Application of RWTD1 gene in prevention and control of sugarcane smut

Abstract

The invention discloses application of an RWTD1 gene in prevention and control of sugarcane smut. The RWTD1 gene is a key factor for regulating sexual coordination of pathogenic bacteria and development of pathogenic symptoms (black whips). Through functional verification, pathogenic bacteria cannot complete the whole life history after RWTD1 gene deletion: even if the pathogenic bacteria can infect hosts, plants cannot be induced to form black whips and generate teliospore, so that a disease transmission chain is cut off. On the basis, the RWTD1 gene, the encoding protein thereof or a related regulation and control sequence are used as prevention and control targets, various application ways including RNA interference, gene editing, chemical inhibition and disease-resistant breeding are provided, and a core molecular basis and a brand new solution are provided for developing a new efficient, accurate and environment-friendly sugarcane smut prevention and control technology.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control technology, and particularly relates to a... RWTD1 Application of genes in the control of sugarcane smut. Background Technology

[0002] sugar cane( Saccharum officinarum Sugarcane (L.) is one of the world's most important sugar and bioenergy crops, and its production is of strategic significance for ensuring sugar security and sustainable energy development. my country is a major sugarcane producer, with sugarcane production accounting for approximately 90% of the country's total sugar production. However, sugarcane production is constantly threatened by various diseases, among which *Ustilago maydis* (sugarcane smut fungus) is a major threat. Sporisorium scitamineum Sugarcane smut, caused by [unspecified pathogen], is a devastating systemic fungal disease that is prevalent in sugarcane-growing regions worldwide, causing severe yield and sugar content losses. The typical symptom of this disease is the abnormal differentiation of the meristem at the apex of the sugarcane stalk induced by the pathogen, forming a black, whip-like structure (commonly known as the "black whip"). The black whip produces numerous teliospores, which, upon maturation, are dispersed by wind, becoming the primary source of infection for the following year's disease spread. In addition to the formation of the black whip, infected plants also exhibit abnormal phenotypes such as premature budding, increased tillering, thin and elongated stems, and narrow, chlorotic leaves, severely impacting the normal growth and development of sugarcane.

[0003] The life cycle of *Ustilago maydis* is complex and polymorphic. Its infection cycle begins with the sexual mating of haploid basidiospores of different mating types (Mat1 and Mat2), forming infective dikaryotic hyphae. These hyphae invade the sugarcane buds, grow and expand within the host tissue, ultimately stimulating the formation of black flagella, where teliospores differentiate and mature. Therefore, sexual mating of the pathogen is a crucial step in establishing infection, while the formation of black flagella and teliospores is the core link in the completion and spread of the disease cycle. Currently, the control of sugarcane smut mainly relies on the selection and planting of resistant varieties. However, the physiological races of the pathogen are prone to variation, often leading to the loss of resistance in previously resistant varieties. For example, the once widely planted resistant varieties YueTang 85-177 and XinTaiTang 10 became susceptible varieties due to changes in their physiological races (Shen Wankuan, 2004). Chemical control has limited effectiveness and poses environmental residue risks, while biological control is still in the research and development stage and has not yet been widely applied. Therefore, in-depth analysis of the pathogenic mechanism of *Ustilago maydis* at the molecular level, especially the key regulatory pathways of its sexual mating and black flagella development, is urgently needed for developing novel, efficient, and durable disease control strategies.

[0004] In our previous research on gene expression patterns before and after sexual mating in *Ustilago maydis*, our team discovered a gene unique to *Ustilago maydis*, named... RWTD1 ( R egulation ofW hipand T eliospore D The evelopment gene 1 is specifically highly expressed in binucleate hyphae formed after sexual mating. This gene encodes a protein predicted to contain four transmembrane domains, suggesting that it may regulate sexual mating in *Ustilago maydis*, but there have been no previous reports on the function of this protein or its homologs.

[0005] Based on this, further research RWTD1 The function of this gene in *Ustilago maydis* not only fills a gap in the functional characterization of the tetraspan membrane protein in this fungus, deepening our understanding of sexual mating, pathogenic mechanisms, and disease symptom formation, but also holds promise for revealing a key new regulatory gene that comprehensively regulates cell membrane-related proteins in the pathogen during sexual mating. Based on this gene, the development of new methods, agents, and products for controlling sugarcane smut by interfering with its function provides a direct theoretical basis and technological foundation. This is of great significance for solving current industry challenges in sugarcane smut control, such as the easy loss of resistant varieties and the lack of effective control methods. Summary of the Invention

[0006] To solve the above-mentioned technical problems, the present invention proposes a... RWTD1 The application of genes in the control of sugarcane smut, this invention clarifies RWTD1 The crucial role of this gene in the pathogenesis of *Ustilago maydis*. Experiments have shown that this gene is essential for sexual mating in the pathogen; more importantly, its function is a key switch driving black flag formation and teliospore differentiation. In mating strains of the pathogen Mat2... RWTD1 When this gene is deleted, a highly valuable phenotype is produced: the pathogen can still infect the host sugarcane normally, but it completely loses its ability to induce black flagellar formation and produce teliospores within the host. This phenotype provides a precise intervention point for effectively blocking the disease transmission cycle without completely eradicating the pathogen. Furthermore, when both mating strains lack this gene, the incidence of pathogen infection in sugarcane decreases from 91% to 0%, and the infection rate decreases from 95.5% to 7.10%. While reducing the incidence, this significantly reduces or even prevents the formation of pathogen teliospores, which are conducive to disease transmission, thus lowering the initial source of infection.

[0007] To achieve the above objectives, the present invention provides a method for controlling sugarcane smut, the method comprising inhibiting *Ustilago maydis* (sugarcane smut fungus). Sporisorium scitamineum )middle RWTD1 The function and / or expression of the gene, wherein RWTD1 The nucleotide sequence of the gene is shown in SEQ ID No. 1, or it encodes a protein with the same function.

[0008] Furthermore, the inhibition is achieved by applying a substance targeting the sugarcane, the sugarcane growing environment, or *Ustilago maydis*. RWTD1 This can be achieved through RNA interference molecules, antisense nucleic acids, or gene editing systems.

[0009] Furthermore, the inhibition is achieved by applying a compound capable of inhibiting the activity of the RWTD1 protein to sugarcane, the sugarcane growing environment, or *Ustilago maydis*.

[0010] Furthermore, the inhibition resulted in the suppression of black flag formation and / or teliospore development after *Ustilago canis* infection of sugarcane.

[0011] Furthermore, the inhibition resulted in *Ustilago maydis* being able to infect sugarcane but failing to induce the production of black whips.

[0012] This invention also provides RWTD1 The application of genes or their encoded proteins in the control of sugarcane smut, wherein... RWTD1 The nucleotide sequence of the gene is shown in SEQ ID No. 1, or it encodes a protein with the same function.

[0013] The present invention also provides a kit for controlling sugarcane smut, the kit comprising ingredients for inhibiting the growth of *Ustilago maydis*. RWTD1 Active ingredients that target gene function and / or expression, said active ingredients being selected from: RWTD1 RNA interference molecules, antisense nucleic acids, gene editing systems, or compounds that can inhibit the activity of the RWTD1 protein.

[0014] The present invention also provides a recombinant microorganism comprising the ability to express targeted *Ustilago maydis*. RWTD1 An expression vector for an RNA interference molecule or antisense nucleic acid of a gene, or an expression vector containing a protein capable of expressing an inhibitory effect on RWTD1 protein activity, wherein... RWTD1 The nucleotide sequence of the gene is shown in SEQ ID No. 1, or encodes a protein with the same function; the recombinant microorganism is a bacterium or fungus.

[0015] The present invention also provides a molecular marker for identifying or monitoring the control effect of sugarcane smut, wherein the molecular marker is *Smutella spp.* RWTD1 Gene expression levels or functional states, among which RWTD1 Downregulation or inactivation of gene expression indicates effective prevention and treatment.

[0016] The present invention also provides a method for cultivating sugarcane plants resistant to smut, the method comprising introducing into the sugarcane a substance that expresses... RWTD1The constructs of the gene's dsRNA, siRNA, or shRNA are used to produce a targeted strain of *Ustilago maydis* in sugarcane. RWTD1 Interfering RNA in genes.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects: The technical effect of this invention is primarily reflected in its direct and efficient blocking of the transmission cycle of sugarcane smut. This is achieved by inhibiting the growth of sugarcane smut fungus. RWTD1 The gene's function effectively interferes with the sexual mating process of pathogens, weakening their infectivity. More significantly, even if the pathogen successfully colonizes, this intervention specifically and thoroughly inhibits the formation of black flagella and the differentiation of teliospores, thereby eliminating the production of disease-transmitting agents (teliospores) at the source. This unique control phenotype of "infecting but not flagellating" means that the core objective of interrupting the disease cycle and preventing disease spread can be achieved without completely eradicating the pathogen, providing a new path for developing efficient and environmentally friendly sustainable control strategies.

[0018] From a molecular mechanism perspective, the technical effect of this invention stems from precise intervention in key pathogenic pathways of pathogenic bacteria. The RWTD1 protein is located in an intracellular vesicle-like structure; its loss of function disrupts downstream key genes involved in sexual coupling (such as...). PRM1 The normal expression of pheromones leads to the blockage of the pheromone signaling pathway. Meanwhile, RWTD1 The absence of these substances causes pathogens to lose their ability to manipulate hormone balance within the host, preventing the accumulation of growth-promoting auxins and cytokinins at the site of infection. Instead, it may enhance the signaling of host defense-related hormones (such as salicylic acid and jasmonic acid), thereby synergistically enhancing the plant's own immune response while inhibiting symptom development.

[0019] Furthermore, the technical solution of this invention has the outstanding advantages of diverse applications and strong targeting. Based on the specific target RWTD1, various innovative control methods can be derived, including specific small molecule inhibitors, RNA interference biological agents, gene editing breeding, and engineered microorganisms. Compared with traditional broad-spectrum fungicides or strategies relying on single resistant varieties, this precise intervention targeting key pathogenic genes helps reduce the environmental impact of pesticides, delays the development of drug resistance in pathogens, and provides a solid molecular basis and novel genetic resources for breeding new sugarcane varieties with durable disease resistance. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0021] Figure 1 for RWTD1 RT-qPCR validation diagram of gene expression levels in haploid and binucleate hyphae.

[0022] Figure 2 A schematic diagram of the predicted topological structure of the transmembrane domain of the RWTD1 protein.

[0023] Figure 3 Phylogenetic tree of RWTD1 protein and its homologous proteins from other species.

[0024] Figure 4 Electrophoresis image showing the construction and enzyme digestion verification of the pEX1-g6182-RWTD1-GFP fluorescent fusion expression vector. Wherein, M: Gene Ruler TM l kb Plus DNA ladder; 1: GFP fragment; 2: RWTD1 Segment 3: RWTD1 4: GFP fusion fragment; 5: pEX1-g6182; 6: pEX1-g6182-RWTD1-GFP; 7: pEX1-g6182-RWTD1-GFP enzyme digestion verification (enzymes used: Pac I).

[0025] Figure 5 Fluorescence micrographs showing the subcellular localization of RWTD1 protein in different cells of *Ustilago maydis*. A: RWTD1 Bright-field, fluorescence, and overlay images of the protein in haploid basidiospore cells. B: RWTD1 Bright-field, fluorescence, and overlay images of proteins in binucleate hyphae and teliospore cells.

[0026] Figure 6 for RWTD1 Figure 1 shows the molecular validation results of the gene knockout strain. A: Schematic diagram of the PCR primer positions used for knockout strain validation. B: Electrophoresis diagrams of PCR detection of wild-type and knockout strains using the internal gene primers RWTD1-F / RWTD1-R, the left outer primer RWTD1-1.6zF, and the hygromycin gene internal primer hyg-TestR; and using the hygromycin gene internal primer hyg-TestF and the right outer primer RWTD1-1.6yR.

[0027] Figure 7This is a schematic diagram of the construction and validation electrophoresis of the pNGR-ble-RWTD1 complementation vector. A: Fragment amplification schematic. B: M: Gene Ruler. TM l kb Plus DNA ladder; 1: Completion fragment; 2: Hind III: Linearized pNGR-ble; 3: pNGR-ble-RWTD1. C: 1: Control; 2-4: pNGR-ble-RWTD1 colony PCR verification.

[0028] Figure 8 for RWTD1 PCR and RT-qPCR validation results of the reinstated strain. Among them, A: C35- RWTD1 PCR verification of partial gene sequence fragments from the reinstated strain. B: C36- RWTD1 PCR verification of partial gene sequence fragments from the reinstated strain. C:C35- RWTD1 qPCR analysis of gene complementation strains. D: C36- RWTD1 qPCR analysis of gene complementation strains. t-test data are used; * indicates significant data. p <0.05, ** indicates highly significant. p <0.01.

[0029] Figure 9 for RWTD1 Visual representation of the sexual mating phenotype of the gene knockout strain. A: Panoramic view of sexually mating colonies. B: RWTD1 Cell morphology of the knockout strain after sexual mating (optical microscope).

[0030] Figure 10 For missing RWTD1 RT-qPCR analysis of the effects of genes on the expression of key genes involved in sexual mating in *Ustilago canis*. Actin For internal reference genes, relative quantification was used. - Ct The relative expression levels of genes were calculated using a t-test, and differences in the same gene were compared between the control and experimental groups. Data were processed using a t-test; * indicates significance. p <0.05, ** indicates highly significant. p <0.01.

[0031] Figure 11 for RWTD1 Cell morphology of the gene complemented strain after sexual mating (optical microscope).

[0032] Figure 12 for RWTD1 Statistical curve of the occurrence process of smut after inoculation of sugarcane with gene knockout strains.

[0033] Figure 13 for RWTD1 Image of sugarcane plants infected with gene knockout strains.

[0034] Figure 14 for RWTD1 Statistical curve of the occurrence process of smut after inoculation of sugarcane with gene complementation plants.

[0035] Figure 15 for RWTD1 Principal component analysis diagram of transcriptome samples from the sexual mating process of knockout strains.

[0036] Figure 16 The image shows the RT-qPCR validation results of differentially expressed genes in *Ustilago maydis*.

[0037] Figure 17 Wild type and RWTD1 Venn diagram and GO enrichment analysis of differentially expressed genes before and after sexual mating in knockout strains. A: Wild-type strain and... RWTD1 Venn analysis of differentially expressed genes before and after sexual mating in knockout strains. B: GO enrichment analysis of 512 genes differentially expressed only in WT_72h vs WT_0h.

[0038] Figure 18 This is a weighted gene co-expression network analysis diagram of genes related to sexual mating in *Ustilago maydis*. A: Gene clustering analysis of *Ustilago maydis*. Based on gene expression levels, it was divided into 6 gene modules. B: Correlation analysis between gene modules and phenotypes. The MEtuiquoise module showed a positive correlation with WT_72h (r=0.753). p =0.0047).

[0039] Figure 19 Venn analysis and cluster heatmaps of core candidate differentially expressed genes. A: Venn analysis of differentially expressed genes 181_up, GO_152, and MEturquoise_558. B: Cluster heatmap of 37 core differentially expressed genes from the Venn analysis.

[0040] Figure 20 Candidate genes g_006207 , g_003363 and g_005785 A bar chart showing the expression analysis of RT-qPCR at the binucleate hyphae stage.

[0041] Figure 21 A bar chart showing the effect of smut fungus infection on the auxin (IAA) content in sugarcane shoot tip tissue.

[0042] Figure 22This is a bar chart showing the content of four cytokinins in sugarcane shoot tip tissue after infection by *Smutella smut*. A: Trans-zeatin (T-Zeatin) content. B: Zeatin content. C: Zeatin nucleoside (TZR) content. D: Isopentenyl adenosine (IPA) content.

[0043] Figure 23 This is a bar chart showing the effects of *Smutella smut* infection on the content of other plant hormones in sugarcane shoot tip tissue. A: Gibberellin (GA4) content; B: Abscisic acid (ABA) content; C: Salicylic acid (SA) content; D: Jasmonic acid (JA) content.

[0044] Figure 24 Wild-type of *Ustilago maydis* and RWTD1 TaqMan quantitative PCR analysis of the biomass of the deletion mutant in sugarcane. Where A: bE2 Gene quantification standard curve. B: Pathogens in each sample. bE2 Bar chart showing the results of gene copy number quantification. Detailed Implementation

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0047] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0048] This invention focuses on a newly identified tetratransmembrane protein-coding gene in *Ustilago maydis*. RWTD1 Through a series of genetic, cell biological, transcriptomic, and physiological and biochemical experiments, the core role of this gene in regulating the sexual mating, pathogenicity, and development of black whip symptoms and teliospore formation of pathogens was systematically elucidated, and the feasibility of using it as a target for disease control was verified.

[0049] The primer pair sequences used in this invention are as follows: Example 1: RWTD1 Gene mining and bioinformatics analysis After comparing and analyzing the transcriptome sequencing of wild-type strain colonies before sexual mating (haploid basidiospore state) and after mating (binkaryotic hyphae state), we screened out a gene g_000709 that is related to... RWTD1 The gene was expressed at a low level during the basidiospore stage but its expression was significantly upregulated during the binucleate hyphae stage. RT-qPCR validation confirmed that the expression level of this gene in the wild-type binucleate hyphae stage (JG35×JG36) was upregulated by approximately 20% compared to the haploids (JG35, JG36) (results are shown in Figure 1). Figure 1 (As shown). This gene is 780 bp in length, contains one intron and two exons, and encodes a protein of 241 amino acids. It has no known conserved protein domains. Later, it was discovered that it participates in inducing the formation of black flagellates and teliospores, and was named... RWTD1 (Regulation of Whip and Teliospore Development gene 1). This protein is conserved in *Ustilago maydis*, and its domains are predicted to have four transmembrane regions. Its nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0050] SEQ ID NO.1: atgactttcctccataccggtcaaacgcaagcccaagtaaatcagatcctctacaagctcatctacgttcccgaaacggcggatcatcgaacgtggctcctcatcagccaattcgagtgccttctctccctcgtcgccgctttgctcatcatcatcaagaagcagagtttcggaaagctctggttggtttccagaaaggactcttccttcgggtcgatttacgtcaccaatgcgattctctgcctgatcttgggagtggcaagctatctgctggcttggacggtgatgtcgatcctcgaatgggtcgtctcgctattctacgtcgccctgcttgtcttttgcacgcccgccttcggttgtctacccatttttatcgtcgccaactcatttcctcgtcagctgcacacgggcgatttcagcagtacccttcgcacggctgtgatactggtcagcgtattcatgtttgccatgtgtgcctttctcatctccttctgcacggttgtcacgctggacccgctgttccgctcggcgattggcctcaatgtgctgcggaaccaaattccgatcgatgtgaagctcgtcgaacgtcaggatggacaggacgacgtggaagtcgccgcgatagagcaggacgggttgaggatcaatgtggagcatgtggagcaggtcgagcaggtcgagaagcggactctcgggaaaagcctctgccagcacgctctcgagtag SEQ ID NO.2: MTFLHTGQTQAQVNQILYKLIYVPETADHRTWLLISQFECLLSLVAALLIIIKKQSFGKLWLVSRKDSSFGSIYVTNAILCLILGVASYLLAWTVMSILEWVVSLFYVALLVFCTPAFGC LPIFIVANSFPRQLHTGDFSSTLRTAVILVSVFMFAMCAFLISFCTVVTLDPLFRSAIGLNVLRNQIPIDVKLVERQDGQDDVEVAAIEQDGLRINVEHVEQVEQVEKRTLGKSLCQHALE Structural prediction of the RWTD1 protein using online tools revealed that it contains four transmembrane domains (located at amino acids 32-51, 73-99, 105-127, and 147-170, respectively), with its N-terminus and C-terminus both located outside the plasma membrane (transmembrane structure prediction is shown in Figure 1). Figure 2 (As shown). Subcellular localization predicted it to be an integrated membrane protein anchored in the lipid bilayer. Through protein sequence homology alignment and phylogenetic analysis, it was found that the RWTD1 protein of *Ustilago canis* was distantly related to homologous proteins from other *Ustilago canis* species (phylogenetic tree shown). Figure 3 (As shown in the image). The above analysis indicates that RWTD1 is a membrane protein with low conservation in Ustilago may be involved in membrane-related biological processes.

[0051] Example 2: Subcellular localization of RWTD1 protein To investigate the intracellular action site of the RWTD1 protein, we constructed a fusion expression vector of RWTD1 and green fluorescent protein (GFP) and transformed it into *Ustilago maydis* for observation.

[0052] Using cDNA from wild-type strain JG36 as a template, the stop codon was removed and amplified using primers RWTD1-PacI-F / RWTD1-GFP-R. RWTD1 Coding sequence (CDS). Using pEX1-g6182-GFP plasmid as a template, the GFP sequence was amplified using primers GFP-F / GFP-Pac1-R. The two fragments were ligated by fusion PCR and then cloned into a [polymer / polymer / pac1-R]. Pac In the linearized pEX1-g6182-GFP vector, the expression vector pEX1-g6182-RWTD1-GFP with C-terminal fusion was constructed (e.g., ...). Figure 4 (As shown). The successfully constructed vector and the empty vector control (pEX1-g6182-GFP) were transformed into Agrobacterium AGL1, and then transformed into wild-type haploid basidiospores JG35 and JG36 of *Ustilago maydis* using Agrobacterium-mediated transformation. Stable transformants were obtained after hygromycin resistance screening.

[0053] Fluorescence signals were observed using laser confocal scanning microscopy. In basidiospores expressing the RWTD1::GFP fusion protein (JG35-RWTD1::GFP and JG36-RWTD1::GFP), the green fluorescence signal was specifically localized on round or oval vesicle-like structures near the cell membrane (e.g., Figure 5 As shown in A); while in the control strain expressing only free GFP, the fluorescence signal was evenly distributed throughout the cytoplasm and nucleus (as shown in A); Figure 5 As shown in A). Further observation of the binucleate hyphae formed after sexual mating revealed that in the hyphae formed by the RWTD1::GFP fusion expression strain, green fluorescent vesicle structures distributed along the hyphae could still be clearly observed (e.g., ...). Figure 5 As shown in B), while in the binucleate hyphae of the control strain, GFP protein markers were widely distributed in the cytoplasm and nuclear compartments (e.g., as shown in B). Figure 5 As shown in B).

[0054] Furthermore, observation of teliospores collected from black flagella formed by inoculation with RWTD1::GFP strain revealed multiple tiny green fluorescent granules in their cytoplasm, a stark contrast to the uniform green fluorescence in control teliospores (e.g., ...). Figure 5 (As shown in B). The results of this example indicate that the RWTD1 protein is specifically located in membrane-bound intracellular vesicular organelles in the basidiospores, binucleate hyphae, and teliospores of *Ustilago maylis*, suggesting that it may be involved in important cellular processes such as membrane transport or signal transduction.

[0055] Example 3: RWTD1 Construction and validation of gene knockout mutants To explore RWTD1 To understand the biological function of this gene, we used a homologous recombination substitution strategy to construct knockout mutants of this gene in two mating types, Mat1 (JG36) and Mat2 (JG35).

[0056] First, using wild-type strain genomic DNA as a template, PCR amplification was performed separately. RWTD1 The left homologous arm (LB) 1500 bp upstream and the right homologous arm (RB) 1500 bp downstream of the gene coding region were amplified. Simultaneously, the hygromycin B resistance gene was amplified from plasmid pEX1. HYG ) two fragments HYG -up and HYG -down. Then set LB and... HYG -up、 HYG -down was PCR fused with RB to obtain two fragments for homologous DNA replacement.

[0057] The two homologous substitution fragments were introduced into the protoplasts of wild-type haploid basidiospores JG35 and JG36 of *Ustilago maydis* using a polyethylene glycol (PEG)-mediated protoplast transformation method. After two rounds of screening with regeneration medium containing hygromycin B, resistant and stable candidate transformants were obtained.

[0058] Candidate knockout strains were validated using multi-level PCR and RT-qPCR (the locations of the validation primers are shown in the diagram). Figure 6 (As shown in A). Genotyping analysis showed that primers located on the outer side of the left homologous arm were used. RWTD1 PCR using primer hyg-TestR, located within the hygromycin resistance gene, at -1.6F amplified a specific band of approximately 2 kb in the knockout strain, while this band was absent in the wild type (e.g., ...). Figure 6 (As shown in B); using primer hyg-TestF located inside the hygromycin resistance gene and primer located outside the right homologous arm. RWTD1 PCR using -1.6R amplified a specific band of approximately 2 kb in the knockout strain, while this band was absent in the wild type (e.g., Figure 6 (As shown in B). Conversely, using the method targeted at... RWTD1 PCR using primers RWTD1-F / RWTD1-R for detecting the internal sequence of the gene only amplifies a specific target band of approximately 500 bp in wild-type individuals, while non-specific amplification occurs in knockout strains (e.g., ...). Figure 6 (As shown in B). The above results fully demonstrate that we have successfully obtained... RWTD1 Homozygous knockout mutants with complete gene deletion were named Δ35- RWTD1 (Mat2 mating type) and Δ36- RWTD1 (Mat1 mating type).

[0059] Example 4: RWTD1 Construction and validation of gene complementation mutants To confirm RWTD1 The phenotype observed in the knockout strain was indeed due to RWTD1 Caused by gene deletion, and excluding the influence of random mutations, we constructed... RWTD1 Gene replacement strain.

[0060] Using the genomic DNA of the wild-type strain as a template, primers RWTD1-HindIII-F / RWTD1-HindIII-R were used to amplify the sequence containing its own promoter, complete coding sequence, and terminator via PCR. RWTD1 Restore the fragment. Clone the fragment to the previous one. Hind III. In the linearized binary vector pNGR-ble carrying the bleomycin resistance marker, the complementation vector pNGR-ble-RWTD1 was constructed (see schematic diagram of the construction process). Figure 7As shown in Figure A). Verification was performed via enzyme digestion (e.g., Figure 7 After confirming the results (as shown in B) and sequencing, the bacteria were transformed into Agrobacterium tumefaciens AGL1 via electroporation, and positive Agrobacterium engineered strains were obtained by colony PCR verification (e.g., shown in B). Figure 7 (as shown in C).

[0061] Using Agrobacterium-mediated transformation technology, the complementation vector was introduced into... RWTD1 Knockout strain Δ35- RWTD1 and Δ36- RWTD1 Transformants were obtained through bleomycin-resistant plate selection. PCR verification using the specific primers RWTD1-F / RWTD1-R confirmed the positive result for the reintroduced strain C35-. RWTD1 and C36- RWTD1 Both strains amplified bands of the same size as the wild type (approximately 700 bp), while knockout strains did not exhibit this band (e.g., Figure 8 (As shown in A and 8B). RT-qPCR analysis further confirmed that the reinjected strains contained... RWTD1 The mRNA expression of the gene is restored, and even overexpression occurs (e.g.) Figure 8 (As shown in C, 8D).

[0062] Example 5: Missing RWTD1 Effects on sexual mating and filamentous growth of pathogens To clarify RWTD1 To investigate whether the gene is involved in regulating the sexual pairing of *Smuts spores*, we paired knockout strains, replacement strains, and wild-type strains to observe their mycelial formation ability.

[0063] Equal amounts of different mating strains were mixed and inoculated onto YEPSA solid medium and cultured at 28°C for 3-7 days. Phenotypic observation revealed that the wild-type control combination (JG35×JG36) and Δ35- RWTD1 The ×JG36 combination could form white, fluffy, dikaryotic mycelia after 72 hours of cultivation, but Δ35- RWTD1 The ×JG36 combination produced less mycelium than the wild-type strain. However, the JG35×Δ36- RWTD1 Combination and double knockout combination (Δ35- RWTD1 ×Δ36- RWTD1 No binucleate hyphae growth was observed on the surface and edges of the colonies formed, and no hyphal structure was detected under an optical microscope (as observed in the panoramic view and cell morphology of sexually mated colonies, such as...). Figure 9 (As shown in A and 9B). The results of this embodiment indicate that, RWTD1 The gene is essential for the sexual mating and filamentous growth of *Ustilago maydis*, and its function exhibits a significant mating type dependence, specifically in the Mat1 mating type strain. RWTD1Genes play a more crucial role in hyphal formation.

[0064] Example 6: Missing RWTD1 Effects on the expression of key genes in sexually coupled sex To further explore RWTD1 To investigate the molecular mechanism by which deletion leads to defective sexual mating, we used RT-qPCR to detect changes in the expression of several key genes during sexual mating in the knockout strain.

[0065] Wild-type (JG35×JG36) and double knockout mutant (Δ35-) were collected separately. RWTD1 ×Δ36- RWTD1 Total RNA was extracted from bacterial samples 72 hours after sexual mating and reverse transcribed into cDNA. The pheromone precursor gene at the α-mating type locus was selected. mfa1 , mfa2 ) and pheromone receptor gene ( pra1 , pra2 ), transcription factor genes at mating site b ( bE1 , bW1 , bE2 , bW2 ), and pheromone response factor genes prf1 As the detection target, Actin Genes were used as internal controls for expression level analysis.

[0066] The results showed that, compared with the wild type, in RWTD1 During sexual mating of double knockout mutants, all genes at the mating type a1 and b1 loci ( mfa1 , pra1 , bE1 , bW1 The expression levels of both α2 and β2 loci were significantly downregulated. Only α2 and β2 loci showed downregulation. pra2 and bE2 Significantly lowered, mfa2 and bW2 The change in expression levels was not significant. Furthermore, pheromone response factors... prf1 The expression level was also significantly downregulated (expression analysis results are as follows). Figure 10 (As shown). These data indicate that, RWTD1 The absence of [something] severely affects the normal function of the pheromone signaling pathway, causing the generation of the mfa1 signal to be disrupted by the reception of two mating pheromones. pra The inhibition of gene expression at the ) step and the b1 locus may be the direct molecular cause of its loss of sexual mating ability. Meanwhile, mfa2 and bW2 The lack of significant effect on gene expression may be due to Δ35- RWTD1 The reason why binucleate hyphae formation was not completely blocked during sexual mating with wild-type strains.

[0067] Example 7: RWTD1 Gene complementation verifies its function Functional complementation experiments showed that different mating types RWTD1 Equal volumes of gene-replaced strains were inoculated onto YEPSA solid medium and cultured at 28°C for 3-7 days. Microscopic observation revealed that the wild-type control combination (JG35×JG36) and C35- RWTD1 ×C36- RWTD1 All combinations were able to sexually combine and form hyphae after 72 hours of cultivation (e.g. Figure 11 (As shown).

[0068] Example 8: Missing RWTD1 Effects on pathogenicity, induction of black flagella formation and teliospore formation For evaluation RWTD1 To investigate the role of genes in the pathogenesis of *Ustilago maydis*, we conducted standardized pathogenicity tests on knockout mutants and replacement strains.

[0069] A positive control was prepared by soaking sugarcane tissue culture seedlings (variety ROC22) in a mixture of wild-type strains (JG35×JG36). Simultaneously, a JG35×Δ36- strain was used as a positive control. RWTD1, Δ35- RWTD1 ×JG36 and Δ35- RWTD1 ×Δ36- RWTD1 Three combined inoculation treatments were administered. Plant disease incidence was observed and recorded regularly after inoculation. The positive control group began showing black scab symptoms 32 days after inoculation, with a final disease incidence rate of 91.0% (results are shown in Table 1, and the disease progression curve is shown in Figure 2). Figure 12 (As shown). In comparison, JG35×Δ36- RWTD1 Sugarcane plants inoculated with the combined strains showed a delayed onset of black stinger on day 60, and the final disease incidence rate was significantly reduced to 4.6% (results are shown in Table 1, and the disease progression curve is as follows). Figure 12 (As shown). And Δ35- RWTD1 ×JG36 and Δ35- RWTD1 ×Δ36- RWTD1 No black whip-like symptoms were developed in the combined inoculation of sugarcane during the 120-day experimental period. Observation of symptoms in diseased plants showed that all affected sugarcane exhibited typical symptoms of smut, such as thin stems, elongated internodes, and the emergence of black whip-like growths from the tips. There were no significant differences in the appearance of diseased plants among different inoculation treatments (symptom comparison...). Figure 13 (As shown). For surviving plants that did not exhibit black whip symptoms, shoot tip meristem tissue was sectioned and stained with trypan blue. JG35×Δ36- RWTD1 and Δ35- RWTD1 ×Δ36- RWTD1 In the combined sugarcane, 15 and 7 plants, respectively, showed pathogenic fungal hyphae within their tissues under an optical microscope; notably, inoculation with Δ35- RWTD1 Pathogenic fungal hyphae were observed in the tissues of sugarcane infected with the ×JG36 combination. No significant difference in hyphal morphology was observed between wild-type and mutant-infected tissues (hyphae observation is shown in...). Figure 13 (As shown). Based on comprehensive statistics of *Trichoderma purpureus* plants and plants with only mycelium, the infection rate of the wild-type inoculated group was 95.5%, while... RWTD1 The infection rate of the double knockout inoculation combination was only 7.14%, while Δ35- RWTD1 The infection rate of the ×JG36 combination was only 100% (statistical results are shown in Table 1, and the disease occurrence progress curve is as follows). Figure 14 (As shown). This example fully demonstrates that in Mat2 mating type strains... RWTD1 While gene deletion does not affect the pathogen's initial infectivity (it can still colonize), it significantly weakens its ability to induce typical disease symptoms (black whip) and ultimately lead to disease development. Deletion of both mating types completely inhibits the pathogen's infectivity and its ability to induce typical disease symptoms. This indicates... RWTD1 It is a key virulence factor that regulates the infectivity type, induced symptoms, and teliospore formation of *Ustilago maydis*.

[0070] Inoculation with complement strain combinations (such as C36- RWTD1 × JG35 or JG36 × C35- RWTD1 Sugarcane plants with a black scab incidence rate and pathogen infection rate were significantly lower than those with a knockout rate (Δ36-). RWTD1 × Δ35- RWTD1 The mycelium showed significant recovery, although it did not fully reach the wild-type level, but it was able to form typical black whip symptoms, and the hyphae morphology within the whip were normal (results are shown in Table 1). This example fully demonstrates that... RWTD1 The deletion of the gene is the direct cause of sexual mating defects and the inability to form black flagella; the restoration of its function can partially salvage the phenotype.

[0071] Table 1 Wild type, RWTD1 Statistics on inoculation results of knockout strains and replenishment strains 1 Data represent the number of whip-like plants / total number of surviving plants (black whip rate, %). These plants were observed 120 days post-inoculation.

[0072] 2 The data represents the total number of flagellated and mycelial plants / the total number of surviving plants (infection rate, %).

[0073] Example 9: RWTD1 Effects of deletion on the transcriptome of sexual mating process For analysis RWTD1 The molecular mechanisms regulating sexual pairing, we are studying wild-type and... RWTD1 The transcriptomes of the knockout strains at key sexual mating time points were compared and analyzed.

[0074] Samples were collected at 0 hours (when haploid spores were mixed) and 72 hours (diknusic hyphae formation period) of sexual maturation, including: wild-type combination (WT: JG35×JG36) and Mat1 knockout combination (JG36×Δ35-). RWTD1 Mat2 knockout combination (Δ36-) RWTD1 ×JG35) and double knockout combination (Δ36- RWTD1 ×Δ35- RWTD1 Each group consisted of three biological replicates (sample details are shown in Tables 2 and 3). Total RNA was extracted and subjected to high-throughput sequencing. Principal component analysis showed that the replicates were well clustered and the data were reliable (e.g., ...). Figure 15 (As shown). Randomly selected differentially expressed genes were validated by RT-qPCR, and their expression trends were highly consistent with the transcriptome data (e.g., Figure 16 As shown in the figure, this proves the reliability of the data.

[0075] Table 2. Transcriptome samples of *Ustilago maydis* with sexual mating at 0 h. Table 3. Transcriptome samples of *Ustilago maydis* after 72 h of sexual mating. Differential gene expression analysis revealed 1155 differentially expressed genes in the wild-type before and after sexual mating (WT_72h vs WT_0h). Meanwhile... RWTD1 In the knockout context, the number of differentially expressed genes decreased, especially in the double knockout combination where only 507 differentially expressed genes were detected (statistics shown in Table 4). Through Wayne analysis, we screened out 512 genes that were differentially expressed only during sexual mating in the wild-type group, and not in any other group. RWTD1 Knockout genes that do not change in any combination (such as...) Figure 17 As shown in A). These genes are significantly enriched in GO entries such as "membrane integrated components", "membrane intrinsic components", and "hydrolyzed enzyme activity" (e.g., Figure 17 (as shown in B), prompt RWTD1 The absence of these genes affects the normal reprogramming of membrane-related and hydrolytic metabolism-related genes during coordination.

[0076] Furthermore, through weighted gene co-expression network analysis, we identified a gene module (MEturquoise module) that is highly positively correlated with wild-type dikaryotic hyphae formation (e.g., Figure 18(As shown). Cross-analysis was performed on genes in this module, the upregulated portions of the aforementioned 512 differentially expressed genes, and genes enriched in the "membrane integration component," ultimately identifying 37 core candidate genes, all of which were specifically upregulated after sexual mating (Venn diagram and expression heatmap shown). Figure 19 (As shown). This includes RWTD1 itself( g_000709 ). For 3 of them ( g_006207、g_003363 and g_005785 RT-qPCR quantitative analysis showed that the expression levels of these three genes in binucleate hyphae colonies formed by the wild-type strain after 72 h were significantly higher than those in strain combinations that failed to form hyphae (e.g., Figure 20 (As shown). This example reveals from the transcriptional level... RWTD1 How the absence of these genes leads to disordered expression of sexually related genes, and how to screen for potential downstream regulatory targets.

[0077] Table 4. Statistics on the number of differentially expressed genes in *Ustilago maydis*. Example 10: RWTD1 Regulation of plant hormone levels during infection Given that black flag formation involves host developmental reprogramming, we measured the content of various plant hormones in sugarcane shoot tip tissue before and after black flag formation.

[0078] Healthy sugarcane shoot tips were collected (H-30d, H-40d) and inoculated. RWTD1 Knockout combination (JG36×Δ35- RWTD1 Hormone quantification was performed on sugarcane shoot tips 30 and 40 days after inoculation (RWTD1-30d, RWTD1-40d), sugarcane shoot tips 30 days after inoculation with wild type (JG35×JG36) but without black whip formation (WT-30d), and the white, gray, and black parts of the black whip formed by wild type infection.

[0079] The results showed that the contents of auxin (IAA), four cytokinins (trans-zeatin T-Zeatin, zeatin, zeatin nucleoside TZR, and isopentenyl adenosine monophosphate IPA), and abscisic acid (ABA) in the black whip tissue were significantly higher than those in the sugarcane stalk tips that did not form black whips (e.g., ...). Figure 21 , Figure 22 AD, Figure 23 (As shown in B). IAA was found in the highest concentrations in the white and gray parts of the black flagellum; Zeatin, T-Zeatin, and TZR peaked in the black part. In stark contrast, during inoculation... RWTD1In the sugarcane shoot tips of the knockout plants, the levels of these growth-promoting hormones remained consistently low, without any accumulation over time (e.g., Figure 21 , Figure 22 (As shown in AC). Conversely, the defense-related hormones salicylic acid (SA) and jasmonic acid (JA) in... RWTD1 The content was significantly higher in the stem tips of sugarcane infected by knockout plants (e.g. Figure 23 (As shown in C and D). This example demonstrates that successful black flag development requires pathogens (dependent on...). RWTD1 (Function) actively manipulates the host, leading to the accumulation of auxin and cytokinin; while RWTD1 The loss of function prevents pathogens from driving this hormone reprogramming, which may instead enhance the host's defense response (increased SA and JA levels), consistent with its inability to form black whips.

[0080] Example 11: RWTD1 Effect of deletion on pathogen biomass within the host To explore RWTD1 To investigate whether the knockout strain's ability to infect but not produce flagella stems from its restricted growth within the host, we quantitatively measured the biomass of the pathogen in sugarcane tissue.

[0081] Sugarcane shoot tip tissue was extracted 60 days after inoculation (samples included: wild-type JG35×JG36 infected and named V_WT, Δ35- RWTD1 The ×JG36 knockout strain was named V_d35, JG35×Δ36- RWTD1 The knockout strains were named V_d36 and Δ35- RWTD1 ×Δ36- RWTD1 The genomic DNA of *Ustilago maydis* (named V_d35×V_d36) was knocked out by double knockout. The mating type-specific gene of *Ustilago maydis* b was used. bE2 To target the target, TaqMan probes and primers were designed, and absolute quantification was performed using real-time quantitative PCR. First, a... bE2 The standard curve for gene copy number shows a good linear relationship (R0). 2 =0.9989), the amplification efficiency meets the requirements (e.g., Figure 24 (As shown in A).

[0082] The pathogens in each sample were calculated using this standard curve. bE2 Gene copy number. Results showed that the pathogen biomass was highest in wild-type infected tissues (V_WT). All RWTD1 In tissues infected by the knockout combinations, the biomass of the pathogen was significantly lower than that of the wild type. Among them, the double knockout combination (V_d35×V_d36) had the lowest biomass, while the single allele knockout combination (V_d35, V_d36) was in between (e.g., ...). Figure 24(As shown in B). This embodiment demonstrates that, RWTD1 The absence of this substance not only affects symptom development but also significantly inhibits the proliferation and colonization of pathogens within the host, which may be one of the important reasons for its weakened pathogenicity.

Claims

1. A method for controlling sugarcane smut, characterized in that, The method includes inhibiting sugarcane smut fungus ( ). Sporisorium scitamineum )middle RWTD1 The function and / or expression of the gene, wherein RWTD1 The nucleotide sequence of the gene is shown in SEQ ID No. 1, or it encodes a protein with the same function.

2. The method according to claim 1, characterized in that, The inhibition is achieved by applying a substance targeting the sugarcane, the sugarcane growing environment, or *Smuttiformis sugarcane*. RWTD1 This can be achieved through RNA interference molecules, antisense nucleic acids, or gene editing systems.

3. The method according to claim 1, characterized in that, The inhibition is achieved by applying a compound that inhibits the activity of the RWTD1 protein to sugarcane, the sugarcane growing environment, or *Ustilago maydis*.

4. The method according to any one of claims 1-3, characterized in that, The inhibition resulted in the suppression of black flag formation and / or teliospore development after *Ustilago canis* infection of sugarcane.

5. The method according to claim 4, characterized in that, The inhibition resulted in *Ustilago maydis* being able to infect sugarcane but failing to induce the production of black whips.

6. RWTD1 The application of genes or their encoded proteins in the control of sugarcane smut, wherein... RWTD1 The nucleotide sequence of the gene is shown in SEQ ID No. 1, or it encodes a protein with the same function.

7. A kit for controlling sugarcane smut, characterized in that, The kit contains ingredients for inhibiting *Ustilago maydis*. RWTD1 Active ingredients that target gene function and / or expression, said active ingredients being selected from: RWTD1 RNA interference molecules, antisense nucleic acids, gene editing systems, or compounds that can inhibit the activity of the RWTD1 protein.

8. A recombinant microorganism, characterized in that, It contains *Ustilago maydis* capable of expressing targeted sugarcane spores. RWTD1 An expression vector for an RNA interference molecule or antisense nucleic acid of a gene, or an expression vector containing a protein capable of expressing an inhibitory effect on RWTD1 protein activity, wherein... RWTD1 The nucleotide sequence of the gene is shown in SEQ ID No. 1, or encodes a protein with the same function; the recombinant microorganism is a bacterium or fungus.

9. A molecular marker for identifying or monitoring the control efficacy of sugarcane smut, characterized in that, The molecular marker is *Smuts spores*. RWTD1 Gene expression levels or functional states, among which RWTD1 Downregulation or inactivation of gene expression indicates effective prevention and treatment.

10. A method for cultivating sugarcane plants resistant to smut, characterized in that, The method includes introducing an expressive substance into sugarcane. RWTD1 The constructs of the gene's dsRNA, siRNA, or shRNA are used to produce a targeted strain of *Ustilago maydis* in sugarcane. RWTD1 Interfering RNA in genes.

Images