Application of ZmRBG gene in improving 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 ZmRBG gene in improving resistance of corn to southern rust. An early-stage sequencing result shows that the gene ZmRBG is down-regulated after being infected by puccinia multiflora; a VIGS silent vector pCMV201-ZmRBG is constructed for phenotypic analysis, and it is found that a VIGS silent ZmRBG plant shows that the expression level is reduced, a CMV: ZmRBG plant shows aggravated disease symptoms after inoculation, and meanwhile, the biomass of pathogenic bacteria is increased; compared with a control group, the overexpressed gene ZmRBG shows lighter disease symptoms, and the biomass of pathogenic bacteria is remarkably reduced, so that the gene ZmRBG improves the resistance of the corn to the puccinia polypoda, a new gene resource and a technical path are provided for the cultivation of a new variety of the corn for resisting the southern rust disease, and the gene ZmRBG has important theoretical significance and application value.

Description

Technical Field

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

[0002] As the most widely planted and highest-yielding grain and cash crop in my country, maize holds an irreplaceable strategic position in ensuring national food security and supporting livestock farming and industrial processing. However, the frequent occurrence of maize diseases severely restricts its yield stability and quality improvement. With global climate change, adjustments in farming systems, and the promotion and iteration of varieties, previously minor diseases have gradually become major threats, among which southern maize rust has become one of the catastrophic diseases in maize-producing areas of my country. Developing disease-resistant varieties by utilizing maize's own disease resistance has advantages such as long-lasting control effects, environmental friendliness, and low cost, making it the most economical and effective fundamental approach to addressing the damage caused by southern rust.

[0003] Currently, my country's maize breeding efforts against southern rust face a dual bottleneck: a scarcity of resistance resources and a narrow genetic base. Most resistance materials used in current production originate from tropical inbred lines, with very few directly applicable temperate resistance germplasm. Compared to foreign germplasm, my country's maize germplasm lacks highly resistant materials, mainly concentrated in local varieties or P-group materials with tropical lineage, leading to a prominent problem of single-source resistance. Long-term use of a single resistance source easily induces pathogenic variations in the physiological races of the pathogen, causing resistant varieties to lose their resistance. For example, the United States successfully controlled southern rust for 30 years using the resistance gene Rpp9; however, in recent years, due to variations in the physiological races of the pathogen, this gene has largely lost its resistance in regions such as Africa and Hawaii. Recent research further reveals that the *Heterophyllus multifiliis* rust population in my country has differentiated into highly virulent lineages, capable of escaping the recognition of existing resistance genes, further complicating resistance breeding. Therefore, it is urgent to discover new resistance materials and resistance genes to broaden the foundation of maize resistance germplasm and provide core gene resources for resistance breeding.

[0004] With the development of molecular biology techniques, the identification, cloning, and functional verification of resistance genes have become key means to overcome the bottleneck of resistance sources. The cloning and application of plant disease resistance genes provide core targets for precision breeding technologies such as molecular marker-assisted breeding and transgenic breeding, significantly accelerating the breeding process. For example, patent 202510110770.X provides a... ZmPHYLLThe gene can inhibit the germination of *Rhizoctonia solani* spores, thereby improving the resistance of maize plants to southern rust and providing new gene resources for the creation of breeding materials. Patent 202110676913.5 discloses a maize southern rust resistance gene, RppM. Functional annotation, sequence alignment, and expression level analysis of candidate genes within the RppM location region identified two candidate genes that have the function of improving maize resistance to southern rust, providing new resistance source materials and gene resources for maize resistance breeding. Although some resistance genes have been identified and applied, the number of cloned resistance genes is limited, making it difficult to meet the needs of responding to rapid pathogen mutation and breeding broad-spectrum, durable resistant varieties. In summary, discovering new southern rust resistance genes, elucidating their molecular mechanisms of regulating resistance, and applying them to the breeding of resistant varieties through molecular breeding techniques are urgent needs in the field of maize breeding, and are of great significance for ensuring safe maize production and promoting the high-quality development of the seed industry. Summary of the Invention

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

[0006] The technical solution of this invention is implemented as follows: This invention provides ZmRBG Application of genes in improving maize resistance to southern rust.

[0007] Given that the hyphae of *Russula multifiliis* 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... ZmRBG The concentration was reduced after infection with multiple rust fungi.

[0008] Preferably, the above-mentioned improvement in maize resistance to southern rust is achieved by overexpressing... ZmRBG The gene reduces the severity of southern rust by decreasing the biomass of the pathogen within the maize plant. The pathogen of southern rust is *Russula multifiliis*.

[0009] Secondly, the present invention provides ZmRBG Application of gene overexpression vectors in improving maize resistance to diseases caused by *Hemiberlesia lataniae*.

[0010] Preferably, the above ZmRBG The gene's accession number in NCBI is LOC100194217, (MaizeGDB [https: / / maizegdb.org / ] gene ID: Zm00001eb217560 .

[0011] Thirdly, the present invention also provides a method for breeding transgenic maize resistant to southern rust, the steps of which are: constructing a transgenic maize containing... ZmRBG The gene overexpression vector SCmDu::GFP was transferred into tobacco leaves using Agrobacterium-mediated transformation. After the infected tobacco leaves were homogenized in phosphate buffer, they were rubbed onto maize leaves, and transgenic maize plants resistant to southern rust were obtained after cultivation.

[0012] Preferably, the above ZmRBG The gene's accession number in NCBI is LOC100194217.

[0013] Preferably, the above-mentioned infection involves co-infecting tobacco leaves with a bacterial suspension of SCmDu::GFP along with the silencing repressor P22 and the Enhancer protein; and during the immersion of the tobacco leaves, the bacterial suspension of the SCmDu::GFP infectious clone is OD-treated. 600 Adjust to 1.0-1.4, while co-wetting OD 600 The silencing repressors P22 and OD of tomato chlorosis virus were 0.3. 600 The Enhancer protein is 0.3.

[0014] The infection period is 5-15 days. The homogenization ratio is 1 mL of phosphate buffer per 0.5 g of tobacco leaves. The phosphate buffer concentration is 0.01 M and pH=7.0. SCMV-GFP was successfully transferred to maize by friction inoculation, preferably using B73 maize seedlings that have been transplanted for about one week. The seedlings should not be too large.

[0015] Preferably, the maize variety mentioned above is the inbred line B73.

[0016] 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 ( ZmRBG The gene was downregulated after infection with *Russula multifiliis*. Phenotypic analysis of the gene using virus-induced gene silencing (VIGS) showed that VIGS silencing... ZmRBG The plants showed reduced expression levels, unlike the control, CMV: ZmRBG Plants exhibited more severe disease symptoms after inoculation, along with an increase in pathogen biomass; overexpression of the gene ZmRBG Compared with the control, it showed milder disease symptoms and a significant reduction in pathogen biomass, indicating that ZmRBGPositive regulation of maize's defense against *Heterostilbene multiplostomum* rust; the above results indicate that the gene... ZmRBG Improving maize's resistance to *Hemiberlesia lataniae* provides new genetic resources and technical pathways for the breeding of new maize varieties resistant to southern rust, which has important theoretical significance and application value. Attached Figure Description

[0017] 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.

[0018] Figure 1 This 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] Figure 5 Key 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. ZmRBG Gene expression levels and locations; G represents transiently silenced maize plants. ZmRBG Analysis of gene expression levels using Student's t -Assessing differences between groups:** p <0.05; H represents the multi-stalked rust fungus in CMV: ZmRBG - Symptoms develop on silent corn leaves; I for CMV: ZmRBG Assessment of the relative biomass of *Russula multifiliis* in silent seedlings after fungal inoculation.

[0023] Figure 6 Key genes involved in maize's defense against *Stylosporium multistachys* rust; Figure A shows the *Stylosporium multistachys* rust challenged with *Stylosporium multistachys*: ZmRBG Symptoms of overexpression in maize leaves; B represents SMV: ZmRBG In overexpressing plants and controls ZmRBG Analysis of gene expression levels using Student's t -Statistical analysis was performed using the test: *** p<0.001; C is SMV: ZmRBG - Assessment of the relative biomass of maize leaves after overexpression following infection with *Russula multifiliis* fungus.

[0024] 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.

[0025] 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.

[0026] Figure 9 The diagram shows the construction process of the pSCmDu::GFP viral expression plasmid; where Figure A is the vector pSCmDu and Figure B is the recombinant vector pSCmDu::GFP.

[0027] Figure 10 This is a plasmid map of pSCmDu::GFP.

[0028] Figure 11 The images show pSCmDu::GFP inoculation of Nicotiana Bunsenata and transfected maize. Figure A shows the infection phenotype in maize and sorghum 15 days after inoculation, Figure B shows the subcellular localization of Nicotiana Bunsenata, and Figure C shows the fluorescence observation of SCmDu::GFP inoculation of maize and sorghum. Detailed Implementation

[0029] 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.

[0030] 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.

[0031] 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).

[0032] 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. Significant differences were determined using Student's t-test or ordinary one-way ANOVA, with statistical significance defined by p-value. snRNA-seq analysis utilized three biological replicates, while stRNA-seq utilized four biological replicates. For differentially expressed gene identification and GO enrichment analysis, p-values ​​and q-values ​​adjusted for false discovery rate were applied. All statistical analyses were performed using the R programming language.

[0033] Example 1: Establishment of infection of *Russula multistachys* in maize seedling leaves

[0034] Plant material and inoculation and collection of multi-stalked rust fungi:

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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 1 A). 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).

[0039] 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.

[0040] Example 2: Establishment of cell types in maize leaves infected with *Russula multistachys*

[0041] 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.

[0042] 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).

[0043] 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.

[0044] 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).

[0045] 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.

[0046] 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 2 A). 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 3B). 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).

[0047] 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.

[0048] Example 3: Identification of key genes involved in maize's defense against *Heterostilbene multistachys* rust.

[0049] 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 (ZmRBG), Zm00001eb054050, and Zm00001eb165310 were upregulated after infection with *Russula multifiliis*, while Zm00001eb226470 was downregulated. Figure 5 E). 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.

[0050] Application Example 1

[0051] 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 ZmRBG. 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-ZmRBG construct. The recipient maize variety used in this application is the inbred line B73.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] Table 1 Primers used in this application

[0056]

[0057] Phenotypic analysis was performed on the genes related to Example 3 using virus-induced gene silencing. The maize materials developed in this study showed that ZmRBG plants with VIGS silencing exhibited reduced expression levels. Figure 5 G). Unlike the control, CMV:ZmRBG plants showed more severe disease symptoms after inoculation. Figure 5 H), while the biomass of pathogens increases ( Figure 5 I). These results collectively demonstrate that these two genes promote maize resistance to *Hemiberlesia lataniae* through different regulatory mechanisms, and also validate the reliability of the multi-omics data.

[0058] Application Example 2

[0059] Using maize exhibiting mild mosaic symptoms, which was incidentally discovered during a large-scale field survey, as material, the full-length cDNA of the viral genomic RNA was identified and cloned, ligated into the pCB301 plant binary expression vector, and transformed into Agrobacterium tumefaciens, which could infect maize and sorghum. The SCMV-mDu infectious cDNA clone pSCmDu was successfully constructed. Further, GFP coding regions with flanking single enzyme restriction sites were inserted into the viral genome of pSCmDu to obtain pSCmDu::GFP, which facilitated vector linearization and the ligation of exogenous gene coding regions. This system was named SCMV-mDu VOX. The SCMV-mDu VOX system is suitable for efficient protein expression in monocotyledonous C4 plants (such as maize, sorghum, millet, and sugarcane). SCMV-mDu infection in maize and sorghum causes mild symptoms and has minimal interference with plant phenotypes after protein overexpression, making it suitable for expressing proteins and small peptides. The SCMV-mDu VOX system only requires inserting the coding region of the gene under study (ZmRBG) into the vector for efficient and systematic gene expression. The method of this invention is simple, fast, efficient, low-cost, and does not require genetic transformation.

[0060] The plasmid pSCmDu::GFP was constructed using RNA from SCMV-mDu, an isovirus isolate from field-infected maize that causes only mild mosaic symptoms, as a template. The full-length cDNA of its genomic RNA was amplified by RT-PCR and then cloned into the pCB301 vector to obtain the intermediate plasmid pSCmDu. The nucleotide sequence corresponding to the protease cleavage site between NIb / CP at positions 10153-10179 of pSCmDu was doubled, and a GFP coding region sequence digested with Bln I and Stu I was inserted between the doubled sequences to obtain the plasmid pSCmDu::GFP. Figure 9-10 This plasmid can be used as a control vector, and can also be used to linearize the plasmid by double digestion of pSCmDu::GFP with Bln I and Stu I to remove GFP and then insert the foreign gene coding region.

[0061] (1) Plasmid extraction: pSCmDu::GFP plasmid (sequence shown in SEQ ID No.1) can be transformed into E. coli DH5α normally. However, since it is a low-copy plasmid, it is generally recommended to extract about 6 mL of bacterial culture after overnight shaking at 37℃ for plasmid extraction (3-5 mL is generally sufficient for high-copy plasmid extraction). At the same time, refer to the instructions of the plasmid extraction kit to avoid adding too much bacterial culture. The expected concentration of plasmid extraction is about 100 ng / µL (dissolved in 50 µL ddH2O).

[0062] This plasmid map has been provided in one aspect, which can be viewed according to the map ( Figure 10Primers were designed to enrich the GFP insertion site in the plasmid, and a sequencing reaction was performed to verify the plasmid sequence. On the other hand, 10 µL of the extracted pSCmDu::GFP plasmid was transformed into common strains such as Agrobacterium C58C1 or GV3101 to confirm infectivity.

[0063] (2) Vector digestion

[0064] Enzymatic digestion is performed using TAKARA's rapid digestive enzymes (Bln I and Stu I). The digestion system can be set as follows:

[0065] pSCmDu::GFP plasmid (approximately 100 ng / µL): 15 μL; 10×Buffer: 4 μL; Bln I: 2 μL; Stu I: 2 μL; add water to 40 µL; react at 37℃ for 4-6 h (the amount of enzyme used was increased and the reaction time was extended).

[0066] After enzyme digestion, the digestion product needs to be treated at 85℃ for 15 min to denature and inactivate Bln I and Stu I; the product after treatment is used as a linearized vector.

[0067] (3) The exogenous fragment was ligated into the pSCmDu::GFP linearized vector.

[0068] The connection system is as follows:

[0069] ZmRBG gene fragment (150-200 ng): 2 μL; linearized pCB301 vector (~100 ng): 3 μL; 2×Basic Assembly Mix: 5 μL; reaction at 50℃ for 30 min.

[0070] (4) Soaking of native tobacco and grafted corn

[0071] After extracting plasmids (kanamycin resistant) from the selected positive clones, they can be transformed into either Agrobacterium C58C1 or GV3101. Through experimentation, OD500 of the bacterial suspension containing the SCmDu::GFP infectious clone was successfully applied to tobacco leaves. 600 Adjust to 1.0-1.4, while co-wetting OD 600 The silencing repressors P22 and OD of tomato chlorosis virus were 0.3. 600 The Enhancer protein concentration was 0.3 (the above three Agrobacterium tumefaciens bacterial cultures all showed resistance to kanamycin and rifampin; it is essential to co-infiltrate with P22 and Enhancer, otherwise the SCmDu::GFP infectious clone will accumulate at a very low level in tobacco cells, firstly, fluorescence will not be visible under UV light, and the efficiency of corn transfer will also be very low). After at least 5 days, the infiltrated area of ​​the Tobacco Bunsenii leaf showed visible green fluorescence under UV light. Figure 11After 5 to 15 days of soaking, the leaves of *Tobacco Bunsenii* are homogenized and then successfully transferred to corn via friction inoculation (preferably using B73 corn seedlings that have been transplanted for about one week; the seedlings should not be too large; the success rate of this invention's SCmDu::GFP transfer from tobacco to corn is 50%-80%; if there are problems with this transfer step and the efficiency is too low, you can try using a few diseased corn seedlings as the virus source to inoculate the corn seedlings, i.e., corn inoculating corn. This inoculation method will greatly improve the efficiency. For this method, it is necessary to check in advance with RT-PCR to ensure that the corn used as the virus source has not lost the inserted fragment). The ratio for transfer is 0.5 g of leaves added to 1 mL of phosphate inoculation buffer.

[0072] 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 ZmRBG gene expression. Figure 6 B), and after inoculation with *Sclerotium styracifolium*, plants with virus-based gene overexpression (VOX) (SMV:ZmRBG) showed milder disease symptoms compared to the control. Figure 6 A). Further analysis showed that the pathogen biomass in overexpressing plants was significantly reduced, indicating that ZmRBG negatively regulates maize's defense against *Rust moniliforme*. Figure 6 C). These results collectively demonstrate that the ZmRBG gene can enhance maize's resistance to *Hemiberlesia lataniae*, and also validate the reliability of the multi-omics data.

[0073] 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. ZmRBG Application of genes in improving maize resistance to southern rust.

2. The application according to claim 1, characterized in that: The improvement of maize's resistance to southern rust is achieved by overexpressing... ZmRBG The gene reduces the severity of southern rust by decreasing the biomass of the southern rust pathogen in the corn plant.

3. The application according to claim 2, characterized in that: The pathogen causing southern rust is *Russula multifiliis*.

4. ZmRBG Application of gene overexpression vectors in improving maize resistance to diseases caused by *Hemiberlesia lataniae*.

5. The application according to any one of claims 1-4, characterized in that: The ZmRBG The gene's accession number in NCBI is LOC100194217.

6. A method for breeding transgenic maize resistant to southern rust, characterized in that, The steps are as follows: Construct a system containing ZmRBG The gene overexpression vector SCmDu::GFP was transferred into tobacco leaves using Agrobacterium-mediated transformation. After the infected tobacco leaves were homogenized in phosphate buffer, they were rubbed onto maize leaves, and transgenic maize plants resistant to southern rust were obtained after cultivation.

7. The method according to claim 6, characterized in that: The ZmRBG The gene's accession number in NCBI is LOC100194217.

8. The method according to claim 7, characterized in that: The infection process involved co-infecting tobacco leaves with SCmDu::GFP bacterial suspension, along with the silencing repressor P22 and Enhancer protein; the OD of the SCmDu::GFP bacterial suspension... 600 It is 1.0-1.

4.

9. The method according to claim 8, characterized in that: The infection time is 5-15 days, and the homogenization ratio is 1 mL of phosphate buffer per 0.5 g tobacco leaves. The phosphate buffer concentration is 0.01 M and pH=7.

0.

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

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