Maize catalases are recruited by a virus to modulate viral multiplication and infection

Abstract

Given the detrimental effects of excessive reactive oxygen species (ROS) accumulation in plant cells, various antioxidant mechanisms have evolved to maintain cellular redox homeostasis, encompassing both enzymatic components (e.g., catalase, superoxide dismutase) and non‐enzymatic ones. Despite extensive research on the role of antioxidant systems in plant physiology and responses to abiotic stresses, the potential exploitation of antioxidant enzymes by plant viruses to facilitate viral infection remains insufficiently addressed. Herein, we demonstrate that maize catalases (ZmCATs) exhibited up‐regulated enzymatic activities upon sugarcane mosaic virus (SCMV) infection. ZmCATs played crucial roles in SCMV multiplication and infection by catalysing the decomposition of excess cellular H₂O₂ and promoting the accumulation of viral replication‐related cylindrical inclusion (CI) protein through interaction. Peroxisome‐localized ZmCATs were found to be distributed around SCMV replication vesicles in Nicotiana benthamiana leaves. Additionally, the helper component‐protease (HC‐Pro) of SCMV interacted with ZmCATs and enhanced catalase activities to promote viral accumulation. This study unveils a significant involvement of maize catalases in modulating SCMV multiplication and infection through interaction with two viral factors, thereby enhancing our understanding regarding viral strategies for manipulating host antioxidant mechanisms towards robust viral accumulation.

Maize catalase activities are enhanced during SCMV infection. All three catalases are recruited to the SCMV replication complex and play pro‐viral roles by scavenging H₂O₂ and reinforcing the accumulation of viral RNA helicase.

1. INTRODUCTION

Due to their sessile nature, plants have developed multiple defence mechanisms against various biotic and abiotic stresses (Zhou & Zhang, 2020). Reactive oxygen species (ROS) play a pivotal role as signalling molecules in perceiving diverse stimuli and activating plant defence responses (Baxter et al., 2013; Mittler et al., 2022). ROS encompass hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂), the superoxide anion (${\mathrm{O}}_2^{\cdot -}$) and the hydroxyl radical (·OH), with H₂O₂ being the most stable form that prominently triggers downstream reactions (Qi et al., 2018; Waszczak et al., 2018). Intracellular ROS in plants are generated within organelles such as chloroplasts, peroxisomes and mitochondria, while apoplastic ROS are mainly generated through the actions of plasma membrane‐localized NADPH oxidase, peroxidase and amine oxidase (Kadota et al., 2015; Qi et al., 2017; Singh et al., 2016). ROS exert important roles in signal transduction at low levels but cause oxidative damage to macromolecules (DNA, protein, lipids, etc.) at high levels (Camejo et al., 2016; Foyer & Noctor, 2005). In order to maintain the cellular redox homeostasis, several enzymatic components (e.g., catalase, superoxide dismutase, glutathione peroxidase) and non‐enzymatic ones (e.g., vitamin C, vitamin E, glutathione) work in concert as antioxidant defence systems (Gill & Tuteja, 2010; Wrzaczek et al., 2013).

Catalase (CAT) is a highly conserved heme‐containing enzyme, present in virtually all living organisms, that can convert excess H₂O₂ into H₂O and O₂ through a dismutation reaction (Mhamdi et al., 2012). The three catalase isozymes in maize, namely ZmCAT1, ZmCAT2 and ZmCAT3, are encoded by three unlinked genes (Roupakias et al., 1980). These isozymes exhibit a high degree of amino acid sequence similarity (67%–78%) (Guan & Scandalios, 1996). Similar to many other higher plants, all three maize catalases are localized in peroxisomes due to the presence of an uncleaved C‐terminal tripeptide motif (S/TRL) (Jiao, Wang, et al., 2021; Mhamdi et al., 2010). Previous studies have demonstrated the involvement of catalases in response to abiotic stresses and pathogen infections (Yuan et al., 2017; Zhang et al., 2015; Zhou et al., 2018). For instance, CAT2 plays a pivotal role in coordinating the repression of auxin accumulation and jasmonic acid (JA) biosynthesis mediated by salicylic acid (SA), thereby regulating resistance against biotrophs or necrotrophs in Arabidopsis thaliana (Yuan et al., 2017). Furthermore, the interaction between maize chlorotic mottle virus (MCMV) P31 and ZmCAT1 promotes P31 accumulation. By inhibiting catalase activity, P31 down‐regulates the expression of SA‐responsive pathogenesis‐related protein (PR) genes to facilitate MCMV multiplication and infection (Jiao, Tian, et al., 2021). Although catalases have been implicated in various plant viral infections by interacting with viral factors, their potential exploitation for modulating viral replication remains unexplored.

Sugarcane mosaic virus (SCMV) is a member of the genus Potyvirus (family Potyviridae), which causes severe dwarf mosaic disease in maize (Zea mays), sugarcane (Saccharum sinensis) and sorghum (Sorghum vulgare) (Fan et al., 2003; Xiao et al., 1993). Infection by SCMV leads to leaf mosaic, seedling dwarfism and losses of maize yield, while its synergistic infection with MCMV causes the devastating disease maize lethal necrosis (MLN) and results in severe yield losses (Chen, Cao, et al., 2017; Jiao et al., 2022). The genome of SCMV is a single‐stranded, positive‐sense RNA of approximately 10 kb in length, encoding a large polyprotein that can be cleaved into 10 mature proteins (P1 protease, HC‐Pro, P3, 6K1, CI, 6K2, viral genome‐linked protein [VPg], nuclear inclusion a‐protease [NIa‐Pro], nuclear inclusion b [NIb] and coat protein [CP]) by three self‐encoded proteases (Inoue‐Nagata et al., 2022). In addition, a fusion polypeptide P3N‐PIPO (Pretty Interesting Potyviridae ORF) is generated through transcriptional slippage and −1/+2 frameshift within the P3 cistron (Olspert et al., 2015). During the replication of SCMV, 6K2‐VPg‐NIa‐Pro‐induced cytoplasmic vesicles associated with viral replication are observed to specifically target multiple organelles, including the endoplasmic reticulum (ER), Golgi apparatus, peroxisomes and mitochondria (Xie et al., 2021).

Our previous research has demonstrated that the manifestation of mosaic symptoms in SCMV‐infected maize leaves is accompanied by elevated levels of H₂O₂ (Jiang, Du, Wang, et al., 2023). Therefore, we hypothesized that catalases could respond to SCMV infection as a protective mechanism against oxidative stress‐induced cell damage. In agreement with our hypothesis, we observed an increase in catalase activities upon SCMV infection. All three maize catalases catalyse the decomposition of excessive H₂O₂ within plant cells and promote the accumulation of SCMV replication‐related CI protein. Additionally, we found that SCMV HC‐Pro could interact with ZmCATs both in yeast and in planta, enhancing catalase activities to facilitate viral accumulation.

2. RESULTS

2.1. SCMV infection up‐regulates the activity of maize catalases

To investigate whether ZmCATs were responsive to SCMV infection, the first true leaf of 8‐day‐old B73 maize seedlings was inoculated with GFP‐tagged SCMV (SCMV‐GFP), while plants inoculated with phosphate buffer served as controls (mock). The second true leaf (i.e., the first systemically infected leaf [1st SL]) of SCMV‐GFP‐inoculated maize seedlings was collected at 3, 6 and 9 days post‐inoculation (dpi) for reverse transcription‐quantitative PCR (RT‐qPCR) and immunoblot analyses, and the equivalent for mock controls. At the early stage of infection (3 dpi), no distinct symptoms were observed in SCMV‐infected maize seedlings, and virus accumulation remained undetectable through immunoblot analysis (Figure 1a,b). ZmCAT1 mRNA exhibited a nearly 50% reduction, while there were no significant alterations observed in the accumulation of ZmCAT2 and ZmCAT3 mRNA in SCMV‐infected seedlings compared with the mock‐inoculated plants (Figure S1a–c). Subsequently, mild chlorotic and mosaic symptoms emerged at the base of the first SL at 6 dpi, which rapidly progressed upwards by 9 dpi. Consistent with these visible manifestations, green fluorescence indicative of viral distribution was also observed throughout the leaf tissues at 9 dpi (Figure 1a). The SCMV accumulation was also concomitantly increased as analysed by immunoblot, while the expression levels of ZmCATs were down‐regulated at 6 or 9 dpi (Figure S1a–c). Intriguingly, the activity of maize catalases exhibited an increase during SCMV infection (Figure 1c).

FIGURE 1.

SCMV infection up‐regulates the activity of maize catalases. (a) Symptoms in the first systemically infected leaves (1st SLs) of mock‐inoculated or SCMV‐inoculated maize (B73) seedlings in a time course. Scale bars, 1 cm. (b) Immunoblotting analysis of SCMV coat protein (CP) in mock‐inoculated or SCMV‐infected 1st SLs at 3, 6 and 9 days post‐inoculation (dpi). Actin was used as the loading control. (c) The catalase activities of the 1st SLs of mock‐inoculated or SCMV‐inoculated maize seedlings were measured with a spectrophotometer in a time course. The values of catalase activity in mock‐inoculated maize leaves were set to 1. Error bars represent mean ± SEM (n ≥ 5). The asterisks indicate significant differences (two‐tailed Student's t test; **p < 0.01, ***p < 0.001; ns, no significance, p > 0.05). (d) Symptoms in the 1st SLs of SCMV‐inoculated maize (B73) seedlings pretreated with 10 μM methyl viologen (MV) or 20 mM 3‐amino‐1,2,4‐triazole (3‐AT). (e) The relative accumulation levels of SCMV CP mRNA in the 1st SLs at 7 dpi were analysed by reverse transcription‐quantitative PCR. The values of SCMV CP mRNA accumulation in mock (water)‐treated maize leaves were set to 1. Error bars represent mean ± SEM (n ≥ 5). The asterisks indicate significant differences (two‐tailed Student's t test; ****p < 0.0001). (f) The accumulation levels of SCMV CP in the 1st SLs at 7 dpi were analysed through immunoblot. Actin was used to show equal loading. The relative accumulation levels of SCMV CP were quantified using the software ImageJ. The amount of SCMV CP in water‐sprayed maize leaves was set as 1.0.

To characterize the role of catalase in SCMV multiplication, the ROS inducer methyl viologen (MV) and the catalase inhibitor 3‐amino‐1, 2, 4‐triazole (3‐AT) were exogenously applied to 8‐day‐old maize seedlings, respectively, while water‐treated plants served as controls. Then, the seedlings were inoculated with SCMV‐GFP at 4 h after treatment. The H₂O₂ contents in MV‐ or 3‐AT‐treated maize leaves were measured at 4 h after spraying. Similar to the results reported previously (Jiao, Tian, et al., 2021; Jiao, Wang, et al., 2021), the H₂O₂ levels were higher than those in the control plants (Figure S2). Compared with the control plants, MV‐treated maize seedlings displayed milder mosaic symptoms at 6 dpi, while 3‐AT‐treated plants exhibited a photobleaching phenotype in the newly emerged leaves, resulting in the impossibility to distinguish mosaic symptoms (Figure 1d). The fluorescence intensity of GFP in MV‐ or 3‐AT‐treated plants was lower than that in water‐treated seedlings, which was consistent with the decreased accumulations of SCMV RNA and CP detected by RT‐qPCR and immunoblotting (Figure 1e,f). As reported previously, SCMV infection leads to an increase in H₂O₂ levels in maize cells, which can cause cytotoxicity when present at excessive levels (Jiang, Du, Xie, et al., 2023). Therefore, catalase activity may be enhanced during virus multiplication to protect plant cells from oxidative damage, while attenuation of catalase activity results in excess H₂O₂ and impairs virus accumulation. These findings suggest that maize catalases may play essential roles in robust SCMV infection by facilitating the decomposition of excess H₂O₂.

2.2. Maize catalases play a crucial role in facilitating SCMV infection

To further elucidate the role of catalases in SCMV infection, we employed a cucumber mosaic virus (CMV)‐based gene silencing vector to silence ZmCATs as previously described (Wang et al., 2016). A highly conserved fragment of 245 bp (identity = 82.5%) among all three ZmCATs was amplified and cloned into pCMV201‐2b_N81 vector (Figure S3). A 254‐bp DNA fragment of the GFP gene was inserted into pCMV201‐2b_N81 as a control. Maize (B73) seeds were inoculated with the recombinant virus ZMBJ‐CMV harbouring the ZmCAT or GFP fragment using the vascular puncture inoculation method (Wang et al., 2016). The second true leaf (first SL) of the CMV‐inoculated maize seedlings was harvested at 14 dpi for assessing the silencing efficiency of ZmCATs by RT‐qPCR. The RT‐qPCR results showed that the relative expressions of ZmCAT1, ZmCAT2 and ZmCAT3 were reduced to about 64%, 39% and 54%, respectively, in the ZmCATs‐silenced plants compared with that in the control plants (Figure 2a). The CMV‐inoculated maize seedlings were challenge‐inoculated with SCMV at the two‐leaf stage. Silencing of ZmCATs expression did not affect maize growth, and milder mosaic symptoms were observed in the ZmCATs‐silenced seedlings than in the control plants at 8 days post‐SCMV inoculation (Figure 2b). Meanwhile, the SCMV genomic RNA levels and CP accumulation in the ZmCATs‐silenced plants decreased by about 37% and 27%, respectively (Figure 2c,d).

FIGURE 2.

Maize catalases play a crucial role in facilitating SCMV infection. (a) Knockdown efficiency of ZmCATs in the second true leaves of maize seedlings at 14 days post‐inoculation (dpi). The values of ZmCATs accumulation in the control plants were set to 1.0. Error bars represent mean ± SEM (n ≥ 7). The asterisks indicate significant differences (two‐tailed Student's t test; *p < 0.05, **p < 0.01). (b) Silencing of ZmCATs expression via a CMV vector‐attenuated SCMV mosaic symptoms in the first systemically infected leaf (1st SLs) of maize seedlings. Maize plants inoculated with CMV‐GFP were used as the control. All the photographs were taken at 8 days after challenge inoculation (dpi) of SCMV. Scale bars = 2 cm. (c) The relative accumulation levels of SCMV CP mRNA in the 1st SLs at 8 dpi were determined by reverse transcription‐quantitative PCR (RT‐qPCR). The values of SCMV CP mRNA accumulation in the control plants were set to 1.0. Error bars represent mean ± SEM (n ≥ 7). The asterisks indicate significant differences (two‐tailed Student's t test; *p < 0.05). (d) The accumulation levels of SCMV CP in the 1st SLs at 8 dpi were analysed through immunoblot. Actin was used to show equal loading. The relative accumulation levels of SCMV CP were quantified using the software ImageJ. (e) Schematic organization of the recombinant SCMV‐related constructs. The coding sequences of 3×FLAG‐GFP or 3×FLAG‐ZmCAT1/2/3, 3×FLAG‐ZmCAT1^HNRY were inserted between the NIb and CP cistrons to obtain the reconstructed viral infectious clones. (f) Mosaic symptoms in SCMV‐3×FLAG‐GFP or 3×FLAG‐ZmCAT1/2/3‐infected plants. All the maize leaves were photographed at 7 dpi. Scale bars = 2 cm. (g, j) The relative accumulation levels of SCMV genomic RNA in the 1st SLs at 7 dpi were determined by RT‐qPCR. Error bars represent mean ± SEM (n ≥ 5). The asterisks indicate significant differences (two‐tailed Student's t test; *p < 0.05, **p < 0.01, ***p < 0.001). (h) Detection of SCMV CP, 3×FLAG‐GFP and 3×FLAG‐ZmCATs in the 1st SLs at 7 dpi through immunoblotting. Actin was used to show equal loading. The relative accumulation levels of SCMV CP were quantified using the software ImageJ. (i) Mosaic symptoms in SCMV‐3×FLAG‐GFP or 3×FLAG‐ZmCAT1/ZmCAT1^HNRY‐infected plants. All the maize leaves were photographed at 7 dpi. Scale bars = 2 cm. (k) Detection of SCMV CP, 3×FLAG‐GFP, 3×FLAG‐ZmCAT1 and 3×FLAG‐ZmCAT1^HNRY in the 1st SLs at 7 dpi through immunoblotting. Actin was used to show equal loading. The relative accumulation levels of SCMV CP were quantified using the software ImageJ.

In addition, we overexpressed ZmCATs using the SCMV infectious clone. The GFP fragment between NIb and CP cistron on the pSCMV‐GFP vector was excised via enzyme digestion and replaced with the coding sequence of 3×FLAG‐tagged ZmCAT1, ZmCAT2 or ZmCAT3 to obtain the reconstructed vectors designated collectively as pSCMV‐3×FLAG‐ZmCAT1/2/3, with pSCMV‐3×FLAG‐GFP as a control (Figure 2e). The enzyme cutting sites of the viral protease were preserved at both ends of the inserted fragment, so that the inserted protein could be cut and remain free, without affecting virus replication. Subsequently, we used the crude extracts from infiltrated Nicotiana benthamiana leaves harbouring each of these recombinant SCMV infectious clones to mechanically inoculate maize seedlings. As depicted in Figure 2f, plants infected with SCMV‐3×FLAG‐ZmCAT1/2/3 exhibited more pronounced mosaic symptoms on the first SL (the second true leaf) compared to the control plants. The accumulation of SCMV genomic RNA and CP in the SCMV‐3×FLAG‐ZmCAT1/2/3‐infected plants also increased, compared with that in SCMV‐3×FLAG‐GFP‐infected plants (Figure 2g,h). The protein expression of 3×FLAG‐tagged ZmCATs and GFP was also detected through immunoblot (the lower panel in Figure 2h).

To determine if catalase activity is required for its role in promoting SCMV accumulation, we designed a series of catalase mutants by substituting key amino acids involved in heme‐binding. His⁶⁵, Asp¹³⁸, Arg³⁴⁴ and Tyr³⁴⁸ of ZmCAT1 were previously identified as being involved in heme‐binding (Diaz et al., 2012; Fujikawa et al., 2019). These amino acids were substituted with alanine (A) to generate maize catalase mutants (ZmCAT^HNRY) that showed greatly reduced enzyme activity compared to the wild‐type maize catalases (Figure S4a–c). The expression levels of FLAG‐tagged ZmCATs and the mutated catalases were detected through immunoblotting, revealing only slight attenuation of expression due to the point mutations in N. benthamiana leaves (Figure S4d). The fragment 3×FLAG‐ZmCAT1^HNRY was inserted into the enzymatically cleaved pSCMV vector for the construction of the recombinant pSCMV‐ZmCAT1^HNRY vector. As illustrated in Figure 2i, the maize seedlings infected with SCMV‐3×FLAG‐ZmCAT1^HNRY displayed milder mosaic symptoms on the first SL compared to the seedlings inoculated with SCMV‐3×FLAG‐ZmCAT1. The accumulation of SCMV genomic RNA and CP in seedlings infected with SCMV‐3×FLAG‐ZmCAT1^HNRY also exhibited a decrease compared to that in seedlings overexpressing 3×FLAG‐ZmCAT1 via viral vector (Figure 2j). The protein expression of 3×FLAG‐tagged ZmCAT1/ZmCAT1^HNRY and GFP was also detected through immunoblotting (the lower panel in Figure 2k). Collectively, these findings underscore the crucial roles of ZmCATs in facilitating robust SCMV infection.

2.3. Maize catalases can be recruited to SCMV replication complexes

It has been reported that all three ZmCATs are present in peroxisomes within maize cells, and SCMV can specifically target peroxisomes for replication (Jiao, Wang, et al., 2021; Xie et al., 2021). Hence, we presumed that ZmCATs may also localize to the replication complexes of SCMV. For this, GFP‐fused maize catalases were transiently co‐expressed with either the peroxisome marker DsRed‐SKL or SCMV‐mCherry‐6K2 in N. benthamiana leaves. Confocal microscopy observations showed that GFP‐ZmCATs localized in scattered peroxisomes (Figure 3a). However, GFP‐CATs were closely localized to 6K2‐induced virals replication complexes (VRC) with aggregated punctate fluorescence in the presence of SCMV (Figure 3b), while the subcellular localization of GFP alone remained unchanged (Figure S5).

FIGURE 3.

Maize catalases can be recruited to SCMV replication complexes. (a) Subcellular localization of GFP‐tagged ZmCATs in Nicotiana benthamiana leaves with DsRed‐SKL as a peroxisome marker. The fluorescence signals were captured by confocal microscopy at 3 days post‐agroinfiltration (dpi). Scale bars = 10 μm. (b) GFP‐tagged ZmCATs were recruited to the viral replicase complexes marked by mCherry‐6K2 of SCMV. The images were taken using confocal microscopy at 5 dpi. Scale bars = 10 μm.

2.4. Maize catalases promote SCMV replication in maize protoplasts

As ZmCATs can be recruited to the SCMV replication complexes, we supposed that ZmCATs may be involved in regulating SCMV replication. To verify this presumption, maize protoplasts were isolated and co‐transfected with the genomic SCMV RNA (extracted from purified virions) and 3×FLAG‐tagged ZmCATs/ GFP (control). Total RNA was extracted at 20 h after transfection for analysis of viral genomic RNA accumulation by RT‐qPCR. Compared with the control, overexpression of ZmCATs in maize protoplasts significantly enhanced the SCMV (+/−) RNA accumulation (Figure 4a,b). The relative accumulation level of ZmCATs in the transfected maize protoplasts was determined by RT‐qPCR (Figure 4d). We noticed that the overexpression level of ZmCAT1 was far less than those of ZmCAT2 and ZmCAT3, which may be due to the strict regulation of ZmCAT1 expression or the high background expression level of ZmCAT1 in maize mesophyll cells. To ascertain the necessity of catalase activity in facilitating SCMV replication, maize protoplasts were co‐transfected with the genomic SCMV RNA and 3×FLAG‐tagged ZmCATs/ZmCATs^HNRY or 3×FLAG‐GFP (as a control), respectively. The co‐transfection experiments revealed that the promotion roles of the mutated forms of catalases in SCMV replication were significantly attenuated compared with those of the active form of catalases (Figure 4e). Protein expression from the transfected vectors was confirmed through immunoblot analysis (Figure 4c,f). To further prove that catalases play an important role in promoting SCMV replication, maize protoplasts were isolated and transfected with SCMV RNA, and then incubated with MV or 3‐AT for 20 h. The accumulation of viral genomic RNA was quantified by RT‐qPCR. As depicted in Figure 4g, treatment with either MV or 3‐AT significantly impeded SCMV replication, potentially attributed to the excessive accumulation of H₂O₂ or other ROS patterns in maize protoplasts. Collectively, these data suggest that maize catalases enhance SCMV replication in maize protoplasts by scavenging excessive cellular H₂O₂.

FIGURE 4.

Maize catalases promote SCMV replication in maize protoplasts. (a, b) The relative accumulation levels of SCMV (+/−sense strand) RNA in the protoplasts co‐transfected with SCMV‐BJ viral RNA and 3×FLAG‐ZmCATs or 3×FLAG‐GFP (control) were measured by reverse transcription‐quantitative PCR (RT‐qPCR). The values of SCMV RNA accumulation in control were set to 1. (c) Detection of 3×FLAG‐GFP and 3×FLAG‐ZmCATs in the transfected maize protoplasts through immunoblotting with an anti‐FLAG antibody. Actin was used to show equal loading. (d) The relative accumulation level of ZmCATs in the transfected maize protoplasts was determined by RT‐qPCR. The values of ZmCATs accumulation in the control were set to 1. (e) The relative accumulation levels of SCMV (+) RNA in the protoplasts co‐transfected with SCMV genomic RNA and 3×FLAG‐GFP (control), 3×FLAG‐ZmCATs or mutant of 3×FLAG‐ZmCATs were measured by RT‐qPCR. The values of ZmCATs accumulation in the control were set to 1. (f) Detection of 3×FLAG‐GFP, 3×FLAG‐ZmCATs and mutant of 3×FLAG‐ZmCATs in the transfected maize protoplasts through immunoblotting with an anti‐FLAG antibody. Actin was used to show equal loading. (g) Maize protoplasts were inoculated with SCMV RNA extracted from virions and incubated at 25°C for 20 h in the presence of 10 μM methyl viologen (MV) or 20 mM 3‐amino‐1,2,4‐triazole (3‐AT). The relative accumulation levels of SCMV RNA in the protoplasts were analysed by RT‐qPCR. The values of SCMV CP accumulation in water‐treated maize protoplasts were set to 1. Error bars represent mean ± SEM (n ≥ 4). The asterisks above the bars indicate significant differences (two‐tailed Student's t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

2.5. Both SCMV HC‐Pro and CI interact with maize catalases in yeast and in planta

Given the importance of ZmCATs in SCMV multiplication and replication, possible interactions between ZmCAT1 and SCMV proteins were explored through yeast two‐hybrid (Y2H) analysis. As shown in Figure S6, strong interactions were observed between CI/HC‐Pro and ZmCAT1, while P1 and 6K2 exhibited weak association with ZmCAT1. To validate whether SCMV CI and HC‐Pro could interact with the other two maize catalases, the coding sequences of three maize (B73) catalases were cloned into the pGADT7 vector and co‐transformed with pGBKT7‐HC‐Pro/CI into yeast cells for subsequent Y2H analysis. The interactions between all three maize catalases and HC‐Pro/CI were confirmed (Figure 5a,d). These interactions were also verified through luciferase complementation imaging (LCI) assay in planta (Figure 5b,e). Co‐immunoprecipitation assays were performed to further validate these interactions with N. benthamiana tissues co‐expressing 3×FLAG‐HC‐Pro or CI‐3×Myc along with GFP or GFP‐ZmCATs. The crude extracts were precipitated with agarose‐conjugated anti‐GFP antibody, followed by SDS‐PAGE separation of the precipitated proteins for immunoblot analysis. All three GFP‐tagged maize catalases demonstrated co‐precipitation with 3×FLAG‐HC‐Pro/CI‐3×Myc, whereas no interaction was detected in negative controls (Figure 5c,f).

FIGURE 5.

Both SCMV HC‐Pro and CI interact with maize catalases in yeast and in planta. (a, d) SCMV HC‐Pro or CI interacts with ZmCATs in yeast cells. The yeast two‐hybrid (Y2H) Gold cells co‐transformed with pGADT7‐ZmCAT1/2/3 and pGBKT7‐HC‐Pro/ CI were spotted on synthetic dropout (SD)/−Trp−Leu, and −Trp−Leu−His−Ade medium in 10‐fold serial dilutions for 3 days. Yeast cells co‐transformed with AD‐T and BD‐53 served as a positive control, while yeast cells co‐transformed with empty AD vector and BD‐HC‐Pro or CI served as negative controls. (b, e) Luciferase complementation imaging assays were performed to detect the interaction between HC‐Pro/CI and ZmCATs. The Nicotiana benthamiana leaves infiltrated with the indicated constructs were collected for fluorescence imaging at 3 days post‐infiltration. (c, f) Co‐immunoprecipitation of ZmCATs and SCMV HC‐Pro or CI in planta. 3×FLAG‐HC‐Pro or CI‐3×Myc was co‐expressed with GFP‐ZmCAT1/2/3 or GFP in N. benthamiana leaves through agroinfiltration. The cell lysates were immunoprecipitated with anti‐GFP agarose affinity gel. The crude protein extracts and immunoprecipitates were immunoblotted with anti‐GFP or anti‐FLAG/Myc antibodies. The asterisks in red indicate the target bands.

2.6. SCMV HC‐Pro enhances maize catalase activities

Considering the up‐regulation of maize catalases activity by SCMV infection, it is presumed that viral factors may be involved in enhancing catalase activities through protein interaction. To investigate the potential impact of HC‐Pro on maize catalase activities, we co‐expressed 3×FLAG‐GFP or 3×FLAG‐HC‐Pro with 3×Myc‐ZmCATs in N. benthamiana leaves and extracted total proteins from the treated leaf patches for analysis at 3 days after agroinfiltration. As depicted in Figure 6a, co‐expression of ZmCATs with HC‐Pro led to an increase in catalase activities in N. benthamiana leaves. To confirm whether this increase was attributed to enhanced mRNA level of ZmCATs, the relative expression levels of ZmCATs were measured using RT‐qPCR. The expression of ZmCATs was up‐regulated upon co‐expression with HC‐Pro (Figure 6b). Furthermore, immunoblot analysis was performed to assess the protein accumulation of maize catalases. As illustrated in Figure 6c–e, the protein level of catalases increased in the presence of HC‐Pro.

FIGURE 6.

SCMV HC‐Pro enhances maize catalase activities. 3×Myc‐ZmCAT1, 3×Myc‐ZmCAT2 or 3×Myc‐ZmCAT3 was co‐expressed with 3×FLAG‐GFP (control) or 3×FLAG‐HC‐Pro in Nicotiana benthamiana leaves. (a, f) The catalase activity was measured using a spectrophotometer at 3 days post‐agroinfiltration (dpi). The values of catalase activity in the control were set to 1. (b) The relative accumulation levels of ZmCATs in N. benthamiana leaves were measured by reverse transcription‐quantitative PCR at 3 dpi. The relative accumulation levels of ZmCATs in the control were set to 1. Nbactin served as an internal control gene. Error bars represent mean ± SEM (n ≥ 6). The asterisks indicate significant differences (two‐tailed Student's t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (c–e) The total protein was extracted and immunoblotted by an anti‐FLAG antibody or an anti‐Myc antibody. The relative accumulation levels of 3×Myc‐ZmCATs were visualized using the software ImageJ. The accumulation levels of 3×Myc‐ZmCATs in the control were set to 1. Ponceau S staining of RuBisCO large subunit served as a loading control. Rep represents repeated detection of different groups of samples.

To ascertain whether the promotional role of HC‐Pro in catalase activities is contingent upon its activity as a suppressor of RNA silencing (SRS), the arginine at position 184 in the highly conserved FRNK motif of HC‐Pro was substituted with isoleucine to impair its SRS activity as described previously (Xu et al., 2020) (Figure S7). The co‐expression of ZmCATs with HC‐Pro^FINK led to a decrease in catalase activities compared to those observed upon co‐expression with HC‐Pro (Figure 6f). Collectively, these findings demonstrate that HC‐Pro significantly enhances both the maize catalase activities and expression level of ZmCATs, which is contingent upon the SRS activity of HC‐Pro.

2.7. Maize catalases enhance the accumulation of the SCMV CI protein

In contrast to HC‐Pro, SCMV CI did not exert an impact on catalase activities (Figure S8). However, co‐expression of ZmCATs in N. benthamiana tissues led to increased protein accumulation of CI (Figure 7a–c). To investigate whether the elevated levels of CI protein are associated with enhanced transcriptional expression, RT‐qPCR analysis was performed to assess the abundance of CI transcripts. No significant difference in CI mRNA levels was observed (Figure S9), suggesting that the augmented accumulation of CI protein may be attributed to increased protein stability. Furthermore, we examined the effect of MG132 (an inhibitor of the 26S proteasome) or 3‐MA (an autophagy pathway inhibitor) on the accumulation of CI to explore its potential degradation pathways. As shown in Figure 7d, the CI protein level was increased upon treatment with MG132 compared to the control treated with dimethyl sulphoxide (DMSO). Additionally, treatment with 3‐MA did not result in any significant alteration in the protein level of CI (Figure S10). Hence, it can be inferred that the SCMV CI protein undergoes degradation via the 26S proteasome pathway in N. benthamiana tissues, and ZmCATs contribute to enhancing its accumulation.

FIGURE 7.

Maize catalases enhance the accumulation of the SCMV CI protein. (a–c) CI‐3×Myc was co‐expressed with 3×FLAG‐ZmCATs or GUS‐3×FLAG (control) in Nicotiana benthamiana leaves. The total protein was extracted and detected by an anti‐FLAG antibody or an anti‐Myc antibody. The relative accumulation levels of CI‐3×Myc were quantified using the ImageJ software. The accumulation levels of CI‐3×Myc in the control were set to 1. Actin was used to show equal loading. (d) The stability of the SCMV CI protein was affected via ubiquitination pathway. CI‐3×Myc was transiently co‐expressed with GUS‐3×FLAG (control) in N. benthamiana leaves for 72 h after infiltration, and the infiltrated leaves were treated with dimethyl sulphoxide (DMSO) (control), MG132 (50 μM) at 12 h before harvest. The relative accumulation levels of CI‐3×Myc were detected through immunoblotting with an anti‐Myc antibody and quantified using the software ImageJ. The accumulation levels of CI‐3×Myc in DMSO‐treated leaf regions were set to 1. Actin was set as the control of equal loading. Rep represents repeated detection of different groups of samples.

3. DISCUSSION

During plant–virus interactions, the generation of ROS as a defence response is commonly considered an early reaction to transmit viral stimuli into plant resistance (Király et al., 2021; Vranova et al., 2002). Hydrogen peroxide, possessing a long half‐life, acts as a stable and crucial messenger in regulating both plant development and defence. However, excessive H₂O₂ leads to organelle autophagy and cell death. Consequently, antioxidant responses are triggered to eliminate cellular oxidative stress (Smirnoff & Arnaud, 2019; Waszczak et al., 2018). Nevertheless, the role of antioxidant systems in plant–virus interactions remains unclear. In this study, we observed down‐regulation of ZmCAT transcript levels during SCMV infection (Figure S1a–c), while maize catalase activities were increased in SCMV‐infected cells (Figure 1c). Inhibition of catalase activity or induction of ROS accumulation also hindered SCMV accumulation and replication in maize plants and protoplasts (Figures 1d–f and 4g). Moreover, transient silencing of ZmCATs in maize seedlings using a CMV‐based gene silencing vector alleviated SCMV infection (Figure 2a–d). Overexpression of ZmCATs via the SCMV infectious clone promoted viral accumulation (Figure 2f–h). These findings suggest that maize catalases play a pro‐viral role in SCMV infection by scavenging excessive H₂O₂. Although previous studies have primarily focused on the involvement of catalase in viral infection (Inaba et al., 2011; Jiao, Tian, et al., 2021; Yang et al., 2020), limited reports have been published regarding its impact on viral replication (Jiao, Wang, et al., 2021). In this study, we observed peroxisome‐localized maize catalases surrounding SCMV replication complexes (Figure 3a,b). Overexpression of ZmCATs enhanced SCMV replication in maize protoplasts, and this effect was compromised when the enzyme activities of ZmCATs were attenuated (Figure 4a,b,e). Therefore, ZmCATs could enhance SCMV replication by mitigating cellular oxidative damage caused by excessive H₂O₂. Similarly, all three maize catalases can also localize to MCMV replication sites and are essential for viral replication (Jiao, Wang, et al., 2021). Thus, it is plausible that catalases play a common role in virus replication. However, the mechanism underlying the localization of catalases to viral replication complexes remains elusive. We hypothesize that maize catalases may be recruited to SCMV replication sites through interaction with 6K2, because a weak interaction between ZmCAT1 and 6K2 was observed in the Y2H assay (Figure S6).

Despite the pivotal role of ROS in triggering plant defence responses, emerging studies have unveiled their advantageous impact on viral infection and replication (Foo et al., 2022). For instance, red clover necrotic mosaic virus replication necessitates a ROS burst mediated by respiratory burst oxidase homologue B (Hyodo et al., 2017). Barley stripe mosaic virus (BSMV) infection induces chloroplast oxidative stress to facilitate viral replication (Wang et al., 2021). Consequently, the manipulation of ROS and antioxidant systems by viruses varies among different host–virus systems. For example, BSMV γb protein enhances viral infection by interacting with host NADPH‐dependent thioredoxin reductase C (NTRC), thereby disrupting chloroplast antioxidant defence through interference with the NTRC‐2‐Cys Prx interaction (Wang et al., 2021). Tomato chlorosis virus (ToCV) p27 directly binds to Solanum lycopersicum catalases to inhibit catalase‐mediated anti‐ToCV processes (Sun et al., 2023). In this study, we demonstrated that SCMV HC‐Pro interacted with maize antioxidant catalases (Figure 5a–c) and enhanced their enzyme activities to facilitate virus accumulation (Figure 6a), which may provide an explanation for the observed increase in catalase activity upon SCMV infection. Moreover, the enhancement of catalase activities by HC‐Pro was dependent on its SRS activity (Figure 6f). Intriguingly, ZmCATs were also involved in the defence response by impairing the SRS activity of SCMV HC‐Pro, indicating that although CATs were recruited to promote virus infection, they could still limit the robust accumulation of SCMV to achieve a balance between the host and virus (Figure S11). In this article, our primary focus was on elucidating the promotional effect of ZmCATs on viral infection, while further exploration is required to understand how ZmCATs attenuate the SRS activity of HC‐Pro. ZmCATs also interacted with the SCMV CI protein (Figure 5d–f) and promoted CI accumulation (Figure 7a–c). It remains to be determined if ZmCATs enhanced CI accumulation through impeding protein degradation via the 26S proteasome pathway in N. benthamiana tissues (Figure 7d). The helicase activity of CI has been characterized, and its involvement in viral replication has been demonstrated by several reverse genetic experiments (Sorel et al., 2014). CI is also implicated in viral cell‐to‐cell and long‐distance movement, possibly through interaction with the viral P3N‐PIPO protein (Sorel et al., 2014). Therefore, maize catalases may positively modulate SCMV multiplication and infection by enhancing the accumulation of CI.

In recent years, increasing evidence has shown that catalases play an essential role in host resistance and are commonly targeted by factors from other pathogens. For instance, two effectors PsCRN63 and PsCRN115 (for crinkling‐ and necrosis‐inducing proteins) secreted by Phytophthora sojae manipulate plant programmed cell death and H₂O₂ homeostasis to disturb host immune responses through interacting with host catalases (Zhang et al., 2015). In addition, effector Avh113 of P. sojae interacts with soybean transcription factor DPB and induces DPB degradation, thereby inhibiting downstream CAT1‐induced cell death and enhancing host susceptibility to Phytophthora (Zhu et al., 2023). Thus, due to the essential role of catalase in regulating cellular homeostasis and response to stresses, it deserves further exploration for its functional dissection and more focus during molecular breeding for disease resistance.

In this study, we reveal that maize catalases are recruited by SCMV to modulate viral multiplication. During SCMV infection, maize catalase activities are enhanced significantly. All three maize catalases (ZmCATs) can be recruited to the SCMV replication complex and play pro‐viral roles in viral multiplication by scavenging excess H₂O₂ and reinforcing the protein accumulation of viral RNA helicase. HC‐Pro interacts with ZmCATs and enhances the enzymatic activity of ZmCATs to facilitate SCMV accumulation (Figure 8). Altogether, our results provide implications for understanding the manipulation of host antioxidant factors by viruses to promote virus multiplication and infection.

FIGURE 8.

A proposed working model indicates that maize catalases are recruited by sugarcane mosaic virus (SCMV) to modulate viral multiplication and infection. During SCMV infection, maize catalase (ZmCATs) activities are enhanced, and all three ZmCATs can be recruited to the viral replication complex (VRC). All three maize catalases play pro‐viral roles in SCMV accumulation and replication by scavenging excess H₂O₂ and reinforcing the protein accumulation of viral RNA helicase. SCMV HC‐Pro interacts with ZmCATs and enhances the enzymatic activity of ZmCATs to facilitate viral accumulation. The arrows indicate positive regulatory actions, while lines with bars represent negative regulatory actions. The dotted arrows indicate that the results need further investigation. CI, cylindrical inclusion protein; HC‐Pro, helper component‐protease.

4. EXPERIMENTAL PROCEDURES

4.1. Plant growth conditions and virus inoculation

Maize (inbred line B73) seedlings and N. benthamiana plants were grown in growth chambers (with a photoperiod of 16 h light at 24°C/8 h dark at 22°C) for virus inoculation and agroinfiltration. SCMV strain BJ was from the previously reported source (Fan et al., 2003). SCMV‐GFP infectious clone was provided by Dr Yule Liu (Tsinghua University). N. benthamiana leaves were agroinfiltrated with SCMV‐GFP (OD₆₀₀ = 1.2) and harvested for virus inoculation at 5 days after infiltration. Crude extracts from SCMV‐BJ‐infected maize leaf tissues or N. benthamiana leaves agroinfiltrated with SCMV‐GFP were homogenized in 0.01 M phosphate buffer (pH 7) at 1:5 (wt/vol) ratio. The first true leaves of maize seedlings (two‐leaf stage) were rub‐inoculated with the crude extracts.

4.2. Plasmid construction

The plasmids of ZmCATs used in this study were constructed as previously described (Jiao, Tian, et al., 2021; Jiao, Wang, et al., 2021). For the overexpression assay via SCMV expression vector, the coding sequence of 3×FLAG‐tagged ZmCAT1, ZmCAT2 or ZmCAT3 replaced the GFP fragment between the NIb and CP cistrons of pSCMV‐GFP to construct the vectors designated collectively as pSCMV‐3×FLAG‐ZmCAT1/2/3, respectively. The fragment 3×FLAG‐GFP was also inserted into the vector as a control. The enzyme cutting sites of viral protease were preserved at both ends of the inserted fragment, so that the inserted protein can be released and detected through immunoblot, without affecting the normal replication of the virus. The coding region of HC‐Pro and CI was amplified by PCR from SCMV‐BJ isolate. For the Y2H assays, pGBKT7 was used to construct BD‐HC‐Pro and BD‐CI. For the luciferase complementation imaging (LCI) assays, pNluc and pCluc were used to construct N‐terminal Nluc fusion constructs of HC‐Pro or CI and C‐terminal Cluc fusion constructs of ZmCATs (Chen et al., 2008). For the transient expression assays, 3×FLAG‐HC‐Pro and CI‐3×Myc were based on pGD vector (Goodin et al., 2002). All the constructs were confirmed through sequencing. All the primers used for plasmid construction were listed in Table S1.

4.3. Cucumber mosaic virus‐induced gene silencing in maize

For the ZMBJ‐CMV‐based virus‐induced gene silencing assay, a 245‐bp DNA fragment of ZmCAT3 representing a conserved partial sequence of all three ZmCATs was amplified and inserted into pCMV201‐2b_N81. A 254‐bp DNA fragment of GFP was also amplified and inserted into pCMV201‐2b_N81 as a control. Then, the recombinant plasmid vectors were introduced into Agrobacterium tumefaciens C58C1. A. tumefaciens cultures carrying pCMV101, pCMV301, pCMV201‐2b_N81:ZmCATs or pCMV201‐2b_N81:GFP were infiltrated into N. benthamiana plants as described previously (Wang et al., 2016). At 5 days after infiltration, the maize seeds (inbred line B73) were puncture‐inoculated with the crude extracts from infiltrated leaf patches. Maize seedlings were grown in growth chambers (with a photoperiod of 16 h light at 22°C/8 h dark at 18°C) and challenge‐inoculated with SCMV at the two‐leaf stage.

4.4. Isolation and transfection of maize protoplasts

Maize protoplasts were isolated from the seedlings of inbred line B73 and transfected as described previously (Zhu et al., 2014). Maize protoplasts with equal concentration were transfected with 10 μg plasmids and 2 μg SCMV‐BJ viral RNA from purified virions for investigating the role of maize catalase in SCMV replication. Maize protoplasts were transfected with 2 μg SCMV‐BJ viral RNA from purified virions and incubated with 20 mM 3‐AT (Sigma‐Aldrich) or 10 μM MV (Sigma‐Aldrich) at 25°C under light. The transfected protoplasts were harvested at 18–20 h post‐transfection (hpt) for RNA extraction followed by RT‐qPCR analysis. Protoplasts in five tubes of the same treatment were pooled for protein extraction followed by immunoblot analysis.

4.5. Y2H assay

The Y2H assay was performed using the GAL4 system as described in the manufacturer's protocol (Clontech). The reconstructed prey vectors and bait vectors were co‐transformed into the yeast strain Y2HGold (Clontech) by using the lithium acetate/polyethylene glycol method. All the transformed cells were plated onto SD/−Leu/−Trp and SD/−Ade/−His/−Leu/−Trp media and cultured at 30°C for 72 h. Interactions between different protein combinations were confirmed by observing the yeast growth conditions on SD/−Ade/−His/−Leu/−Trp plates.

4.6. Agrobacterium‐mediated transient expression and immunoblot analysis

The agroinfiltration was performed as described previously (Jiao, Tian, et al., 2021). The agroinfiltrated N. benthamiana leaf tissues were harvested at 3 days after infiltration (dpi) and homogenized in liquid nitrogen. Total protein was extracted using the buffer (220 mM Tris–HCl, pH 7.4, 250 mM sucrose, 1 mM MgCl₂, 50 mM KCl, 5% β‐mercaptoethanol [β‐ME]) and measured by the method of Bradford (1976). For the protein degradation assay, 50 μM MG132 (an inhibitor of the 26S proteosome system; Sigma‐Aldrich) or an equal volume of DMSO (as control) with 10 mM MgCl₂ were infiltrated into the pre‐agroinfiltrated N. benthamiana leaves. 3‐MA (5 mM, an inhibitor of autophagy pathway; Sigma‐Aldrich) or an equal volume of double‐distilled water (as control) with 10 mM MgCl₂ were infiltrated into the pre‐agroinfiltrated N. benthamiana leaves. The treated leaf regions were collected at 12 h post‐infiltration (hpi) for immunoblot analysis.

The protein immunoblot assays were performed as previously described (Cao et al., 2012). Detections of proteins through immunoblot were conducted by using antibodies specific for SCMV coat protein (CP) (Xia et al., 2016), monoclonal antibodies for green fluorescent protein (GFP) (MBL 598‐7; MBL Beijing Biotech Co.) and plant β‐actin (CW0264M; CWBIO) at a dilution of 1:5000, monoclonal antibodies for c‐Myc (A5598; Sigma‐Aldrich) and FLAG (A8592; Sigma‐Aldrich) at a dilution of 1:10,000. The relative expression levels of individual proteins upon immunoblot analysis were quantified using the software ImageJ (http://imagej.net/).

4.7. Luciferase complementation imaging (LCI) assay

LCI assays were performed as previously described (Chen et al., 2008). A. tumefaciens GV3101 strains containing the expression constructs were infiltrated into fully expanded N. benthamiana leaves. The leaves were collected at 3 dpi and sprayed with 1 mM luciferin (Invitrogen). The chemiluminescence pictures were taken 15 min after exposure by a low‐light cooled imaging apparatus (iXon, Andor Technology).

4.8. Co‐immunoprecipitation

Co‐immunoprecipitation (Co‐IP) assays were performed as described previously (Chen, Yan, et al., 2017). N. benthamiana leaves agroinfiltrated with expression vectors were collected and ground in liquid nitrogen at 3 dpi. Total proteins were extracted from approximately 1.5 g ground powder in 1.5 mL extraction buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% vol/vol glycerol, 0.1% vol/vol Triton X‐100, 5% β‐ME, 1 mM protease inhibitor cocktail [Sigma‐Aldrich] and 1 mM phenylmethylsulfonyl fluoride [PMSF]). The supernatants were filtered through 0.45‐μm filters and equal amounts of total protein were incubated with 30 μL anti‐GFP beads (LabLead) for 1.5 h at 4°C on a rotator. The immunoprecipitates were rinsed four times with ice‐cold IP buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% vol/vol glycerol, 0.1% vol/vol Triton X‐100) and analysed by immunoblot.

4.9. RNA extraction and RT‐qPCR analysis

Total RNA was extracted through TRIzol reagent (Invitrogen) and treated with RNase‐free DNase I (TaKaRa Bio Inc.) as instructed by the manufacturers. A total RNA of 2 μg was used to synthesize the first‐strand cDNA with oligo(dT) primers or random primers and M‐MLV reverse transcriptase (Promega). The gene fragments were amplified using SYBR Premix ExTaq reagents (TaKaRa Bio Inc.) with the primers shown in Table S2. The maize ubiquitin gene was used as the internal control, and the data were analysed using the 2^−ΔΔCt method (Livak & Schmittgen, 2002). The statistical significance of the data was analysed by using the software GraphPad Prism v. 8.0 (GraphPad Software Inc.).

4.10. Detection of catalase activities

The catalase activity was determined using the method described previously (Aebi, 1984; Jiao, Tian, et al., 2021).

4.11. Chemical treatments

Maize seedlings at two‐leaf stage were sprayed with 20 mM 3‐AT or 10 μM MV dissolved in 0.2% vol/vol Triton X‐100. Plants sprayed with 0.2% vol/vol Triton X‐100 (diluted in double‐distilled water) were used as controls.

4.12. H₂O₂ measurement in maize leaves

Measurement of H₂O₂ content in maize leaves was performed as instructed by the manual of Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen). The fluorescent signal was excited at a wavelength of 530 ± 12.5 nm and captured at a wavelength of 590 ± 12.5 nm.

4.13. Confocal microscopic observation

All the subcellular localization observation assays were performed under a laser scanning confocal microscope (SP8; Leica). The fluorescence of GFP was excited at 488 nm and then captured at 495–545 nm. The fluorescence of mCherry/DsRed was excited at wavelength of 552 nm and detected at 562–620 nm. For co‐localization observation, each channel was scanned and image was captured sequentially.

4.14. GFP imaging

GFP fluorescence was photographed using a Canon PowerShot SX20 IS digital camera at 3 dpi with irradiation of a UV light (Black Ray model B 100AP; Ultraviolet Products).

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no competing interests.

Supporting information

FIGURE S1. Sugarcane mosaic virus (SCMV) infection affected the expression levels of ZmCATs. (a–c) The relative accumulation levels of ZmCATs in the first systemically infected leaves of mock‐inoculated or SCMV‐inoculated maize (B73) seedlings were measured by reverse transcription‐quantitative PCR in a time course. Error bars represent mean ± SEM (n ≥ 5). The asterisks indicate significant differences (two‐tailed Student’s t test; **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, no significance).

FIGURE S2. The contents of H₂O₂ in the first true leaves of maize seedlings pretreated with 10 μM methyl viologen (MV) or 20 mM 3‐amino‐1, 2, 4‐triazole (3‐AT) at 4 h after spray were measured as instructed by the manual of Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen). Error bars represent mean ± SEM (n ≥ 6). The letters above the bars indicate significant differences (one‐way analysis of variance followed by Tukey’s multiple comparisons test).

FIGURE S3. Alignment of the nucleotide sequences of ZmCATs for CMV‐based virus‐induced gene silencing assay. The software DNAMAN was used. The GenBank accession numbers of ZmCATs are shown as follows: ZmCAT1, NM_001254879.2; ZmCAT2, NM_001111840.2; ZmCAT3, NM_001363892.1.

FIGURE S4. Analysis of maize catalase activities and protein expression. (a–c) 3×FLAG‐ZmCATs, inactivated (by mutation) 3×FLAG‐ZmCATs and 3×FLAG‐GFP (CK) were transiently expressed in Nicotiana benthamiana leaves. The catalase activity was measured with a spectrophotometer at 3 days after agroinfiltration. The values of catalase activity in CK were set to 1. Error bars represent mean ± SEM (n ≥ 3). The letters above the bars indicate significant differences (one‐way analysis of variance followed by Tukey’s multiple comparisons test). (d) The accumulation levels of 3×FLAG‐GFP, 3×FLAG‐ZmCATs and inactivated (by mutation) 3×FLAG‐ZmCATs in N. benthamiana leaves were analysed through immunoblot with an anti‐FLAG antibody.

FIGURE S5. Subcellular localization of GFP in Nicotiana benthamiana leaves in the presence of SCMV‐mCherry‐6K2. Images were taken by confocal microscopy at 5 days after agroinfiltration. Scale bars = 10 μm.

FIGURE S6. Analysis of the possible interactions of sugarcane mosaic virus (SCMV) proteins with ZmCAT1 through yeast two‐hybrid assay. P1, HC‐Pro, P3, P3N‐PIPO, 6K1, CI, 6K2, VPg, NIa‐Pro, Nib, and CP were fused to vector pGBKT7, and ZmCAT1 was fused to vector pGADT7. Y2H Gold yeast cells were transformed with different combinations of plasmids and were plated on selective medium (without Trp and Leu or without Trp, Leu, His and Ade) in 10‐fold serial dilutions for positive interaction screen at 3 days after transformation. Yeast cells co‐transformed with AD‐T and BD‐53 served as a positive control, while yeast cells co‐transformed with the empty BD and AD‐ZmCAT1 were set as a negative control.

FIGURE S7. Suppressor of RNA silencing activity of wild type and mutants of SCMV HC‐Pro in Agrobacterium co‐infiltration assay. GFP was co‐expressed with either wild‐type or mutant HC‐Pro in Nicotiana benthamiana leaves, with β‐glucuronidase (GUS) as a negative control. High fluorescence in the agroinfiltrated patches indicated high suppressor of RNA silencing activity of HC‐Pro.

FIGURE S8. Maize catalase activities were not affected by sugarcane mosaic virus (SCMV) CI. 3×Myc‐ZmCAT1, 3×Myc‐ZmCAT2 or 3×Myc‐ZmCAT3 was co‐expressed with 3×FLAG‐GFP or CI‐3×FLAG in Nicotiana benthamiana leaves, respectively. The total protein was extracted for catalase activity measurement. Data information: error bars represent mean ± SEM (n ≥ 6). The asterisks indicate significant differences (two‐tailed Student’s t test; ns, no significance).

FIGURE S9. The relative accumulation of sugarcane mosaic virus (SCMV) CI was not altered by maize catalases. CI‐3×Myc was co‐expressed with 3×FLAG‐ZmCATs or GUS‐3×FLAG in Nicotiana benthamiana leaves, respectively. The relative accumulation levels of SCMV CI in N. benthamiana leaves were measured by reverse transcription‐quantitative PCR at 3 days after agroinfiltration. Error bars represent mean ± SEM (n ≥ 5). The letters above the bars indicate significant differences (one‐way analysis of variance followed by Tukey’s multiple comparisons test).

FIGURE S10. The stability of sugarcane mosaic virus (SCMV) CI was not affected via autophagy pathway. CI‐3×Myc was transiently co‐expressed with GUS‐3×FLAG (served as a control) in Nicotiana benthamiana leaves for 72 h after infiltration, and the infiltrated leaf areas were treated with water (control) or 3‐methyladenine (3‐MA) (5 mM) at 12 h before harvest. The relative accumulation levels of CI‐3×Myc were detected by immunoblot with an anti‐Myc antibody and quantified using the ImageJ software. Actin was set to show equal loading of samples.

FIGURE S11. Maize catalases attenuated SCMV HC‐Pro SRS activity. (a) GUS‐3×Myc, 3×Myc‐ZmCAT1, 3×Myc‐ZmCAT2 or 3×Myc‐ZmCAT3 were co‐expressed with GFP and 3×FLAG‐HC‐Pro in Nicotiana benthamiana leaves, respectively. The GFP fluorescence was photographed at 3 days after agroinfiltration with irradiation of a UV light. (b) The relative accumulation levels of GFP in the infiltrated leaf patches were measured by reverse transcription‐quantitative PCR at 3 days after agroinfiltration. Error bars represent mean ± SEM (n ≥ 7). The asterisks indicate significant differences (two‐tailed Student’s t test; ** p < 0.01; *** p < 0.001). (c) Immunoblot analysis of the protein expression in leaf regions depicted in panel. Coomassie brilliant blue (CBB) staining of the RuBisCO large subunit served as a loading control.

TABLE S1. Primers used for plasmid construction.

TABLE S2. Primers used for reverse transcription‐quantitative PCR analysis.

Tian, Y. , Jiao, Z. , Qi, F. , Ma, W. , Hao, Y. , Wang, X. et al. (2024) Maize catalases are recruited by a virus to modulate viral multiplication and infection. Molecular Plant Pathology, 25, e13440. Available from: 10.1111/mpp.13440

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available in the Supporting Information of this article.