Application of PRM1 gene in prevention and control of sugarcane smut

Abstract

The invention discloses an application of a PRM1 gene in prevention and control of sugarcane smut, and the gene encodes a pheromone regulated multi-transmembrane protein which is a key factor for regulating sexual coordination and pathogenicity of pathogenic bacteria. Experiments show that inhibition or knockout of the PRM1 gene can significantly weaken the sexual cooperation ability of pathogenic bacteria, hinder the formation of binuclear hyphae and cause the reduction of the pathogenicity of the binuclear hyphae. In a molecular mechanism, PRM1 plays a role by influencing expression of a key gene of a pheromone signaling pathway. On the basis, the PRM1 gene and the encoding protein thereof are determined as a new target for preventing and treating the sugarcane smut, and can be used for developing novel green prevention and control technologies and products such as RNA interference preparations, specific inhibitors, gene editing breeding and engineering microorganisms, and a brand-new molecular basis and solution are provided for effectively controlling the sugarcane smut.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control technology, and particularly relates to a... PRM1 Application of genes in the control of sugarcane smut. Background Technology

[0002] sugar cane( Saccharum officinarum Sugarcane smut (L.) is a globally important sugar and energy crop, and its stable production is crucial for national sugar security and agricultural economy. However, the production of sugarcane smut fungus (L.) is problematic. Sporisorium scitamineum Sugarcane smut, caused by the pathogen, is one of the major diseases hindering the healthy development of the sugarcane industry. A typical characteristic of this disease is that the apical meristem of the infected sugarcane plant is induced by the pathogen to abnormally differentiate into a black whip-like structure, known as the "black whip." The black whip is filled with the pathogen's teliospores, which, upon maturation, are dispersed by air currents, becoming the most important source of primary and secondary infection in the disease cycle. This leads to the rapid spread of the disease in the field, causing severe yield and sugar content losses.

[0003] The pathogenicity of *Ustilago maydis* is closely related to its complex life cycle, one of the core steps of which is the sexual mating of haploid basidiospores of different mating types (Mat1 and Mat2) to form infectious binucleate hyphae. This process is mainly regulated by genes at mating type loci (a and b). Specifically, the pheromone precursor (e.g., Mfa) and its receptor (Pra) encoded by locus a are responsible for intercellular recognition and attraction, while the heterodimeric transcription factor (bE / bW) encoded by locus b regulates subsequent filamentous growth and pathogenicity. Therefore, sexual mating of the pathogen is not only a necessary step in completing its life cycle but also the logical starting point for establishing infection and causing disease. Disrupting this process will fundamentally undermine the pathogen's pathogenic basis.

[0004] Currently, the control of sugarcane smut mainly relies on the breeding of resistant varieties and cultivation management measures. 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 Yue Tang 85-177 and Xin Tai Tang 10, which were once widely planted in sugarcane areas of my country, became susceptible varieties due to changes in their physiological races, leaving disease control in a passive position. Chemical control is limited by issues such as unstable efficacy, environmental residues, and the potential risk of pathogen resistance. Therefore, in-depth analysis of the sexual mating regulatory network of *Smutella spores* at the molecular level, and the discovery of its key node genes as new targets for control, is an urgent theoretical and practical need for developing green, efficient, and sustainable disease control technologies.

[0005] Previous research, in exploring the key regulatory factor RWTD1 in sexual mating of *Ustilago maydis*, screened a batch of candidate genes that are highly associated with RWTD1 expression patterns and specifically highly expressed during the sexual mating stage through comparative transcriptomics and weighted gene co-expression network analysis. Among them, [the gene...] g_006207 It attracted our attention because its expression was significantly upregulated in the binucleate hyphae stage and it was annotated as a "pheromone-regulated multispanning membrane protein," and we named it accordingly. PRM1 Bioinformatics analysis predicts that the PRM1 protein contains four transmembrane domains, classifying it as an integrated membrane protein, suggesting its potential involvement in cell membrane-related signal sensing or substance transport processes. In model organisms such as *Saccharomyces cerevisiae*, the homologous protein Prm1p has been confirmed as a key factor regulating plasma membrane fusion and sexual coordination. However, in *Ustilago maydis* and even plant pathogenic fungi, PRM1 There have been no reports on whether genes are involved in or how they regulate sexual mating and pathogenic processes.

[0006] Based on this, we will conduct in-depth research and clarify. PRM1 The biological function of the gene in *Ustilago maydis* not only fills a research gap in this field and deepens our understanding of the molecular mechanisms of sexual coordination in pathogens, but more importantly, it holds promise for revealing a key target located downstream of the pheromone signaling pathway that controls hyphal fusion and growth. Based on this target, we can develop a gene that interferes with... PRM1 Novel control strategies, such as designing specific inhibitors or gene silencing technologies, hold promise for precise intervention in the early stages of pathogen infection—specifically, the sexual mating stage—thereby preventing the formation of infectious dikaryotic hyphae. This has significant scientific value and application potential for addressing current industry challenges in sugarcane smut control, such as the scarcity of targets and the limited range of technological approaches. Summary of the Invention

[0007] To solve the above-mentioned technical problems, the present invention proposes a... PRM1 The application of genes in the control of sugarcane smut is demonstrated experimentally in this invention. PRM1 The gene is specifically highly expressed during the stage of sexual mating in pathogens to form binucleate hyphae, and is a core factor regulating this critical pathogenesis. Specifically, the deletion... PRM1 The gene severely impairs the sexual mating ability of the pathogen, inhibits the formation of dikaryotic hyphae, and ultimately significantly weakens its pathogenicity. Further molecular mechanism studies have shown that... PRM1 Its function is closely related to the pheromone signaling pathway, and its absence will cause pheromone precursor genes (…). mfa ) and receptor gene ( praThe expression of ) is significantly downregulated, thereby disrupting the signal transduction process of sexual coordination.

[0008] 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 PRM1 The function and / or expression of the gene, wherein PRM1 The gene encodes a pheromone-regulated multi-transmembrane protein.

[0009] Furthermore, the inhibition is achieved by applying a substance targeting the sugarcane, the sugarcane growing environment, or *Ustilago maydis*. PRM1 This can be achieved through RNA interference molecules, antisense nucleic acids, or gene editing systems.

[0010] Furthermore, the inhibition is achieved by applying a compound capable of inhibiting the activity of the PRM1 protein to sugarcane, the sugarcane growing environment, or *Ustilago maydis*.

[0011] Furthermore, the inhibition results in a reduction or loss of the sexual mating ability and / or binucleate hyphae formation ability of *Ustilago canis*.

[0012] Furthermore, the inhibition specifically reduces or eliminates the sexual mating ability of Mat1 mating type *Smuts spp.*

[0013] Furthermore, the inhibition leads to downregulation of the expression of pheromone precursor genes and / or pheromone receptor genes in *Ustilago maydis*.

[0014] This invention also provides PRM1 The application of genes or their encoded proteins in the control of sugarcane smut, wherein... PRM1 The gene encodes a pheromone-regulated multi-transmembrane protein.

[0015] The present invention also provides a kit for controlling sugarcane smut, the kit comprising ingredients for inhibiting the growth of *Ustilago maydis*. PRM1 Active ingredients that target gene function and / or expression, said active ingredients being selected from: PRM1 RNA interference molecules, antisense nucleic acids, gene editing systems, or compounds that can inhibit the activity of the PRM1 protein.

[0016] The present invention also provides a recombinant microorganism comprising the ability to express targeted *Ustilago maydis*. PRM1 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 protein activity of PRM1, wherein... PRM1 The gene encodes a pheromone-regulated multi-transmembrane protein.

[0017] 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... PRM1 The constructs of the gene's dsRNA, siRNA, or shRNA are used to produce a targeted strain of *Ustilago maydis* in sugarcane. PRM1 Interfering RNA in genes.

[0018] 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 inhibition of the sexual mating process of sugarcane smut fungus. This is achieved by interfering with or inhibiting... PRM1 The function of the gene can significantly weaken or even completely block the sexual mating of different mating types of basidiospores in *Ustilago maydis*, preventing the formation of infectious dikaryotic hyphae. Experiments have confirmed that... PRM1 Gene knockout mutants, especially strains lacking double mating type, essentially lose their ability to sexually mate, thereby curbing the production of infectious mycelia from the source and achieving early control effects.

[0019] From a mechanistic perspective, the technical effect of this invention stems from the precise interference with the pathogenic pheromone signaling pathway. PRM1, a pheromone-regulated membrane protein, loses function when its function is impaired, leading to the formation of downstream pheromone precursor genes (…). mfa1 , mfa2 ) and receptor gene ( pra1 , pra2 The expression of PRM1 was significantly downregulated, disrupting the intercellular recognition and signal transduction necessary for sexual mating. This disruption of the core signaling pathway not only explains its phenotypic defects but also validates the reliability of PRM1 as a key node, providing a clear molecular basis for the development of highly specific inhibitors.

[0020] In disease control applications, the strategy of this invention has advantages in both high efficiency and targeted application. Because... PRM1 Genes are indispensable in the sexual mating of pathogens, and intervention targeting this gene can effectively reduce the infection success rate and the severity of disease in the field. Compared with traditional fungicides that target pathogen growth or spore germination, control strategies that act on the early stages of sexual mating may help alleviate selective pressure and delay the development of resistance. Furthermore, this target opens up a feasible path for developing novel green control products based on RNA interference, gene editing, or specific small molecule compounds, providing an important new tool for the integrated management of sugarcane smut. Attached Figure Description

[0021] 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.

[0022] Figure 1 Candidate genes g_006207 ( PRM1 RT-qPCR validation diagram of expression level in the binucleate mycelial stage of *Ustilago canis*.

[0023] Figure 2 This is a bioinformatics analysis diagram of the PRM1 protein. A: Schematic diagram of predicted transmembrane domains of the PRM1 protein. B: Phylogenetic tree of the PRM1 protein with homologous proteins from other species.

[0024] Figure 3 This diagram illustrates a strategy for constructing homologous DNA replacement fragments for gene knockout.

[0025] Figure 4 for PRM1 Molecular validation results of gene knockout strains. A: Schematic diagram of PCR primer positions used for knockout strain validation. B: Electrophoresis diagram of PCR detection of wild-type and knockout strains using the internal gene primers PRM1-F / PRM1-R. C: Electrophoresis diagram of PCR detection of wild-type and knockout strains using the left outer primer PRM1-1.6F and the hygromycin gene inner primer Hyg-226R. D: Electrophoresis diagram of PCR detection of wild-type and knockout strains using the hygromycin gene inner primer Hyg-225F and the right outer primer PRM1-1.6R. E: PRM1 Bar chart showing RT-qPCR analysis of gene transcription levels in wild-type and knockout strains.

[0026] Figure 5 for PRM1 Images showing the sexual mating phenotype of gene knockout strains. A: Panoramic view of sexually mated colonies. B: Enlarged view of a sexually mated colony. C: PRM1 Cell morphology of the knockout strain after sexual mating (optical microscope).

[0027] Figure 6 For missing PRM1 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. - CtThe 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.

[0028] Figure 7 for PRM1 Statistical curve of the occurrence process of smut after inoculation of sugarcane with gene knockout strains.

[0029] Figure 8 For wild type or PRM1 Image of sugarcane plants infected with a gene knockout strain. A: Wild type and... PRM1 Comparison of disease symptoms in grafted plants of gene knockout strain. B: Wild type and... PRM1 Observation of mycelial staining in the growth point tissue of gene knockout strain inoculated plants. Detailed Implementation

[0030] 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.

[0031] 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.

[0032] 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.

[0033] This invention focuses on a newly identified key gene in *Ustilago maydis*— PRM1 Through systematic genetic, molecular biological and pathological experiments, the core role of this gene in regulating the sexual mating and pathogenicity of pathogens was elucidated for the first time, and its feasibility as a new target for disease control was verified.

[0034] The primer pair sequences used in this invention are as follows: Example 1: PRM1 Gene mining and bioinformatics analysis In comparison with wild type and RWTD1 After comparing and analyzing the transcriptomes of the sexual mating process in the knockout strains, we screened out a strain that was similar to... RWTD1 Candidate genes with highly correlated co-expression patterns and significantly upregulated at the binucleate hyphae stage g_006207RT-qPCR validation confirmed that the expression level of this gene was significantly upregulated at 72 hours of sexual mating in wild-type individuals (binukaryotic hyphae stage) compared to 0 hours (haploid stage), while... RWTD1 During sexual mating of the knockout strain, the upregulation trend disappeared (results as follows). Figure 1 (As shown). This gene is 2910 bp in length, has no introns, and encodes a protein composed of 969 amino acids. Through database comparison, this protein was annotated as a "Pheromone-regulated multispanning membrane protein," hence its name. PRM1 Its nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0035] SEQ ID NO.1: SEQ ID NO.2: MASQQIVDDPPSYLHQDHLRSPTYTQSTFVPANPSTDMPSHSKPPILQPWLGLRARLFLSPISIPLISLLFVGTRLWMSSNDANNSVSSAKTNLLSACDAVEGTASLAASFPHFLAATTNLQLAQSVTSTVHGAARVFDLSMVAIEKVLVYIVNSYKSLFMCFMELLVRGSLAVLIEAVQLISQAITAAAQGIRLAIQESVEGVNAILSTAVGAINDVVGVFGQHVNPPHIAVPSLTALENITLPHEIQDGLLKLNATLPTLQQLKQTMDSLIQTPFEEMRREVNATLGAFEFNHTLLPVPQMRNVTFCDRIDTSPLDELGEELRGAARWGLIVLALVAVVVMLIGVGWEWWKWQREVRAVERTRGVWLAQHREAPSKEDGDDQRDVLKTENLISLLAISRHPLISSFSLRICQRVGIKTRRAQNRLAWLLCFLTHPASLACIFTGLLGLLSVLLQSILIHRLADNSSSSINTSLTHLSSDVIDLINDHSRNASIEFSTSANAIILQVESELNQHVFRWVDTTTSTMNATLNQFVDGITESLESTFGGTPFNAPLQTFVQCVVGQKVAGIGKALTWIHDNAHVNFSLVPPGVLVLGEDQQEAVLAPVREAMLGGAGGGGDGVVGNVVGRYLKHLQQEKVMFAVLVGVYGVILCVGLVVVLYASAVERRGQDGRDGEEVHHDSDVMEEKLRGDLQGGPAAGVGWWRGMPRPSLSRFRSKPAPLSSQPVEPTHSLQNVAARRTSLAHSNRSARTHVTKDSISYPFQIHHSLNTSSSPSTRPRQPTPLRQASTGDRNTVLSLRQEAPLPPAAAEQRSSWLSFLATHSHDDHSAAPETQQQEEGAQERFERLFGCSPVASPTTTKFDQHVSAPAASRSANVDDGDRRYRETLDLRPVQDWIGSKSPIPATAADRTSEVELTRSSSVIDGEMKGKAEGESSFAFANTAQIRPPQHQQQQQRTVGSPQSISFYAW Structural prediction of the PRM1 protein using online tools revealed that it contains four transmembrane domains (located at amino acids 57-78, 330-349, 427-455, and 639-662, respectively), with its N-terminus and C-terminus both located outside the membrane (transmembrane structure prediction is shown in Figure 1). Figure 2 As shown in Figure A). Subcellular localization predicted it to be an integrated membrane protein anchored in the lipid bilayer. Through protein sequence homology alignment and phylogenetic analysis, the PRM1 protein of *Ustilago maydis* was found to be similar to that of *Ustilago graminea* (as shown in Figure A). Sporisorium graminicola The homologous proteins of these proteins are most closely related, with an amino acid identity of up to 86% (phylogenetic tree as shown). Figure 2 (As shown in B). The above analysis indicates that PRM1 is a relatively evolutionarily conserved membrane protein that may be involved in membrane-related biological processes.

[0036] Example 2: PRM1 Construction and validation of gene knockout mutants To investigate PRM1 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).

[0037] First, using wild-type strain genomic DNA as a template, PCR amplification was performed separately. PRM1 The left homologous arm (LB) 1500 bp upstream and the right homologous arm (RB) 1500 bp downstream of the coding region were amplified. Simultaneously, the hygromycin B resistance gene (HYG) fragment was amplified from plasmid pEX1. These three fragments were sequentially ligated and cloned into a plasmid using seamless cloning technology. Xho I and Eco A knockout vector was constructed from the pSUC2 vector backbone digested with RI double enzymes. Finally, using this vector as a template, a complete homologous substitution fragment containing the left homologous arm-HYG-right homologous arm was amplified by PCR (strategy diagram shown in Figure 1). Figure 3 (As shown).

[0038] The aforementioned 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.

[0039] Candidate knockout strains were validated using multi-level PCR and RT-qPCR (the locations of the validation primers are shown in the diagram). Figure 4(As shown in A). Genotyping analysis showed that PCR using primer PRM1-1.6F (located on the outer side of the left homologous arm) and primer Hyg-226R (located inside the hygromycin resistance gene) amplified a specific 3441 bp band in the knockout strain, while this band was absent in the wild type (as shown in A). Figure 4 (As shown in C); PCR was performed using primer Hyg-225F, located inside the hygromycin resistance gene, and primer PRM1-1.6R, located outside the right homologous arm. A specific band of 2553 bp was amplified in the knockout strain, while this band was not present in the wild type (as shown in C). Figure 4 (As shown in D). Conversely, using the method targeted at... PRM1 PCR using primers PRM1-F / PRM1-R for detecting the internal gene sequence only amplified the 2910 bp target band in the wild type, with no amplification product in the knockout strain (e.g., Figure 4 (As shown in B). Phenotypic identification was performed by RT-qPCR, and the results showed that no phenotypic markers were detected in the knockout strains. PRM1 mRNA expression of genes (e.g.) Figure 4 (As shown in E). The above results fully demonstrate that we have successfully obtained... PRM1 Homozygous knockout mutants with complete gene deletion were named Δ35- PRM1 (Mat2 mating type) and Δ36- PRM1 (Mat1 mating type).

[0040] Example 3: Effects of PRM1 deletion on sexual mating and filamentous growth of pathogens To clarify PRM1 To determine whether the gene is involved in regulating the sexual pairing of *Smuts spores*, we paired the knockout strain with the wild-type strain and observed their hyphal formation ability.

[0041] Equal amounts of different mating types of bacterial 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- PRM1 Both the ×JG36 and ×JG35 combinations formed abundant, white, fluffy dikaryotic mycelia after 72 hours of cultivation, with no significant difference in mycelial abundance. However, the JG35×Δ36 combination... PRM1 Only a very small number of hyphae were observed at the colony edges in the combination. The most significant phenotypic difference appeared in the double knockout combination (Δ35- PRM1 ×Δ36- PRM1 In the study, no binucleate hyphae growth was observed on the surface and edges of the colonies, and no hyphal structures were detected under an optical microscope (panoramic view, local magnification, and cell morphology observation of sexually mated colonies are shown in the image). Figure 5 (As shown). Furthermore, PRM1No significant differences were found in the morphology of haploid basidiospores from the knockout strain compared to the wild type. The results of this example indicate that... PRM1 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. PRM1 Genes play a more crucial role in hyphal formation.

[0042] Example 4: Effects of PRM1 deletion on expression of key genes for sexual mating To further explore PRM1 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.

[0043] Wild-type (JG35×JG36) and double knockout mutant (Δ35-) were collected separately. PRM1 ×Δ36- PRM1 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.

[0044] The results showed that, compared with the wild type, in PRM1 During sexual mating of double knockout mutants, all detected pheromone system genes ( mfa1 , mfa2 , pra1 , pra2 The expression levels of all of these genes were significantly downregulated. In the b-site gene, bW2 and bE2 The expression level of surged abnormally, and [[ID=1o8]]bW1 and bE1 The expression of [certain substances] showed a downward trend. It is worth noting that the core pheromone response factor [was affected]. prf1 No significant difference was found in the expression levels between the two groups (expression level analysis results are as follows). Figure 6 (As shown). These data indicate that, PRM1 The lack of pheromone severely affects the normal function of the pheromone signaling pathway, leading to signal generation ( mfa ) and receiving ( praThe impaired gene expression at the β site may be the direct molecular cause of the loss of sexual mating ability. Simultaneously, the dysregulation of gene expression patterns at the β site also suggests… PRM1 It may be involved in coordinating signal integration between the two mating loci, a and b.

[0045] Example 5: Effect of PRM1 deletion on pathogenicity For evaluation PRM1 To investigate the role of genes in the pathogenesis of *Ustilago maydis*, we conducted a standardized pathogenicity assay on the knockout mutant.

[0046] A positive control was used, consisting of sugarcane tissue culture seedlings (variety ROC22) soaked in a mixture of wild-type strains (JG35×JG36). Simultaneously, a Δ35- PRM1 ×Δ36- PRM1 Double knockout combination inoculation treatment was used. Plant disease incidence was observed and recorded regularly after inoculation. The positive control group began showing black whip symptoms 46 days after inoculation, with a final disease incidence of 90.12%. In contrast, the inoculated... PRM1 The sugarcane plants with the double knockout mutant showed a delayed first appearance of black stingers on day 56, and the final disease incidence rate was significantly reduced to 54.43% (disease progression curve as shown in the figure). Figure 7 (As shown). Observation of symptoms in diseased plants showed that all diseased sugarcane exhibited typical symptoms of smut, such as thin stems, elongated internodes, and the emergence of black whip-like growths at the tips. There were no significant differences in the appearance of diseased plants among different inoculation treatments (symptom comparisons are shown). Figure 8 (As shown in A). For surviving plants that did not exhibit black whip symptoms, tissue sections were taken from the shoot tip meristem and stained with trypan blue. Under an optical microscope, pathogenic hyphae were observed within the tissues, and no significant difference in hyphal morphology was observed between wild-type and mutant-infected tissues (hyphae observation is shown in A). Figure 8 (As shown in B). Based on the combined statistics of *Trichoderma purpureus* plants and plants with only mycelium, the infection rate of the wild-type inoculated group was 95.06%, while... PRM1 The infection rate in the double knockout inoculation group was 55.70% (statistical results are shown in Table 1). This example fully demonstrates that... PRM1 Although 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, indicating that... PRM1 It is a key virulence factor that regulates the pathogenicity of *Ustilago maydis*.

[0047] Table 1 Wild type and PRM1 Statistics on the inoculation results of knockout strains 1Data represent the number of whip-like plants / total number of surviving plants (black whip rate, %). These plants were observed 120 days post-inoculation.

[0048] 2 The data represents the total number of flagellated and mycelial plants / the total number of surviving plants (infection rate, %).

[0049] In summary, this invention, through a series of embodiments, has for the first time identified and confirmed that... PRM1 This gene plays an indispensable role in the sexual mating and pathogenicity of *Ustilago maydis*. It influences the formation of dikaryotic hyphae by regulating the pheromone signaling pathway, thereby determining the pathogenicity of the fungus. Therefore, [[ID=I28]]PRM Genes and their encoded proteins can serve as ideal molecular targets for developing novel control technologies for sugarcane smut. Interference can be achieved through the design of specific inhibitors, RNA interference molecules, or gene editing strategies. PRM1 Its function can effectively block the sexual mating process of pathogens, thereby curbing the occurrence and development of diseases in the early stages of infection, providing a new approach and a solid scientific foundation for developing efficient and precise green disease control products.

[0050] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for controlling sugarcane smut, characterized in that, The method includes inhibiting sugarcane smut fungus ( ). Sporisorium scitamineum )middle PRM1 The function and / or expression of the gene, wherein PRM1 The gene encodes a pheromone-regulated multi-transmembrane protein.

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 sugarcane smut fungus. PRM1 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 PRM1 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 a reduction or loss of the sexual mating ability and / or binucleate hyphae formation ability of *Ustilago canis*.

5. The method according to claim 4, characterized in that, The inhibition specifically reduces or eliminates the sexual mating ability of Mat1 mating type *Smuts spores*.

6. The method according to claim 5, characterized in that, The inhibition resulted in downregulation of the expression of pheromone precursor genes and / or pheromone receptor genes in *Ustilago canis*.

7. PRM1 The application of genes or their encoded proteins in the control of sugarcane smut, wherein... PRM1 The gene encodes a pheromone-regulated multi-transmembrane protein.

8. A kit for controlling sugarcane smut, characterized in that, The kit contains ingredients for inhibiting *Ustilago maydis* (a type of mold). PRM1 Active ingredients that target gene function and / or expression, said active ingredients being selected from: PRM1 RNA interference molecules, antisense nucleic acids, gene editing systems, or compounds that can inhibit the activity of the PRM1 protein.

9. A recombinant microorganism, characterized in that, It contains *Ustilago maydis* capable of expressing targeted sugarcane spores. PRM1 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 protein activity of PRM1, wherein... PRM1 The gene encodes a pheromone-regulated multi-transmembrane protein.

10. A method for cultivating sugarcane plants resistant to smut, characterized in that, The method includes introducing an expressive substance into sugarcane. PRM1 The constructs of the gene's dsRNA, siRNA, or shRNA are used to produce a targeted strain of *Ustilago maydis* in sugarcane. PRM1 Interfering RNA in genes.

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