A pathogenic factor of sugarcane smut and its application

Abstract

This invention discloses a pathogenic factor of sugarcane smut and its application. This invention discovers... SsPHACA Application in regulating sexual mating, SDS tolerance, and pathogenicity of *Ustilago maydis*. This invention utilizes the principle of homologous recombination, employing a PEG-mediated protoplast transformation method to... SsPHACA Knockout and replacement were performed, resulting in knockout mutants and replacement strains. The results showed... SsPHACA Knockout resulted in a reduced ability of *Ustilago canescens* to form dimycelia through sexual mating, increased susceptibility to SDS, and decreased pathogenicity, indicating... SsPHACA This gene plays a crucial role in regulating the sexual mating, SDS tolerance, and pathogenicity of *Ustilago maydis*. This invention provides a novel target gene for the development of effective fungicides.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a pathogenic factor of *Ustilago maydis* and its application. Background Technology

[0002] Sugarcane smut is caused by *Ustilago maydis* (Suaeda salsa). Sporisorium scitamineum This disease, caused by [a specific pathogen], affects sugarcane yield and sugar accumulation. The pathogen produces teliospores through sexual reproduction, which are spread by air currents and infect the shoot apical meristem of sugarcane, leading to plant malformation and sugar loss. Current control methods mainly include chemical agents (such as difenoconazole and triadimefon), agricultural management (such as crop rotation and removal of diseased plants), and the breeding of resistant varieties. However, long-term reliance on chemical agents easily leads to drug resistance, agricultural measures are costly and have limited effectiveness, and the development of resistant varieties is time-consuming and susceptible to variations in pathogen races. Therefore, there is an urgent need to develop novel environmentally friendly control strategies based on molecular targets.

[0003] The presence of key pathogenic genes in pathogenic bacteria is of paramount importance. It can not only elucidate the pathogenic mechanism, but also lay the foundation and provide direction for the establishment of detection methods, as well as the development of vaccines and targeted drugs.

[0004] Currently, there are few research reports on pathogenic genes of *Ustilago maydis*. Relevant reports include: SsCI68230 is related to the sexual binding and pathogenicity of *Ustilago maydis*, and SsRss1 participates in the pathogen's response to salicylic acid and makes a significant contribution to salicylic acid tolerance and pathogenicity. SsCyp86 It plays an important role in the sexual reproduction and pathogenic infection process of *Ustilago maydis*. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a pathogenic factor of sugarcane smut fungus and its application.

[0006] The first objective of this invention is to provide products that knock out or silence the pathogenic proteins of *Smuts smut* for use in the treatment of sugarcane smut.

[0007] A second objective of this invention is to provide products that knock out or silence the pathogenic gene of *Smutus canis* for use in the treatment of sugarcane smut.

[0008] A third objective of this invention is to provide the application of pathogenic proteins of *Smuts spp.* in regulating the pathogenicity of *Smuts spp.*.

[0009] The fourth objective of this invention is to provide the application of pathogenic genes of *Smuts spp.* in regulating the pathogenicity of *Smuts spp.*

[0010] The fifth objective of this invention is to provide the application of pathogenic proteins of *Smuts spp.* in regulating sexual coordination in *Smuts spp.*

[0011] The sixth objective of this invention is to provide the application of pathogenic genes of *Smuts spp.* in regulating sexual mating in *Smuts spp.*

[0012] The seventh objective of this invention is to provide the application of pathogenic proteins of *Ustilago maydis* in regulating the mycelial growth of *Ustilago maydis*.

[0013] The eighth objective of this invention is to provide the application of pathogenic genes of *Smuts spp.* in regulating the mycelial growth of *Smuts spp.*

[0014] The ninth objective of this invention is to provide the application of pathogenic proteins of *Smuts candelilla* in regulating SDS tolerance in *Smuts candelilla*.

[0015] The tenth objective of this invention is to provide the application of pathogenic genes of *Smuts canis* in regulating the SDS tolerance of *Smuts canis*.

[0016] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0017] This invention provides another gene capable of regulating the sexual mating, SDS tolerance, and pathogenicity of *Smuts spp.*, named [gene name missing]. SsPHACA Its DNA nucleotide sequence is shown in SEQ ID NO:1. The open reading frame of this DNA sequence consists of 1595 nucleotides, including one intron sequence. The amino acid sequence of the SsPhacA protein it encodes is shown in SEQ ID NO:2, consisting of 531 amino acids, with one conserved p450 domain and one transmembrane structure. It can be used in the field of genetic engineering for plant resistance to sugarcane smut.

[0018] The SsPHACA The gene and SsPhacA protein are pathogenic factors of sugarcane smut caused by *Ustilago maydis*.

[0019] Therefore, this invention claims protection for the following applications:

[0020] The application of products that knock out or silence the pathogenic protein of *Smuts smutus* in the treatment of sugarcane smut, wherein the amino acid sequence of the pathogenic protein is shown in SEQ ID NO:2.

[0021] The application of products that knock out or silence the pathogenic gene of *Smutus canis* in the resistance to sugarcane smut, wherein the pathogenic gene is the gene encoding the pathogenic protein of *Smutus canis* with an amino acid sequence as shown in SEQ ID NO:2.

[0022] The application of pathogenic protein of *Ustilago maydis* in regulating the pathogenicity of *Ustilago maydis*, wherein the amino acid sequence of the pathogenic protein is shown in SEQ ID NO:2.

[0023] The application of the pathogenic gene of *Ustilago maydis* in regulating the pathogenicity of *Ustilago maydis*, wherein the pathogenic gene is the gene encoding the pathogenic protein of *Ustilago maydis* with an amino acid sequence as shown in SEQ ID NO:2.

[0024] The application of pathogenic protein of *Ustilago maydis* in regulating sexual coordination of *Ustilago maydis*, wherein the amino acid sequence of the pathogenic protein is shown in SEQ ID NO:2.

[0025] The application of the pathogenic gene of *Ustilago maydis* in regulating the sexual pairing of *Ustilago maydis*, wherein the pathogenic gene is the gene encoding the pathogenic protein of *Ustilago maydis* with the amino acid sequence shown in SEQ ID NO:2.

[0026] The application of pathogenic protein of *Ustilago maydis* in regulating the mycelial growth of *Ustilago maydis*, wherein the amino acid sequence of the pathogenic protein is shown in SEQ ID NO:2.

[0027] The application of the pathogenic gene of *Ustilago maydis* in regulating the mycelial growth of *Ustilago maydis*, wherein the pathogenic gene is the gene encoding the pathogenic protein of *Ustilago maydis* with the amino acid sequence shown in SEQ ID NO:2.

[0028] The application of pathogenic protein of *Ustilago maydis* in regulating SDS tolerance of *Ustilago maydis*, wherein the amino acid sequence of the pathogenic protein is shown in SEQ ID NO:2.

[0029] The application of the pathogenic gene of *Ustilago maydis* in regulating the SDS tolerance of *Ustilago maydis*, wherein the pathogenic gene is the gene encoding the pathogenic protein of *Ustilago maydis* with the amino acid sequence shown in SEQ ID NO:2.

[0030] As a specific implementation, the nucleotide sequence of the encoding gene is shown in SEQ ID NO:1.

[0031] Based on this, the present invention also claims protection for:

[0032] The application of reagents for detecting the pathogenic protein or pathogenic gene of *Smutus canis* in screening drugs for resistance to sugarcane smut or *Smutus canis*.

[0033] A method for screening drugs to control sugarcane smut caused by *Ustilago maydis*, comprising detecting the pathogenic protein or the pathogenic gene in sugarcane samples inoculated or infected with sugarcane smut after treatment with the drug, wherein the pathogenic protein is the pathogenic protein of *Ustilago maydis*, and the pathogenic gene is the pathogenic gene of *Ustilago maydis*.

[0034] A method for evaluating the ability of a drug to reduce the pathogenicity of *Ustilago maydis* involves detecting pathogenic proteins or pathogenic genes of *Ustilago maydis* treated with the drug, wherein the pathogenic protein is a pathogenic protein of *Ustilago maydis*, and the pathogenic gene is a pathogenic gene of *Ustilago maydis*.

[0035] Preferably, the expression of the pathogenic protein or pathogenic gene is detected, and if its expression is reduced, the drug is effective.

[0036] More preferably, the expression status of the pathogenic gene is detected by real-time quantitative PCR, and the expression status of the pathogenic protein is detected by Western blot.

[0037] And a low-pathogenic sugarcane smut fungus, by knocking out the pathogenic protein or the pathogenic gene.

[0038] The application of inhibitors of the pathogenic proteins or pathogenic genes of *Smutus canis* in the preparation of drugs for the prevention and control of *Smutus canis* or sugarcane smut disease, and the detection of pathogenic proteins or pathogenic genes of *Smutus canis* treated with the drug.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] This invention discovered SsPHACA Application in regulating sexual mating, SDS tolerance, and pathogenicity of *Ustilago maydis*. This invention utilizes the principle of homologous recombination, employing a PEG-mediated protoplast transformation method to... SsPHACA Knockout and replacement were performed, resulting in knockout mutants and replacement strains. The results showed... SsPHACA The knockout mutant exhibits reduced sexual mating ability to form two mycelia, increased SDS susceptibility, and decreased pathogenicity, indicating that... SsPHACA This gene plays a crucial role in regulating the sexual mating, SDS tolerance, and pathogenicity of *Ustilago maydis*. This invention provides a novel target gene for the development of effective fungicides. Attached Figure Description

[0041] Figure 1 This is a vector diagram of pDNA-HPT.

[0042] Figure 2 This is a schematic diagram illustrating the predicted domain structure of the SsPhacA protein.

[0043] Figure 3 for SsPHACA Expression spectrum during the sexual mating stage.

[0044] Figure 4 for SsPHACA A. Electrophoresis diagrams of PCR and RT-qPCR verification of knockout mutants and complement strains.

[0045] Figure 5 for SsPHACA The knockout effect on sexual mating and mycelial growth ability.

[0046] Figure 6 for SsPHACA The effects of knockout on SDS stress survival, from left to right: OD 600 The bacterial solution was 1.0, and it was prepared according to a 10... 0 10 -1 10 -2 10 -3 Diluted growth conditions.

[0047] Figure 7 for SsPHACA The impact of knockout on pathogenicity. A: Symptoms of disease; B: Incidence statistics. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0049] The wild type of sugarcane smut fungus used in the following examples MAT-1 and MAT-2 Disclosed in Transcriptome analysis of Sporisorium scitamineum The fungi were isolated, identified, and preserved by the Fungal Laboratory of South China Agricultural University in the paper "Reveals critical environmental signals for fungal sexual mating and filamentous growth".

[0050] The culture media used in the following examples were prepared as follows:

[0051] YePS liquid medium preparation: Weigh 20 g of peptone, 20 g of sucrose and 10 g of yeast extract, then add deionized water to a final volume of 1 L, mix well and dispense into containers, and sterilize at 121℃ for 20 min; YePS solid medium preparation: Add 2% agar powder to YePS medium and sterilize at 121℃ for 20 min.

[0052] The pEASY-COM vector, constructed and preserved in the inventor's laboratory, consists of a GPA promoter and an anti-bleomycin gene fusion DNA fragment inserted at the HindIII restriction site of the pEASY-Blunt vector. This vector is available to the public from the applicant. The pDNA-HPT vector, carrying the anti-hygromycin gene (HPT), was constructed and preserved in the inventor's laboratory; its vector diagram is shown below. Figure 1 The vector is disclosed in the literature "Establishment of a gene knockout method for DNA double fragment transformation by protoplast of *Ustilago canis*" (Cai Enping, Mei Dan, Zhang Xiaomeng, et al. *Acta Mycosystema Sinica*, 2020, 39(12):14. DOI:10.13346 / j.mycosystema.200273), and the public can obtain it from the applicant.

[0053] Example 1 SsPHACA The acquisition

[0054] Transcriptomic analysis of the sexual reproductive stage of *Ustilago maydis* revealed a gene encoding a cytochrome P450 enzyme. SsPHACA The expression level of α is significantly upregulated during the sexual mating phase.

[0055] Will get SsPHACA Its nucleotide sequence is shown in SEQ ID NO:1, with a length of 1595 bp, and it encodes the SsPhacA protein, with an amino acid sequence shown in SEQ ID NO:2, with a length of 497 amino acids.

[0056] The SsPhacA protein sequence (amino acid sequence shown in SEQ ID NO:2) was submitted to the SMART online analysis platform (http: / / smart.embl-heidelberg.de / ) for protein domain prediction. The prediction results showed that the SsPhacA protein encoded by this sequence contains 497 amino acid residues, and its structural features include a transmembrane region and two highly conserved cytochrome P450 functional domains (see...). Figure 2 Based on the above-mentioned structural domain composition characteristics, SsPhacA It is a conserved member of the cytochrome P450 enzyme family.

[0057] Example 2 SsPHACA Expression profile analysis during the sexual mating stage

[0058] I. Experimental Methods

[0059] Wild-type cells were treated with YePS liquid medium. MAT-1 and MAT-2 The strain was cultured overnight, and when the OD of the culture medium... 600When the OD value reaches 1.5–2.0 and the growth states of the two strains are basically the same, the bacterial cells are collected by centrifugation. Subsequently, the bacterial cells are washed once with sterile deionized water, and the washed bacterial cells are resuspended in appropriate sterile water to adjust the OD value of the bacterial solution. 600 Value up to 1.0.

[0060] Next, equal volumes MAT-1 and MAT-2 The bacterial cultures were thoroughly mixed and spread onto YePS agar plates. Total RNA was extracted from the mixed bacterial cells at 0, 12, 24, 36, 48, and 60 h after sexual mating, and the RNA was analyzed using RT-qPCR during the sexual mating process of *Ustilago maydis*. SsPHACA transcriptional expression levels; ACTIN The gene was used as an internal reference gene, and the primers are shown in Table 1.

[0061] Table 1: RT-qPCR Primer Information

[0062]

[0063] RT-qPCR analysis was performed using ChamQ Universal SYBR qPCR Master Mix. The reaction mixture consisted of 10.0 μL of 2 × ChamQ Universal SYBR qPCR Master Mix, 0.8 μL of forward primers (5 μM), 0.8 μL of reverse primers (5 μM), 1 μL of diluted cDNA, and deionized water to a final volume of 20 μL. The reaction procedure is shown in Table 2.

[0064] Table 2:

[0065]

[0066] Three biological replicates were set up, and the results are expressed as the mean, with the error bar representing the standard error. Data analysis used the Tukey test for significance analysis.

[0067] II. Experimental Results

[0068] RT-qPCR results are shown below Figure 3 The results showed that SsCYP86 Genes in wild type MAT-1 and MAT-2 Expression levels were significantly upregulated 24 hours after mixing, peaking at 48 hours, and then gradually decreased. The results indicate that... SsCYP86 Genes play an important role in the sexual mating and mycelial growth of *Ustilago maydis*.

[0069] Example 3 SsPHACA Knockout construction

[0070] I. Experimental Methods

[0071] (i) Construction using homologous recombination technology SsPHACA Knockout strains.

[0072] (1) Amplification of the right homologous arm and the left homologous arm

[0073] First, using wild-type strains of sugarcane smut fungus. MAT-1 Using genomic DNA as a template, specific primers were designed for PCR amplification to obtain the homologous arm DNA sequence of the target gene.

[0074] For the left homologous arm, a forward primer was used. SsPHACA -LB-F and reverse primers SsPHACA Amplification was performed using the -LB-R primer (which incorporates a 15-25 bp sequence fused to the upstream end of the HPT-up primer at its 5' end) to obtain the left homologous arm (LB); for the right homologous arm, a reverse primer was used. SsPHACA -RB-R and forward primers SsPHACA Amplification was performed using RB-F (the 5' end of which incorporates a 15-25 bp sequence downstream of the HPT-down primer) to obtain the right homologous arm RB. Relevant primers are shown in Table 3.

[0075] Table 3: SsPHACA left and right homologous arm primer information

[0076]

[0077] PCR amplification reaction system: 12.5 μL 2× Phanta® Max Buffer, 0.5 μL dNTP (10 mM), 1 μL Forward primers (5 μM), 1 μL Reverse primers (5 μM), 1 μL DNA template (10 ng / μL), 0.5 μL Phanta Max Super-Fidelity DNA Polymerase, and deionized water to a final volume of 25 μL. The PCR amplification reaction procedure is shown in Table 4.

[0078] Table 4:

[0079]

[0080] (2) Amplification of the hygromycin resistance gene fragments HPT-up and HPT-down

[0081] Using the pDNA-HPT vector as a template, PCR amplification was performed using the forward primer pDNA-LB-F and the reverse primer LB-226-R of HPT-up to obtain the DNA sequence of the upstream fragment (HPT-up) of the hygromycin resistance gene. Using the pDNA-HPT vector as a template, PCR amplification was performed using the forward primer RB-225-F and the reverse primer pDNA-RB-R of HPT-up to obtain the DNA sequence of the downstream fragment (HPT-down) of the hygromycin resistance gene. The relevant primers are shown in Table 5.

[0082] Table 5: Primer information for the left and right homologous arms of the hygromycin resistance gene

[0083]

[0084] The PCR amplification reaction system and procedure are the same as above.

[0085] (3) Perform fusion PCR amplification

[0086] After completing the above amplification, the PCR products were identified by gel electrophoresis. Once confirmed to be correct, each product was diluted 10-fold.

[0087] Using 1 μL of diluted left homologous arm LB and HPT-up as templates, the forward primer of the left homologous arm was used. SsPHACA Fusion PCR amplification was performed using LB-F and HPT-up reverse primer LB-226-R to obtain fusion PCR product 1 (nucleotides as shown in SEQ ID NO: 15).

[0088] Take 1 μL of diluted right homologous arm RB and HPT-down as templates, and use the right homologous arm reverse primer. SsPHACA Fusion PCR amplification was performed using the forward primers RB-225-F and HPT-down to obtain fusion PCR product 2 (nucleotides as shown in SEQ ID NO:16).

[0089] The PCR amplification reaction system and procedure were the same as above. The PCR products were identified by gel electrophoresis. Fusion PCR product 1 and fusion PCR product 2 were recovered by gel electrophoresis and stored at -20°C for later use.

[0090] (4) Construction of knockout mutants

[0091] Wild type MAT-1 and MAT-2 Using the strain as a background, fusion PCR product 1 and fusion PCR product 2 were co-transformed into protoplasts via PEG-mediated protoplast transformation, and homologous recombination was used to knock out the strains. SsPHACA Knockout mutants were successfully obtained. ssphacA Δ-1 and ssphacA Δ -2 During the screening process, hygromycin B (Hyg B) was used for resistance screening to ensure the accuracy of mutants.

[0092] (two) SsPHACA Construction of complement strains

[0093] SsPHACA The principle of in situ replacement is similar to that of homologous recombination knockout. This is because each homologous recombination knockout mutant contains a DNA insertion fragment of approximately 3.0 kb (including the antihygromycin gene). HPT (Sequence included), therefore, the antihygromycin gene HPT Partial sequences were knocked out, targeting the bleomycin resistance gene ( ZEO R The resistance selection gene is then used, followed by PEG-mediated protoplast transformation to introduce two homologous recombination DNA fragments for complementation into the knockout mutant protoplasts. HPT A partial sequence of a gene.

[0094] (1) Using efficient DNA seamless cloning technology to... SsPHACA The specific method for cloning to the pEASY-COM vector is as follows:

[0095] First, using wild-type genomic DNA as a template, a complement primer with a 5' end-lined vector terminal sequence was introduced to backfill the sequence. SsPHACA -COM-F and SsPHACA -COM-R for PCR amplification SsPHACA DNA fragment replacement (including) SsPHACA (Gene promoter, full-length gene, and terminator), relevant primers are shown in Table 6.

[0096] Table 6: SsPHACA Primer information recovery

[0097]

[0098] The PCR amplification reaction system and procedure are the same as above;

[0099] Secondly, the pEASY-COM vector was linearized by using HindIII as the restriction site;

[0100] Finally, the linearized carrier is combined with the above-mentioned SsPHACA The inserted DNA fragment was mixed at a molar ratio of 1:2 and incubated at 37°C for 30 min under the catalysis of recombinase ClonExpress® II (Vazyme) to complete cloning, thus constructing the in situ addition vector pEASY-COM-SsPHACA .

[0101] (2) Amplification of fused DNA fragments

[0102] pEASY-COM- SsPHACA Using the vector as a template and primers COM-LB-F and COM-LB-R, a complete PCR amplification was performed to produce a single molecule consisting of the left homologous arm sequence. SsPHACA And a DNA fragment 1 fused with a portion of the anti-bleomycin gene (nucleotides as shown in SEQ ID NO:19). pEASY-COM- SsPHACA Using the vector as a template, primers COM-RB-F and COM-RB-R were used to amplify a DNA fragment 2 (nucleotides shown in SEQ ID NO: 20) consisting of a partial bleomycin resistance gene fused with a right homologous arm sequence. The relevant primers are shown in Table 7.

[0103] Table 7: Information on Universal Complementation Primers

[0104]

[0105] The PCR amplification reaction system and procedure are the same as above.

[0106] (3) PEG-mediated protoplast transformation

[0107] DNA fragment 1 and DNA fragment 2 were jointly transferred into ssphacA Δ -1 or ssphacA Δ -2 Corresponding replacement strains were obtained from the mutant cells. ssphacA Δ / PHACA-1 and ssphacA Δ / PHACA-2 .

[0108] (three) SsPHACA Identification of knockout and replacement strains

[0109] (1) PCR amplification detection

[0110] Wild type of sugarcane smut ( MAT-1 and MAT-2 ), SsPHACA Knockout ( ssphacA Δ -1 and ssphacA Δ -2 mutants) and complement strains ( ssphacA Δ / PHACA-1 and ssphacA Δ / PHACA-2 Using genomic DNA as a template, detection primers were used. SsPHACA -inside-F / SsPHACA-inside-R (inside, according to) SsPHACA (Specific primers designed from the knocked-out DNA) and peripheral detection primers SsPHACA -outside-F / SsPHACA -outside-R (outside, according to SsPHACA Specific primers designed from the DNA sequences upstream of the left homologous arm and downstream of the right homologous arm were used for PCR amplification. The primers are shown in Table 8.

[0111] Table 8: SsPHACA Information on endogenous and peripheral primers for detection

[0112]

[0113] The PCR reaction system consisted of 12.5 μL of 2× Green Taq Mix (Vazyme), 1 μL of DNA, 1 μL each of forward primers (5 μM) and reverse primers (5 μM), and deionized water to a final volume of 25 μL. The reaction procedure is shown in Table 9.

[0114] Table 9:

[0115]

[0116] (2) RT-qPCR detection

[0117] RT-qPCR analysis was performed using ChamQ Universal SYBR qPCR Master Mix to detect wild-type *Ustilago maydis*. MAT-1 and MAT-2 ), SsPHACA Knockout ( ssphacA Δ -1 and ssphacA Δ -2 mutants) and complement strains ( ssphacA Δ / PHACA-1 and ssphacA Δ / PHACA-2 )of SsPhacA The expression status, and the specific detection method is as follows:

[0118] Will MAT-1 , MAT-2 , ssphacA Δ -1 , ssphacA Δ -2 , ssphacA Δ / PHACA-1 and ssphacA Δ / PHACA-2After growing the cells in YePS solid medium for 24 h, the bacterial cells were collected, and total RNA was extracted from the mixed bacterial cells according to the method in Example 2. The RNA was then detected by RT-qPCR. SsPHACA transcriptional expression levels; ACTIN Genes are used as internal reference genes.

[0119] II. Experimental Results

[0120] The results showed that both wild-type and complemented transformants could be amplified by internal detection primers to extract the target DNA fragment, while ssphacA Δ -1 and ssphacA Δ -2 The mutant cannot be amplified to produce the target DNA fragment; however, both wild-type and knockout transformants can be amplified to produce target DNA fragments of different sizes by external detection primers (see...). Figure 4 (A in the middle).

[0121] RT-qPCR analysis results (see) Figure 4 B) shows that SsPHACA exist ssphacA Δ -1 and ssphacA Δ -2 No signal was detected in the mutant, but in the replacement strain ssphacA Δ / PHACA-1 and ssphacA Δ / PHACA-2 The signal was detected in the medium-sized cells and was indistinguishable from that of the wild-type cells.

[0122] The above results indicate that the system was successfully built. SsPHACA Knockout and reintroduction of strains.

[0123] Example 4 ssphacA Sexual mating and hyphal growth of Δ mutants

[0124] I. Experimental Methods

[0125] YePS liquid medium was used for MAT-1 , MAT-2 , ssphacA Δ -1 , ssphacA Δ -2 , ssphacA Δ / PHACA-1 as well as ssphacA Δ / PHACA-2 The strains were incubated overnight, and the OD values ​​of the culture media for each strain were adjusted accordingly. 600 Once the OD value reached 1.5–2.0 and the growth status became uniform, the bacterial cells were collected by centrifugation. Subsequently, the bacterial cells were washed once with sterile deionized water, and the washed bacterial cells were resuspended in appropriate sterile water to adjust the OD of the bacterial solution. 600Value up to 1.0. According to the following combination methods: MAT-1 × MAT-2 , ssphacA Δ -1 × ssphacA Δ -2 and ssphacA Δ / PHACA-1 × ssphacA Δ / PHACA-2 Equal volumes of different mating strains were mixed. After mixing, 1.5 µL of the bacterial solution was spotted onto YePS solid medium. After the bacterial solution dried, the medium was placed in a constant temperature environment at 28°C for 1–2 days for static incubation. Subsequently, the sexual mating and mycelial growth of *Ustilago maydis* were recorded by photography.

[0126] II. Experimental Results

[0127] See results Figure 5 The results showed that MAT-1 × MAT-2 ,and ssphacA Δ / PHACA-1 × ssphacA Δ / PHACA- 2 The combination can form fluffy white hyphae, while ssphacA Δ -1 × ssphacA Δ -2 The ability of the mycelium to form white hyphae was significantly reduced. This phenomenon indicates that... SsPHACA It plays a key role in regulating the sexual mating and mycelial growth of *Ustilago maydis*. SsPHACA The knockout of the substance significantly inhibited the sexual mating and mycelial growth of *Ustilago maydis*.

[0128] Example 5 SsPHACA Effects on survival under sodium dodecyl sulfate (SDS) stress

[0129] I. Experimental Methods

[0130] Incubate overnight in YePS liquid medium. MAT-1 , ssphacA Δ -1 and ssphacA Δ / PHACA-1 strain, waiting for its OD 600 When the bacterial cells were between 1.5 and 2.0 μg / mL and in a generally consistent growth state, they were collected by centrifugation, washed once with sterile deionized water, and then resuspended in sterile deionized water to adjust their OD value. 600 The initial concentration was 1.0; then the bacterial culture was serially diluted 10-fold to 10. 0 10 -1 10 -2 10 -3Next, 1.5 μL of bacterial culture was spotted onto YePSA medium (prepared fresh) with or without 0.011% SDS, dried, and cultured at 28°C for 3–4 days; the growth of sugarcane smut fungus under SDS stress was recorded by photograph.

[0131] II. Experimental Results

[0132] The results showed that, compared to wild type and ssphacA Δ / PHACA-1 strain, ssphacA Δ -1 The growth of the mutant was inhibited on YePSA medium containing 0.011% SDS (see [link]). Figure 6 ),show SsPHACA Membrane stability is affected by regulating membrane repair-related pathways (such as lipid metabolism, membrane protein synthesis, or vesicle transport).

[0133] Example 6 SsPHACA Effects on pathogenicity

[0134] I. Experimental Methods

[0135] YePS liquid medium was used for MAT-1 , MAT-2 , ssphacA Δ -1 , ssphacA Δ -2 , ssphacA Δ / PHACA-1 and ssphacA Δ / PHACA-2 The strains were incubated overnight, and the OD values ​​of the culture media for each strain were adjusted accordingly. 600 Once the OD value reached 1.5–2.0 and the growth status was basically uniform, the bacterial cells were collected by centrifugation, and the supernatant culture medium was discarded. Subsequently, sterile deionized water was added to resuspend the bacterial cells, and the OD value was adjusted to 1.5–2.0. 600 The value is 1.0. Equal volumes of different mating types of *Smutsia candel* were mixed according to the following combination: MAT-1 × MAT-2 (wild type), ssphacA Δ -1 × ssphacA Δ -2 ( ssphacA Δ) and ssphacA Δ / PHACA-1 × ssphacA Δ / PHACA-2 ( ssphacA Δ / PHACA Inject 0.5 mL of the mixed bacterial solution near the growing point of the stem of infected sugarcane variety XTT22 seedlings (at the five-leaf stage).

[0136] Twelve sugarcane seedlings were injected each time, with three biological replicates. Afterward, the inoculated sugarcane seedlings were placed in a greenhouse for 3–6 months, during which time the appearance of black whip symptoms was observed, and disease incidence was recorded.

[0137] II. Experimental Results

[0138] The results showed that inoculation with wild-type strains and SsPHACA Replacement strains ( ssphacA Δ / PHACA The sugarcane seedlings all showed typical black whip symptoms (see...). Figure 7 (A in the text), and the incidence rate is over 70% (see A in the text). Figure 7 (B in the middle).

[0139] In contrast, vaccination ssphacA Δ -1 × ssphacA Δ -2 In sugarcane seedlings, the frequency of typical whip-like symptoms was significantly reduced, with an incidence rate of only about 6% (see...). Figure 7 (A in 7 and B in 7). The results show that... SsPHACA It is crucial for the pathogenicity of sugarcane smut, and is an essential gene for the pathogen's pathogenesis.

[0140] Example 7 Using SsPHACA Gene expression and SsPhacA protein expression were used as targets for screening and evaluating drugs against sugarcane smut.

[0141] Sugarcane smut fungus was placed in YePS medium and incubated overnight at 28°C with shaking at 200 rpm. The culture medium was incubated until the OD value of the culture medium was... 600 When the value reaches 1.5–2.0, the mycelia are collected and divided into several small portions. The compound to be screened or the candidate drug is added to each portion, and the mixture is cultured for several hours. Then, bacterial RNA or protein is extracted, and real-time quantitative PCR is used for detection. SsPHACA The expression status of the gene was detected by Western blot.

[0142] If the candidate drug inhibits SsPHACA The expression of the gene or SsPhacA protein within the bacteria... SsPHACA The expression levels of the gene and SsPhacA protein were significantly reduced, or even absent.

[0143] The obtained compound was reused using the method of Example 6 to determine whether the pathogenicity of wild-type sugarcane smut fungus to sugarcane was reduced in the presence of this compound or candidate drug, and to further determine the compound's effect against sugarcane smut fungus.

[0144] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of a pathogenic protein of Sphacelotheca sacchari in modulating the pathogenicity of Sphacelotheca sacchari, characterized in that, The pathogenic protein has an amino acid sequence as shown in SEQ ID NO: 2, and knocking out or silencing the pathogenic protein inhibits the pathogenicity of the smut fungus.

2. Use of a pathogenicity gene of S. sacchari in modulating the pathogenicity of S. sacchari, characterized in that, The pathogenic gene is a coding gene of a pathogenic protein of the smut fungus with an amino acid sequence as shown in SEQ ID NO: 2, and knocking out or silencing the pathogenic gene inhibits the pathogenicity of the smut fungus.

3. Use of a pathogenic protein of S. sacchari in the regulation of sexual compatibility of S. sacchari, characterized in that, The pathogenic protein has an amino acid sequence as shown in SEQ ID NO: 2, and knocking out or silencing the pathogenic protein inhibits the sexual conjugation of the smut fungus.

4. Use of a pathogenic gene of S. sacchari in the regulation of sexual compatibility of S. sacchari, characterized in that, The pathogenic gene is a coding gene of a pathogenic protein of the smut fungus with an amino acid sequence as shown in SEQ ID NO: 2, and knocking out or silencing the pathogenic gene inhibits the sexual conjugation of the smut fungus.

5. Use of a pathogenic protein of S. sacchari in the regulation of hyphal growth of S. sacchari, characterized in that, The pathogenic protein has an amino acid sequence as shown in SEQ ID NO: 2, and knocking out or silencing the pathogenic protein inhibits the hyphal growth of the smut fungus.

6. Use of a pathogenic gene of S. sacchari in regulating the mycelial growth of S. sacchari, characterized in that, The pathogenic gene is a coding gene of a pathogenic protein of the smut fungus with an amino acid sequence as shown in SEQ ID NO: 2, and knocking out or silencing the pathogenic gene inhibits the hyphal growth of the smut fungus.

Images