Maize Ethylene Response Factor ZmERF061 Is Required for Resistance to Exserohilum turcicum

Plants have evolved a series of sophisticated defense mechanisms to help them from harm. Ethylene Response Factor (ERF) plays pivotal roles in plant immune reactions, however, its underlying mechanism in maize with a defensive function to Exserohilum turcicum (E. turcicum) remains poorly understood. Here, we isolated and characterized a novel ERF transcription factor, designated ZmERF061, from maize. Phylogenetic analysis revealed that ZmERF061 is a member of B3 group in the ERF family. qRT-PCR assays showed that the expression of ZmERF061 is significantly induced by E. turcicum inoculation and hormone treatments with salicylic acid (SA) and methyl jasmonate (MeJA). ZmERF061 was proved to function as a nucleus-localized transcription activator and specifically bind to the GCC-box element. zmerf061 mutant lines resulted in enhanced susceptibility to E. turcicum via decreasing the expression of ZmPR10.1 and ZmPR10.2 and the activity of antioxidant defense system. zmerf061 mutant lines increased the expression of the SA signaling-related gene ZmPR1a and decreased the expression of the jasmonic acid (JA) signaling-related gene ZmLox1 after infection with E. turcicum. In addition, ZmERF061 could interact with ZmMPK6-1. These results suggested that ZmERF061 plays an important role in response to E. turcicum and may be useful in genetic engineering breeding.


INTRODUCTION
Northern corn leaf blight (NCLB) caused by Exserohilum turcicum (E. turcicum) is one of the most destructive fungal diseases of maize in the world (Leach et al., 1977;Galiano-Carneiro and Miedaner, 2017). Heavy infections of NCLB can result in yield losses of greater than 50% (Fajemisin and Hooker, 1974;Raymundo and Hooker, 1981;Perkins and Pedersen, 1987;Ding et al., 2015). Additionally, NCLB may cause a reduction of feeding value and increase the incidence of maize to stalk rot (Hooker et al., 1965;Fajemisin and Hooker, 1974). Host plant resistance is the most common strategy used to control NCLB through the deployment of qualitative and quantitative resistance. Several qualitative Ht genes such as Ht1, Ht2, Ht3, Htn1, HtM, HtP, HtNB, ht4, and rt have already been identified and mapped in maize (Ullstrup, 1963;Gevers, 1975;Hooker, 1977Hooker, , 1981Bentolila et al., 1991;Robbins and Warren, 1993;Simcox and Bennetzen, 1993;Carson, 1995;Ogliari et al., 2005;Hurni et al., 2015;Galiano-Carneiro and Miedaner, 2017). However, these Ht genes can quickly get ineffective and result in significant yield losses due to the emergence of new, virulent races (Welz and Geiger, 2000;McDonald and Linde, 2002). Quantitative resistance is considered to provide more durable disease and more useful in breeding process than qualitative resistance (St Clair, 2010). Therefore, it is vital to identify the important genes conferring quantitative resistance and elucidate their molecular mechanism for improving disease resistance of maize cultivars against E. turcicum.
Plants have evolved a series of sophisticated defense mechanisms to cope with the invading pathogens during their life span (Jones and Dangl, 2006;Dangl et al., 2013;Fu and Dong, 2013;Bigeard et al., 2015;Birkenbihl et al., 2017). In plant immunity system, there are two layers of immune responses, called pathogen/microbe-associated molecular pattern (PAMP or MAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) (Cui et al., 2015;Couto and Zipfel, 2016;Zipfel and Oldroyd, 2017). During PTI and ETI responses, plants trigger a variety of immune reactions including the accumulation of reactive oxygen species (ROS), the activation of mitogen-activated protein kinase (MAPK) signaling pathway, and the expression of pathogenesis-related (PR) genes (Tsuda and Katagiri, 2010;Pieterse et al., 2012;Meng and Zhang, 2013;Baxter et al., 2014). In addition, ETI is often associated with programmed cell death (PCD), also called the hypersensitive response (HR), which occurs at the site of infection and prevents further invasion by the pathogen (Cui et al., 2015).
ERF genes can coordinately integrate the salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) signaling pathways or antagonize them, to finely modulate the defense response to pathogens (Berrocal-Lobo and Molina, 2004;Zarei et al., 2011;Zhang et al., 2015Zhang et al., , 2016Wang et al., 2018). AtERF1, AtERF96, or ORA59 has been shown to positively regulate the Arabidopsis defense against B. cinerea through the JA/ET signaling pathway and negatively modulate immunity against Pseudomonas syringae pv. tomato (Pst) DC3000 through the SA signaling pathway (Berrocal-Lobo et al., 2002;Pré et al., 2008;Catinot et al., 2015). It was also found that AtERF15 is involved in resistance to Pst DC3000 and B. cinerea via the SA and JA/ET signaling pathways . VqERF112, VqERF114, and VqERF072 acted as positive regulators of plant resistance against Pst DC3000 and B. cinerea through integrating the SA and JA/ET signaling pathways . AtERF11 positively regulated Arabidopsis resistance to Pst DC3000 by directly activating the transcription of AtBT4, which depends on the SA and ET signaling pathways (Zheng et al., 2019).
Recently, several ERF genes have shown to regulate the expression of their target genes through interaction with other proteins . ORA59 physically interacted with RELATED TO AP2.3 (RAP2.3) to increase the plant resistance against Pectobacterium carotovorum (Kim et al., 2018). GmERF5 and GmERF113 interacted with a BASIC HELIX-LOOP-HELIX TF (GmbHLH) to improve the soybean resistance against Phytophthora sojae (P. sojae) (Dong et al., 2015;Zhao et al., 2017). AtERF6 could interact with AtMPK6 and directly be phosphorylated by AtMPK6. The phosphorylation of AtERF6 increased its protein stability and thus constitutively activated defense genes Wang et al., 2013).
A large number of ERF genes have been shown to regulate plant resistance against pathogens in many species, however, its underlying mechanism in maize with a defensive function to E. turcicum remains poorly understood. Previously, we identified a maize ERF gene whose expression was specifically induced by E. turcicum inoculation. Therefore, we isolated and characterized ZmERF061 from maize B73. ZmERF061 was proved to function as a nucleus-localized transcription activator and specifically bind to the GCC-box element. zmerf061 mutant lines resulted in decreased resistance to E. turcicum. In addition, ZmERF061 could interact with ZmMPK6-1. These results suggested that ZmERF061 plays an important role in response to E. turcicum and may be useful in genetic engineering breeding.

Plant Materials and Treatments
Exserohilum turcicum (mixed races), the seeds of the maize inbred lines Mo17 (resistant to E. turcicum), Huobai (resistant to E. turcicum), and B73 were obtained from Maize Breeding Team in Jilin Agricultural University, Changchun, China. The seedlings were grown in a glasshouse at 25 • C under long-day (16 h light/8 h dark) and 70% relative humidity conditions. For hormone treatments, the seedlings of maize inbred line B73 were sprayed with 0.1 mM of methyl jasmonate (MeJA) and 0.5 mM of SA at the three-leaf stage. For E. turcicum inoculation, the seedlings of maize inbred line Mo17 and Huobai were inoculated with three drops of conidial suspensions at the six-leaf stage according to the method of Zang et al. (2020). The conidial suspensions were adjusted to 1 × 10 5 conidia ml −1 . The leaves were sampled at 0, 2, 5, 10, and 24 h after hormone treatments and were collected at 0, 10, 24, and 72 h after E. turcicum inoculation, respectively. The leaves were frozen in liquid nitrogen and stored at -80 • C for the subsequent quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The special primers used for assays are listed in Supplementary Table S1.

qRT-PCR Analysis
Total RNA was extracted from maize leaves using TRIzol reagent (Invitrogen, China) according to the manufacturer's instruction. Total RNA (1 µg) was used to reverse transcribe into complementary DNA (cDNA) with ReverTra Ace R qPCR RT Kit (TOYOBO, Japan) following the manufacture's instruction. qRT-PCR was performed using SYBR Mixture system (TOYOBO, Japan) on a QuantStudio 3 instrument (Thermo, United States). A maize Actin gene, ZmTub (GRMZM2G066191), was used as an internal control to normalize the data. The relative expression levels of genes were analyzed using the 2 − CT method. The experimental data were determined using three independent biological repeats, and the significance analysis was performed using Student's t-test ( * P < 0.05, * * P < 0.01). Bars indicate standard error of the mean.

Cloning and Bioinformatics Analysis of ZmERF061
The full-length coding sequence of ZmERF061 was isolated from the leaves of B73 by reverse transcription PCR (RT-PCR). The PCR product was cloned into the pMD18-T vector (TaKaRa, China), and the sequence was verified by sequencing (Sangon, China). ERF sequences from different species were downloaded from the NCBI database 1 , and the phylogenetic tree was built with MEGA 5.0 software using the neighbor joining (NJ) method. The amino acid sequence alignment was performed by DNAMAN software. The nucleic acid sequence and protein sequence of ZmERF061 were analyzed using ExPASy 2 database.

Yeast Two-Hybrid Assay
The full-length coding sequence of ZmERF061 was inserted into pGBKT7 vector to generate the bait plasmid (pGBKT7-ZmERF061). The coding sequence of ZmMPK6-1 was cloned into pGADT7 vector to generate prey plasmid (pGADT7-ZmMPK6-1). The prey and bait plasmids were co-transformed into the yeast strain Y 2 H according to the manufacturer's instructions (Clontech, United States). After selection on SD/-Trp/-Leu medium for 3 days at 30 • C, the transformants were grown on SD/-Trp/-Leu/-His/-Ade medium containing X-α-Gal (20 µg ml −1 ). Yeast cells carrying the pGBKT7-p53 and pGADT7-SV40 plasmids were used as positive controls, and yeast cells harboring the pGBKT7-Lam and pGADT7-SV40 plasmids were used as negative controls.

Subcellular Localization and Bimolecular Fluorescence Complementation Assays
For subcellular localization of ZmERF061, fusion expression vector ZmERF061-green fluorescent protein (GFP) was constructed by inserting full-length coding sequence of ZmERF061 into the pCAMBIA1300 vector. For bimolecular fluorescence complementation (BiFC) assays, the full-length coding sequences of ZmERF061 and ZmMPK6-1 were fused into pUC-SPYCE and pUC-SPYNE vectors, respectively. The plasmids were transiently expressed in Nicotiana benthamiana (N. benthamiana) leaves by Agrobacteriummediated method (Liu et al., 2010). The fluorescence signal in cells was photographed by a laser confocal microscope (Leica TCS SP2, Germany).

Yeast One-Hybrid Assay
Yeast one-hybrid assays were used to examine the binding of ZmERF061 to a GCC-box element and were performed according to the Matchmaker Gold Yeat One-Hybrid Library Screening System (Clontech, United States). The full-length coding sequence of ZmERF061 was cloned into the pGADT7 vector containing a GAL4 transcription activation domain, to generate the prey plasmid (pGADT7-ZmERF061). The synthesized DNA fragments harboring four tandem copies of the GCC-box (ATCCATAAGAGCCGCCACTAAAATAAGACCGATCAA) and mGCC (ATCCATAAGATCCTCCACTAAAATAAGACCGATC AA) were cloned into the pAbAi vector as bait plasmids (pAbAi-4 × GCC and pAbAi-4 × mGCC), respectively. The pGADT7-ZmERF061 plasmid was co-transformed with pAbAi-4 × GCC and pAbAi-4 × mGCC plasmids, into Y 1 H Gold yeast strain, respectively. The co-transformation yeasts were determined on SD/-Leu/-Ura medium supplemented with 200 ng ml −1 of AbA or 300 ng ml −1 of AbA and cultured at 30 • C for 3 days. Positive (pGAD-rec-53 + pAbAi-p53) and negative (pGADT7 + pAbAi) controls were processed in the same manner.

Luciferase Activity Assay
The full-length coding sequence of ZmERF061 was cloned into the pGreenII 62-SK vector as effector, and four tandem copies of the GCC-box (ATCCATAAGAGCCGCCACTAAAATAAGACCGATCAA) were ligated into the pGREENII0800-LUC vector as reporter (4 × GCC-LUC). The effector and reporter plasmids were, respectively transferred into Agrobacterium tumefaciens GV3101 and co-transformed into N. benthamiana leaves by Agrobacterium-mediated method (Liu et al., 2010). The LUC activity was determined using commercial dual-LUC reaction reagents (Promega, United States) according to the previous report (Ma et al., 2018). Empty pGreenII 62-SK vector co-transformed with 4 × GCC-LUC was used as the negative control.

Pathogen Response Assays of zmerf061 Mutant Lines
Loss-of-function zmerf061 mutant lines (zmerf061 UFMu mutant, mu1014012) were obtained from the Maize Genetics Cooperation Stock Center. The homozygous mutant lines were obtained from self-fertilizing and identified by PCR. Then, two homozygous T 4 zmerf061 mutant lines, named zmerf061-1 and zmerf061-2, were confirmed by qRT-PCR and used for further analyses. Artificial inoculation procedures were performed according to the methods described by Zang et al. (2020). The living ear leaves of zmerf061 mutant lines were infected with E. turcicum agar disks, and the detached leaves from inoculated plants were pictured at 5 days post-inoculation (dpi) with a Nikon D7000 camera for disease assays. The relative lesion area was evaluated using the Photoshop CS3 software according to Cui et al. (2009).

Detection of Enzyme Activities
For the enzyme activity assays, the fresh leaves (about 0.1 g) of W22 wild-type (WT) plants and zmerf061 mutant lines were harvested 24 h after inoculation with E. turcicum conidial suspension, and the plants that were treated with water served as control. The superoxide dismutase (SOD) and peroxidase (POD) activities were measured following the methods that described by Li et al. (2015).

Cloning and Characterization of ZmERF061
ZmERF061 (GenBank Accession no. XM008670839), the ERF gene, was isolated from total RNA of maize by RT-PCR. Sequence analysis revealed that ZmERF061 contains a 1,071bp open reading frame (ORF) encoding a polypeptide of 356 amino acids (aa) with predicted molecular mass of 37.783 kDa (pI 4.84). The results from searching the database 3 indicated that ZmERF061 is located on chromosome 2 and does not have signal peptide. ZmERF061 contains a typical AP2/ERF domain, with conserved alanine (A) and aspartic acid (D) in it, suggesting that it belongs to the ERF family (Sakuma et al., 2002). The AP2/ERF domain contains conserved YRG and RAYD elements, which have been shown to play a vital role in GCC-box binding activity and protein interaction, respectively (Mazarel et al., 2002). ZmERF061 contains a conserved PXXSPXSP (X represents any amino acid) motif in the C-terminal region, which is believed to act as MPK phosphorylation sites . Additionally, ZmERF061 also possessed a nuclear targeting signal (NLS) sequence "AANKRKRQQL" (Figure 1). Blast search in NCBI revealed that ZmERF061 shares 67.75, 65.38, and 52.75% identity to SbERF104 (Protein ID: XP021319691), SiERF105 (Protein ID: XP004976417), and OsERF105 (Protein ID: XP015635116), respectively (Figure 2A). The phylogenetic tree analysis indicated that ZmERF061 belongs to B3 group (Sakuma et al., 2002). The prediction of the three-dimensional structure based on SWISS-MODEL database revealed that the ZmERF061 has a long C-terminal α-helix (α) surrounded by a three-stranded anti-parallel β-sheet (from β1 to β3) ( Figure 2B).

Expressions of ZmERF061 Responds to Pathogen Infection and Hormone Induction
To characterize the potential role of ZmERF061 in plant defense reaction, the expression profiles of ZmERF061 in both resistant maize inbred line Mo17 and susceptible maize inbred line Huobai following inoculation with E. turcicum were analyzed by qRT-PCR. The expression level of ZmERF061 in maize inbred Huobai was increased at 10 h but rapidly decreased at 24 h (0.69-fold) and 72 h (0.46-fold) compared with Huobai, however a significant upregulation of ZmERF061 expression is detected in the leaves from 10 to 72 h after E. turcicum in maize inbred line Mo17 ( Figure 3A). These results indicated that ZmERF061 may play an important role in maize defense response to E. turcicum.
ERF genes are involved in a variety of defense signaling hormones, such as SA and JA. In our qRT-PCR assay, the transcript levels of ZmERF061 were analyzed in maize inbred line B73 after the application of SA and MeJA treatments. The expression of ZmERF061 was decreased at 2 h, then rapidly increased from 5 to 24 h, and peaked at 5 h by 9.96-fold after the application of MeJA treatment. In SA-treated plants, the expression level of ZmERF061 was lower at 10 h but peaked 3.06fold higher at 24 h than that in the control. These results indicated that ZmERF061 responds to E. turcicum inoculation and is involved in the JA and SA signaling pathways (Figures 3B,C).

Subcellular Localization of ZmERF061
To elucidate the biological role of ZmERF061, the subcellular localization was analyzed in planta. The coding sequence of ZmERF061 was fused to GFP and was transiently expressed in leaf epidermal cells of N. benthamiana. The ZmERF061-GFP fusion protein was solely localized in nucleus, while the control GFP protein was located in both nucleus and cytoplasm. This result indicated that ZmERF061 is a nucleus-localized protein (Figure 4).

ZmERF061 Binds to GCC-Box Element and Functions as a Transcriptional Activator
Previous studies have demonstrated that some ERF genes can bind to the GCC-box element (Guo et al., 2004;Franco-Zorrilla et al., 2014;Sun et al., 2016). A yeast one-hybrid assay was performed to investigate the binding characteristics of ZmERF061 to the GCC-box element. The yeast cells transfected with pGADT7-ZmERF061 and pAbAi-4 × GCC could grow on SD/-Leu/-Ura medium containing 200 ng ml −1 of AbA or 300 ng ml −1 of AbA. By contrast, yeast cells harboring the mutant bait cannot grow normally. These data suggested that ZmERF061 specifically binds to the GCC-box element ( Figure 5A).
To determine whether ZmERF061 plays a role in transcription activation or inhibition, we performed a transient LUC assay. As shown in Figure 5B, the relative LUC activity of tobacco leaves transfected with the ZmERF061 effector and 4 × GCC-LUC reporter was approximately 3.55-fold higher than that of the control, indicating that ZmERF061 can activate the reporter gene transcription. These results demonstrated that ZmERF061 is able to bind to the GCC-box element and functions as a transcription activator.

zmerf061 Mutant Lines Decreased the Resistance to Exserohilum turcicum
To explore the role of ZmERF061 in mediating the maize resistance to E. turcicum, loss-of-function zmerf061 UFMu mutant lines were obtained from the Maize Genetics Cooperation Stock Center. Homozygous zmerf061 mutant lines were obtained from self-fertilizing and were characterized by PCR. Two homozygous T 4 zmerf061 mutant lines, named zmerf061-1 and zmerf061-2, were confirmed by qRT-PCR. qRT-PCR analysis revealed that the expression levels of ZmERF061 have about 0.22-fold and 0.14-fold declines in zmerf061-1 and zmerf061-2 mutant lines, respectively ( Figure 6B). WT plants and two independent T 4 zmerf061 mutant lines were inoculated with E. turcicum to examine whether ZmERF061 is involved in pathogen resistance. The detached leaves from inoculated plants were pictured with a Nikon D7000 camera for disease assays. At 5 dpi, E. turcicum-caused lesions were significantly smaller on the leaves of the WT plants compared with the zmerf061 mutant lines, indicating that zmerf061 mutant lines decreased resistance to E. turcicum (Figures 6A,C). These results demonstrate that ZmERF061 positively regulates the maize resistance against E. turcicum.

ZmERF061 Mutant Lines Attenuated Exserohilum turcicum-Induced Defense Response
To further investigate the physiological changes in zmerf061 mutant lines after infection with E. turcicum, we analyzed the activities of two important antioxidant enzymes, including SOD and POD. Under both the mock treatment and at 24 h after infection with E. turcicum, both SOD and POD activities were significantly decreased in zmerf061 mutant lines compared with WT plants (Figures 7A,B). These results indicate that ZmERF061 improves maize resistance against E. turcicum through affecting SOD and POD activities.
To examine whether the increased susceptibility to E. turcicum in zmerf061 mutant lines was associated with transcription changes of defense-related genes, we measured the expression levels of two defense-related genes [i.e., ZmPR10.1 (GRMZM2G112488) and ZmPR10.2 (GRMZM2G112538)] in the WT plants and zmerf061 mutant lines after E. turcicum inoculation. Under both the mock treatment and at 24 h inoculation with E. turcicum, the expression levels of ZmPR10.1 and ZmPR10.2 were significantly lower in zmerf061 mutant lines than in the WT plants (Figures 7C,D). These results showed that ZmERF061 is involved in maize resistance to E. turcicum through regulating the expression of defense-related genes.
To explore whether ZmERF061 is required for SA-induced and JA-induced defense response, the expression levels of SAand JA-responsive genes were analyzed by qRT-PCR after E. turcicum inoculation. Under both the mock treatment and at 24 h after infection with E. turcicum, the expression levels of SA-responsive gene, ZmPR1a (GRMZM2G465226), were significantly lower in zmerf061 mutant lines than in the WT plants ( Figure 7E). By contrast, the expression levels of the JA signaling-related gene, ZmLox1 (GRMZM2G156861), were increased after E. turcicum inoculation in zmerf061 mutant lines compared with the WT plants ( Figure 7F). These results demonstrated that ZmERF061 may regulate resistance against E. turcicum via the SA signaling pathway.

ZmERF061 Interacted With ZmMPK6-1
Recently, several B3 group ERF TFs have been shown to interact with MPK6 and involve in MAPK signaling cascade (Bethke et al., 2009;Wang et al., 2013). Amino acid sequence analysis has suggested that ZmERF061 contains putative MPK phosphorylation sites at its C terminus (Fujimoto et al., 2000). The samples were collected at 0, 2, 5, 10, and 24 h after the initiation of treatments. Transcript levels were normalized to ZmTub (GRMZM2G066191). The relative expression levels of genes were analyzed using the 2 -CT method. The experimental data were determined using three independent biological replicates, and the significance analysis was performed using Student's t-test (*P < 0.05, **P < 0.01). Bars indicate standard error of the mean. Thus, we anticipated that ZmERF061 might interact with ZmMPK6-1 (GRMZM2G002100), which shares high identity to AtMPK6 (At2g43790).
To confirm the interaction between ZmERF061 and ZmMPK6-1, a BiFC assay was performed in leaf epidermal cells of N. benthamiana. As shown in Figure 8B, yellow fluorescence was displayed in leaf epidermal cells of N. benthamiana co-transformed with N-terminal yellow fluorescent protein (nYFP)-tagged ZmMPK6-1 and C-terminal YFP (cYFP)-tagged ZmERF061. These results indicated that ZmERF061 physically interacts with ZmMPK6-1.

DISCUSSION
ERF TFs play critical roles in response to pathogen infection in plants. In maize, a total of 76 predicted ERF genes have been identified (Hao et al., 2020). In the present study, a novel ERF gene, ZmERF061, was firstly isolated and functionally characterized in maize. Sequence analysis showed that ZmERF061 is characterized as a member of B3 group in the ERF family (Figure 2A). Nucleus-localized ZmERF061 plays a positive role in plant resistance to E. turcicum (Figures 4, 6). The full-length coding sequence of ZmERF061 was fused with the GAL4 transcription activation domain of vector pGADT7 to generate the prey plasmid (pGADT7-ZmERF061). Four tandem copies of the GCC-box and mGCC-box mutants were cloned into the pAbAi vector and used as bait. The yeast transformants were incubated for 3 days at 30 • C on SD/-Leu/-Ura medium with 200 ng ml -1 of AbA or 300 ng ml -1 of AbA. Positive (pGAD-rec-53 + pAbAi-p53) and negative (pGADT7 + pAbAi) controls were processed in the same manner. (B) Transcription activity of ZmERF061. The full-length coding sequence of ZmERF061 was cloned into the pGreenII 62-SK vector as effector, and four tandem copies of the GCC-box were ligated into the pGREENII0800-LUC vector as reporter. Relative luciferase activity detected by transient co-transformation with reporter and effector into N. benthamiana. The experiment was performed using three independent biological replicates and analyzed using Student's t-tests (**P < 0.01). Bars indicate standard error of the mean.
Previous studies have shown that ERF genes function in plants' immune response through modulating diverse hormone signaling molecules, such as SA, JA, and ET (Zarei et al., 2011; The relative lesion areas of ZmERF061 mutant lines and wild-type (WT) leaves at 5 dpi. The experiment was performed using three independent biological replicates and analyzed using Student's t-tests (**P < 0.01). Bars indicate standard error of the mean. Zhang et al., 2016). Generally, ERF TFs regulate the Arabidopsis defense against necrotrophic pathogens through the JA/ET signaling pathway and negatively modulate immunity against (hemi)biotrophic pathogens through the SA signaling pathway (Berrocal-Lobo et al., 2002;Pré et al., 2008;Catinot et al., 2015). However, our present study demonstrated that relative expression levels of ZmERF061 were significantly induced by SA and the expression levels of SA-responsive defense gene (ZmPR1a) were decreased in zmerf061 mutant lines after infection with E. turcicum, indicating that ZmERF061 may be involved in E. turcicum resistance via the SA signaling pathway (Figures 3B,  7E). This is different from the functions of AtERF1, AtERF5, AtERF6, ORA59, or AtERF96, which have been reported to play positive roles in disease resistance against B. cinerea by promoting the JA/ET signaling pathway (Berrocal-Lobo et al., 2002;Lorenzo et al., 2003;Pré et al., 2008;Moffat et al., 2012;Catinot et al., 2015). In addition, we demonstrated that the expression levels of JA-responsive defense gene (ZmLox1) were significantly induced in zmerf061 mutant lines after infection with E. turcicum, indicating that ZmERF061 is involved in modulating immune response through antagonizing the SA and JA/ET signaling pathways ( Figure 7F). How ZmERF061 modulates the JA and SA signaling pathways to improve plant resistance is an interesting question for future studies.
To ensure survival and negate the adverse effects of ROS, plants have evolved a complete antioxidant defense system to remove extra ROS (Radwan et al., 2010;Hu et al., 2017). POD and SOD are key antioxidant enzymes to help scavenge extra ROS in plants, so that ROS are maintained at a low level to improve the plant resistance against pathogens (Mengiste, 2012). Overexpression of GmSnRK1.1 in soybean showed enhanced resistance to P. sojae through increasing the activity levels of SOD and POD, and GmSnRK1.1-RNAi plants exhibited opposite patterns . Our studies also showed that both SOD and POD activities were significantly lower in zmerf601 mutant lines after infection with E. turcicum than those in the WT plants ( Figures 7A,B), suggesting that ZmERF061 may improve the resistance to pathogen in maize via regulating the plants' antioxidant defense system.
Recently, some ERF genes have shown to be involved in plant defense reactions through interacting with other proteins Dong et al., 2015). GmERF5 and GmERF113 interacted with GmbHLH to improve soybean resistance against P. sojae (Dong et al., 2015;Zhao et al., 2017). ORA59 could enhance Arabidopsis resistance against Pectobacterium carotovorum through interaction with RAP2.3 (Kim et al., 2018). In addition, several ERF genes can interact with MPK genes and are the substrates of pathogen-responsive MAPK signaling cascade (Popescu et al., 2009;Son et al., 2012;Cao et al., 2018). AtERF6 or AtERF104 could interact physically with AtMPK6 and be phosphorylated by AtMPK6 (Bethke et al., 2009;Wang et al., 2013). The upregulation of AtERF6 or AtERF104 in response to B. cinerea depends on AtMPK6 signaling cascade. Here, we found that ZmERF061 interacted physically with ZmMPK6-1 in yeast cells and N. benthamiana (Figure 8). We speculated that ZmERF061 may play an important role downstream of the ZmMPK6-1 signaling cascade in regulating maize defense against E. turcicum.
An increasing number of evidence indicated that ERF TFs can specifically bind to the GCC-box element to activate the expression of defense-related genes. AtERF1, ORA59, AtERF6, The mock-treated WT sample was set to unity. Transcript levels of ZmPR10.1, ZmPR10.2, ZmPR1a, and ZmLox1 were normalized to ZmTub (GRMZM2G066191). The relative expression levels of genes were analyzed using the 2 -CT method. The experiment was performed using three independent biological replicates and analyzed using Student's t-tests (**P < 0.01). Bars indicate standard error of the mean.
or AtERF96 could directly bind to the GCC-box element in the promoter of AtPDF1.2 to enable its transcription activity (Berrocal-Lobo et al., 2002;Lorenzo et al., 2003;Pré et al., 2008;Moffat et al., 2012;Catinot et al., 2015). GmERF3, TiERF1, or VaERF057 also conferred the ability to bind to the GCCbox element (Liang et al., 2008;Zhang et al., 2009;Sun et al., 2016). In this study, we found that ZmERF061 specifically binds to the GCC-box element by Y 1 H Gold yeast strain and in planta (Figure 5). These results suggested that ZmERF061 may directly activate the expression of downstream defenserelated genes by interacting with the GCC-box element in their promoter regions. It will be helpful to characterize the direct target genes that are regulated by ZmERF061 during immune response against E. turcicum.
In conclusion, we isolated and characterized a novel ERF gene, ZmERF061, which was a nucleus-localized transcription activator and could specifically bind to the GCC-box element. The expression of ZmERF061 was induced by E. turcicum, SA, and MeJA. We also demonstrated that ZmERF061 plays a positive role in modulating plant resistance to E. turcicum through regulating the expression of downstream defense-related genes and antioxidant defense system. Moreover, we found that ZmERF061 can interact with ZmMPK6-1. These data will be important to elucidate the function and regulatory mechanisms of ZmERF061 in maize and provide a reference for breeding disease-resistant varieties.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.

AUTHOR CONTRIBUTIONS
LJ and WGY conceived and designed the experiments, contributed reagents, materials, and analysis tools. ZZ, ZW, and FZ performed the experiments and drafted the manuscript. WY, JC, and XR analyzed the data. All authors contributed to the article and approved the submitted version.