloroplast protein ferredoxin 1 (NbFd1) inhibited the expression of abscisic acid (ABA) synthesis‐related genes and reduced the content of ABA to promote potato virus X (PVX) infection (Yang et al. 2020). Overexpression of oxygen‐evolving enhancer 2 (VpPsbP) enhanced susceptibility to Plasmopara viticola in grapevine and up‐regulated the transcript levels of 2‐Cys‐peroxiredoxin (2PRX), ascorbate peroxidase (APX), superoxide dismutase (FeSOD and FeSOD3) and glutathione reductase (GR) (Liu, Chen, et al. 2021). The accumulation of these two photosynthesis‐related genes can affect the expression levels of other genes. Therefore, the down‐regulated expression of some proteins after viral infection may be related to the impaired photosynthesis of maize in this study, rather than ubiquitination modification. The specific mechanism of action needs to be further explored.

Sugar metabolism was reported to be involved in plant photosynthesis, energy metabolism and immune response (Kanwar and Jha 2019). The expression of glucose‐, sucrose‐, fructose‐, starch‐ and SWEET‐related transcripts are up‐regulated in SCMV‐infected sugarcane plants (Akbar et al. 2021). Tomato leaf curl New Delhi virus (ToLCNDV) infection increases the accumulation of sucrose, glucose and fructose, and significantly attenuates starch synthesis in potato (Kumar et al. 2023). Our results showed that most of the fructose and mannose metabolism‐related proteins were up‐regulated and possessed up‐regulated DUSs after viral infection (Figure 5d and Figure S5). We hypothesised that viruses use plant carbon to promote proliferation by enhancing host sugar metabolism. By contrast, host plants increase ubiquitination levels to decrease this process for combating virus infections.

Photorespiration is the process by which a plant takes in oxygen and releases carbon dioxide and is generally considered a wasteful cycle (Shi and Bloom 2021). However, it is worth noting that photorespiration can eliminate the toxic 2‐phosphoglycolate (2‐PG) generated by the oxygenase reaction of Rubisco, regulate reactive oxygen species (ROS) homeostasis and affect defence hormone synthesis, which plays an important role in the plant immune response (Jiang et al. 2023). As a C4 plant, maize has a low photorespiration rate (Bräutigam and Gowik 2016). The results of proteome and ubiquitinome analyses revealed that the proteins related to the glyoxylate and dicarboxylate metabolism pathway showed significant differences in protein expression and ubiquitination level between virus‐infected and non‐infected groups (Figure S6). GOX is one of the key enzymes in the photorespiration pathway and is localised in the peroxisome (Dellero et al. 2016). The accumulation of glycolate in the maize zmgo1 mutant is significantly elevated and plants could not survive in normal air (Zelitch et al. 2009). H₂O₂ accumulation and callose deposition were decreased in GOX‐silenced N. benthamiana plants and GOX T‐DNA insertion mutants of Arabidopsis thaliana , resulting in impaired nonhost resistance (Rojas et al. 2012). The resistance of GOX2‐silenced tomato plants to Pseudomonas syringae infection is significantly down‐regulated, and the content of H₂O₂ is decreased (Ahammed et al. 2018). Decreased expression of the GOX1 gene in rice increased resistance to Xanthomonas oryzae pv. oryzae and up‐regulated expression of defence regulators (Chern et al. 2013). P8 of rice dwarf virus (RDV) was reported to interact with GOX in rice, resulting in the translocation of P8 into peroxisomes, which promotes viral replication (Zhou et al. 2007). In this study, we found that the protein level of ZmGOX1 (A0A3L6E0R4) was significantly down‐regulated in S+M versus mock‐inoculation (PBS), which had up‐regulated DUSs in five different comparisons (Figure S6). The results of CMV‐VIGS assays indicated that ZmGOX1 plays an important role in resistance to SCMV and MCMV infection in maize (Figure 5). Moreover, the expression of ZmGOX1 was up‐regulated after Na₂S treatment, improving the resistance of maize to virus infections (Figure 7). The results of SCMV‐based VOX assays showed that the ZmGOX1 mutants at Kub sites possessed higher anti‐SCMV activity than that of ZmGOX1, especially ZmGOX1^RR (Figure 6). These results showed that virus infections decreased the protein level of ZmGOX1 by up‐regulating its ubiquitination level to suppress the antiviral response in maize. However, the mechanism underlying the antiviral roles of ZmGOX1 needs to be further investigated.

Hydroxypyruvate reductase (HPR) gene encodes an enzyme with hydroxypyruvate reductase, glyoxylate reductase (GR) and D‐glycerate dehydrogenase enzymatic activities (Givan and Kleczkowski 1992). In this study, we found that the expression of ZmHPR1 (B4FLP0) was altered after viral infection, which had up‐regulated DUSs in M versus PBS, S+M versus PBS, S+M versus S and S+M versus M (Figure S6). The expression of ZmHPR2 (A0A1D6KCW2) was up‐regulated in virus‐infected maize plants, which had significantly up‐regulated DUSs in S+M versus M (Figure S6). Rice OsHPR1 and OsHPR2 proteins are localised in peroxisomes and the cytosol, respectively (Ye et al. 2014). Different subcellular localisation may be responsible for the different expression patterns and functions of ZmHPR1 and ZmHPR2 in response to virus infection. The VIGS results showed that ZmHPR2‐ and ZmHPR1‐silenced maize plants showed increased resistance to virus infections (Figure 5). Glyoxylate is produced in plant peroxisomes and is highly toxic to plant cells (Lu et al. 2014). The glyoxylate level in rice OsGR knockout mutants was increased but the phenotype did not change, indicating that glyoxylate accumulation caused by lack of OsGR does not cause toxicity to rice (Zhang et al. 2020). The syringolide signalling receptor P34 can interact with NADPH‐dependent HPR to inhibit its function and induce the hypersensitive response (HR) (Okinaka et al. 2002). Arabidopsis HPR2 protein can bind to salicylic acid (SA) and may play an important role in immune responses (Manohar et al. 2015). Therefore, we speculate that the antiviral role of HPR may be related to HR and/or the SA‐related pathway.

Taken together, ubiquitination is widely involved in the antiviral response of maize. After virus infections, the ubiquitination levels of proteins in maize were increased. The results of proteome and ubiquitinome analyses showed that most of the DAPs related to carbon fixation in photosynthetic organisms, photosynthesis, fructose and mannose metabolism and glyoxylate and dicarboxylate metabolism were changed at protein levels and had up‐regulated DUSs. Furthermore, the antiviral roles of three candidate genes, ZmGOX1, ZmHPR1 and ZmHPR2, were investigated using the CMV‐VIGS approach, and the stability and VOX assays verified that ubiquitination modification was involved in the regulation of ZmGOX1 protein levels and its anti‐SCMV activity (Figure 8). Our study provides novel insights into the role of ubiquitination modification in plant–virus interaction and candidate genes for disease resistance breeding.

FIGURE 8.

A proposed model for the involvement of ubiquitination in resistance to virus infection in maize. MCMV, maize chlorotic mottle virus; SCMV, sugarcane mosaic virus. The accumulation of the proteins in the pink boxes was up‐regulated after virus infection. The accumulation of the proteins in the blue boxes was down‐regulated after virus infection; red circles represent up‐regulated differentially ubiquitinated sites (DUSs); blue circles represent down‐regulated DUSs. The protein in the red font plays antiviral roles in maize, which was verified through virus‐induced gene silencing (VIGS) and virus‐mediated overexpression assays. The proteins in the green font facilitate virus infections in maize, which were verified through VIGS assays.

4. Experimental Procedures

4.1. Plant Materials and Treatments

Maize ( Zea mays ) inbred line B73 plants were cultured in the growth chambers (28°C/22°C, 16 h/8 h, day/night). The source preparation and inoculation method of SCMV and MCMV single and synergistic infection (S+M) referred to our previous report (Hao et al. 2023). At 9 dpi, the first systemically infected leaves were harvested for proteome and ubiquitinome analysis. For MG132 inhibitor treatment, the leaves of maize at the three‐leaf stage were sprayed with 50 μM MG132 (MedChemExpress) dissolved in 1% (vol/vol) DMSO or a 1% (vol/vol) DMSO solution as control. MG132‐treated and control maize plants were inoculated with the viruses 24 h after spraying. At 7 dpi, the first systemically infected leaves were harvested for measurement of viral accumulation.

4.2. 26S Proteasome Activity Assay

The double antibody sandwich method (Hengyuan Biological) was employed to quantify the 26S proteasome activity in plant samples. Briefly, maize leaves were ground in PBS and the tissue fragments were removed by centrifugation at 4°C at 3000 g for 30 min, then each sample was diluted with assay buffer. The samples and standard products were added to the designated wells, incubated at 37°C for 30 min, and then washed five times with the washing solution. Then the enzyme‐labelled reagent was added to each well, incubated at 37°C for 30 min, and then washed five times with the washing solution. Finally, the chromogenic substrate was added and incubated in the dark for 15 min then the stop solution was added and the absorbance was measured at 450 nm.

4.3. Protein Extraction

Proteome and ubiquitinome sequencing were performed on PBS‐, SCMV‐, MCMV‐ and S+M‐inoculated maize samples (PTM BioLabs Co. Ltd., Hangzhou, China). Maize leaf samples were ground in liquid nitrogen and transferred to centrifuge tubes. Four volumes of lysis buffer (0.7 M sucrose, 0.1 M KCl, 0.5 M Tris–HCl, pH 7.5 and 50 mM EDTA, 10 mM dithiothreitol [DTT], 1% protease inhibition cocktail, 50 μM PR‐619) were added into the centrifuge tubes. After ultrasonic cracking, an equal volume of Tris‐saturated phenol (pH 8.0) was added. Then, we collected the supernatant into a new centrifuge tube after centrifugation at 5500 g for 10 min at 4°C. Proteins were precipitated by adding five volumes of ammonium sulphate‐saturated methanol and incubated for at least 6 h. The supernatant was discarded after centrifugation at 4°C for 10 min. The remaining precipitate was washed with ice‐cold methanol, followed by ice‐cold acetone three times. The protein was redissolved in 8 M urea and the protein concentration was determined with a bicinchoninic acid (BCA) kit (Beyotime Biotechnology) according to the manufacturer's instructions. Each sample pooled with at least 10 plants was performed for three independent biological replicates.

4.4. Trypsin Digestion

Equal volume of protein from different samples was used, and 5 mM DTT was added, and reduction reaction performed at 56°C. After 30 min, 11 mM iodoacetamide (IAM) was added, and the alkylation was continued for 15 min at room temperature in darkness. Then the protein sample was diluted with 200 mM tetraethylammonium bromide (TEAB). Finally, trypsin was added to the protein solution at a mass ratio of 1:50 (trypsin/protein) for the enzymatic digestion overnight.

4.5. Enrichment of Ubiquitinated Peptides

Peptides were dissolved in IP buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris–HCl, 0.5% NP‐40, pH 8.0), and the supernatant was incubated with pre‐washed antibody beads (PTM Biolabs) at 4°C overnight with gentle shaking. Then the beads were washed for four times with IP buffer and twice with double‐distilled water. The bound peptides were eluted from the beads with 0.1% trifluoroacetic acid. Finally, the eluted fractions were combined and vacuum‐dried. The obtained peptides were cleaned with C18 Zip Tips (Millipore).

4.6. 4D Mass Spectrometry

The solvent B (0.1% formic acid in acetonitrile) was used to separate peptides that were dissolved in solvent A (0.1% formic acid and 2% acetonitrile) by NanoElute Ultra Performance Liquid Chromatography system (Bruker Daltonics) at a constant flow rate of 450 nL/min. For the ubiquitinome analysis, the gradient settings were as follows: 0–40 min, 6%–22% B; 40–52 min, 22%–30% B; 52–56 min, 30%–80% B; 56–60 min, 80% B. For the proteome analysis, the gradient settings were as follows: 0–70 min, 6%–24% B; 70–82 min, 24%–32% B; 82–86 min, 32%–80% B; 86–90 min, 80% B. The separated peptides were injected into the capillary source for ionisation and then into the timsTOF Pro 2 (Bruker Daltonics) mass spectrometer for analysis. The data acquisition was set to Parallel Accumulated Serial Fragmentation (PASEF) mode and dynamic exclusion time was set to 30 s.

4.7. Database Search and Bioinformatics Analysis

Maxquant search engine (v.1.6.15.0) was used for tandem mass spectrometry (MS/MS) data processing as described previously with minor changes (Cai et al. 2020). The Zea mays UniProt database (63,235 sequences) concatenated with the reverse decoy database was used for MS/MS data search. Label‐free quantification (LFQ) and unique peptides for quantification were selected. DAPs and DUSs were identified with a reference threshold of fold change > 1.5 or < 0.67, p < 0.05. Eggnog‐mapper software (v. 2.1.6) (http://eggnog‐mapper.embl.de/) was used for GO analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/tools/kaas/) was used for KEGG pathway enrichment analysis. WoLF PSORT software (https://wolfpsort.hgc.jp/) was used to predict and analyse the subcellular structure of the proteins. In the nine‐quadrant diagram, for multiple Kub sites of the same protein, the fold change of the ubiquitination level at each Kub site was independently calculated and paired with the fold change of the protein expression. Transcriptome data used in this study were obtained from our previous study (Hao et al. 2023).

4.8. Parallel Reaction Monitoring Analysis

Protein extraction, trypsin digestion and enrichment of ubiquitinated peptides were performed as above mentioned, but the formula of the lysis buffer was adjusted to add 50 mM nicotinamide and 3 μM trichostatin A. The solvent B (0.1% formic acid and 90% acetonitrile) was used to separate peptides that were dissolved in solvent A (0.1% formic acid and 2% acetonitrile) by EASY‐nLC 1200 UPLC system (Thermo Scientific) at a constant flow rate of 500 nL/min. For the analysis of PRM targeting ubiquitinated peptides, the gradient settings were as follows: 0–40 min, 8%–22% B; 40–52 min, 22%–32% B; 52–56 min, 32%–80% B; 56–60 min, 80% B. Full MS detection was performed in the scan range of 480–1200 m/z with a resolution on the Orbitrap Exploris 480 MS instrument (Thermo Fisher Scientific) set to 300% for automatic gain control (AGC) and 50 ms for the maximum injection time. For secondary MS, Orbitrap scanning resolution was set to 15,000 with AGC at 100%, maximum injection time at 220 ms, and isolation window at 2.0 m/z. The data were analysed using the Skyline 21.1 software (https://skyline.ms/) for the PRM RAW files. For the analysis of PRM targeting proteins, the gradient settings were as follows: 0–40 min, 6%–20% B; 40–52 min, 20%–28% B; 52–56 min, 28%–80% B; 56–60 min, 80% B. Full MS detection was performed in the scan range of 430–1125 m/z. For secondary MS, isolation window was set at 1.6 m/z. Other parameters were the same as before, and the data were analysed using the Skyline 21.2 software (https://skyline.ms/).

4.9. CMV‐VIGS Assay in Maize

The CMV‐VIGS assays were performed according to a previous report (Wang et al. 2016). The first true leaf of three‐leaf stage gene‐silenced maize plants was challenged by inoculation with SCMV and/or MCMV. The first systemically infected leaves were collected at 7 dpi.

4.10. The Stability Assays of ZmGOX1 and Its Mutants

Coding sequences for ZmGOX1 (Zm00001eb300130_T002) were amplified from maize cDNA templates. The pGD vector was used for transient expression (Goodin et al. 2002). The fragments of the coding sequences of ZmGOX1, ZmGOX1^K136R, ZmGOX1^K361R and ZmGOX1^RR were inserted into the pGD‐3×Flag vector and the construction of pGD‐3×Flag was described previously (Sun et al. 2018). All the primers used for plasmid construction are listed in Table S1.

For the protein degradation assay, the pGD‐3×Flag‐ZmGOX1 recombinant vectors were transiently expressed in N. benthamiana leaves for 72 h after infiltration, and the infiltrated leaves were treated with 50 μM MG132 or an equal volume of DMSO with 10 mM MgCl₂ at 12 h before harvest. For protein stability assays, N. benthamiana leaves were agroinfiltrated with pGD‐3×Flag‐ZmGOX1, pGD‐3×Flag‐ZmGOX1^K136R, pGD‐3×Flag‐ZmGOX1^K361R and pGD‐3×Flag‐ZmGOX1^RR (OD₆₀₀ = 1.0) and harvested at 3 days after infiltration.

4.11. SCMV‐Based VOX Assay

The modified infectious clones pSCMV‐3×Flag‐ZmGOX1, pSCMV‐3×Flag‐ZmGOX1^K136R, pSCMV‐3×Flag‐ZmGOX1^K361R and pSCMV‐3×Flag‐ZmGOX1^RR were generated by replacing GFP in the pSCMV‐GFP vector with 3×Flag‐ZmGOX1, 3×Flag‐ZmGOX1^K136R, 3×Flag‐ZmGOX1^K361R and 3×Flag‐ZmGOX1^RR, respectively. All the primers used for plasmid construction were listed in Table S1. The N. benthamiana leaves were agroinfiltrated with pSCMV‐GFP or its derivative viruses, including pSCMV‐3×Flag‐ZmGOX1, pSCMV‐3×Flag‐ZmGOX1^K136R, pSCMV‐3×Flag‐ZmGOX1^K361R and pSCMV‐3×Flag‐ZmGOX1^RR (OD₆₀₀ = 1.5), and harvested at 5 days after infiltration for virus inoculation of maize. At 5 dpi, the first systemically infected leaves of maize were harvested for measurement of viral accumulation and 3×Flag‐ZmGOX1 protein levels.

4.12. Sodium Sulphide Treatment

Maize plants were sprayed with double‐distilled water, 1 mM or 2 mM Na₂S aqueous solution at 24 h before and after PBS, SCMV, MCMV and S+M inoculation. The first systemically infected leaves in PBS‐inoculated maize plants were harvested to measure ZmGOX1 gene expression level at 24 h, 72 h and 120 h after the last water or Na₂S spray. The first systemically infected maize leaves were harvested at 4 dpi for measurement of viral accumulation and ZmGOX1 gene expression level.

4.13. RT‐qPCR

Total RNA was extracted from the first systemically infected maize leaves by TRIzol reagent (Vazyme), which was reverse transcribed to cDNA by HiScript III RT SuperMix (+gDNA wiper) (Vazyme). Real‐time qPCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme) with a StepOne Plus real‐time PCR system (Applied Biosystems). The relative gene expression was calculated by the 2^−ΔΔCT method (Livak and Schmittgen 2001). ZmUBI (XM_008647047) was used as an internal control. The specific primers for RT‐qPCR assays were designed according to maize nucleotide sequences (Table S1). The experiments were performed with three biological replicates.

4.14. Western Blot Analysis

Total proteins were extracted from the first systemically infected maize leaves under different treatments for analysis of ubiquitination level. The proteins were separated with 7.5% SDS‐PAGE and transferred onto 0.22 μm nitrocellulose (NC) membrane (Biosharp). The membranes were blocked with blocking solution (Solarbio) for 1 h. Anti‐ubiquitin monoclonal antibody (PTM BIO) was used at a dilution of 1:2000. Horseradish peroxidase (HRP)‐conjugated goat anti‐mouse secondary antibody (ABclonal) was used at a dilution of 1:10,000. Finally, conjugates immobilised on the membrane were detected by Chemiluminescence Gel Imager (Tanon) through ECL solution (Millipore).

Western blot analysis was performed to determine the accumulation of SCMV CP, MCMV CP, ZmGOX1 and its mutants at Kub sites according to the above protocol with minor modifications. Briefly, the proteins were separated and transferred onto 0.2 μm polyvinylidene fluoride (PVDF) membranes (Sangon Biotech). Anti‐SCMV or MCMV monoclonal antibody (LV BAO) was used at a dilution of 1:5000. Anti‐Flag monoclonal antibody (Solarbio) was used at a dilution of 1:10,000. Anti‐mouse IgG HRP antibody (ABclonal) was used at a dilution of 1:10,000.

4.15. Immunoprecipitation

The total protein was incubated with 25 μL of anti‐Flag magnetic beads (ABclonal) at 4°C on a rotator for 2 h. Magnetic beads were collected after centrifugation and rinsed three times with IP buffer. The magnetic beads were boiled in the 1 × SDS‐PAGE sample loading buffer for 10 min and analysed using western blotting. Anti‐Flag monoclonal antibody (Solarbio) was used at a dilution of 1:10000. Anti‐ubiquitin monoclonal antibody (PTM BIO, Hangzhou, China) was used at a dilution of 1:2000.

Author Contributions

Huiyan Guo was involved in validation, writing – original draft preparation. Xue Dong was involved in formal analysis and investigation. Kaiqiang Hao, Jinxiu Guo, Shixue Zhao and Lijun Sang were involved in investigation and visualisation. Xinran Gao and Jian Li were involved in investigation, visualisation and validation. Zhiping Wang and Mengnan An were involved in writing – review and editing. Zihao Xia and Yuanhua Wu were involved in conceptualization, methodology and funding acquisition.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: The expression of ubiquitination pathway related genes and proteins in maize plants and 26S proteasome activity assay. (a) The expression of ubiquitination pathway related genes in maize plants. (b) The expression of proteasome subunit proteins in maize plants. (c) The assay of 26S proteasome activity. The colour scale (blue to red) represents low to high gene expression intensities. The asterisk indicates the DUSs. Red asterisks represent up‐regulated DUSs. PBS solution, PBS; S, SCMV; M, MCMV; S+M, SCMV and MCMV co‐infection. The statistical significances were determined using one‐way analysis of variance followed by Duncan's multiple comparison test (p value < 0.05).

Figure S2: Functional categorizations of DAPs in five different comparisons. (a) GO analyses of DAPs. (b) KEGG enrichment analyses of DAPs. (c) Classification of subcellular location of DAPs.

Figure S3: Scatter plot of the DAPs in proteome and ubiquitinome data based on their fold change values in four comparisons. Scatter plot in S versus PBS (a), M versus PBS (b), S+M versus PBS (c) and S+M versus S (d). The coloured dots indicate proteins with fold change values of either > 1.5 or < 0.67 in DAPs and DUSs. Ps, proteins; Us, ubiquitinated sites. The blue dotted line represents the trend line.

Figure S4: GO and KEGG analysis of DAPs between proteome and ubiquitinome in four comparisons. GO and KEGG analysis in S versus PBS (a), M versus PBS (b), S+M versus PBS (c) and S+M versus S (d).

Figure S5: The complex association between enriched KEGG pathways in S versus PBS (a), M versus PBS (b), S+M versus PBS (c) and S+M versus S (d). Plots showing DAPs of different KEGG pathways detected in proteomics, and the dot colours indicate fold change values. The asterisk indicates the DUSs. Red asterisks represent up‐regulated DUSs. Blue asterisks represent down‐regulated DUSs. Yellow asterisks represent both up‐regulated and down‐regulated DUSs.

Figure S6: Diagram showing the crosstalk of glyoxylate metabolism pathway and the expression levels of corresponding proteins. LFQ intensity values were used to generate the heat map. Red asterisks represent up‐regulated DUSs, and the black asterisk indicates unchanged.

Figure S7: Proteome and ubiquitinome data were verified by PRM analysis. (a) The protein levels determined by PRM analysis. (b) The Kub sites determined by PRM analysis.

Figure S8: The expression level of ZmGOX1 gene in maize plants treated with Na₂S at 24 h, 72 h and 120 h. Lowercase letters indicate statistical difference between treatments. The statistical significances were determined using one‐way analysis of variance followed by Duncan's multiple comparison test (p value < 0.05).

Figure S9: The expression of autophagy‐related genes at the transcriptional and protein level. (a) The expression of autophagy‐related genes at the transcriptional level. (b) The expression of autophagy‐related genes at the protein level.

Table S1: Specific primers used for qPCR, VIGS and overexpression assays.

Table S2: Summary of all proteins.

Table S3: Basic information of the identified proteins.

Table S4: DAPs only in S+M versus PBS.

Table S5: Summary of ubiquitinome database.

Table S6: Basic information of the identified proteins possessed DUSs.

Table S7: The different expression patterns of multiple pathway‐related proteins in maize response to viral infection.

Table S8: The peptide and ubiquitylation lysine sites verified by PRM analysis.

Guo, H. , Dong X., Hao K., et al. 2025. “Proteome and Ubiquitinome Analyses Reveal the Involvement of Ubiquitination in Resistance to Maize Lethal Necrosis.” Molecular Plant Pathology 26, no. 9: e70147. 10.1111/mpp.70147.

Data Availability Statement

The mass spectrometry proteome data and ubiquitinome data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD054963 and PXD054971.