Application of ZmXET1 gene in regulating and controlling resistance of corn to southern rust disease

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to application of a ZmXET1 gene to regulation and control of resistance of corn to southern rust. Mononuclear RNA sequencing and space transcriptome sequencing are integrated, it is displayed that the gene ZmXET1 is up-regulated after being infected by the puccinia polypoda, phenotypic analysis is conducted on the gene through virus-induced gene silencing, and it is found that after the puccinia polypoda is inoculated, a gene silencing plant shows light disease symptoms compared with a contrast, the biomass of pathogenic bacteria in the silencing plant is remarkably reduced, and it is found that the pathogenic bacteria in the silent plant are remarkably reduced. The overexpressed gene ZmXET1 shows heavier disease symptoms compared with a control group, and the biomass of pathogenic bacteria is remarkably increased, which indicates that ZmXET1 negatively regulates and controls the defense of the corn to the puccinia polypoda; the results jointly show that the gene ZmXET1 can improve the resistance of the corn to the southern rust disease. The application provides a new gene resource and a technical path for the cultivation of a new variety of southern rust resistance of corn, and has important theoretical significance and application value.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, and specifically relates to a gene that regulates maize's resistance to southern rust. Background Technology

[0002] corn( Zea mays Southern corn rust (SCR) is a globally important food crop and a vital source of food, feed, and industrial products. However, due to changes in temperature and humidity caused by global warming, and the increased spread of pathogens through inter-regional seed transport and agricultural machinery operations, southern corn rust has become one of the most devastating diseases affecting summer corn production in southern my country and the Huang-Huai-Hai Plain, posing a serious threat to the corn industry. SCR is caused by *Styrax multiplostiformis*, a fungus that causes southern corn rust. This disease leads to severe yield losses, typically between 30% and 50%, and in severe cases, can result in yield reductions exceeding 50%, negatively impacting both yield and quality, and posing a serious challenge to global food security. The pathogen multiplies in humid environments, infects corn leaves, and spreads rapidly within the plant, impairing photosynthesis and hindering growth.

[0003] Currently, the main control methods for SCR in maize production include chemical control, agricultural cultivation measures, and breeding of disease-resistant varieties, but all have significant limitations. Breeding and promoting disease-resistant varieties is the most economical, effective, and environmentally friendly core method for controlling maize SCR, while the discovery and utilization of disease-resistant genes are the foundation and key to disease-resistant breeding. With the development of molecular biology techniques, research on plant disease resistance mechanisms has moved from traditional phenotypic identification to the gene level. A large number of genes related to crop disease resistance have been cloned and their functions verified, providing support for the application of precision breeding technologies such as molecular marker-assisted breeding and gene editing.

[0004] The pathogenicity of SCR in maize involves a complex interaction between the pathogen and the immune system. The genomic characteristics of *S. multistachys* reveal a variety of effector proteins that manipulate host physiology to promote infection. *S. multistachys* initially infects the leaf surface, then penetrates the plant, progressing through various tissue layers. Studies have shown that different tissues and cell types exhibit differentiated immune responses at different stages of infection, and genetic factors play a crucial role. ZmREM1.3 Positive regulation of maize's defense against *Rhizoctonia solani* via a salicylic acid / jasmonic acid-mediated signaling pathway (Wang S, Chen Z, Tian L, et al. Comparative proteomics combined with analyses of transgenic plants reveal) ZmREM1.3Mediates maizeresistance to southern corn rust. Plant Biotechnol J. 2019 Nov;17(11):2153-2168.). Patent 202211640747.4 discloses maize ZmlecRK-G2 This gene and its application: mutants of this gene reduce maize's resistance to southern rust, laying the foundation for broadening the germplasm of maize disease-resistant resources. Furthermore, among the few reported maize southern rust resistance genes, some have a narrow resistance spectrum, exhibiting resistance only to specific physiological races, making them ill-suited to cope with the rapid mutation of the pathogen and limiting their application in breeding. Therefore, the current field of maize southern rust resistance breeding urgently needs to continue exploring resistance genes with broad-spectrum resistance and stable genetic effects, providing new gene resources and technical pathways for the breeding of new maize varieties resistant to southern rust, which has significant theoretical and practical value. Summary of the Invention

[0005] To address the above problems, this invention proposes a... ZmXET1 Application of genes in regulating maize resistance to southern rust.

[0006] The technical solution of this invention is implemented as follows: This application integrates single-nuclear RNA sequencing and spatial transcriptome sequencing to discover that the JA-mediated signaling pathway is involved in the infection of *Russula multifiliis* in maize leaves. Given that *Russula multifiliis* hyphae invade maize leaves through stomata or epidermal cells and absorb nutrients through mesophyll cells, the focus was on identifying key genes by analyzing DEGs shared in mesophyll and epidermal cells at 24 h and 48 h time points. The results showed that... ZmXET1 Upregulation occurred after infection with multiple rust fungi.

[0007] Based on this, the present invention provides a ZmXET1 Application of genes in regulating maize resistance to southern rust.

[0008] Preferably, the above ZmXET1 The gene's accession number in NCBI is LOC542312 (MaizeGDB [https: / / maizegdb.org / ] gene ID: Zm00001eb226470 ).

[0009] Preferably, the above-mentioned regulation of maize resistance to southern rust is to reduce the incidence of southern rust and reduce the biomass of the southern rust pathogen in maize plants, the pathogen being *Russula multifiliis*.

[0010] Preferably, the above-mentioned reduction in the severity of southern rust and decrease in the biomass of *Symplocos rubra* fungi within maize plants is achieved through silencing... ZmXET1It is achieved through genes.

[0011] After inoculation with *Russula multiflora*, gene-silenced plants (CMV: ZmXET1 Compared to the control, the silent plants exhibited milder disease symptoms, and the pathogen biomass within them was significantly reduced, indicating that... ZmXET1 Negative regulation of maize's defense against multi-stem rust fungus.

[0012] Secondly, this invention provides a method for improving maize's resistance to southern rust by silencing [the virus] in maize plants. ZmXET1 Genes, thereby improving corn's resistance to southern rust; ZmXET1 The gene's accession number in NCBI is LOC542312.

[0013] Thirdly, the present invention also provides a method for breeding transgenic maize resistant to southern rust, comprising the steps of: constructing ZmXET1 Gene silencing vector pCMV201- ZmXET1 The bacteria were transferred into tobacco leaves using Agrobacterium-mediated transformation. The infected tobacco leaves were homogenized in phosphate buffer and centrifuged. The supernatant was then microinjected into maize embryos, and transgenic maize plants were obtained after cultivation.

[0014] Agrobacterium tumefaciens GV3101 was transformed using a ternary plasmid system (pCMV101 / pCMV201 / pCMV302), with pCMV201-GFP used as a control. The bacterial suspension (OD) was then... 600 =0.8) Incubate for 3 h before infiltration. Three-week-old *Nicotiana benthamiana* leaves (positions 3-4) are infiltrated with a syringe and maintained in a controlled environment chamber. Infected leaf tissue (3-5 days post-inoculation) is homogenized (1 mL / g tissue) in ice-cold 0.01 M phosphate buffer (pH=7.0), centrifuged (4°C, 4500 rpm, 3 min), and the supernatant is then applied to B73 maize embryos via microinjection (15 μL / seed). A precision inoculation needle (60° insertion angle, 1-2 mm depth) is used to minimize embryo damage. Seeds are germinated for 3 days in the dark at 25°C on moistened filter paper, then transferred to soil and retained under 20°C / 18°C (16 h light / 8 h dark) conditions.

[0015] Preferably, the above ZmXET1 The gene's accession number in NCBI is LOC542312. The silencing vector pCMV201- was constructed. ZmXET1 forward primer ZmXET1 The CMV-F sequence is shown in SEQ ID No. 1, and the reverse primer... ZmXET1 -CMV-R is shown in SEQ ID No. 2.

[0016] Preferably, the infection time is 3-5 days, the phosphate buffer concentration is 0.01 M, pH=7.0, and the homogenization ratio is 1-1.5 mL of phosphate buffer per g of tobacco leaves. The microinjection ratio is 15-20 μL of supernatant injected into each immature embryo, using an inoculation needle with a 60° insertion angle and a depth of 1-2 mm into the maize immature embryo.

[0017] Preferably, the maize variety mentioned above is the inbred line B73.

[0018] The present invention has the following beneficial effects: This invention integrates single-nuclear RNA sequencing and spatial transcriptome sequencing, revealing that the JA-mediated signaling pathway is involved in the infection of *Russula multifiliis* in maize leaves. Given that *Russula multifiliis* hyphae invade maize leaves through stomata or epidermal cells and absorb nutrients through mesophyll cells, the focus was on identifying key genes by recognizing shared DEGs in mesophyll and epidermal cells at 24 h and 48 h time points. The results showed… Zm00001eb217560 ( ZmXET1 The gene was upregulated after infection with *Russula multifiliis*, and phenotypic analysis of the gene using virus-induced gene silencing (VIGS) showed... ZmXET1 Gene expression was reduced by approximately 70%, and after inoculation with *Russula multiflora*, gene-silenced plants (CMV: ZmXET1 Compared to the control, the plants exhibited milder disease symptoms, and the pathogen biomass in the silenced plants was significantly reduced; the overexpressed gene... ZmXET1 Compared with the control, it showed more severe disease symptoms and a significant increase in pathogen biomass, indicating that ZmXET1 Negative regulation of maize's defense against *Heterostilbene stalk*; the above results collectively indicate that genes... ZmXET1 This study demonstrates the ability to regulate maize resistance to *Hemiberlesia lataniae*. It provides new genetic resources and technical pathways for the breeding of new maize varieties resistant to southern rust, and has significant theoretical and practical value. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1This study established a model for the infection of *Rust flocculationis* in maize leaves. Figure A shows the symptom development of maize leaves inoculated with *Rust flocculationis* at 24 h, 48 h, 72 h, and 10 d post-inoculation (scale bar = 1 cm). Figure B shows the monitoring of the infection process of *Rust flocculationis* by WGA-AF488 staining. Fungal hyphae are stained with WGA-AF488, and substomatal vesicles are marked with green fluorescence. Dashed circles represent stomata indicating urediniospore germination and hyphal penetration (scale bar = 100 μm). DIC and differential interference contrast were used. Figure C shows the evaluation of WGA signal by measuring the area density of green fluorescence. *** indicates the results obtained through one-way ANOVA. p The significance between the two samples was <0.001, n=30 leaves; D represents the relative fungal biomass assessment in maize leaves inoculated with *Russula multistachys*, and "*", "**", and "**" respectively indicate the significance between the two samples. p <0.05、 p <0.01 and p <0.0001, ns indicates not significant, and error bars represent the mean ± standard deviation.

[0021] Figure 2 This is a transcriptome map of *Russula multifiliis* infection in maize leaf cells. A shows the workflow analysis of snRNA and stRNA sequencing of *Russula multifiliis*-infected maize leaves at 24 h and 48 h post-inoculation. B shows cell expression analysis using snRNA-seq sequencing, followed by dimensionality reduction and clustering; the X and Y axes represent the data mapped after dimensionality reduction. C shows the identification of cell types in different snRNA-seq clusters using a relevant marker gene database. D shows GO enrichment analysis of identified mesophyll cell genes; the X-axis represents enrichment factors, and the Y-axis indicates GO entries. E is a bubble chart of marker genes identified in different cell types; the X-axis lists marker genes, the Y-axis represents leaf cell types, the size of the dots indicates the frequency of gene expression in all cells of that tissue, and the color reflects the expression level of the gene in the studied tissue.

[0022] Figure 3 This section presents the quality analysis of snRNA sequencing results; where A is a bar chart showing the median gene number distribution and cell number in each cell of each snRNA sequencing sample, and B is the correlation analysis between snRNA sequencing samples.

[0023] Figure 4 The identification of the top two high-confidence marker genes in different tissue clusters; where A is the UMAP visualization of the expression patterns of cell cluster-specific marker genes, with color representing their relative expression level in the cell cluster, and B is the RNA in situ hybridization verification of representative cell type-specific marker genes for the inferred cell type.

[0024] Figure 5Key genes involved in maize's defense against *Strombus multidus* rust were identified. Figure A shows the DEGs volcano plots of different maize leaf cell types 24 h after *Strombus multidus* infection. Each column represents a specific leaf tissue. Selection criteria: |log2FC|>0.5 and... p -<0.05; B is a DEGs volcano plot of different maize cell types 48 h after infection with *Russula multifiliis*; C is a GO enrichment bubble plot of differentially expressed gene sets across various plant tissues. p <0.05; D is a Venn diagram showing the number of DEGs in mesophyll and epidermal cells under different treatments, with red numbers representing the number of shared key genes across two time points and cell types; E is a heatmap of the relative expression of key genes in different tissues and time points, with color indicating TPM normalization; F is the data obtained by stRNA-seq at 24 h post-inoculation in the treatment and control groups. ZmXET1 Gene expression levels and locations; G represents CMV: ZmXET1 Silent plants and control ZmXET1 Analysis of gene expression levels using Student's t -Statistical analysis was performed using the test: *** p <0.001; H represents the CMV of the multi-stalk rust challenge: ZmXET1 Symptoms of silent corn leaves; I for CMV: ZmXET1 - Relative biomass assessment of silent maize leaves after infection with *Russula multistachys* fungus.

[0025] Figure 6 Phenotypic analysis of virus-mediated overexpression vectors; Figure A shows SMV challenged with *Russula multiplystilts*. ZmXET1 Symptoms of overexpression in maize leaves; B represents SMV: ZmXET1 In overexpressing plants and controls ZmXET1 Analysis of gene expression levels using Student's t -Statistical analysis was performed using the test: *** p <0.001; C is SMV: ZmXET1 - Assessment of the relative biomass of maize leaves after overexpression following infection with *Russula multifiliis* fungus.

[0026] Figure 7 The relative expression levels and number of expressing DEGs with the highest log2FC observed in different cell types under different treatments are shown.

[0027] Figure 8 This study aims to determine the cellular distribution, expression level, and spatial expression location of three key genes obtained through snRNA-seq and stRNA-seq. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0030] This application uses R scripts to generate charts. Box plots, stacked bar charts, pie charts, bubble charts, and other graphs are generated using ggplot2 v3.5.1 (https: / / ggplot2.tidyverse.org / ). All heatmaps are generated using ComplexHeatmap v2.20.1 (https: / / jokergoo.github.io / ComplexHeatmap-reference / book / index.html).

[0031] Statistical Analysis: Preprocessing of snRNA-seq and stRNA-seq data in this application involved dimensionality reduction and standardization. Data from reproducible biological experiments are presented as mean ± standard deviation. VIGS experimental design used a sample size of 6 per group for statistical analysis. Student's... t Statistical significance is determined by a test or a simple one-way ANOVA to identify significant differences. p Value definition. snRNA-seq analysis includes three biological replicates, while stRNA-seq utilizes four biological replicates. For differentially expressed gene identification and GO enrichment analysis, [the following was applied]. p Value and error detection rate adjustment q Values. All statistical analyses were performed using the R programming language.

[0032] Example 1: Establishment of infection of *Russula multistachys* in maize seedling leaves

[0033] Plant material and inoculation and collection of multi-stalked rust fungi: The isolate PP.CN1.0 of *Russula multifiliis* was collected from infected maize (Zhengdan 958 variety) leaves at the Henan Academy of Agricultural Sciences Experimental Station and transferred to 2 mL centrifuge tubes using sterile toothpicks. A homogeneous spore suspension was then prepared in 0.01% Tween solution (diluted with ddH2O), yielding a pale yellow to light brown solution. For manual inoculation, 20–40 μL aliquots were gently applied along the leaf axis from base to tip using a pipette tip, ensuring no epidermal damage. The inoculated leaves were then fogged in a humidity-controlled room at 25°C in the dark for 12–24 h, and then transferred to standard photoperiod conditions after incubation.

[0034] Maize seedlings of the resistant inbred line Qi319 were cultured in a light incubator at the Henan Academy of Agricultural Sciences under controlled conditions of a 14-hour light / 10-hour dark photoperiod and a temperature of 20-25°C. When the maize reached the V3 growth stage, *Russula multifiliis* spores were suspended in a 0.01% Tween 20 solution at a concentration of approximately 1 × 10⁻⁶. 6 Spores / mL were collected and the spore suspension was evenly sprayed onto all leaf surfaces using a small spray bottle. A parallel control group (simulated, MK) was established using a suspension without *Russula multifiliis* (Tween 20 diluted to 0.01% in ddH2O). Inoculated leaves from leaf sheath to leaf tip were then collected at 24 h and 48 h post-inoculation for further analysis.

[0035] Fluorescent WGA procedure: Plant tissue samples were collected and destained using a bleaching solution (glacial acetic acid: anhydrous ethanol = 1:1) until the leaves became translucent and chlorophyll-free. The destained tissue was then treated with chloral hydrate for 1–2 days. The resulting samples were rinsed twice in 50% ethanol for 15 minutes each time, followed by rinsing 1–2 times in distilled water for 10 minutes each time. The samples were then transferred to 1 M potassium hydroxide solution and placed in a boiling water bath for 20–30 minutes to soften the leaf tissue and promote the penetration of the fluorescent dye into the mesophyll cells. The samples were then soaked in 50 mM Tri-HCl (pH 7.0–7.4) for 30 minutes, stained in the dark with 20 µg / mL WGA staining solution for over 10 minutes, and rinsed 2–3 times with distilled water for 10 minutes each time. Observation and photography were then performed under the green fluorescent protein channel.

[0036] This embodiment aims to determine the key early time point of infection in *Russula multifiliis* and the disease progression in maize leaves. Therefore, the resistant inbred line Qi319 was inoculated with *Russula multifiliis* spores, and infection progression was assessed using phenotypic and histological analyses at multiple time points. No visible symptoms were observed on Qi319 leaves at 24 h, 48 h, or 72 h post-inoculation. Figure 1A). However, fluorescence microscopy using wheat germ lectin staining showed successful fungal infection 24 h post-inoculation, evidenced by the formation of substomatal vesicles, a hallmark structure of rust fungal invasion, indicating that the pathogen successfully entered the intercellular spaces of plant tissues either directly through the leaf epidermis or via stomata. Figure 1 B). This is accompanied by the development of primary hyphae and the formation of haustoria (B). Figure 1 B). At 48 h post-inoculation, increased branching and colonization of fungal hyphae in the intercellular spaces, as well as haustoria formation, were observed, promoting nutrient uptake from the host cells. By 72 h post-inoculation, fungal growth had further advanced, and significant changes occurred in the cellular environment, such as stomatal distortion, although no visible symptoms were yet observed. Figure 1 B). Ten days after inoculation, scattered mature lesion masses appeared on the leaf surface (Spore masses). Figure 1 A). Quantitative analysis using WGA staining and fungal biomass further confirmed the gradual increase in fungal load from 48–72 h post-inoculation, and statistical analysis also supported this. Figure 1 CD).

[0037] The selection of 24 h and 48 h post-inoculation as key early time points is based on previous research, which defines 48 h post-inoculation as a critical threshold for early nutrient invasion, after which extensive host defense responses (such as phenolic deposition and organelle destruction) begin to appear and become dominant by 72 h post-inoculation. Therefore, the transition from initial fungal establishment to early host involvement can be captured at 48 h post-inoculation, making this time point particularly informative for studying pre-defense molecular responses. This establishes 24 h and 48 h post-inoculation as key early time points for studying the early infection of *Russula multifiliis* in maize leaves.

[0038] Example 2: Establishment of cell types in maize leaves infected with *Russula multistachys*

[0039] Nuclear isolation and single-cell library preparation: Leaf fragments were immersed in cryoprotectant (5 mL / sample: 1.5 mL sterile water, 2.5 mL glycerol, 1 mL FBS) and placed on ice for 5 min. The cryoprotectant was then removed by tilting the tube at a 45-degree angle and gently aspirating most of the solution with a pipette tip close to the surface. After brief centrifugation at 100×g and 4°C, the remaining solution was carefully aspirated from the bottom of the tube using a 1 mL sterile syringe and discarded. The resulting sample was then rapidly frozen in liquid nitrogen. Nuclear isolation began by homogenizing the tissue in 2 mL of ice-cold EZ lysis buffer using a glass Dounce homogenizer (Sigma, Cat # D8938), using pestles A and B 25 times each. After adding 3 mL of buffer, the sample was incubated on ice for 5 min and centrifuged at 500 g for 5 min at 4°C. The washed nuclear precipitate was treated with nuclear suspension buffer [NSB; 1× PBS, 0.01% BSA, 0.1% RNase inhibitor (Clontech, Cat. no. 2313A)], filtered through a 35 μm filter membrane (Corning-Falcon, Cat.#. 352235), and quantified for snRNA-seq.

[0040] Single-cell sequencing: Single-nuclear suspensions were prepared in PBS / 0.04% BSA and processed on a Chromium Controller using the Chromium Next GEMSingle Cell 3' Reagent Kits v3.1 (10×Genomics). GEMs were generated via chip loading (Chromium Next GEM Chip G). After nuclear cleavage and RNA barcoding following GEM-based reverse transcription, cDNA libraries were constructed and quality controlled using Qubit 4.0 and Agilent 2100. Sequencing was then performed on an Illumina NovaSeq 6000 with >50,000 PE150 reads per nucleus (Biomarker Technologies Corporation, BMKGENE, Beijing, China).

[0041] Raw data and assays from 10×Genomics snRNA-seq were processed: Sequencing data were aligned to a maize B73 reference genome using 10×Cell Ranger v7.0 (STAR ​​aligner-engineered), and gene expression quantification was performed using cell barcode-gene pairings with unique molecular identifiers (UMIs). Nucleated cell barcodes were filtered through the internal quality control workflow in Cell Ranger v7.0, and subsequent analyses (clustering, cell type annotation, and differential expression profiling) were performed only on validated nuclear populations using Seurat v4.0.1.

[0042] Dimensionality Reduction: To facilitate unsupervised clustering and cell type identification, principal component analysis (PCA) was applied to the merged tissue sample set for dimensionality reduction. For data visualization, Seurat was used to further reduce the dimensionality of all nuclei, and t-SNE was employed to project the cells into 2D space. This process included: 1) calculating gene expression values ​​using the Log-Normalize method of the "Normalization" function in Seurat; 2) performing PCA on the normalized expression values ​​and selecting the top principal components for clustering and *t*-SNE analysis; and 3) identifying clusters based on the clustering method using a weighted shared nearest neighbor graph. The Find All Markers function in Seurat (v4.0.1) was used with default parameters and "bimod" (likelihood ratio test) to determine the marker genes for each cluster. The top 10 genes were selected as marker genes by filtering the Find Markers results (Fold Change > 1.5 and FDR < 0.1).

[0043] Correlation analysis between tRNA-seq and snRNA-seq: Multimodal Intersection Analysis (MIA) was used to assess the relationships between data obtained using tRNA-seq and snRNA-seq. MIA is a multimodal integration method that annotates cellular subpopulations in spatial transcriptome data by detecting significant overlap between characteristic marker genes identified in single-cell subclusters and marker genes enriched in spatial transcriptome regions.

[0044] This embodiment established the transcriptomic profile of *Strombus multifiliis* infecting maize leaves at different time points. Therefore, single-nuclear transcriptome and spatial transcriptome sequencing were performed on samples collected at 24 h and 48 h post-inoculation. Figure 2A). A total of 12 single-cell samples were collected, including leaves treated with *Russula multiplystiltae* (PP24 h and PP48 h) and simulated treatments (MK24 h and MK48 h) at two time points. Nuclei were isolated and filtered to construct snRNA-seq libraries, which were then sequenced using 10×Genomics technology. This yielded a total of 131,601 high-quality single cells, with an average of 30,424 genes detected per sample and a median of 977 genes detected per cell. Figure 3 A). snRNA-seq data showed a strong correlation among the three biological replicates of each sample, validating the robustness of the results. Figure 3 B). After data standardization and linear dimensionality reduction, various resolution parameters were tested to determine the optimal settings for cell clustering. These settings were then applied using a uniform manifold approximation and projection to ensure accurate clustering and visualization of cell types, thereby identifying 16 major cell clusters (B). Figure 2 B).

[0045] These 16 clusters can be divided into 8 different cell types. Specifically, the marker genes for clusters 0, 1, 2, 5, 9, and 10 were identified as mesophyll cells, while clusters 3 and 6 were classified as epidermal cells, clusters 4, 7, and 8 were associated with bundle sheath cells, and clusters 11, 12, 13, 14, and 15 were labeled as seeding cells, accompanying cells, parenchyma, vascular tissue, and guard cells, respectively. Seeding cells and guard cells are both specialized epidermal cell types. Figure 2 C). To further explore the functional roles of different cell types, gene ontology enrichment analysis was performed on the largest cell type group—mesophyll cells. The results showed that differentially expressed genes in mesophyll cells are mainly involved in oxylipin biosynthesis, (1,3)-β-D-glucan biosynthesis, and photosynthesis. Figure 2 D). Several marker genes associated with specific cell types were also identified. Figure 2 E). For example, Zm00001eb158810 and Zm00001eb362640 were identified as marker genes for mesophyll cells, while Zm00001eb232100 and Zm00001eb333330 were highly expressed in epidermal cells, and Zm00001eb387500 and Zm00001eb173960 were highly expressed in guard cells. Figure 4 A). To further validate these candidate marker genes in vivo, RNA in situ hybridization was performed, confirming the reliability of the identified cell type-specific marker genes in distinguishing maize leaf cell types. Figure 4 B). Therefore, expression profiles of maize leaves were constructed using single-cell data, and subcellular types and their associated marker genes were successfully identified.

[0046] Example 3: Identification of key genes involved in maize's defense against *Heterostilbene multistachys* rust.

[0047] To identify key regulatory genes involved in maize's defense against *Strombus multistachys* rust, snRNA-seq and stRNA-seq were used to determine the DEGs involved in the maize defense process. Initially, snRNA-seq was used to identify DEGs across different cell types at different time points after infection. The results showed that 24 h after infection, the expression of a large number of genes changed in maize leaves, particularly in mesophyll, epidermis, and bundle sheath cells. Figure 5 A; Figure 7 AB). In contrast, the number of DEGs in guard cells was unexpectedly low ( ). Figure 5 A), which may be attributed to the relative abundance of other cell types identified in the snRNA-seq analysis. Furthermore, the number of DEGs in each cell type decreased significantly at 48 h post-inoculation, and DEGs were undetectable in guard cells compared to the 24 h time point. Figure 5 B; Figure 7 These findings suggest that the early infection phase of *D. multistachys* in maize leaves represents the most complex stage of defense activation. Therefore, to explore the functional roles of DEGs, GO enrichment analysis was performed for each cell type, revealing significant activation of functional pathways in all cell types at 24 h post-inoculation, including protein folding, protein stability, mRNA processing, and chaperone cofactor-dependent protein refolding. Figure 5 In stark contrast, many functional pathways were not enriched 48 hours after vaccination (C). Figure 5 C). At this later point in time, the main functional responses observed were the response to injury, regulation of JA-mediated signaling pathways, oxylipin biosynthesis, and lipid oxidation (C). Figure 5 (C) indicates that the JA-mediated signaling pathway is involved in the infection of *Russula multifiliis* in maize leaves. Given that *Russula multifiliis* hyphae invade maize leaves through stomata or epidermal cells and absorb nutrients through mesophyll cells, this study focused on identifying key genes by recognizing shared DEGs in mesophyll and epidermal cells at 24 h and 48 h time points. The results showed that five core genes were shared across both cell types at these two time points (C). Figure 5 D). Among them, five genes: Zm00001eb123630, Zm00001eb217560, Zm00001eb054050, and Zm00001eb165310 were upregulated after infection with *Rust hygroscopicus*, while Zm00001eb226470 (ZmXET1) was downregulated. Figure 5E). Subsequently, snRNA-seq and stRNA-seq were used to investigate the spatial expression of these genes, and the results showed that these five genes were predominantly highly expressed in most spatial locations in the PP24 and PP48 samples. Figure 5 F; Figure 8 However, no significant changes in expression levels were observed in some spatial locations, suggesting spatial variability in cell tissues during maize's defense response.

[0048] Application examples

[0049] VIGS assay: The VIGS assay was performed using pCMV101, pCMV201, and pCMV302 vectors to reconstruct the tripartite ZMBJ-CMV genome, establishing a VIGS platform targeting ZmXET1. Silencing fragments were designed using the SGN VIGS portal (https: / / vigs.solgenomics.net) and engineered primers were used (Table 1). The fragments were then cloned into pCMV201 to obtain the pCMV201-ZmXET1 construct. The recipient maize variety used in this application is the inbred line B73.

[0050] Agrobacterium tumefaciens GV3101 was transformed using a ternary plasmid system (pCMV101 / pCMV201 / pCMV302), with pCMV201-GFP used as a control. The bacterial suspension (OD) was then... 600 =0.8) Incubate for 3 h before infiltration. Three-week-old *Nicotiana benthamiana* leaves (positions 3-4) are infiltrated with a syringe and maintained in a controlled environment chamber. Infected leaf tissue (3-5 days post-inoculation) is homogenized (1 mL / g tissue) in ice-cold 0.01 M phosphate buffer (pH=7.0), centrifuged (4°C, 4500 rpm, 3 min), and the supernatant is then applied to B73 maize embryos via microinjection (15 μL / seed). A precision inoculation needle (60° insertion angle, 1-2 mm depth) is used to minimize embryo damage. Seeds are germinated for 3 days in the dark at 25°C on moistened filter paper, then transferred to soil and retained under 20°C / 18°C (16 h light / 8 h dark) conditions.

[0051] qRT-PCR analysis: Total RNA was isolated using Trizol reagent, and approximately 2 μg of RNA was used for cDNA synthesis via reverse transcription using HiScript III RT Super Mix (+gDNA wiper). qRT-PCR was then performed on a QuantStudio 5 system (ThermoFisher Scientific, USA) in a 25 μL reaction mixture containing 12.5 μL LightCycler SYBR Green I Master Mix, 2 μL diluted cDNA (1:5), 8.9 μL distilled H2O, 0.8 μL forward primer (10 mM), and 0.8 μL reverse primer (10 mM). The primers used are listed in Supporting Information Table 1. By comparing 2... -ΔΔCT Methods: Real-time PCR data were analyzed to quantify relative gene expression. Three biological replicates were performed for each sample, and three technical replicates were performed for PCR analysis. Student's t-test was used to assess statistical significance.

[0052] Biomass quantification of *P. multiplostomum*: A standard curve was generated by cloning the reference genes ZmUbi (maize) and PpTub (*P. multiplostomum*) into the pMD19-T vector. A series of plasmid dilutions (100 ng / μL, 10 ng / μL, 1 ng / μL, 10 μ ... -1 ng / μL, 10 −2 ng / μL, 10 −3 ng / μL, 10 −4 Using ng / μL as a template for qRT-PCR, the cycle threshold-DNA concentration relationship was calibrated using gene-specific primers. Genomic DNA was extracted from CMV-silenced and control plants inoculated with *Russula multiflora* at 12–14 days post-treatment, and qRT-PCR analysis was performed using pathogen / host reference primers for absolute DNA quantification. The relative biomass ratio (*Russula multiflora* DNA / maize DNA) between the experimental and control groups was statistically analyzed. The relevant primers designed in this project are shown in Supplementary Table 1.

[0053] Table 1 Primers used in this application

[0054]

[0055] Phenotypic analysis was performed on the genes related to Example 3 using virus-induced gene silencing. The maize materials developed in this study showed a reduction of approximately 70% in ZmXET1 gene expression. Figure 5G), and after inoculation with *Russula multifiliis*, plants with virus-induced gene silencing (VIGS) (CMV:ZmXET1) showed milder disease symptoms compared to the control. Figure 5 H). Further analysis showed that the pathogen biomass in silent plants was significantly reduced, indicating that ZmXET1 negatively regulates maize's defense against *Rhizoctonia solani*. Figure 5 These results collectively demonstrate that the gene ZmXET1 can regulate maize resistance to *Hemiberlesia lataniae*, and also validate the reliability of the multi-omics data.

[0056] Phenotypic analysis of the genes related to Example 3 was performed using a virus-mediated overexpression vector. The maize material developed in this study showed an approximately 10-fold increase in ZmXET1 gene expression. Figure 6 B), and after inoculation with *Sclerotium styracifolium*, plants with virus-based gene overexpression (VOX) (SMV:ZmXET1) showed more severe disease symptoms compared to the control. Figure 6 A). Further analysis showed a significant increase in pathogen biomass within silent plants, indicating that ZmXET1 negatively regulates maize's defense against *Rust hygrophorus*. Figure 6 C). These results collectively demonstrate that the gene ZmXET1 can regulate maize resistance to *Hemiberlesia lataniae*, and also validate the reliability of the multi-omics data.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. ZmXET1 Application of genes in regulating maize resistance to southern rust.

2. The application according to claim 1, characterized in that: The ZmXET1 The gene's accession number in NCBI is LOC542312.

3. The application according to claim 2, characterized in that: The regulation of maize resistance to southern rust involves reducing the severity of southern rust and decreasing the biomass of the southern rust pathogen in maize plants. The pathogen is *Russula multifiliis*.

4. The application according to claim 3, characterized in that: The reduction of the severity of southern rust and the decrease in the biomass of *Symplocos rubra* fungi within maize plants are achieved through silencing... ZmXET1 It is achieved through genes.

5. A method for improving maize's resistance to southern rust, characterized in that: Silent within the corn plant ZmXET1 Genes, thereby improving corn's resistance to southern rust; ZmXET1 The gene's accession number in NCBI is LOC542312.

6. A method for breeding transgenic maize resistant to southern rust, characterized in that, The steps are: build ZmXET1 Gene silencing vector pCMV201- ZmXET1 The bacteria were transferred into tobacco leaves using Agrobacterium-mediated transformation. The infected tobacco leaves were homogenized in phosphate buffer and centrifuged. The supernatant was then microinjected into maize embryos, and transgenic maize plants were obtained after cultivation.

7. The method according to claim 6, characterized in that: The ZmXET1 The gene's accession number in NCBI is LOC542312.

8. The method according to claim 7, characterized in that: The infection time was 3-5 days, the phosphate buffer concentration was 0.01 M, pH=7.0, and the homogenization ratio was 1-1.5 mL of phosphate buffer per gram of tobacco leaves.

9. The method according to claim 8, characterized in that: The microinjection ratio is 15-20 μL of supernatant injected into each immature embryo, using an inoculation needle inserted at a 60° angle and injected into the immature maize embryo at a depth of 1-2 mm.

10. The method according to claim 9, characterized in that: The maize variety in question is inbred line B73.

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