Molecular mechanism of ZmWRKY36 mediated maize resistance to Bipolaris maydis

Molecular mechanism of ZmWRKY36 mediated maize resistance to Bipolaris maydis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular mechanism of ZmWRKY36 mediated maize resistance to Bipolaris maydis Shu Li, Ying Ding, Haiyue Yu, Na Ning, Hongliang Wu, Chaotian Liu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7738242/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Jan, 2026 Read the published version in BMC Plant Biology → Version 1 posted 11 You are reading this latest preprint version Abstract Background WRKY transcription factors (TFs) represent one of the largest families of transcriptional regulators in plants, play crucial role in plant responses to both biotic and abiotic stresses. Studies have shown that WRKY family members modulate the expression of disease resistance genes, hormone synthesis genes and signal transduction genes, thereby mediating plant resistance against diverse pathogens including fungi, bacteria, and viruses. Although reports have indicated the presence of 120 WRKY family protein in maize ( Zea mays L), research on the molecular regulatory mechanism underlying disease resistance mediated by maize WRKY genes remains limited. In this study, we identified the transcription factor gene ZmWRKY36 in maize and investigated its function in maize’s response to infection by Bipolaris maydis -the causal agent of southern corn leaf blight. This work aims to provide a theoretical and experimental basis for exploring maize disease resistant genes and enriching functional studies of WRKY TFs in different crops. Result We identified ZmWRKY36 as a nuclear-localized transcription factor in maize. To explore its biological function in resistance to B. maydis , we constructed Z mWRKY36 -silenced (FoMV: ZmWRKY36 ) and ZmWRKY36 -overexpressed (FoMV: ZmWRKY36 -VOX) maize plants using virus-induced gene silencing (VIGS) and transient overexpressed (VOX) systems, respectively. Disease resistance assays revealed that transiently silenced FoMV: ZmWRKY36 plants exhibited enhanced resistance to B. maydis infection and suppressed chitin-induced reactive oxygen species (ROS) burst, whereas transiently overexpressed FoMV: ZmWRKY36 -VOX plants showed the opposite results. Additionally, overexpressed of ZmWRKY36 upregulated the expression of disease-related genes, suggesting that ZmWRKY36 positively regulate maize resistance to B. maydis . Further functional characterization demonstrated that ZmWRKY36 possesses transcriptional activation activity. Transcriptome analysis of ZmWRKY36 silenced and overexpressed plants revealed that the differentially expressed genes (DEGs) were mainly enriched in pathways related to cellular structure composition, metabolic synthesis and photosynthesis. Promoter analysis of these DEGs identified 105 genes containing W-box elements the core binding motif of WRKY TFs, which suggested that these pathways and target genes are involved in mediating maize resistance to B. maydis . Conclusions These results demonstrate that transcription factor ZmWRKY36 positively regulates maize resistance to B. maydis and identify its potential downstream target genes. This study provides insights into the regulatory role of ZmWRKY36 in maize defense responses and lays a foundation for further dissecting WRKY-mediated disease resistance networks in maize. Maize Bipolaris maydis WRKY TFs W-box VIGS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Background Maize is a globally significant crop serving as food, feed, and industrial raw materials, and its yield and quality directly impact global food security and agricultural economic growth [ 1 ]. Southern corn leaf blight, caused by the pathogen Bipolaris maydis ( B. maydis ), is a devastating fungal disease that frequently outbreaks in major maize-producing regions worldwide, posing a persistent and severe threat to the maize industry [ 2 , 3 ]. For a long time, chemical control has been able to effectively curb the spread of diseases to a certain extent. However, this approach is associated with multiple drawbacks, including environmental pollution, pesticide residue, and increased pathogen resistance. On the other hand, maize resistance to this pathogen is primarily governed by QTLs and is highly susceptible to environmental fluctuations [ 4 , 5 ]. Cultivating and popularizing disease-resistant varieties thus represent the most economical, safe, and sustainable strategy for managing this disease [ 6 , 7 ]. Therefore, further exploring the key genes in maize that regulate resistance to the southern corn leaf blight and dissecting their underlying molecular mechanism of disease resistance remains critical challenges that need to be addressed in the current plant pathology and crop breeding research. During the long-term co-evolution between plants and pathogens, a complex and precise immune regulatory network has been established [ 8 ]. Among its components, transcription factors (TFs), as a key regulator of gene expression, play a core role in activating disease resistance signaling pathways and initiating the defense response [ 9 – 11 ]. WRKY TFs constitute one of the largest families of transcriptional regulators in plants [ 12 ]. Their most distinctive structural feature is the highly conserved WRKY domain, which consists of approximately 60 amino acid residues [ 13 ]. Specifically, their N-terminus contains the characteristic conserved heptapeptide sequence WRKYGQK, while the C-terminus forms a zinc finger structure (C 2 H 2 ) through the coordination of cysteine (Cys) and histidine (His) residues. This structure enables specific binding to the (T)TGAC(C/T) (W-box) cis-acting element in the promoter region of downstream target gene, thereby regulating the expression of the target gene [ 14 ]. WRKY TFs are classified into three major groups based on the number of WRKY domains and the type of zinc finger structure. Group Ⅰ members contain two WRKY domains and C 2 H 2 type zinc finger structures. Group Ⅱ members possess only one WRKY domain and a C 2 H 2 type zinc finger structure, and are further subdivided into five subgroup (Ⅱa to Ⅱe) based on sequence differences. Group Ⅲ members feature a single WRKY domain and a unique C 2 HC type zinc finger structure [ 15 , 16 ]. This structural diversity lays the foundation for functional specialization, allowing WRKY TFs to play specific regulatory roles in distinct biotic stress response pathways. The WRKY TF gene family has attracted considerable attention due to its extensive involvement in plant responses to both biotic and abiotic stresses [ 17 ]. Numerous studies have confirmed that WRKY TFs typically form a complex regulatory network by participating in signaling pathways mediated by Salicylic acid (SA), Jasmonic acid (JA), and Ethylene (ET) [ 18 ]. In plant disease resistance responses, WRKY members enhance resistance to diverse pathogens such as fungi, bacteria and viruses by activating the expression of pathogenesis-related ( PR ) genes [ 19 ], regulating genes involved in secondary metabolites synthesis [ 20 ] or modulating immune receptor genes, thereby improving the basic immune level of plants. For instance, OsWRKY36 regulates the expression of the phenylalanine ammonia-lyase gene, thereby influencing the synthesis of lignin and the thickness of the parenchyma tissue, and consequently affecting the disease resistance of rice [ 21 ]. Under drought stress, leaves of MaWRKY80 overexpressing transgenic Arabidopsis exhibit lower reactive oxygen species (ROS) levels [ 22 ]. AtWRKY33 enhances resistance to Botrytis cinerea by regulating the JA signaling pathway [ 23 ]. OsWRKY45 improves its resistance to rice bacterial leaf blight by participating in the SA signaling process [ 24 ]. These findings have provided significant insights into the role of WRKY TFs in plant disease resistance response. To date, 120 WRKY TFs encoding genes distributed across 10 chromosomes have been identified in maize [ 25 , 26 ]. Although significant progress has been made in WRKY TFs reseach, the specific functions and regulatory network mechanisms of these factors in the southern corn leaf blight resistance remain unclear. In-depth exploration of WRKY TFs mechanism in maize not only helps to reveal the molecular basis of maize responses to biotic and abiotic stresses, but also provides important theoretical support and candidate gene resources for improving maize disease resistance, yield and quality via genetic engineering. In this study, we successfully identified a nuclear-localized transcription factor in maize, designated ZmWRKY36 . Functional analyses indicated that ZmWRKY36 positively regulates maize resistance to B. maydis and possesses transcriptional activation activity. These characteristics highlight its potentially value for research on the southern corn leaf blight resistance regulation. As a transcriptional activator, ZmWRKY36 may regulate the expression of a suite of downstream disease resistant-related genes, thereby constructing a multi-level disease defense system. Further studies are needed to explore the specific regulatory network between ZmWRKY36 and its target genes, its interactions with other signaling pathways, and the resistance effects in different genetic backgrounds. Results Bioinformatics analysis of ZmWRKY36 ZmWRKY36 (GRMZM2G054125) is a maize gene with 690 bp CDS, encoding a 229 amino acids protein, belonging to the WRKY TF gene family. ZmWRKY36 contains the canonical WRKYGQK core motif and C 2 H 2 type zinc finger structure, consistent with the structural characteristics of group Ⅱ members in the WRKY TF family (Fig. 1A, Supplementary Fig. 1). This domain mainly undertakes specific binding to downstream target genes and directing the regulation of their transcriptional expression. It also participates in the construction of complex regulatory networks, playing a critical role in signal integration and precise modulation of plant physiological processes. Through multiple sequence alignments with the reported WRKY proteins from 6 plant species, we further confirmed that the WRKY domain of ZmWRKY36 is highly evolutionarily conserved [ 27 ]. In particular, key amino acids in the WRKYGQK sequence and the zinc finger structure are invariant, suggesting that it may exert transcriptional regulatory functions by recognizing the W-box cis-acting element. Within the WRKY group Ⅱ subfamily, further subdivision into five groups, named Ⅱa, Ⅱb, Ⅱc, Ⅱd, and Ⅱe, is based on gene structure, amino acid sequence similarity, and phylogenetic relationships. The WRKYGQK motif of the ZmWRKY36 is invariant, and the protein contains potential pathogen response-related features, specifically, antioxidant-related cysteine residues, consistent with the conserved traits of group Ⅱe. To further determine the functional characteristics of ZmWRKY36 and its Ⅱe subfamily, we performed domain analysis and motif prediction of ZmWRKY36 and 16 previously reported Ⅱe subgroup genes (Fig. 1B). NCBI domains prediction showed that all WRKY Ⅱe members harbor a canonical WRKY domains. A total of 10 conserved motifs were identified by using MEME for motif prediction, named motif 1 to motif 10, and their lengths ranged from 6 to 50 amino acids. Among them, motif 1 and motif 2 constitute the WRKY domain, which is consistent with the results of the above domain analysis. These results further confirmed the structural conservatism of WRKY Ⅱe subgroup members. To explore the evolutionary origin of ZmWRKY36 and clarify its potential homologs, we performed a BLAST analysis of ZmWRKY36 amino acid sequence against the NCBI (National Center for Biotechnology Information) database. The results showed that 5 genes were identified homology to ZmWRKY36 in maize. To further analyze its phylogenetic position, we selected representative members of the WRKY family from 6 distinct plant species and constructed a phylogenetic tree. The phylogenetic tree results indicated that ZmWRKY36 was closest in evolutionary distance to OsWRKY56 , with amino acid sequence homology as high as 95%. Additionally, ZmWRKY36 and AtWRKY75 , a gene well-characterized for its role in disease resistance regulation in Arabidopsis thaliana , cluster in the same evolutionary branch (Fig. 1C). This evolutionary feature suggested that ZmWRKY36 may exert similar biological functions in maize disease resistance signaling pathway to those homologous proteins, providing a theoretical basis for its role in plant immune responses. ZmWRKY36 positively regulates maize resistance to B. maydis As the WRKY domain mediates responses to biotic stresses, we examined whether ZmWRKY36 is involved in maize defense against B. maydis . Quantitative real-time PCR (RT-qPCR) was used to detect ZmWRKY36 expression levels at six time points post B. maydis infection: 12, 24, 48, 72, 96, and 120 hours post infection (hpi). Results showed that ZmWRKY36 was significantly upregulated following B. maydis infection, with its expression peaking at 12 hpi (Fig. 2A). This indicated that ZmWRKY36 positively responses to B. maydis in maize. To determine whether ZmWRKY36 is involved in the maize resistance to B. maydis , we generated transient ZmWRKY36 -silenced and -overexpressed maize plants used Foxtail mosaic virus (FoMV) mediated virus-induced gene silencing (VIGS) and virus-mediated gene overexpressed (VOX) system respectively. After infiltrating maize leaves with Agrobacterium suspension containing the VIGS and VOX constructs, we quantified ZmWRKY36 expression. The relative expression levels of ZmWRKY36 in FoMV: ZmWRKY36 silenced plants were consistently below 0.5, significantly lower than that in the FoMV:V control (Fig. 2B). On the contrary, ZmWRKY36 expression in FoMV: ZmWRKY36 -VOX overexpressed plants were significantly upregulated, more than 100-fold higher than that in the FoMV: GFP -VOX control (Fig. 2C). For disease resistance phenotyping, we first validated ZmWRKY36 expression in silenced and overexpressed plants following rubbing inoculation on maize leaves. Compared with FoMV:V plants, ZmWRKY36 transcription was significantly reduced in FoMV: ZmWRKY36 plants. In contrast, ZmWRKY36 was markedly upregulated in FoMV: ZmWRKY36 -VOX compared to the control FoMV: GFP -VOX (Fig. 2A, B). Subsequently, we performed in vitro B. maydis inoculation on these plants. FoMV: ZmWRKY36 -silenced plants exhibited significantly larger lesion areas and higher B. maydis fungal biomass than FoMV:V, indicating that the silencing of ZmWRKY36 increases the susceptibility to B. maydis (Fig. 2D, E, F). Conversely, FoMV: ZmWRKY36 -VOX- overexpressed plants showed significantly smaller lesions and fungal biomass than FoMV: GFP -VOX, indicating that the overexpression of ZmWRKY36 enhances maize resistance to B. maydis (Fig. 2G, H, I). These results preliminarily suggested that ZmWRKY36 may positively regulate maize resistance to B. maydis . ZmWRKY36 affects the release of reactive oxygen species in maize To further dissect the functional role of ZmWRKY36 in modulating maize immune responses, we conducted a detailed analysis of chitin-induced reactive oxygen species (ROS) burst dynamics, a hallmark event in plant pattern-triggered immunity (PTI), using FoMV:V, FoMV: ZmWRKY36 , FoMV: GFP -VOX, and FoMV: ZmWRKY36 -VOX plants. FoMV: ZmWRKY36 showed significantly weaker ROS bursts compared to FoMV:V, while FoMV: ZmWRKY36 -VOX showed significantly stronger ROS bursts than FoMV: GFP -VOX (Fig. 2J, K). Additionally, the ROS burst peaked at 5 min post chitin treatment in FoMV: ZmWRKY36 , compared to 2.5 min in FoMV: ZmWRKY36 -VOX plants. Collectively, these findings not only confirm that ZmWRKY36 functions as a positive regulator of maize resistance to B. maydis , but also provide novel insights into its mechanistic role in the maize immune defense response. Such regulation of ROS homeostasis is likely a key component of ZmWRKY36 -mediated immune signaling, highlighting its potential as a target for molecular breeding strategies aimed at enhancing maize disease resistance. ZmWRKY36 regulates the expression levels of pathogenesis-related gene To further explore the role of ZmWRKY36 in maize defense mechanism against B. maydis , we focused on the expression of disease-related genes ( PR ) in maize. The selected PR genes included ZmPR1 , ZmPR3 , ZmPR4 , ZmPR5 , and ZmPR10 , all of which are involved in the defense response process of plants. The relevant analyses were conducted in maize plants of ZmWRKY36 silenced and overexpressed. In the ZmWRKY36 silencing group, the expression levels of ZmPR1 , ZmPR3 , ZmPR4 , ZmPR5 , and ZmPR10 in ZmWRKY36 -silenced (FoMV: ZmWRKY36 ) plants were significantly downregulated compared to the FoMV:V plants (Fig. 3A). This indicated that the absence of ZmWRKY36 led to reduced expression of these key PR genes. In the overexpression group, comparison between FoMV: ZmWRKY36 -VOX plants and the FoMV: GFP -VOX plants showed significant upregulation of the same five PR genes (Fig. 3B). Subsequently, we analyzed the expression levels of these PR genes following B. maydis infection. Data revealed that in the FoMV: ZmWRKY36 plants, their expression were significantly lower compared to the FoMV:V plants, conversely, in the FoMV: ZmWRKY36 -VOX plants, their expression were significantly higher compared with the FoMV: GFP -VOX plants (Fig. 3C, D). This pattern was consistent with our previous phenotypic observations. In conclusion, these results indicated that ZmWRKY36 regulates the expression of PR genes. The positive correlation between ZmWRKY36 transcriptional level and the expression of these PR genes further validates the positive responsiveness of ZmWRKY36 to B. maydis infection. This response not only affected the expression of ZmWRKY36 , but also exerted a cascading effect on the expression of other PR genes during maize defense against B. maydis . These findings provide an important foundation for a deeper understanding of maize’s complex defense mechanism. ZmWRKY36 is a nuclear localized protein To explore the biological function of ZmWRKY36 and determine its subcellular localization, we constructed a ZmWRKY36 -Green Fluorescent Protein (GFP) fusion protein. Agrobacterium-mediated transient transformation was used to heterologously express this fusion protein (with the free GFP as a control) in N. benthamiana. Subcellular localization was observed using a laser confocal microscope (Zeiss Confocal LSM 980). In the control group, GFP signal was detected in both the cytoplasm and nucleus. In contrast, the GFP- ZmWRKY36 fusion protein showed specific enrichment, with its fluorescence signal colocalizing completely with the nuclear red fluorescence marker (Fig. 4A). This indicated that ZmWRKY36 localizes to the nucleus in N. benthamiana. To further verify the subcellular localization characteristics of ZmWRKY36 in a homologous system, we transformed the GFP- ZmWRKY36 fusion protein and free GFP into maize protoplasts. Confocal microscopy revealed that while GFP was distributed in both the cytoplasm and nucleus, GFP- ZmWRKY36 fusion protein was specifically localized to the nucleus, consistent with the observation from N. benthamiana transient expression system (Fig. 4B). Furthermore, to confirm the expression status of ZmWRKY36 , we performed Western blot analysis to detect protein expression in the aforementioned N. benthamiana and maize protoplasts. The results showed that the obtained bands were consistent with the expected size of the target gene (Fig. 4C, D). Collectively, these results confirmed the nuclear localization of ZmWRKY36 , providing a critical spatial basis for its role in regulating downstream gene expression. ZmWRKY36 exhibits transcriptional activation activity Given that WRKY TFs typically possess transcriptional activity, we assayed the transcriptional activity of ZmWRKY36 in both maize protoplasts and yeast systems. In maize protoplasts, we constructed a GAL4- ZmWRKY36 fusion protein and co-transfected it with a luciferase reporter vector containing the GAL4-TATA transcriptional regulatory element. GAL4-VP16 (positive control) and GAL4 (negative control) were included for comparison. Transcriptional activity was evaluated by the ratio of Firefly Luciferase (FLuc) to Reniferase Luciferase (RLuc) signal. The LUC/REN ratio in GAL4- ZmWRKY36 and reporter group was significantly higher than that in the GAL4 negative control group, indicating that ZmWRKY36 has transcriptional activation activity (Fig. 5A, B). In the yeast system, similar as the positive control pGBKT7-VP16, pGBKT7- ZmWRKY36 grew on SD/-Trp-His solid medium and formed blue colonies on the medium supplemented with X-α-gal. In contrast, the negative control failed to grow or produce blue color. This indicated that ZmWRKY36 can independently activate the transcription of the downstream reporter gene (Fig. 5C). Transcriptome analysis of ZmWRKY36 transient silenced and overexpressed materials Revealing the regulatory pathway of ZmWRKY36 in maize resistance to B. maydis is of crucial significance for understanding the complex interaction mechanism between plants and pathogens. To explore this regulatory pathway, we conducted transcriptome analysis on the silenced material (FoMV:V vs FoMV: ZmWRKY36 ) and overexpressed material (FoMV: GFP -VOX vs FoMV: ZmWRKY36 -VOX). All the plants showed high silencing and overexpression efficiencies (Supplementary Fig. 2A, B). To ensure the accuracy and reliability of the experimental results, three biological replicates were set for each group of materials in the transcriptome. Results from transcriptome analysis indicated that the majority of genes exhibited stable expression across different treated materials. In the silenced plants, a total of 1074 genes were commonly expressed under both conditions. Specifically, 1118 genes were detected in FoMV:V, while 1103 genes were identified in FoMV: ZmWRKY36 (Fig. 6A). Based on this, we conducted statistical analysis on the DEGs between the groups. In the data from ZmWRKY36 -silenced materials, a total of 1127 DEGs were identified, including 601 upregulated genes and 526 downregulated genes (Fig. 6B). To gain deeper insights into the functional roles of these DEGs, we performed Gene Ontology (GO) functional enrichment analysis, classifying them into biological processes (BP), cellular components (CC), and molecular functions (MF). In the silenced material group, DEGs were significantly enriched in biological pathways such as plant carbohydrate metabolism, particularly in cell wall polysaccharide synthesis, cell structural composition, as well as related molecular binding and catalytic functions (Fig. 6C, D). Furthermore, to reveal the key metabolic pathways and signal transduction pathways involved in ZmWRKY36 expression, we conducted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on ZmWRKY36 silenced and overexpressed plants. The results showed that DEGs were significantly enriched in multiple pathways. In ZmWRKY36 silenced plants, upregulated genes were enriched in the plant-pathogen interaction pathway, while downregulated genes mainly reflected the redistribution of plant metabolic resources (Fig. 6E, F). These results suggested that ZmWRKY36 may play an important regulatory role in maize resistance to B. maydis . In the overexpressed plants, 9591 genes were detected in FoMV: GFP -VOX, and 9448 genes were detected in FoMV: ZmWRKY36 -VOX. A total of 9192 genes were commonly expressed under both conditions (Fig. 7A). Among them, 9847 DEGs were identified, including 4873 upregulated genes and 4974 downregulated genes (Fig. 7B). In the overexpressed group, GO functional enrichment analysis showed that the DEGs were mainly enriched in the structural aspects of photosynthetic membrane systems, ion binding and transport, as well as enzyme catalysis (Fig. 7C, D). These results indicated that ZmWRKY36 may be involved in the regulation of physiological processes such as plant growth and development, disease resistance defense, and photosynthesis, which further highlights the core role of ZmWRKY36 in the regulatory network of maize resistance to B. maydis . Additionally, KEGG pathway enrichment results showed that the upregulated genes in overexpressed plants were significantly enriched in photosynthesis and carbon fixation, while the downregulated pathways involved protein processing, signal transduction, and other metabolism or structure related pathways (Fig. 7E, F). These results indicated that ZmWRKY36 may participate in signal transduction processes through regulating metabolism or by synthesize lipid derivatives with defensive functions to enhance resistance. Identification of downstream target genes regulated by ZmWRKY36 Through intersection analysis of two groups of DEGs, we identified a total of 206 genes that had opposite expression trends under the two treatments. Specifically, 84 genes were upregulated in the overexpressed group while being downregulated in the silenced group, and 122 genes showed the reverse trend (Fig. 8A, B). These genes are likely target genes directly or indirectly regulated by ZmWRKY36 . Notably, as a member of the WRKY TFs family, the transcriptional regulatory function of ZmWRKY36 lies in its specific binding to the conserved cis-acting elements in the promoter region of target genes. Its interaction with the W-box domain constitutes a key molecular mechanism for regulating downstream target genes to achieve physiological functions. Therefore, we used Tbtools software to extract the 2000 bp upstream promoter sequences of the 206 DEGs with opposite expression trends, the cis-acting elements within these promoter sequences were computationally predicted utilizing PlantCARE. The results showed that the promoter regions of 105 genes contained W-box sequences (Supplementary Fig. 3). We performed cluster heatmap analysis on the cis-acting elements in these DEGs and selected 14 genes containing multiple W-box elements, which were classified them by abiotic stress, biotic stress, plant hormone response, development-related, and light response (Fig. 8C). Among them, Zm00001d005783 and Zm00001d039384 possessed more biotic stress-related elements. This series of findings demonstrated that these DEGs carrying W-box elements are highly likely to be downstream target genes directly regulated by ZmWRKY36 . ZmWRKY36 precisely regulates the transcriptional expression levels of these genes and plays an important regulatory role in the resistance response of maize to B. maydis . Verification of ZmWRKY36 transcriptome data The expression patterns of four candidate target genes using RT-qPCR. These genes exhibited opposite expression trends in the ZmWRKY36 silenced and overexpressed materials. The results showed that their expression trends were consistent with the transcriptome sequencing data (R² >0.9), confirming the reliability of the transcriptome sequencing results (Fig. 9A, B). Discussion In the long-term co-evolution between plants and pathogens, TFs serve as the core regulatory hubs of gene expression, and play pivotal roles in disease resistance signaling pathways [ 28 ]. In this study, we identified ZmWRKY36 as a nuclear localized TF in maize and used VIGS and VOX systems to demonstrate its crucial role in resistance to B. maydis . These finding provided key insights into the molecular mechanism underlying maize disease resistance. Transient silencing of ZmWRKY36 significantly increased maize susceptibility to B. maydis , and inhibited chitin-induced ROS bursts. Conversely, transient overexpressed of ZmWRKY36 effectively enhanced the maize's resistance and promote ROS accumulation. These phenotypic differences directly indicated that ZmWRKY36 plays an active role in the early signaling pathways that activate the plant immune response. Importantly, ZmWRKY36 , as a key regulator in maize, not only responds significantly to the invasion of B. maydis , but also enhances the resistance level of maize through a positive regulatory mechanism. The core evidence for this discovery came from the expression pattern of the PR gene: in the ZmWRKY36 silenced plants, the transcriptional level of the PR gene was significantly downregulated, but upregulated in the overexpressed plants. Further correlation analysis revealed that the transcriptional level of ZmWRKY36 was highly positively correlated with the expression level of PR genes. This confirmed the positive response characteristic of ZmWRKY36 to B. maydis infection, but also revealed that ZmWRKY36 might affect the expression of multiple key genes in maize defense network through a cascade regulatory effect, providing a new molecular perspective for understanding the complex disease resistance mechanism of maize [ 29 ]. However, the specific regulatory network and molecular mechanism by which ZmWRKY36 confers B. maydis resistance require further exploration. Notably, ZmWRKY36 ’s transcriptional activation activity open new avenues for understanding its disease resistance mechanism. Within the molecular regulatory network of plant disease resistance responses, the activation state of TFs constitutes a crucial step in initiating downstream defense signal transduction [ 30 ]. The transcriptional activation activity of ZmWRKY36 further endows it with the potential to regulate the downstream target gene network [ 31 , 32 ]. ZmWRKY36 is capable of specifically binding to target genes, thereby initiating or enhancing the expression of a series of downstream genes. This process may involve the coordinated expression of multiple functional modules, such as the expression of plant pathogenesis-related protein genes, hormone signaling pathway-related [ 33 ], and driving an extracellular ROS burst [ 32 ], consequently forming a complex gene expression regulatory network and providing important molecular support for plants to establish an effective disease resistance defense system. Therefore, in-depth research on the transcriptional activation activity of ZmWRKY36 is expected to provide new insights and experimental foundations for clarifying the fine regulatory mechanism of plant disease resistance responses. Collectively, this result delineated ZmWRKY36 ’s role as an anti-pathogen regulatory node: it initiate the cascade amplification of the immune signal, and enhances maize defense by directly regulating expression of genes. Transcriptome analysis of ZmWRKY36 further revealed a complex metabolic regulatory network. GO enrichment analysis of DEG in ZmWRKY36 silenced and overexpressed plants showed significant enrichment in pathways related to photosynthesis, which are associated with the composition of cell wall polysaccharides and the structure of photosynthetic membranes. This suggested that ZmWRKY36 may participate in the disease resistance process through multiple pathways. On one hand, strengthening physical barriers by increasing cell wall thickness and remodeling cell structure [ 34 , 35 ]; on the other hand, regulating immune responses via energy supply or synergism with phytohormones and other metabolites [ 36 ]. This “photosynthesis-disease resistance” cross-correlation provides a new perspective for exploring the “growth and defense” balance in plants [ 37 , 38 ], and implied that ZmWRKY36 may coordinating energy metabolism and defense responses to enable maize adaptation to pathogen stress [ 39 ]. KEGG pathway analysis further supported this functional speculation. After silencing of ZmWRKY36 , DEGs were significantly enriched in the plant-pathogen interaction pathway, suggesting that silencing relieves inhibition of the basic defense pathway and triggers stress responses. After overexpression of ZmWRKY36 , DEGs were enriched in photosynthesis, protein processing and signal transduction, likely reflecting negative feedback regulation of the overactivated defense signals to maintain cellular homeostasis [ 40 ]. These results implied that ZmWRKY36 enhances the resistance to B. maydis by regulating downstream genes involved in energy metabolism, photosynthesis, and defense signal transduction. WRKY TFs typically bind to W-box elements in target gene promoters [ 41 ], thus, screening and verification of the ZmWRKY36 target gene will clarify its resistance mechanism. In this study, we identified W-box elements in the promoter regions of 105 DEGs from transcriptome data. Techniques such as chromatin immunoprecipitation sequencing (ChIP-seq) [ 42 ] or electrophoretic mobility shift assays (EMSA) [ 43 ] can be used to identify direct ZmWRKY36 targets. Specific binding of ZmWRKY36 to a target gene would directly confirm its role in activating disease resistance genes via transcriptional regulation. This targeted study will clarify the role of ZmWRKY36 in regulating the resistance network of maize to B. maydis , and may reveal novel resistance genes. For instance, ZmWRKY36 might be involved in signaling molecules that are related to ROS [ 44 ], as well as the photosynthetic pathway into an integrated immune regulatory network. Furthermore, functional diversity of the target genes may explain the multiple roles of ZmWRKY36 . Some of the target genes might be cellulose synthase genes that strengthen cell wall to enhance physical defense; some genes might regulate secondary metabolites such as flavonoids to enhance chemical defense [ 45 ], and some might mediate the transmission of immune signals to amplify the defense response [ 46 ]. Coordinated expression of these target genes collectively constitutes the comprehensive disease resistance response mediated by ZmWRKY36 . Additionally, analyzing the number and distribution of W-box in the promoter regions of target genes [ 47 ], may reveal ZmWRKY36 regulatory specificity and efficiency: high W-box abundance might correlate with stronger transcriptional activation, while specific-positions W-boxes might synergize with other cis-acting element [ 48 , 49 ], providing insights into ZmWRKY36’s functional divergence under different stresses. In conclusion, according to the results of this study, we proposed a hypothetical working model for ZmWRKY36 during B. maydis infection (Fig. 10). Under the infection of pathogen, ZmWRKY36 may bind to the cis-acting elements in the promoter of the target gene. It may accelerate the clearance of ROS, regulate the disease resistance genes or the photosignal transduction pathways. Then ZmWRKY36 enhances the resistance of maize to the southern corn leaf blight. Based on the unique W-box binding property of WRKY TFs, key target genes were screened through molecular interaction experiments, which will lay a crucial foundation for in-depth exploration of the mechanism of ZmWRKY36 . Future research will focus on systematically exploring the downstream target genes of ZmWRKY36 . By using ChIP-seq and EMSA technologies to identify direct W-box binding targets, and verifying its regulatory effect through the dual luciferase reporter system [ 50 ]. These studies will clarify the ZmWRKY36 -mediated disease resistance network in maize, and providing more clear targets and theoretical support for maize disease-resistant molecular breeding, accelerating the development of new resistant varieties. Conclusions ZmWRKY36 is a group Ⅱ WRKY TF, containing a conserved WRKY domain and C 2 H 2 type zinc finger domain. Its expression is significantly upregulated in maize upon B. maydis infection, and it positively regulates resistance to the southern corn leaf blight. ZmWRKY36 localizes to the nucleus of N. benthamiana and maize protoplasts, possesses transcriptional activation activity. Transcriptome analysis showed that DEGs from the transient silencing and overexpression of ZmWRKY36 were mainly enriched in pathways related to cellular structure, metabolite synthesis and photosynthesis. Fourteen DEGs with opposite expression trends were identified with multiple W-box elements, which are likely downstream genes regulated by ZmWRKY36 . Our fingdings provide a theoretical basis for the molecular mechanism of resistance to B. maydis in maize, and enrich genetic materials for maize disease-resistant breeding. Ultimately, this work will facilitate the development of high-yield, disease-resistant maize varieties to safeguard global food security. Materials and Methods Plant materials, fungal strains and growth conditions The experimental materials used in this study included the maize inbred line B73 (wild type from CIMMYT) and N. benthamiana (laboratory storage). The soil mixture was prepared with vermiculite and nutrient soil at a ratio of 3:1, which was placed in pots (10 × 8 cm in depth). B73 seeds were sown in these pots and cultivated in a greenhouse under a 14-h light/10-h dark photoperiod. Greenhouse conditions were maintained at 24°C during the day and 20°C at night, with relative humidity controlled within the range of 50%-60%. N. benthamiana seeds were sown in the same soil mixture. Upon germination, individual seedlings were transplanted to fresh soil and grown under the same environmental conditions as maize. The B. maydis strain used was 4-4-3 strain preserved in the laboratory, which was grown on oat agar medium and incubated at 25°C for fourteen days until sporulation occurred. Gene structure analysis and phylogenetic tree construction ZmWRKY36 was analyzed with sixteen WRKY Ⅱe subfamily proteins. Their domains and motifs were predicted using NCBI ( https://www.ncbi.nlm.nih.gov/ ) and MEME ( https://meme-suite.org/meme/tools/meme ), followed by analysis with Tbtools software. For phylogenetic tree construction, thirty protein sequences of highly homologous were selected from six plant species: Zea mays , Arabidopsis thaliana , Glycine max , Gossypium hirsutum , Triticum aestivum , and Oryza sativa . These protein sequences were retrieved from NCBI based on their respective gene identifiers. Subsequently, the homologous protein sequences were imported into MEGA11. To ensure robust homology analysis, multiple sequence alignment was performed using the Muscle algorithm. To enhance clarity and interpretability, the phylogenetic tree was constructed via the neighbor-joining method in MEGA11 and optimized using iTOL ( https://itol.embl.de/ ). The gene information used in this study for homology analysis, domain and motif analysis were listed in Supplementary Tables 1 and 2. Analysis of expression patterns The spore suspension of B. maydis was prepared at a concentration of 1×10⁵ spores/mL and sprayed onto the leaves of 14-day-old B73 seedlings. Inoculated leaves were analyzed at 12, 24, 48, 72, 96, and 120 hours post infection (hpi) using HiScript Ⅲ RT SuperMix (Vazyme, R323, China), with ZmActin as the internal reference for qRT-PCR. The 2 −ΔΔCt method [ 51 ] was used to calculate the relative expression levels of ZmWRKY36 at different time points, and the quantitative data were presented as mean ± standard error of the mean (SEM). All experiments were conducted with three biological replicates to ensure the accuracy and reliability of the results. The primers used in this study were listed in Supplementary Table 3. Construction of Agrobacterium -mediated maize VIGS and VOX plants Foxtail mosaic virus (FoMV) was utilized to construct the virus-induced gene silencing (VIGS) and virus-mediated overexpression (VOX) systems in maize[ 52 ]. A 300 bp fragment from the C-terminal region of the ZmWRKY36 CDS was selected as the silencing fragment, while the full-length CDS (including the termination codon) was chosen as the overexpression fragment. Homologous recombination was used to clone the ZmWRKY36 silenced fragment and full-length CDS into the pFoMV-pCAMBIA1380 silencing and overexpression vectors at restriction enzyme sites, thereby obtaining the VIGS and VOX plasmids. These plasmids were introduced into Agrobacterium tumefaciens GV3101 via chemical transformation. The Agrobacteria were grown overnight in LB liquid medium with shaking, which cells were collected by centrifugation and resuspended in a suspension buffer (10 mM MgSO₄, 100 µM acetosyringone) to adjust an OD₆₀₀ to 1.0. Then, the prepared Agrobacterium suspensions were infiltrated at a point approximately 2–3 mm above the coleoptile of 4-day-old seedlings. Fourteen days post-infiltration, plants exhibiting mosaic phenotypes were selected, and RT-qPCR was performed to assess the silencing and overexpression efficiency of ZmWRKY36 . Collect the fourth to sixth leaves that show obvious mosaic symptoms, as well as the leaves from the silencing and overexpression plants,and place them in 50 ml tubes with desiccant at the bottom. Perform overnight freeze-drying treatment, stored at -20°C for subsequent inoculation experiments. Approximately 100 mg of freeze-dried plant material was ground in 50 mM potassium phosphate buffer (pH 7.0). Once the second leaf of the plants had fully expanded, a layer of silicon carbide powder was sprinkled on the leaf surface. The suspension was gently spread over the leaf surface using fingers to perform the inoculation. After inoculation, the leaves were left undisturbed for 20 min to facilitate viral infection, then rinsed with water to remove excess silicon carbide. Inoculated plants were transferred to the greenhouse, where symptoms were observed over a 14–21 day period. These plants were subsequently used for in vitro or spray inoculation with B. maydis to evaluate disease resistance. Pathogenicity analysis Pathogenicity analysis The fourth or fifth leaf displaying mosaic phenotypes after rub inoculation was selected and placed flat in a 25 × 25 cm petri dish lined with moist filter paper. The B. maydis spore suspension was adjusted to a concentration of 1×10⁵ spores/ml with 0.02% Tween 20, and 10 µl of this suspension was applied to each inoculation site on the maize leaves. After sealing the petri dish with plastic wrap, it was incubated at 25°C with 95% humidity under a 14-h light/10-h dark cycle for 4–5 days to assess disease development. Lesion areas were measured using ImageJ software. The leaf fragment (4–5 cm in size) from the center of the lesion was cut, and the total DNA (including that from maize leaves and B. maydis ) was extracted using the CTAB method. Relative fungal biomass was calculated via the 2 −ΔΔCt method. Reactive oxygen species assay Following rub inoculation, the third leaf with the distinct mosaic phenotype was selected, and 4 mm-diameter discs were punched from the both sides of veins. These leaf discs were placed in a 90 mm petri dish containing sterile water and incubated in the dark overnight. For the assay, three leaf discs were placed in a 1.5 ml tube containing the reaction mixture, which consisted of 100 µl of luminol (Bio-Rad Immun-Star horseradish peroxidase substrate), 1 µl of horseradish peroxidase (HRP), and 1 µl of 1 mM flg22. Luminescence signals were detected using a Glomax 20/20 luminometer (Promega) following treatment with 1 µl of 10 mM chitin or ddH₂O (as control). Measurements were recorded every 10 s over a 20 min period, and each sample analyzed was repeated three times. Subcellular localization assay In N. benthamiana experiments, free Green Fluorescent Protein (GFP) and the nuclear red fluorescence marker were introduced into Agrobacterium tumefaciens EHA105 via chemical transformation. Overnight-cultured Agrobacteria were collected and resuspended in a solution containing 1 M MgCl₂, 1 M MES, and 100 mM acetosyringone to adjust the OD₆₀₀ to 1.0. The suspension was injected into the third or fourth N. benthamiana leaves using a 1 ml sterile syringe (without needle). GFP served as the control, with at least three plants per treatment. Plants were incubated in a 25°C greenhouse for 36 h. For maize protoplast experiments, free GFP and GFP- ZmWRKY36 plasmids were co-transfected with the nuclear red fluorescence marker into protoplasts. They were used for confocal microscopy observation after dark incubation for 12–16 h Confocal microscopy The fluorescence signals were detected using the ZEISS confocal microscope 980. The Green Fluorescent Protein (GFP) and the nuclear red fluorescence marker signals were imaged at 488 nm and 587 nm excitation lasers respectively. The lens magnification was 20 times, and the scanning resolution was 1024 px × 1024 px. Transcriptional activity analysis ZmWRKY36 was fused with GAL4 binding domain and co-transfected into maize protoplasts with a reporter vector containing the GAL-TATA element and Firefly Luciferase (FLuc). GAL4-VP16 and GAL4 served as positive and negative controls. After overnight dark incubation, protoplast protein extracts were analyzed for Firefly Luciferase (Luc) and Reniferase Luciferase (Ren) activities using the microplate reader. Transcriptional activity was evaluated by the ratio of Firefly Luciferase (FLuc) to Reniferase Luciferase (RLuc) signal. In the yeast system, the bait plasmid pGBKT7- ZmWRKY36 was transformed into AH109 competent cells, with pGBKT7 and pGBKT7-VP16 as negative and positive controls. Transformants were plated on SD/-Trp medium for 2–3 days. Then, single colonies were serially diluted (1×, 10×, 100×, 1000×) and spotted onto SD/-Trp, SD/-Trp-His, and SD/-Trp-His + X-α-gal media to verify transcriptional activity. Transcriptome sequencing In this study, the following four samples were selected for RNA-Seq: FoMV:V, FoMV: ZmWRKY36 , FoMV: GFP -VOX, and FoMV: ZmWRKY36 -VOX. For transient silencing, FoMV:V served as the control and FoMV: ZmWRKY36 as the experimental group. For transient overexpression, controls was FoMV: GFP -VOX and FoMV: ZmWRKY36 -VOX as experimental group. The fourth leaves of plants showing mosaic phenotypes after rub inoculation were selected. RT-qPCR confirmed the silencing and overexpression efficiency of ZmWRKY36 , and leaves with stable expression levels were chosen. Three replicates per treatment were sent to Novogene Co., Ltd. for transcriptome sequencing. Quantification and statistical analysis This study employed Student's t-test (with *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) for statistical analysis. The data represent SEM ± the mean. The detailed information of the statistical analysis is provided in the legend. Declarations Ethics approval and consent to participate All materials used in this study complies with institutional, national, and international guidelines. The maize inbred B73 was provided by CIMMYT. N. benthamiana and pathogen strain was kept and bred by our laboratory. Competing interests The authors declare no competing interests. Funding This work was financially supported by the National Natural Science Foundation of China, International Cooperation and Exchange Project (32061143033). Author Contribution ZQ and WD conceived and designed the experiments. Shu performed the research and writing original draft. Ying, HY, Na, HL, CT, RY provided help during research, ZQ and WD revised the manuscript. All authors approved the manuscript and consent to publication this manuscript Acknowledgements We thank Dr. Steven A. Whitham (Department of Plant Pathology & Microbiology, Iowa State University) for providing plasmid pFoMV-EV-pCAMBIA1380-IS5. Data Availability The datasets generated and analyzed during the current study are available in the NCBI repository, PRJNA1320840. References Wang ZY, Wang XM. Current status and management strategies for corn pests and diseases in China. Plant Prot. 2019;45(1):1–11. Lv GZ, Chen J, Bai JK, Wang CP. The current situation, trend and prevention and control measures of corn pests and diseases in our country. Plant Prot. 1997;1(4):20–1. Zhou NH, Liu SQ. Further study on the effect of the southern corn leaf blight on Bipolaris maydis. BMC Plant Biol. 1983;25(1):39–46. Chen G, Xiao Y, Dai S, Dai Z, Wang X, Li B, et al. Genetic basis of resistance to southern corn leaf blight in the maize multi-parent population and diversity panel. 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Supplementary Files SupplementaryTable1.docx SupplementaryTable2.docx SupplementaryTable3.docx SupplementaryFigure1.png SupplementaryFigure2.png SupplementaryFigure3.png Cite Share Download PDF Status: Published Journal Publication published 05 Jan, 2026 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 23 Oct, 2025 Reviews received at journal 19 Oct, 2025 Reviews received at journal 16 Oct, 2025 Reviewers agreed at journal 11 Oct, 2025 Reviewers agreed at journal 11 Oct, 2025 Reviewers agreed at journal 10 Oct, 2025 Reviewers invited by journal 10 Oct, 2025 Editor assigned by journal 10 Oct, 2025 Editor invited by journal 10 Oct, 2025 Submission checks completed at journal 10 Oct, 2025 First submitted to journal 10 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":1982617,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBioinformatics analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZmWRKY36\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Protein structure of ZmWRKY36. The protein structure was obtained from UniProt and visualized using IBS 3.0. \u003cstrong\u003eB\u003c/strong\u003e Domain analysis and motif prediction of WRKY Ⅱe subfamily in maize. A total of 17 proteins including ZmWRKY36 were analyzed for domains (NCBI) and conserved motifs (MEME). \u003cstrong\u003eC\u003c/strong\u003e Phylogenetic tree analysis of ZmWRKY36. The phylogenetic tree was constructed using the neighbor-joining method in MEGA11 with 30 highly homologous protein sequences from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eGlycine max\u003c/em\u003e, \u003cem\u003eGossypium hirsutum\u003c/em\u003e, \u003cem\u003eZea mays\u003c/em\u003e, \u003cem\u003eTriticum aestivum\u003c/em\u003e, and \u003cem\u003eOryza sativa\u003c/em\u003e. ZmWRKY36 (GRMZM2G054125) is highlighted in red.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7738242/v1/3a55073ee51207a19b280d38.png"},{"id":94404705,"identity":"295f70a9-d7ad-43fe-9893-712cd07c690f","added_by":"auto","created_at":"2025-10-27 14:01:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3759751,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eZmWRKY36\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003epositively regulates maize resistance to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. maydis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e RT-qPCR analysis of \u003cem\u003eZmWRKY36\u003c/em\u003e expression in response to \u003cem\u003eB. maydis \u003c/em\u003einfection. Water-treated plants served as controls, samples were detected at 0 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h after \u003cem\u003eB. maydis \u003c/em\u003einfection. Values represent the mean ± standard deviation of three biological replicates. \u003cstrong\u003eB\u003c/strong\u003e Silencing efficiency of FoMV:\u003cem\u003eZmWRKY36 \u003c/em\u003eplants was determined after injection. \u003cstrong\u003eC\u003c/strong\u003e Transient overexpressed efficiency of FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX plants was determined after injection. \u003cstrong\u003eD, E, F\u003c/strong\u003eIdentification of disease-resistant phenotypes, lesion area, and fungal biomass of \u003cem\u003eZmWRKY36\u003c/em\u003e-silenced material inoculated in vitro with \u003cem\u003eB. maydis\u003c/em\u003e. \u003cstrong\u003eG, H, I\u003c/strong\u003e Identification of disease resistance phenotypes, lesion area, and fungal biomass statistics of \u003cem\u003eZmWRKY36\u003c/em\u003e-overexpressed material inoculated in vitro with \u003cem\u003eB. maydis\u003c/em\u003e. \u003cstrong\u003eJ, K\u003c/strong\u003e ROS assay of FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e and FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX plants. Data contain mean ± standard error of three replicates. (* p ≤0.05; ** p ≤ 0.01; *** p ≤0.001)\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7738242/v1/dfed89ffa32cb6960ef24b92.png"},{"id":94405159,"identity":"ee8147a1-5f35-440f-b158-48f898f33ae2","added_by":"auto","created_at":"2025-10-27 14:01:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":633539,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes in ZmWRKY36-silenced materials and ZmWRKY36-overexpressed materials. A\u003c/strong\u003eRT-qPCR analyses showing the expression of \u003cem\u003eZmPR1\u003c/em\u003e, \u003cem\u003eZmPR3\u003c/em\u003e, \u003cem\u003eZmPR4\u003c/em\u003e, \u003cem\u003eZmPR5\u003c/em\u003e and \u003cem\u003eZmPR10\u003c/em\u003e in FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e. \u003cstrong\u003eB\u003c/strong\u003e RT-qPCR analyses showing the expression of \u003cem\u003eZmPR1\u003c/em\u003e, \u003cem\u003eZmPR3\u003c/em\u003e, \u003cem\u003eZmPR4\u003c/em\u003e, \u003cem\u003eZmPR5\u003c/em\u003eand \u003cem\u003eZmPR10\u003c/em\u003e in FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX. \u003cstrong\u003eC\u003c/strong\u003e RT-qPCR analyses showing the expression of \u003cem\u003eZmPR1\u003c/em\u003e, \u003cem\u003eZmPR3\u003c/em\u003e, \u003cem\u003eZmPR4\u003c/em\u003e, \u003cem\u003eZmPR5\u003c/em\u003eand \u003cem\u003eZmPR10\u003c/em\u003e in FoMV:\u003cem\u003eZmWRKY36 \u003c/em\u003efollowing infection with \u003cem\u003eB. maydis\u003c/em\u003e. \u003cstrong\u003eD\u003c/strong\u003e RT-qPCR analyses showing the expression of \u003cem\u003eZmPR1\u003c/em\u003e, \u003cem\u003eZmPR3\u003c/em\u003e, \u003cem\u003eZmPR4\u003c/em\u003e, \u003cem\u003eZmPR5\u003c/em\u003e and \u003cem\u003eZmPR10\u003c/em\u003ein FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX following infection with \u003cem\u003eB. maydis\u003c/em\u003e. Data contain mean ± standard error of three replicates (* p ≤0.05; ** p ≤ 0.01; *** p ≤0.001).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7738242/v1/23564a8e446083eb9817590f.png"},{"id":94403228,"identity":"04a352cb-0b88-435f-ad06-d5b174a30144","added_by":"auto","created_at":"2025-10-27 14:00:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2476534,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZmWRKY36 protein is located in nucleus. A\u003c/strong\u003e Free GFP, GFP-\u003cem\u003eZmWRKY36\u003c/em\u003e, and the nuclear red fluorescent marker were transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e EHA105 and introduced into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Confocal microscopy observation were conducted after 36 h. The scale is 5 micrometers. \u003cstrong\u003eB\u003c/strong\u003e Free GFP, GFP-\u003cem\u003eZmWRKY36\u003c/em\u003e, and the nuclear red fluorescent marker were transformed into maize protoplasts. Confocal microscopy observation were conducted after cultivating in the dark for 12 h. The scale is 5 micrometers. \u003cstrong\u003eC\u003c/strong\u003e The expression of GFP, GFP-ZmWRKY36 protein in \u003cem\u003eN. benthamiana \u003c/em\u003ewere detected through Western blot analysis.\u003cem\u003e \u003c/em\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cem\u003e \u003c/em\u003eThe expression of GFP, GFP-ZmWRKY36 protein in maize protoplasts were detected through Western blot analysis.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7738242/v1/1cfa2314c2b3c0d3ce5af3e7.png"},{"id":94403716,"identity":"b2bcc11c-98ba-4788-87fb-f1fce3730469","added_by":"auto","created_at":"2025-10-27 14:00:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1333622,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptional activity of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZmWRKY36\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in maize protoplasts and yeast system. A\u003c/strong\u003eThe structure diagram of the vector in the dual-luciferase reporter assay system. \u003cstrong\u003eB\u003c/strong\u003e The combined construct GAL4-\u003cem\u003eZmWRKY36\u003c/em\u003e, GAL4-VP16, and GAL4 were transformed into maize protoplasts. The activities of Firefly Luciferase (FLuc) and Reniferase Luciferase (RLuc)were detected on the microplate reader. Transcriptional activity was evaluated by the ratio of Firefly Luciferase (FLuc) to Reniferaseluciferase (RLuc) signal. \u003cstrong\u003eC\u003c/strong\u003e The single colony plates of pGBKT7-\u003cem\u003eZmWRKY36\u003c/em\u003e, pGBKT7, and pGBKT7-VP16 were transferred to SD/-Trp, SD/-Trp-His and SD/-Trp-His + X-α-gal solid media for transcriptional activity verification. Data contain mean ± standard error of three replicates.(* p ≤ 0.05; ** p ≤0.01; *** p ≤ 0.001).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7738242/v1/ed61c7b92c38ca071af97d93.png"},{"id":94405143,"identity":"bd254bd1-827d-4c28-896c-0daedc2c987f","added_by":"auto","created_at":"2025-10-27 14:01:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1073366,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZmWRKY36\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e silenced plants. A\u003c/strong\u003eVenn diagram of DEGs in \u003cem\u003eZmWRKY36\u003c/em\u003e silenced plants. \u003cstrong\u003eB\u003c/strong\u003e Volcano plot analysis of DEGs in \u003cem\u003eZmWRKY36\u003c/em\u003e silenced plants (p value ≤0.05, |log2 fold change| ≥ 1). \u003cstrong\u003eC\u003c/strong\u003e GO enriched pathways of downregulated DEGs in \u003cem\u003eZmWRKY36\u003c/em\u003e silenced plants. \u003cstrong\u003eD\u003c/strong\u003eGO enriched pathways of upregulated DEGs in \u003cem\u003eZmWRKY36\u003c/em\u003e silenced plants. \u003cstrong\u003eE\u003c/strong\u003eKEGG enriched pathways of upregulated DEGs in \u003cem\u003eZmWRKY36\u003c/em\u003e silenced plants. \u003cstrong\u003eF\u003c/strong\u003eKEGG enriched pathways of downregulated DEGs in \u003cem\u003eZmWRKY36\u003c/em\u003e silenced plants.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7738242/v1/d5fcdd9f58ba73d33de25914.png"},{"id":94405022,"identity":"b28958e9-ded2-47c1-8b8b-57c70b3121c5","added_by":"auto","created_at":"2025-10-27 14:01:30","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1309581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZmWRKY36\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e overexpressed plants. A\u003c/strong\u003eVenn diagram of DEGs in \u003cem\u003eZmWRKY36\u003c/em\u003e overexpressed plants. \u003cstrong\u003eB\u003c/strong\u003e Volcano plot analysis of DEGs in \u003cem\u003eZmWRKY36\u003c/em\u003e overexpressed plants (p value ≤0.05, |log2 fold change| ≥ 1). \u003cstrong\u003eC\u003c/strong\u003e GO enriched pathways of downregulated DEGs in \u003cem\u003eZmWRKY36\u003c/em\u003e overexpressed plants. \u003cstrong\u003eD\u003c/strong\u003e GO enriched pathways of upregulated DEGs in \u003cem\u003eZmWRKY36\u003c/em\u003eoverexpressed plants. \u003cstrong\u003eE\u003c/strong\u003e KEGG enriched pathways of upregulated DEGs in \u003cem\u003eZmWRKY36\u003c/em\u003eoverexpressed plants. \u003cstrong\u003eF\u003c/strong\u003e KEGG enriched pathways of downregulated DEGs in \u003cem\u003eZmWRKY36\u003c/em\u003eoverexpressed plants.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7738242/v1/506b2909bd2094d3d69faca5.png"},{"id":94489329,"identity":"3c736d8a-1481-463b-a40c-6705f73cebd0","added_by":"auto","created_at":"2025-10-27 17:04:12","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1092773,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome analysis of DEGs in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZmWRKY36\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e silenced and overexpressed plants. A\u003c/strong\u003eThe number of DEGs in \u003cem\u003eZmWRKY36\u003c/em\u003e silenced and overexpressed plants. \u003cstrong\u003eB \u003c/strong\u003eThe number of DEGs with opposite expression trends in \u003cem\u003eZmWRKY36\u003c/em\u003e silenced and overexpressed plants. The expression was upregulated in the overexpressed group, while downregulated in the silenced group. The expression was downregulated in the silenced group, while upregulated in the overexpressed group. \u003cstrong\u003eC\u003c/strong\u003e Statistics of promoter cis-acting elements of 14 DEGs.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7738242/v1/feb6df6252bea5469c7cd4d2.png"},{"id":94405119,"identity":"ed65044b-b218-4d18-8605-6450e0209a2f","added_by":"auto","created_at":"2025-10-27 14:01:36","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":380373,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRT-qPCR confirmed the results of the transcriptome analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZmWRKY36\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e silenced and overexpressed plants. \u003c/strong\u003eRelative expression of four randomly selected DEGs in \u003cem\u003eZmWRKY36\u003c/em\u003e silenced and overexpressed transcriptomes via RT-qPCR. \u003cstrong\u003eA\u003c/strong\u003eVerification of the upregulated and downregulated expression genes in the silented material. \u003cstrong\u003eB\u003c/strong\u003e Verification of the upregulated and downregulated expression genes in the overexpressed materials. Data contain mean ± standard error of three replicates. (* p ≤ 0.05; ** p ≤0.01; *** p ≤ 0.001).\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-7738242/v1/ec3716419125de48f6326740.png"},{"id":94404789,"identity":"df324a3b-3e1b-491e-8ccf-662163c47dae","added_by":"auto","created_at":"2025-10-27 14:01:20","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":912140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe hypothetical functional model of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZmWRKY36\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in maize under \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. maydis \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003einfection. \u003c/strong\u003eUnder the infection of \u003cem\u003eB. maydis\u003c/em\u003e, \u003cem\u003eZmWRKY36 \u003c/em\u003emay bind to the cis-acting elements in the promoter of the target gene. Accelerate the clearance of reactive oxygen species, regulate the disease resistance genes or the photosignal transduction pathway. Then enhances maize resistance to the southern corn leaf blight.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-7738242/v1/8c515fb6f917106dcebb06a3.png"},{"id":100069111,"identity":"4d0a3635-f44e-4390-af80-9edd047c2e84","added_by":"auto","created_at":"2026-01-12 16:09:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15863370,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7738242/v1/5231e1aa-ccfa-4789-b21a-4b26eb6b92d3.pdf"},{"id":94405153,"identity":"af06ffe1-5b7c-475a-8fe9-08a7c4a8165c","added_by":"auto","created_at":"2025-10-27 14:01:40","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16879,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7738242/v1/13f0273ccb5fe34e09e981d8.docx"},{"id":94404346,"identity":"6b320c77-bc72-40e0-a961-da5864acf7f8","added_by":"auto","created_at":"2025-10-27 14:01:00","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18700,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7738242/v1/8f807a594dc6f6ec8284bf63.docx"},{"id":94404657,"identity":"368422bf-ebf0-4298-8ec5-7f52b32741ae","added_by":"auto","created_at":"2025-10-27 14:01:10","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16616,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7738242/v1/df0161c1532ec207721056cf.docx"},{"id":94404836,"identity":"2e449b00-d4fd-4679-baae-f2924160bd35","added_by":"auto","created_at":"2025-10-27 14:01:22","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":251282,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7738242/v1/ff83072f20a23b5065fdd093.png"},{"id":94405174,"identity":"c2304ec0-bccb-4f31-b460-1d72a8dd71d0","added_by":"auto","created_at":"2025-10-27 14:01:41","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":188723,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7738242/v1/aba0a8e63067651792b10783.png"},{"id":94404543,"identity":"2261125f-9d63-4178-9372-f7a0dfea1f23","added_by":"auto","created_at":"2025-10-27 14:01:08","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":995641,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7738242/v1/0500a782028c746188ca83a0.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular mechanism of ZmWRKY36 mediated maize resistance to Bipolaris maydis","fulltext":[{"header":"Background","content":"\u003cp\u003eMaize is a globally significant crop serving as food, feed, and industrial raw materials, and its yield and quality directly impact global food security and agricultural economic growth [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Southern corn leaf blight, caused by the pathogen \u003cem\u003eBipolaris maydis\u003c/em\u003e (\u003cem\u003eB. maydis\u003c/em\u003e), is a devastating fungal disease that frequently outbreaks in major maize-producing regions worldwide, posing a persistent and severe threat to the maize industry [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. For a long time, chemical control has been able to effectively curb the spread of diseases to a certain extent. However, this approach is associated with multiple drawbacks, including environmental pollution, pesticide residue, and increased pathogen resistance. On the other hand, maize resistance to this pathogen is primarily governed by QTLs and is highly susceptible to environmental fluctuations [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Cultivating and popularizing disease-resistant varieties thus represent the most economical, safe, and sustainable strategy for managing this disease [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, further exploring the key genes in maize that regulate resistance to the southern corn leaf blight and dissecting their underlying molecular mechanism of disease resistance remains critical challenges that need to be addressed in the current plant pathology and crop breeding research.\u003c/p\u003e\u003cp\u003eDuring the long-term co-evolution between plants and pathogens, a complex and precise immune regulatory network has been established [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Among its components, transcription factors (TFs), as a key regulator of gene expression, play a core role in activating disease resistance signaling pathways and initiating the defense response [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. WRKY TFs constitute one of the largest families of transcriptional regulators in plants [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Their most distinctive structural feature is the highly conserved WRKY domain, which consists of approximately 60 amino acid residues [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Specifically, their N-terminus contains the characteristic conserved heptapeptide sequence WRKYGQK, while the C-terminus forms a zinc finger structure (C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e) through the coordination of cysteine (Cys) and histidine (His) residues. This structure enables specific binding to the (T)TGAC(C/T) (W-box) cis-acting element in the promoter region of downstream target gene, thereby regulating the expression of the target gene [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. WRKY TFs are classified into three major groups based on the number of WRKY domains and the type of zinc finger structure. Group Ⅰ members contain two WRKY domains and C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e type zinc finger structures. Group Ⅱ members possess only one WRKY domain and a C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e type zinc finger structure, and are further subdivided into five subgroup (Ⅱa to Ⅱe) based on sequence differences. Group Ⅲ members feature a single WRKY domain and a unique C\u003csub\u003e2\u003c/sub\u003eHC type zinc finger structure [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This structural diversity lays the foundation for functional specialization, allowing WRKY TFs to play specific regulatory roles in distinct biotic stress response pathways.\u003c/p\u003e\u003cp\u003eThe WRKY TF gene family has attracted considerable attention due to its extensive involvement in plant responses to both biotic and abiotic stresses [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Numerous studies have confirmed that WRKY TFs typically form a complex regulatory network by participating in signaling pathways mediated by Salicylic acid (SA), Jasmonic acid (JA), and Ethylene (ET) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In plant disease resistance responses, WRKY members enhance resistance to diverse pathogens such as fungi, bacteria and viruses by activating the expression of pathogenesis-related (\u003cem\u003ePR\u003c/em\u003e) genes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], regulating genes involved in secondary metabolites synthesis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] or modulating immune receptor genes, thereby improving the basic immune level of plants. For instance, \u003cem\u003eOsWRKY36\u003c/em\u003e regulates the expression of the phenylalanine ammonia-lyase gene, thereby influencing the synthesis of lignin and the thickness of the parenchyma tissue, and consequently affecting the disease resistance of rice [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Under drought stress, leaves of \u003cem\u003eMaWRKY80\u003c/em\u003e overexpressing transgenic Arabidopsis exhibit lower reactive oxygen species (ROS) levels [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. \u003cem\u003eAtWRKY33\u003c/em\u003e enhances resistance to \u003cem\u003eBotrytis cinerea\u003c/em\u003e by regulating the JA signaling pathway [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. \u003cem\u003eOsWRKY45\u003c/em\u003e improves its resistance to rice bacterial leaf blight by participating in the SA signaling process [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These findings have provided significant insights into the role of WRKY TFs in plant disease resistance response.\u003c/p\u003e\u003cp\u003eTo date, 120 WRKY TFs encoding genes distributed across 10 chromosomes have been identified in maize [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Although significant progress has been made in WRKY TFs reseach, the specific functions and regulatory network mechanisms of these factors in the southern corn leaf blight resistance remain unclear. In-depth exploration of WRKY TFs mechanism in maize not only helps to reveal the molecular basis of maize responses to biotic and abiotic stresses, but also provides important theoretical support and candidate gene resources for improving maize disease resistance, yield and quality via genetic engineering.\u003c/p\u003e\u003cp\u003eIn this study, we successfully identified a nuclear-localized transcription factor in maize, designated \u003cem\u003eZmWRKY36\u003c/em\u003e. Functional analyses indicated that \u003cem\u003eZmWRKY36\u003c/em\u003e positively regulates maize resistance to \u003cem\u003eB. maydis\u003c/em\u003e and possesses transcriptional activation activity. These characteristics highlight its potentially value for research on the southern corn leaf blight resistance regulation. As a transcriptional activator, \u003cem\u003eZmWRKY36\u003c/em\u003e may regulate the expression of a suite of downstream disease resistant-related genes, thereby constructing a multi-level disease defense system. Further studies are needed to explore the specific regulatory network between \u003cem\u003eZmWRKY36\u003c/em\u003e and its target genes, its interactions with other signaling pathways, and the resistance effects in different genetic backgrounds.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eBioinformatics analysis of\u003c/b\u003e \u003cb\u003eZmWRKY36\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eZmWRKY36\u003c/em\u003e (GRMZM2G054125) is a maize gene with 690 bp CDS, encoding a 229 amino acids protein, belonging to the WRKY TF gene family. \u003cem\u003eZmWRKY36\u003c/em\u003e contains the canonical WRKYGQK core motif and C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e type zinc finger structure, consistent with the structural characteristics of group Ⅱ members in the WRKY TF family (Fig.\u0026nbsp;1A, Supplementary Fig.\u0026nbsp;1). This domain mainly undertakes specific binding to downstream target genes and directing the regulation of their transcriptional expression. It also participates in the construction of complex regulatory networks, playing a critical role in signal integration and precise modulation of plant physiological processes. Through multiple sequence alignments with the reported WRKY proteins from 6 plant species, we further confirmed that the WRKY domain of \u003cem\u003eZmWRKY36\u003c/em\u003e is highly evolutionarily conserved [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In particular, key amino acids in the WRKYGQK sequence and the zinc finger structure are invariant, suggesting that it may exert transcriptional regulatory functions by recognizing the W-box cis-acting element.\u003c/p\u003e\u003cp\u003eWithin the WRKY group Ⅱ subfamily, further subdivision into five groups, named Ⅱa, Ⅱb, Ⅱc, Ⅱd, and Ⅱe, is based on gene structure, amino acid sequence similarity, and phylogenetic relationships. The WRKYGQK motif of the \u003cem\u003eZmWRKY36\u003c/em\u003e is invariant, and the protein contains potential pathogen response-related features, specifically, antioxidant-related cysteine residues, consistent with the conserved traits of group Ⅱe. To further determine the functional characteristics of \u003cem\u003eZmWRKY36\u003c/em\u003e and its Ⅱe subfamily, we performed domain analysis and motif prediction of \u003cem\u003eZmWRKY36\u003c/em\u003e and 16 previously reported Ⅱe subgroup genes (Fig.\u0026nbsp;1B). NCBI domains prediction showed that all WRKY Ⅱe members harbor a canonical WRKY domains. A total of 10 conserved motifs were identified by using MEME for motif prediction, named motif 1 to motif 10, and their lengths ranged from 6 to 50 amino acids. Among them, motif 1 and motif 2 constitute the WRKY domain, which is consistent with the results of the above domain analysis. These results further confirmed the structural conservatism of WRKY Ⅱe subgroup members.\u003c/p\u003e\u003cp\u003eTo explore the evolutionary origin of \u003cem\u003eZmWRKY36\u003c/em\u003e and clarify its potential homologs, we performed a BLAST analysis of ZmWRKY36 amino acid sequence against the NCBI (National Center for Biotechnology Information) database. The results showed that 5 genes were identified homology to \u003cem\u003eZmWRKY36\u003c/em\u003e in maize. To further analyze its phylogenetic position, we selected representative members of the WRKY family from 6 distinct plant species and constructed a phylogenetic tree. The phylogenetic tree results indicated that \u003cem\u003eZmWRKY36\u003c/em\u003e was closest in evolutionary distance to \u003cem\u003eOsWRKY56\u003c/em\u003e, with amino acid sequence homology as high as 95%. Additionally, \u003cem\u003eZmWRKY36\u003c/em\u003e and \u003cem\u003eAtWRKY75\u003c/em\u003e, a gene well-characterized for its role in disease resistance regulation in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, cluster in the same evolutionary branch (Fig.\u0026nbsp;1C). This evolutionary feature suggested that \u003cem\u003eZmWRKY36\u003c/em\u003e may exert similar biological functions in maize disease resistance signaling pathway to those homologous proteins, providing a theoretical basis for its role in plant immune responses.\u003c/p\u003e\u003cp\u003e\u003cb\u003eZmWRKY36\u003c/b\u003e \u003cb\u003epositively regulates maize resistance to\u003c/b\u003e \u003cb\u003eB. maydis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs the WRKY domain mediates responses to biotic stresses, we examined whether \u003cem\u003eZmWRKY36\u003c/em\u003e is involved in maize defense against \u003cem\u003eB. maydis\u003c/em\u003e. Quantitative real-time PCR (RT-qPCR) was used to detect \u003cem\u003eZmWRKY36\u003c/em\u003e expression levels at six time points post \u003cem\u003eB. maydis\u003c/em\u003e infection: 12, 24, 48, 72, 96, and 120 hours post infection (hpi). Results showed that \u003cem\u003eZmWRKY36\u003c/em\u003e was significantly upregulated following \u003cem\u003eB. maydis\u003c/em\u003e infection, with its expression peaking at 12 hpi (Fig.\u0026nbsp;2A). This indicated that \u003cem\u003eZmWRKY36\u003c/em\u003e positively responses to \u003cem\u003eB. maydis\u003c/em\u003e in maize.\u003c/p\u003e\u003cp\u003eTo determine whether \u003cem\u003eZmWRKY36\u003c/em\u003e is involved in the maize resistance to \u003cem\u003eB. maydis\u003c/em\u003e, we generated transient \u003cem\u003eZmWRKY36\u003c/em\u003e-silenced and -overexpressed maize plants used Foxtail mosaic virus (FoMV) mediated virus-induced gene silencing (VIGS) and virus-mediated gene overexpressed (VOX) system respectively. After infiltrating maize leaves with Agrobacterium suspension containing the VIGS and VOX constructs, we quantified \u003cem\u003eZmWRKY36\u003c/em\u003e expression. The relative expression levels of \u003cem\u003eZmWRKY36\u003c/em\u003e in FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e silenced plants were consistently below 0.5, significantly lower than that in the FoMV:V control (Fig.\u0026nbsp;2B). On the contrary, \u003cem\u003eZmWRKY36\u003c/em\u003e expression in FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX overexpressed plants were significantly upregulated, more than 100-fold higher than that in the FoMV:\u003cem\u003eGFP\u003c/em\u003e-VOX control (Fig.\u0026nbsp;2C).\u003c/p\u003e\u003cp\u003eFor disease resistance phenotyping, we first validated \u003cem\u003eZmWRKY36\u003c/em\u003e expression in silenced and overexpressed plants following rubbing inoculation on maize leaves. Compared with FoMV:V plants, \u003cem\u003eZmWRKY36\u003c/em\u003e transcription was significantly reduced in FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e plants. In contrast, \u003cem\u003eZmWRKY36\u003c/em\u003e was markedly upregulated in FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX compared to the control FoMV:\u003cem\u003eGFP\u003c/em\u003e-VOX (Fig.\u0026nbsp;2A, B). Subsequently, we performed in vitro \u003cem\u003eB. maydis\u003c/em\u003e inoculation on these plants. FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-silenced plants exhibited significantly larger lesion areas and higher \u003cem\u003eB. maydis\u003c/em\u003e fungal biomass than FoMV:V, indicating that the silencing of \u003cem\u003eZmWRKY36\u003c/em\u003e increases the susceptibility to \u003cem\u003eB. maydis\u003c/em\u003e (Fig.\u0026nbsp;2D, E, F). Conversely, FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX- overexpressed plants showed significantly smaller lesions and fungal biomass than FoMV:\u003cem\u003eGFP\u003c/em\u003e-VOX, indicating that the overexpression of \u003cem\u003eZmWRKY36\u003c/em\u003e enhances maize resistance to \u003cem\u003eB. maydis\u003c/em\u003e (Fig.\u0026nbsp;2G, H, I). These results preliminarily suggested that \u003cem\u003eZmWRKY36\u003c/em\u003e may positively regulate maize resistance to \u003cem\u003eB. maydis\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eZmWRKY36\u003c/b\u003e \u003cb\u003eaffects the release of reactive oxygen species in maize\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further dissect the functional role of \u003cem\u003eZmWRKY36\u003c/em\u003e in modulating maize immune responses, we conducted a detailed analysis of chitin-induced reactive oxygen species (ROS) burst dynamics, a hallmark event in plant pattern-triggered immunity (PTI), using FoMV:V, FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e, FoMV:\u003cem\u003eGFP\u003c/em\u003e-VOX, and FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX plants. FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e showed significantly weaker ROS bursts compared to FoMV:V, while FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX showed significantly stronger ROS bursts than FoMV:\u003cem\u003eGFP\u003c/em\u003e-VOX (Fig.\u0026nbsp;2J, K). Additionally, the ROS burst peaked at 5 min post chitin treatment in FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e, compared to 2.5 min in FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX plants. Collectively, these findings not only confirm that \u003cem\u003eZmWRKY36\u003c/em\u003e functions as a positive regulator of maize resistance to \u003cem\u003eB. maydis\u003c/em\u003e, but also provide novel insights into its mechanistic role in the maize immune defense response. Such regulation of ROS homeostasis is likely a key component of \u003cem\u003eZmWRKY36\u003c/em\u003e-mediated immune signaling, highlighting its potential as a target for molecular breeding strategies aimed at enhancing maize disease resistance.\u003c/p\u003e\u003cp\u003e\u003cb\u003eZmWRKY36\u003c/b\u003e \u003cb\u003eregulates the expression levels of pathogenesis-related gene\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further explore the role of \u003cem\u003eZmWRKY36\u003c/em\u003e in maize defense mechanism against \u003cem\u003eB. maydis\u003c/em\u003e, we focused on the expression of disease-related genes (\u003cem\u003ePR\u003c/em\u003e) in maize. The selected \u003cem\u003ePR\u003c/em\u003e genes included \u003cem\u003eZmPR1\u003c/em\u003e, \u003cem\u003eZmPR3\u003c/em\u003e, \u003cem\u003eZmPR4\u003c/em\u003e, \u003cem\u003eZmPR5\u003c/em\u003e, and \u003cem\u003eZmPR10\u003c/em\u003e, all of which are involved in the defense response process of plants. The relevant analyses were conducted in maize plants of \u003cem\u003eZmWRKY36\u003c/em\u003e silenced and overexpressed. In the \u003cem\u003eZmWRKY36\u003c/em\u003e silencing group, the expression levels of \u003cem\u003eZmPR1\u003c/em\u003e, \u003cem\u003eZmPR3\u003c/em\u003e, \u003cem\u003eZmPR4\u003c/em\u003e, \u003cem\u003eZmPR5\u003c/em\u003e, and \u003cem\u003eZmPR10\u003c/em\u003e in \u003cem\u003eZmWRKY36\u003c/em\u003e-silenced (FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e) plants were significantly downregulated compared to the FoMV:V plants (Fig.\u0026nbsp;3A). This indicated that the absence of \u003cem\u003eZmWRKY36\u003c/em\u003e led to reduced expression of these key \u003cem\u003ePR\u003c/em\u003e genes. In the overexpression group, comparison between FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX plants and the FoMV:\u003cem\u003eGFP\u003c/em\u003e-VOX plants showed significant upregulation of the same five \u003cem\u003ePR\u003c/em\u003e genes (Fig.\u0026nbsp;3B). Subsequently, we analyzed the expression levels of these \u003cem\u003ePR\u003c/em\u003e genes following \u003cem\u003eB. maydis\u003c/em\u003e infection. Data revealed that in the FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e plants, their expression were significantly lower compared to the FoMV:V plants, conversely, in the FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX plants, their expression were significantly higher compared with the FoMV:\u003cem\u003eGFP\u003c/em\u003e-VOX plants (Fig.\u0026nbsp;3C, D). This pattern was consistent with our previous phenotypic observations.\u003c/p\u003e\u003cp\u003eIn conclusion, these results indicated that \u003cem\u003eZmWRKY36\u003c/em\u003e regulates the expression of \u003cem\u003ePR\u003c/em\u003e genes. The positive correlation between \u003cem\u003eZmWRKY36\u003c/em\u003e transcriptional level and the expression of these \u003cem\u003ePR\u003c/em\u003e genes further validates the positive responsiveness of \u003cem\u003eZmWRKY36\u003c/em\u003e to \u003cem\u003eB. maydis\u003c/em\u003e infection. This response not only affected the expression of \u003cem\u003eZmWRKY36\u003c/em\u003e, but also exerted a cascading effect on the expression of other \u003cem\u003ePR\u003c/em\u003e genes during maize defense against \u003cem\u003eB. maydis\u003c/em\u003e. These findings provide an important foundation for a deeper understanding of maize\u0026rsquo;s complex defense mechanism.\u003c/p\u003e\u003cp\u003e\u003cb\u003eZmWRKY36\u003c/b\u003e \u003cb\u003eis a nuclear localized protein\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo explore the biological function of \u003cem\u003eZmWRKY36\u003c/em\u003e and determine its subcellular localization, we constructed a \u003cem\u003eZmWRKY36\u003c/em\u003e-Green Fluorescent Protein (GFP) fusion protein. Agrobacterium-mediated transient transformation was used to heterologously express this fusion protein (with the free GFP as a control) in \u003cem\u003eN. benthamiana.\u003c/em\u003e Subcellular localization was observed using a laser confocal microscope (Zeiss Confocal LSM 980). In the control group, GFP signal was detected in both the cytoplasm and nucleus. In contrast, the GFP-\u003cem\u003eZmWRKY36\u003c/em\u003e fusion protein showed specific enrichment, with its fluorescence signal colocalizing completely with the nuclear red fluorescence marker (Fig.\u0026nbsp;4A). This indicated that \u003cem\u003eZmWRKY36\u003c/em\u003e localizes to the nucleus in \u003cem\u003eN. benthamiana.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo further verify the subcellular localization characteristics of \u003cem\u003eZmWRKY36\u003c/em\u003e in a homologous system, we transformed the GFP-\u003cem\u003eZmWRKY36\u003c/em\u003e fusion protein and free GFP into maize protoplasts. Confocal microscopy revealed that while GFP was distributed in both the cytoplasm and nucleus, GFP-\u003cem\u003eZmWRKY36\u003c/em\u003e fusion protein was specifically localized to the nucleus, consistent with the observation from \u003cem\u003eN. benthamiana\u003c/em\u003e transient expression system (Fig.\u0026nbsp;4B).\u003c/p\u003e\u003cp\u003eFurthermore, to confirm the expression status of \u003cem\u003eZmWRKY36\u003c/em\u003e, we performed Western blot analysis to detect protein expression in the aforementioned \u003cem\u003eN. benthamiana\u003c/em\u003e and maize protoplasts. The results showed that the obtained bands were consistent with the expected size of the target gene (Fig.\u0026nbsp;4C, D). Collectively, these results confirmed the nuclear localization of \u003cem\u003eZmWRKY36\u003c/em\u003e, providing a critical spatial basis for its role in regulating downstream gene expression.\u003c/p\u003e\u003cp\u003e\u003cb\u003eZmWRKY36\u003c/b\u003e \u003cb\u003eexhibits transcriptional activation activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven that WRKY TFs typically possess transcriptional activity, we assayed the transcriptional activity of \u003cem\u003eZmWRKY36\u003c/em\u003e in both maize protoplasts and yeast systems. In maize protoplasts, we constructed a GAL4-\u003cem\u003eZmWRKY36\u003c/em\u003e fusion protein and co-transfected it with a luciferase reporter vector containing the GAL4-TATA transcriptional regulatory element. GAL4-VP16 (positive control) and GAL4 (negative control) were included for comparison. Transcriptional activity was evaluated by the ratio of Firefly Luciferase (FLuc) to Reniferase Luciferase (RLuc) signal. The LUC/REN ratio in GAL4-\u003cem\u003eZmWRKY36\u003c/em\u003e and reporter group was significantly higher than that in the GAL4 negative control group, indicating that \u003cem\u003eZmWRKY36\u003c/em\u003e has transcriptional activation activity (Fig.\u0026nbsp;5A, B). In the yeast system, similar as the positive control pGBKT7-VP16, pGBKT7-\u003cem\u003eZmWRKY36\u003c/em\u003e grew on SD/-Trp-His solid medium and formed blue colonies on the medium supplemented with X-α-gal. In contrast, the negative control failed to grow or produce blue color. This indicated that \u003cem\u003eZmWRKY36\u003c/em\u003e can independently activate the transcription of the downstream reporter gene (Fig.\u0026nbsp;5C).\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscriptome analysis of\u003c/b\u003e \u003cb\u003eZmWRKY36\u003c/b\u003e \u003cb\u003etransient silenced and overexpressed materials\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRevealing the regulatory pathway of \u003cem\u003eZmWRKY36\u003c/em\u003e in maize resistance to \u003cem\u003eB. maydis\u003c/em\u003e is of crucial significance for understanding the complex interaction mechanism between plants and pathogens. To explore this regulatory pathway, we conducted transcriptome analysis on the silenced material (FoMV:V vs FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e) and overexpressed material (FoMV:\u003cem\u003eGFP\u003c/em\u003e-VOX vs FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX). All the plants showed high silencing and overexpression efficiencies (Supplementary Fig.\u0026nbsp;2A, B). To ensure the accuracy and reliability of the experimental results, three biological replicates were set for each group of materials in the transcriptome.\u003c/p\u003e\u003cp\u003eResults from transcriptome analysis indicated that the majority of genes exhibited stable expression across different treated materials. In the silenced plants, a total of 1074 genes were commonly expressed under both conditions. Specifically, 1118 genes were detected in FoMV:V, while 1103 genes were identified in FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e (Fig.\u0026nbsp;6A). Based on this, we conducted statistical analysis on the DEGs between the groups. In the data from \u003cem\u003eZmWRKY36\u003c/em\u003e-silenced materials, a total of 1127 DEGs were identified, including 601 upregulated genes and 526 downregulated genes (Fig.\u0026nbsp;6B). To gain deeper insights into the functional roles of these DEGs, we performed Gene Ontology (GO) functional enrichment analysis, classifying them into biological processes (BP), cellular components (CC), and molecular functions (MF). In the silenced material group, DEGs were significantly enriched in biological pathways such as plant carbohydrate metabolism, particularly in cell wall polysaccharide synthesis, cell structural composition, as well as related molecular binding and catalytic functions (Fig.\u0026nbsp;6C, D). Furthermore, to reveal the key metabolic pathways and signal transduction pathways involved in \u003cem\u003eZmWRKY36\u003c/em\u003e expression, we conducted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on \u003cem\u003eZmWRKY36\u003c/em\u003e silenced and overexpressed plants. The results showed that DEGs were significantly enriched in multiple pathways. In \u003cem\u003eZmWRKY36\u003c/em\u003e silenced plants, upregulated genes were enriched in the plant-pathogen interaction pathway, while downregulated genes mainly reflected the redistribution of plant metabolic resources (Fig.\u0026nbsp;6E, F). These results suggested that \u003cem\u003eZmWRKY36\u003c/em\u003e may play an important regulatory role in maize resistance to \u003cem\u003eB. maydis\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eIn the overexpressed plants, 9591 genes were detected in FoMV:\u003cem\u003eGFP\u003c/em\u003e-VOX, and 9448 genes were detected in FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX. A total of 9192 genes were commonly expressed under both conditions (Fig.\u0026nbsp;7A). Among them, 9847 DEGs were identified, including 4873 upregulated genes and 4974 downregulated genes (Fig.\u0026nbsp;7B). In the overexpressed group, GO functional enrichment analysis showed that the DEGs were mainly enriched in the structural aspects of photosynthetic membrane systems, ion binding and transport, as well as enzyme catalysis (Fig.\u0026nbsp;7C, D). These results indicated that \u003cem\u003eZmWRKY36\u003c/em\u003e may be involved in the regulation of physiological processes such as plant growth and development, disease resistance defense, and photosynthesis, which further highlights the core role of \u003cem\u003eZmWRKY36\u003c/em\u003e in the regulatory network of maize resistance to \u003cem\u003eB. maydis\u003c/em\u003e. Additionally, KEGG pathway enrichment results showed that the upregulated genes in overexpressed plants were significantly enriched in photosynthesis and carbon fixation, while the downregulated pathways involved protein processing, signal transduction, and other metabolism or structure related pathways (Fig.\u0026nbsp;7E, F). These results indicated that \u003cem\u003eZmWRKY36\u003c/em\u003e may participate in signal transduction processes through regulating metabolism or by synthesize lipid derivatives with defensive functions to enhance resistance.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIdentification of downstream target genes regulated by\u003c/b\u003e \u003cb\u003eZmWRKY36\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThrough intersection analysis of two groups of DEGs, we identified a total of 206 genes that had opposite expression trends under the two treatments. Specifically, 84 genes were upregulated in the overexpressed group while being downregulated in the silenced group, and 122 genes showed the reverse trend (Fig.\u0026nbsp;8A, B). These genes are likely target genes directly or indirectly regulated by \u003cem\u003eZmWRKY36\u003c/em\u003e. Notably, as a member of the WRKY TFs family, the transcriptional regulatory function of \u003cem\u003eZmWRKY36\u003c/em\u003e lies in its specific binding to the conserved cis-acting elements in the promoter region of target genes. Its interaction with the W-box domain constitutes a key molecular mechanism for regulating downstream target genes to achieve physiological functions. Therefore, we used Tbtools software to extract the 2000 bp upstream promoter sequences of the 206 DEGs with opposite expression trends, the cis-acting elements within these promoter sequences were computationally predicted utilizing PlantCARE. The results showed that the promoter regions of 105 genes contained W-box sequences (Supplementary Fig.\u0026nbsp;3). We performed cluster heatmap analysis on the cis-acting elements in these DEGs and selected 14 genes containing multiple W-box elements, which were classified them by abiotic stress, biotic stress, plant hormone response, development-related, and light response (Fig.\u0026nbsp;8C). Among them, \u003cem\u003eZm00001d005783\u003c/em\u003e and \u003cem\u003eZm00001d039384\u003c/em\u003e possessed more biotic stress-related elements. This series of findings demonstrated that these DEGs carrying W-box elements are highly likely to be downstream target genes directly regulated by \u003cem\u003eZmWRKY36\u003c/em\u003e. \u003cem\u003eZmWRKY36\u003c/em\u003e precisely regulates the transcriptional expression levels of these genes and plays an important regulatory role in the resistance response of maize to \u003cem\u003eB. maydis\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eVerification of\u003c/b\u003e \u003cb\u003eZmWRKY36\u003c/b\u003e \u003cb\u003etranscriptome data\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe expression patterns of four candidate target genes using RT-qPCR. These genes exhibited opposite expression trends in the \u003cem\u003eZmWRKY36\u003c/em\u003e silenced and overexpressed materials. The results showed that their expression trends were consistent with the transcriptome sequencing data (R\u0026sup2; \u0026gt;0.9), confirming the reliability of the transcriptome sequencing results (Fig.\u0026nbsp;9A, B).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the long-term co-evolution between plants and pathogens, TFs serve as the core regulatory hubs of gene expression, and play pivotal roles in disease resistance signaling pathways [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In this study, we identified \u003cem\u003eZmWRKY36\u003c/em\u003e as a nuclear localized TF in maize and used VIGS and VOX systems to demonstrate its crucial role in resistance to \u003cem\u003eB. maydis\u003c/em\u003e. These finding provided key insights into the molecular mechanism underlying maize disease resistance. Transient silencing of \u003cem\u003eZmWRKY36\u003c/em\u003e significantly increased maize susceptibility to \u003cem\u003eB. maydis\u003c/em\u003e, and inhibited chitin-induced ROS bursts. Conversely, transient overexpressed of \u003cem\u003eZmWRKY36\u003c/em\u003e effectively enhanced the maize's resistance and promote ROS accumulation. These phenotypic differences directly indicated that \u003cem\u003eZmWRKY36\u003c/em\u003e plays an active role in the early signaling pathways that activate the plant immune response. Importantly, \u003cem\u003eZmWRKY36\u003c/em\u003e, as a key regulator in maize, not only responds significantly to the invasion of \u003cem\u003eB. maydis\u003c/em\u003e, but also enhances the resistance level of maize through a positive regulatory mechanism. The core evidence for this discovery came from the expression pattern of the \u003cem\u003ePR\u003c/em\u003e gene: in the \u003cem\u003eZmWRKY36\u003c/em\u003e silenced plants, the transcriptional level of the \u003cem\u003ePR\u003c/em\u003e gene was significantly downregulated, but upregulated in the overexpressed plants. Further correlation analysis revealed that the transcriptional level of \u003cem\u003eZmWRKY36\u003c/em\u003e was highly positively correlated with the expression level of \u003cem\u003ePR\u003c/em\u003e genes. This confirmed the positive response characteristic of \u003cem\u003eZmWRKY36\u003c/em\u003e to \u003cem\u003eB. maydis\u003c/em\u003e infection, but also revealed that \u003cem\u003eZmWRKY36\u003c/em\u003e might affect the expression of multiple key genes in maize defense network through a cascade regulatory effect, providing a new molecular perspective for understanding the complex disease resistance mechanism of maize [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, the specific regulatory network and molecular mechanism by which \u003cem\u003eZmWRKY36\u003c/em\u003e confers \u003cem\u003eB. maydis\u003c/em\u003e resistance require further exploration.\u003c/p\u003e\u003cp\u003eNotably, \u003cem\u003eZmWRKY36\u003c/em\u003e\u0026rsquo;s transcriptional activation activity open new avenues for understanding its disease resistance mechanism. Within the molecular regulatory network of plant disease resistance responses, the activation state of TFs constitutes a crucial step in initiating downstream defense signal transduction [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The transcriptional activation activity of \u003cem\u003eZmWRKY36\u003c/em\u003e further endows it with the potential to regulate the downstream target gene network [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. \u003cem\u003eZmWRKY36\u003c/em\u003e is capable of specifically binding to target genes, thereby initiating or enhancing the expression of a series of downstream genes. This process may involve the coordinated expression of multiple functional modules, such as the expression of plant pathogenesis-related protein genes, hormone signaling pathway-related [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and driving an extracellular ROS burst [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], consequently forming a complex gene expression regulatory network and providing important molecular support for plants to establish an effective disease resistance defense system. Therefore, in-depth research on the transcriptional activation activity of \u003cem\u003eZmWRKY36\u003c/em\u003e is expected to provide new insights and experimental foundations for clarifying the fine regulatory mechanism of plant disease resistance responses. Collectively, this result delineated \u003cem\u003eZmWRKY36\u003c/em\u003e\u0026rsquo;s role as an anti-pathogen regulatory node: it initiate the cascade amplification of the immune signal, and enhances maize defense by directly regulating expression of genes.\u003c/p\u003e\u003cp\u003eTranscriptome analysis of \u003cem\u003eZmWRKY36\u003c/em\u003e further revealed a complex metabolic regulatory network. GO enrichment analysis of DEG in \u003cem\u003eZmWRKY36\u003c/em\u003e silenced and overexpressed plants showed significant enrichment in pathways related to photosynthesis, which are associated with the composition of cell wall polysaccharides and the structure of photosynthetic membranes. This suggested that \u003cem\u003eZmWRKY36\u003c/em\u003e may participate in the disease resistance process through multiple pathways. On one hand, strengthening physical barriers by increasing cell wall thickness and remodeling cell structure [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]; on the other hand, regulating immune responses via energy supply or synergism with phytohormones and other metabolites [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This \u0026ldquo;photosynthesis-disease resistance\u0026rdquo; cross-correlation provides a new perspective for exploring the \u0026ldquo;growth and defense\u0026rdquo; balance in plants [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and implied that \u003cem\u003eZmWRKY36\u003c/em\u003e may coordinating energy metabolism and defense responses to enable maize adaptation to pathogen stress [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. KEGG pathway analysis further supported this functional speculation. After silencing of \u003cem\u003eZmWRKY36\u003c/em\u003e, DEGs were significantly enriched in the plant-pathogen interaction pathway, suggesting that silencing relieves inhibition of the basic defense pathway and triggers stress responses. After overexpression of \u003cem\u003eZmWRKY36\u003c/em\u003e, DEGs were enriched in photosynthesis, protein processing and signal transduction, likely reflecting negative feedback regulation of the overactivated defense signals to maintain cellular homeostasis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. These results implied that \u003cem\u003eZmWRKY36\u003c/em\u003e enhances the resistance to \u003cem\u003eB. maydis\u003c/em\u003e by regulating downstream genes involved in energy metabolism, photosynthesis, and defense signal transduction.\u003c/p\u003e\u003cp\u003eWRKY TFs typically bind to W-box elements in target gene promoters [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], thus, screening and verification of the \u003cem\u003eZmWRKY36\u003c/em\u003e target gene will clarify its resistance mechanism. In this study, we identified W-box elements in the promoter regions of 105 DEGs from transcriptome data. Techniques such as chromatin immunoprecipitation sequencing (ChIP-seq) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] or electrophoretic mobility shift assays (EMSA) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] can be used to identify direct \u003cem\u003eZmWRKY36\u003c/em\u003e targets. Specific binding of \u003cem\u003eZmWRKY36\u003c/em\u003e to a target gene would directly confirm its role in activating disease resistance genes via transcriptional regulation. This targeted study will clarify the role of \u003cem\u003eZmWRKY36\u003c/em\u003e in regulating the resistance network of maize to \u003cem\u003eB. maydis\u003c/em\u003e, and may reveal novel resistance genes. For instance, \u003cem\u003eZmWRKY36\u003c/em\u003e might be involved in signaling molecules that are related to ROS [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], as well as the photosynthetic pathway into an integrated immune regulatory network.\u003c/p\u003e\u003cp\u003eFurthermore, functional diversity of the target genes may explain the multiple roles of \u003cem\u003eZmWRKY36\u003c/em\u003e. Some of the target genes might be cellulose synthase genes that strengthen cell wall to enhance physical defense; some genes might regulate secondary metabolites such as flavonoids to enhance chemical defense [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], and some might mediate the transmission of immune signals to amplify the defense response [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Coordinated expression of these target genes collectively constitutes the comprehensive disease resistance response mediated by \u003cem\u003eZmWRKY36\u003c/em\u003e. Additionally, analyzing the number and distribution of W-box in the promoter regions of target genes [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], may reveal \u003cem\u003eZmWRKY36\u003c/em\u003e regulatory specificity and efficiency: high W-box abundance might correlate with stronger transcriptional activation, while specific-positions W-boxes might synergize with other cis-acting element [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], providing insights into ZmWRKY36\u0026rsquo;s functional divergence under different stresses.\u003c/p\u003e\u003cp\u003eIn conclusion, according to the results of this study, we proposed a hypothetical working model for \u003cem\u003eZmWRKY36\u003c/em\u003e during \u003cem\u003eB. maydis\u003c/em\u003e infection (Fig.\u0026nbsp;10). Under the infection of pathogen, \u003cem\u003eZmWRKY36\u003c/em\u003e may bind to the cis-acting elements in the promoter of the target gene. It may accelerate the clearance of ROS, regulate the disease resistance genes or the photosignal transduction pathways. Then \u003cem\u003eZmWRKY36\u003c/em\u003e enhances the resistance of maize to the southern corn leaf blight. Based on the unique W-box binding property of WRKY TFs, key target genes were screened through molecular interaction experiments, which will lay a crucial foundation for in-depth exploration of the mechanism of \u003cem\u003eZmWRKY36\u003c/em\u003e. Future research will focus on systematically exploring the downstream target genes of \u003cem\u003eZmWRKY36\u003c/em\u003e. By using ChIP-seq and EMSA technologies to identify direct W-box binding targets, and verifying its regulatory effect through the dual luciferase reporter system [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. These studies will clarify the \u003cem\u003eZmWRKY36\u003c/em\u003e-mediated disease resistance network in maize, and providing more clear targets and theoretical support for maize disease-resistant molecular breeding, accelerating the development of new resistant varieties.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e\u003cem\u003eZmWRKY36\u003c/em\u003e is a group Ⅱ WRKY TF, containing a conserved WRKY domain and C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e type zinc finger domain. Its expression is significantly upregulated in maize upon \u003cem\u003eB. maydis\u003c/em\u003e infection, and it positively regulates resistance to the southern corn leaf blight. \u003cem\u003eZmWRKY36\u003c/em\u003e localizes to the nucleus of \u003cem\u003eN. benthamiana\u003c/em\u003e and maize protoplasts, possesses transcriptional activation activity. Transcriptome analysis showed that DEGs from the transient silencing and overexpression of \u003cem\u003eZmWRKY36\u003c/em\u003e were mainly enriched in pathways related to cellular structure, metabolite synthesis and photosynthesis. Fourteen DEGs with opposite expression trends were identified with multiple W-box elements, which are likely downstream genes regulated by \u003cem\u003eZmWRKY36\u003c/em\u003e. Our fingdings provide a theoretical basis for the molecular mechanism of resistance to \u003cem\u003eB. maydis\u003c/em\u003e in maize, and enrich genetic materials for maize disease-resistant breeding. Ultimately, this work will facilitate the development of high-yield, disease-resistant maize varieties to safeguard global food security.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003ePlant materials, fungal strains and growth conditions\u003c/h2\u003e\u003cp\u003eThe experimental materials used in this study included the maize inbred line B73 (wild type from CIMMYT) and \u003cem\u003eN. benthamiana\u003c/em\u003e (laboratory storage). The soil mixture was prepared with vermiculite and nutrient soil at a ratio of 3:1, which was placed in pots (10 \u0026times; 8 cm in depth). B73 seeds were sown in these pots and cultivated in a greenhouse under a 14-h light/10-h dark photoperiod. Greenhouse conditions were maintained at 24\u0026deg;C during the day and 20\u0026deg;C at night, with relative humidity controlled within the range of 50%-60%. \u003cem\u003eN. benthamiana\u003c/em\u003e seeds were sown in the same soil mixture. Upon germination, individual seedlings were transplanted to fresh soil and grown under the same environmental conditions as maize. The \u003cem\u003eB. maydis\u003c/em\u003e strain used was 4-4-3 strain preserved in the laboratory, which was grown on oat agar medium and incubated at 25\u0026deg;C for fourteen days until sporulation occurred.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGene structure analysis and phylogenetic tree construction\u003c/h3\u003e\n\u003cp\u003eZmWRKY36 was analyzed with sixteen WRKY Ⅱe subfamily proteins. Their domains and motifs were predicted using NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and MEME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/tools/meme\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), followed by analysis with Tbtools software. For phylogenetic tree construction, thirty protein sequences of highly homologous were selected from six plant species: \u003cem\u003eZea mays\u003c/em\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eGlycine max\u003c/em\u003e, \u003cem\u003eGossypium hirsutum\u003c/em\u003e, \u003cem\u003eTriticum aestivum\u003c/em\u003e, and \u003cem\u003eOryza sativa\u003c/em\u003e. These protein sequences were retrieved from NCBI based on their respective gene identifiers. Subsequently, the homologous protein sequences were imported into MEGA11. To ensure robust homology analysis, multiple sequence alignment was performed using the Muscle algorithm. To enhance clarity and interpretability, the phylogenetic tree was constructed via the neighbor-joining method in MEGA11 and optimized using iTOL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The gene information used in this study for homology analysis, domain and motif analysis were listed in Supplementary Tables\u0026nbsp;1 and 2.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAnalysis of expression patterns\u003c/h2\u003e\u003cp\u003eThe spore suspension of \u003cem\u003eB. maydis\u003c/em\u003e was prepared at a concentration of 1\u0026times;10⁵ spores/mL and sprayed onto the leaves of 14-day-old B73 seedlings. Inoculated leaves were analyzed at 12, 24, 48, 72, 96, and 120 hours post infection (hpi) using HiScript Ⅲ RT SuperMix (Vazyme, R323, China), with ZmActin as the internal reference for qRT-PCR. The 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] was used to calculate the relative expression levels of \u003cem\u003eZmWRKY36\u003c/em\u003e at different time points, and the quantitative data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). All experiments were conducted with three biological replicates to ensure the accuracy and reliability of the results. The primers used in this study were listed in Supplementary Table\u0026nbsp;3.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConstruction of\u003c/b\u003e \u003cb\u003eAgrobacterium\u003c/b\u003e\u003cb\u003e-mediated maize VIGS and VOX plants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFoxtail mosaic virus (FoMV) was utilized to construct the virus-induced gene silencing (VIGS) and virus-mediated overexpression (VOX) systems in maize[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. A 300 bp fragment from the C-terminal region of the \u003cem\u003eZmWRKY36\u003c/em\u003e CDS was selected as the silencing fragment, while the full-length CDS (including the termination codon) was chosen as the overexpression fragment. Homologous recombination was used to clone the \u003cem\u003eZmWRKY36\u003c/em\u003e silenced fragment and full-length CDS into the pFoMV-pCAMBIA1380 silencing and overexpression vectors at restriction enzyme sites, thereby obtaining the VIGS and VOX plasmids. These plasmids were introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV3101 via chemical transformation. The Agrobacteria were grown overnight in LB liquid medium with shaking, which cells were collected by centrifugation and resuspended in a suspension buffer (10 mM MgSO₄, 100 \u0026micro;M acetosyringone) to adjust an OD₆₀₀ to 1.0. Then, the prepared Agrobacterium suspensions were infiltrated at a point approximately 2\u0026ndash;3 mm above the coleoptile of 4-day-old seedlings. Fourteen days post-infiltration, plants exhibiting mosaic phenotypes were selected, and RT-qPCR was performed to assess the silencing and overexpression efficiency of \u003cem\u003eZmWRKY36\u003c/em\u003e. Collect the fourth to sixth leaves that show obvious mosaic symptoms, as well as the leaves from the silencing and overexpression plants,and place them in 50 ml tubes with desiccant at the bottom. Perform overnight freeze-drying treatment, stored at -20\u0026deg;C for subsequent inoculation experiments. Approximately 100 mg of freeze-dried plant material was ground in 50 mM potassium phosphate buffer (pH 7.0). Once the second leaf of the plants had fully expanded, a layer of silicon carbide powder was sprinkled on the leaf surface. The suspension was gently spread over the leaf surface using fingers to perform the inoculation. After inoculation, the leaves were left undisturbed for 20 min to facilitate viral infection, then rinsed with water to remove excess silicon carbide. Inoculated plants were transferred to the greenhouse, where symptoms were observed over a 14\u0026ndash;21 day period. These plants were subsequently used for in vitro or spray inoculation with \u003cem\u003eB. maydis\u003c/em\u003e to evaluate disease resistance.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePathogenicity analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003ePathogenicity analysis\u003c/div\u003e\u003cp\u003eThe fourth or fifth leaf displaying mosaic phenotypes after rub inoculation was selected and placed flat in a 25 \u0026times; 25 cm petri dish lined with moist filter paper. The \u003cem\u003eB. maydis\u003c/em\u003e spore suspension was adjusted to a concentration of 1\u0026times;10⁵ spores/ml with 0.02% Tween 20, and 10 \u0026micro;l of this suspension was applied to each inoculation site on the maize leaves. After sealing the petri dish with plastic wrap, it was incubated at 25\u0026deg;C with 95% humidity under a 14-h light/10-h dark cycle for 4\u0026ndash;5 days to assess disease development. Lesion areas were measured using ImageJ software. The leaf fragment (4\u0026ndash;5 cm in size) from the center of the lesion was cut, and the total DNA (including that from maize leaves and \u003cem\u003eB. maydis\u003c/em\u003e) was extracted using the CTAB method. Relative fungal biomass was calculated via the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e\n\u003ch3\u003eReactive oxygen species assay\u003c/h3\u003e\n\u003cp\u003eFollowing rub inoculation, the third leaf with the distinct mosaic phenotype was selected, and 4 mm-diameter discs were punched from the both sides of veins. These leaf discs were placed in a 90 mm petri dish containing sterile water and incubated in the dark overnight. For the assay, three leaf discs were placed in a 1.5 ml tube containing the reaction mixture, which consisted of 100 \u0026micro;l of luminol (Bio-Rad Immun-Star horseradish peroxidase substrate), 1 \u0026micro;l of horseradish peroxidase (HRP), and 1 \u0026micro;l of 1 mM flg22. Luminescence signals were detected using a Glomax 20/20 luminometer (Promega) following treatment with 1 \u0026micro;l of 10 mM chitin or ddH₂O (as control). Measurements were recorded every 10 s over a 20 min period, and each sample analyzed was repeated three times.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSubcellular localization assay\u003c/h2\u003e\u003cp\u003eIn \u003cem\u003eN. benthamiana\u003c/em\u003e experiments, free Green Fluorescent Protein (GFP) and the nuclear red fluorescence marker were introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e EHA105 via chemical transformation. Overnight-cultured Agrobacteria were collected and resuspended in a solution containing 1 M MgCl₂, 1 M MES, and 100 mM acetosyringone to adjust the OD₆₀₀ to 1.0. The suspension was injected into the third or fourth \u003cem\u003eN. benthamiana\u003c/em\u003e leaves using a 1 ml sterile syringe (without needle). GFP served as the control, with at least three plants per treatment. Plants were incubated in a 25\u0026deg;C greenhouse for 36 h. For maize protoplast experiments, free GFP and GFP-\u003cem\u003eZmWRKY36\u003c/em\u003e plasmids were co-transfected with the nuclear red fluorescence marker into protoplasts. They were used for confocal microscopy observation after dark incubation for 12\u0026ndash;16 h\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eConfocal microscopy\u003c/h2\u003e\u003cp\u003eThe fluorescence signals were detected using the ZEISS confocal microscope 980. The Green Fluorescent Protein (GFP) and the nuclear red fluorescence marker signals were imaged at 488 nm and 587 nm excitation lasers respectively. The lens magnification was 20 times, and the scanning resolution was 1024 px \u0026times; 1024 px.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eTranscriptional activity analysis\u003c/h2\u003e\u003cp\u003e\u003cem\u003eZmWRKY36\u003c/em\u003e was fused with GAL4 binding domain and co-transfected into maize protoplasts with a reporter vector containing the GAL-TATA element and Firefly Luciferase (FLuc). GAL4-VP16 and GAL4 served as positive and negative controls. After overnight dark incubation, protoplast protein extracts were analyzed for Firefly Luciferase (Luc) and Reniferase Luciferase (Ren) activities using the microplate reader. Transcriptional activity was evaluated by the ratio of Firefly Luciferase (FLuc) to Reniferase Luciferase (RLuc) signal. In the yeast system, the bait plasmid pGBKT7-\u003cem\u003eZmWRKY36\u003c/em\u003e was transformed into AH109 competent cells, with pGBKT7 and pGBKT7-VP16 as negative and positive controls. Transformants were plated on SD/-Trp medium for 2\u0026ndash;3 days. Then, single colonies were serially diluted (1\u0026times;, 10\u0026times;, 100\u0026times;, 1000\u0026times;) and spotted onto SD/-Trp, SD/-Trp-His, and SD/-Trp-His\u0026thinsp;+\u0026thinsp;X-α-gal media to verify transcriptional activity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eTranscriptome sequencing\u003c/h2\u003e\u003cp\u003eIn this study, the following four samples were selected for RNA-Seq: FoMV:V, FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e, FoMV:\u003cem\u003eGFP\u003c/em\u003e-VOX, and FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX. For transient silencing, FoMV:V served as the control and FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e as the experimental group. For transient overexpression, controls was FoMV:\u003cem\u003eGFP\u003c/em\u003e-VOX and FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX as experimental group. The fourth leaves of plants showing mosaic phenotypes after rub inoculation were selected. RT-qPCR confirmed the silencing and overexpression efficiency of \u003cem\u003eZmWRKY36\u003c/em\u003e, and leaves with stable expression levels were chosen. Three replicates per treatment were sent to Novogene Co., Ltd. for transcriptome sequencing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eQuantification and statistical analysis\u003c/h2\u003e\u003cp\u003eThis study employed Student's t-test (with *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) for statistical analysis. The data represent SEM\u0026thinsp;\u0026plusmn;\u0026thinsp;the mean. The detailed information of the statistical analysis is provided in the legend.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eAll materials used in this study complies with institutional, national, and international guidelines. The maize inbred B73 was provided by CIMMYT. \u003cem\u003eN. benthamiana\u003c/em\u003e and pathogen strain was kept and bred by our laboratory.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was financially supported by the National Natural Science Foundation of China, International Cooperation and Exchange Project (32061143033).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZQ and WD conceived and designed the experiments. Shu performed the research and writing original draft. Ying, HY, Na, HL, CT, RY provided help during research, ZQ and WD revised the manuscript. All authors approved the manuscript and consent to publication this manuscript\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eWe thank Dr. Steven A. Whitham (Department of Plant Pathology \u0026amp; Microbiology, Iowa State University) for providing plasmid pFoMV-EV-pCAMBIA1380-IS5.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and analyzed during the current study are available in the NCBI repository, PRJNA1320840.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang ZY, Wang XM. Current status and management strategies for corn pests and diseases in China. Plant Prot. 2019;45(1):1\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLv GZ, Chen J, Bai JK, Wang CP. 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J Vis Exp. 2021(168):e62277.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Maize, Bipolaris maydis, WRKY TFs, W-box, VIGS","lastPublishedDoi":"10.21203/rs.3.rs-7738242/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7738242/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eWRKY transcription factors (TFs) represent one of the largest families of transcriptional regulators in plants, play crucial role in plant responses to both biotic and abiotic stresses. Studies have shown that WRKY family members modulate the expression of disease resistance genes, hormone synthesis genes and signal transduction genes, thereby mediating plant resistance against diverse pathogens including fungi, bacteria, and viruses. Although reports have indicated the presence of 120 WRKY family protein in maize (\u003cem\u003eZea mays\u003c/em\u003e L), research on the molecular regulatory mechanism underlying disease resistance mediated by maize WRKY genes remains limited. In this study, we identified the transcription factor gene \u003cem\u003eZmWRKY36\u003c/em\u003e in maize and investigated its function in maize\u0026rsquo;s response to infection by \u003cem\u003eBipolaris maydis\u003c/em\u003e-the causal agent of southern corn leaf blight. This work aims to provide a theoretical and experimental basis for exploring maize disease resistant genes and enriching functional studies of WRKY TFs in different crops.\u003c/p\u003e\u003ch2\u003eResult\u003c/h2\u003e\u003cp\u003eWe identified \u003cem\u003eZmWRKY36\u003c/em\u003e as a nuclear-localized transcription factor in maize. To explore its biological function in resistance to \u003cem\u003eB. maydis\u003c/em\u003e, we constructed Z\u003cem\u003emWRKY36\u003c/em\u003e-silenced (FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e) and \u003cem\u003eZmWRKY36\u003c/em\u003e-overexpressed (FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX) maize plants using virus-induced gene silencing (VIGS) and transient overexpressed (VOX) systems, respectively. Disease resistance assays revealed that transiently silenced FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e plants exhibited enhanced resistance to \u003cem\u003eB. maydis\u003c/em\u003e infection and suppressed chitin-induced reactive oxygen species (ROS) burst, whereas transiently overexpressed FoMV:\u003cem\u003eZmWRKY36\u003c/em\u003e-VOX plants showed the opposite results. Additionally, overexpressed of \u003cem\u003eZmWRKY36\u003c/em\u003e upregulated the expression of disease-related genes, suggesting that \u003cem\u003eZmWRKY36\u003c/em\u003e positively regulate maize resistance to \u003cem\u003eB. maydis\u003c/em\u003e. Further functional characterization demonstrated that \u003cem\u003eZmWRKY36\u003c/em\u003e possesses transcriptional activation activity. Transcriptome analysis of \u003cem\u003eZmWRKY36\u003c/em\u003e silenced and overexpressed plants revealed that the differentially expressed genes (DEGs) were mainly enriched in pathways related to cellular structure composition, metabolic synthesis and photosynthesis. Promoter analysis of these DEGs identified 105 genes containing W-box elements the core binding motif of WRKY TFs, which suggested that these pathways and target genes are involved in mediating maize resistance to \u003cem\u003eB. maydis\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThese results demonstrate that transcription factor \u003cem\u003eZmWRKY36\u003c/em\u003e positively regulates maize resistance to \u003cem\u003eB. maydis\u003c/em\u003e and identify its potential downstream target genes. This study provides insights into the regulatory role of \u003cem\u003eZmWRKY36\u003c/em\u003e in maize defense responses and lays a foundation for further dissecting WRKY-mediated disease resistance networks in maize.\u003c/p\u003e","manuscriptTitle":"Molecular mechanism of ZmWRKY36 mediated maize resistance to Bipolaris maydis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-26 08:32:30","doi":"10.21203/rs.3.rs-7738242/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-23T20:53:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-20T03:33:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-16T09:49:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46230903897837417049307114364307211085","date":"2025-10-11T18:15:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"131946971364889790864231456527815454115","date":"2025-10-11T07:27:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"316198231480641920976018080481989852158","date":"2025-10-10T11:26:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-10T11:18:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-10T11:12:54+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-10T11:01:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-10T07:58:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-10-10T07:54:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c0d5090c-613e-43a1-b1c2-4803533695ff","owner":[],"postedDate":"October 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:01:19+00:00","versionOfRecord":{"articleIdentity":"rs-7738242","link":"https://doi.org/10.1186/s12870-025-07961-1","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2026-01-05 15:57:25","publishedOnDateReadable":"January 5th, 2026"},"versionCreatedAt":"2025-10-26 08:32:30","video":"","vorDoi":"10.1186/s12870-025-07961-1","vorDoiUrl":"https://doi.org/10.1186/s12870-025-07961-1","workflowStages":[]},"version":"v1","identity":"rs-7738242","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7738242","identity":"rs-7738242","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}