Application of pathogenic factors of sugarcane smut fungus in regulating pathogenicity of sugarcane smut fungus

Abstract

The application discloses application of a pathogenic factor of a sugarcane smut fungus in regulating pathogenicity of the sugarcane smut fungus. An amino acid sequence of the pathogenic protein SsCyp64 is shown as SEQ ID NO: 2, and a pathogenic gene SsCYP64 coding the amino acid sequence is shown as SEQ ID NO: 1. The application obtains a knockout mutant by a PEG-mediated protoplast transformation method in a wild type of the sugarcane smut fungus SsCYP64 , and obtains a back complement strain by respectively back complementing the gene in the knockout mutant. It is found that the knockout mutant has weakened ability of forming a double mycelium by sexual cooperation, increased sensitivity to H2O2 and SDS stress tolerance, and decreased pathogenicity, which indicates that SsCYP64 the pathogenic factor plays an important role in regulating sexual cooperation, oxidation resistance, SDS tolerance and pathogenicity of the sugarcane smut fungus. The application provides a target gene for developing an effective fungicide.

Description

Technical Field

[0001] This invention relates to the field of sugarcane disease technology, and more specifically, to the application of pathogenic factors of *Ustilago maydis* in regulating the pathogenicity of *Ustilago maydis*. Background Technology

[0002] By sugarcane smut fungus ( Sporisorium scitamineum Sugarcane smut is a devastating disease in sugarcane production. The teliospores produced by the pathogen through sexual reproduction can be spread over long distances by air currents. After infecting the plant, they form black whip-like structures, leading to severe yield reduction.

[0003] Currently, sugarcane smut is mainly controlled through chemical, agricultural, biological, and resistant variety screening methods, but these approaches still have limitations. Agricultural control measures such as timely irrigation, proper fertilization and hilling, timely removal and burning of diseased plants, maintaining ventilation and light penetration in sugarcane fields, and strengthening crop rotation can reduce the number of pathogens, improve soil structure, increase soil fertility, and enhance sugarcane resistance to some extent. However, these agricultural control measures can only minimize the losses caused by sugarcane smut and require significant investment of manpower and resources. Existing control methods rely on chemical fungicides (such as propiconazole and quinolinone), but long-term use can easily induce resistance.

[0004] While biological control is the most ideal control measure for sugarcane smut, its effectiveness is limited by factors such as sugarcane variety, microecological environment, and actual natural environment. The severity of sugarcane smut is closely related to the resistance level of sugarcane varieties. Therefore, screening and breeding resistant sugarcane varieties is an effective, safe, and environmentally friendly method for controlling sugarcane smut. However, current breeding efforts for resistant sugarcane smut are insufficient, and the long replacement cycle of resistant varieties hinders their widespread adoption. Therefore, there is an urgent need to discover novel molecular targets in the pathogen to develop efficient and sustainable control strategies.

[0005] Cytochrome P450 (CYP) is a superfamily of enzymes belonging to the monooxygenase class, named for its specific absorption peak at 450 nm. Members of this cytochrome family are present in all organisms throughout evolutionary history (from bacteria to humans). Currently, thousands of cytochrome P450 species have been discovered in various organisms, with nearly a hundred identified physiological functions. While members of the cytochrome P450 superenzyme family share some structural similarities, their functions cannot be directly predicted based on their structure. Different members exhibit differences in substrate specificity, catalytic activity, and other characteristics. Summary of the Invention

[0006] The application aims to overcome the deficiencies of the prior art and provide the application of a factor of smut fungus of sugarcane in regulating pathogenicity of smut fungus of sugarcane.

[0007] The first object of the application is to provide the application of an inhibitor of a protein with an amino acid sequence as shown in SEQ ID NO: 2 in the preparation of a medicine against smut fungus of sugarcane or sugarcane smut.

[0008] The second object of the application is to provide the application of an inhibitor of a gene encoding a protein with an amino acid sequence as shown in SEQ ID NO: 2 in the preparation of a medicine against smut fungus of sugarcane or sugarcane smut.

[0009] The third object of the application is to provide the application of a product of knocking out or silencing a pathogenic protein or pathogenic gene of smut fungus of sugarcane in the prevention of sugarcane smut.

[0010] The fourth object of the application is to provide the application of a reagent for detecting a pathogenic protein or pathogenic gene of smut fungus of sugarcane in the screening of a potential medicine for inhibiting pathogenicity of smut fungus of sugarcane.

[0011] The fifth object of the application is to provide the application of a pathogenic protein or pathogenic gene of smut fungus of sugarcane.

[0012] The sixth object of the application is to provide a method for screening a medicine for preventing and treating sugarcane smut caused by smut fungus of sugarcane.

[0013] The seventh object of the application is to provide a method for evaluating the ability of a medicine to reduce pathogenicity of smut fungus of sugarcane.

[0014] The eighth object of the application is to provide a smut fungus of sugarcane with low pathogenicity.

[0015] In order to achieve the above objects, the application is implemented by the following technical solutions:

[0016] The application provides a gene from smut fungus of sugarcane, which has the functions of regulating sexual compatibility, resisting oxidation, tolerating SDS and pathogenicity, and is named SsCyp64. SsCYP64 The DNA nucleotide sequence of the SsCyp64 gene is shown as SEQ ID NO: 1, and the open reading frame of the DNA sequence is composed of 1563 nucleotides, containing one intron sequence.

[0017] The application provides a SsCyp64 protein encoded by the SsCyp64 gene. SsCYP64 The amino acid sequence of the SsCyp64 protein is shown as SEQ ID NO: 2, and the sequence is composed of 394 amino acids, having two conserved p450 domains.

[0018] The SsCyp64 protein has the functions of regulating sexual compatibility, resisting oxidation, tolerating SDS and pathogenicity. SsCYP64The gene and the SsCyp64 protein are pathogenic factors of smut of sugarcane caused by S. sacchari.

[0019] The present application claims the following applications:

[0020] The use of an inhibitor of a protein with an amino acid sequence as shown in SEQ ID NO: 2 in the preparation of a medicine for resisting S. sacchari or smut of sugarcane.

[0021] The use of an inhibitor of a gene encoding a protein with an amino acid sequence as shown in SEQ ID NO: 2 in the preparation of a medicine for resisting S. sacchari or smut of sugarcane.

[0022] Preferably, the nucleotide sequence of the encoding gene is as shown in SEQ ID NO: 1.

[0023] The use of a product of a pathogenic protein or a pathogenic gene of S. sacchari in resisting smut of sugarcane, wherein the pathogenic protein is a protein with an amino acid sequence as shown in SEQ ID NO: 2, and the pathogenic gene is a gene encoding a protein with an amino acid sequence as shown in SEQ ID NO: 2.

[0024] The use of a reagent for detecting a pathogenic protein or a pathogenic gene of S. sacchari in screening a potential medicine for inhibiting pathogenicity of S. sacchari, wherein the pathogenic protein is a protein with an amino acid sequence as shown in SEQ ID NO: 2, and the pathogenic gene is a gene encoding a protein with an amino acid sequence as shown in SEQ ID NO: 2.

[0025] Preferably, screening is performed with the expression of the pathogenic protein or the pathogenic gene as a target, and a medicine is effective if the expression is reduced.

[0026] More preferably, the expression of the pathogenic gene is detected by a real-time quantitative PCR method, and the expression of the pathogenic protein is detected by a Western blot method.

[0027] The use of a pathogenic protein or a pathogenic gene of S. sacchari, wherein the pathogenic protein is a protein with an amino acid sequence as shown in SEQ ID NO: 2, and the pathogenic gene is a gene encoding a protein with an amino acid sequence as shown in SEQ ID NO: 2.

[0028] The use is for regulating pathogenicity of S. sacchari, regulating sexual compatibility of S. sacchari, regulating hyphal growth of S. sacchari, regulating H2O2 tolerance of S. sacchari, or regulating SDS tolerance of S. sacchari.

[0029] The present application claims a method for screening a drug for preventing and treating sugarcane smut caused by Sphacelotheca sacchari, detecting a pathogenic protein or a pathogenic gene in a sugarcane sample inoculated or infected with sugarcane smut treated by the drug, wherein the pathogenic protein is a protein with an amino acid sequence as shown in SEQ ID NO: 2, and the pathogenic gene is a gene encoding the protein with an amino acid sequence as shown in SEQ ID NO: 2.

[0030] Preferably, the expression of the pathogenic protein or the pathogenic gene is detected, and if the expression is reduced, the drug is effective.

[0031] More preferably, the expression of the pathogenic gene is detected by real-time quantitative PCR, and the expression of the pathogenic protein is detected by Western blot.

[0032] The present application also claims a method for evaluating the pathogenic ability of Sphacelotheca sacchari reduced by a drug, detecting a pathogenic protein or a pathogenic gene in Sphacelotheca sacchari treated by the drug, wherein the pathogenic protein is a protein with an amino acid sequence as shown in SEQ ID NO: 2, and the pathogenic gene is a gene encoding the protein with an amino acid sequence as shown in SEQ ID NO: 2.

[0033] Preferably, the expression of the pathogenic protein or the pathogenic gene is detected, and if the expression is reduced, the drug is effective.

[0034] More preferably, the expression of the pathogenic gene is detected by real-time quantitative PCR, and the expression of the pathogenic protein is detected by Western blot.

[0035] The present application also claims a low-pathogenic Sphacelotheca sacchari with a gene encoding a protein with an amino acid sequence as shown in SEQ ID NO: 2 knocked out.

[0036] Preferably, the nucleotide sequence of the gene encoding the protein with an amino acid sequence as shown in SEQ ID NO: 2 is as shown in SEQ ID NO: 1.

[0037] Compared with the prior art, the present application has the following beneficial effects:

[0038] The present application discloses SsCYP64 application in regulating the sexual compatibility, antioxidant, SDS tolerance pathogenicity of Sphacelotheca sacchari. The present application obtains knockout mutants MAT-1 and MAT-2 by knocking out SsCYP64 in wild type sscyp64 Δ -1 and sscyp64 Δ -2 of Sphacelotheca sacchari by PEG-mediated protoplast transformation method, respectively. SsCYP64Complementation of the knockout mutant with the gene respectively to obtain the complementation strain sscyp64 Δ / CYP64-1 and sscyp64 Δ / CYP64-2 The results show that SsCYP64 The ability of sexual mating to form double mycelium of the knockout mutant is weakened, the sensitivity to H2O2 is increased, and the pathogenicity is decreased, indicating that SsCYP64 plays an important role in the process of regulating sexual mating, antioxidant, SDS tolerance and pathogenicity of the sugarcane smut fungus. The present application provides a target gene for developing effective fungicides. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 It is a vector map of pDNA-HPT.

[0040] Figure 2 It is a schematic diagram of SsCyp64 protein domain prediction.

[0041] Figure 3 It is SsCYP64 Expression profile in the sexual mating stage, average value of 3 biological replicates, error line is standard error, significance analysis is carried out by Tukey test.

[0042] Figure 4 It is SsCYP64 PCR and RT-qPCR verification electrophoretogram of the knockout mutant and the complementation strain.

[0043] Figure 5 It is SsCYP64 Effect of knockout on the ability of sexual mating and mycelial growth.

[0044] Figure 6 It is SsCYP64 Effect of knockout on the survival ability under H2O2 stress, from left to right, it is OD 600 of 1.0, according to 10 0 , 10 -1 , 10 -2 , 10 -3 diluted growth.

[0045] Figure 7 It is SsCYP64 Effect of knockout on pathogenicity; A: disease symptoms; B: disease rate statistics. DETAILED DESCRIPTION

[0046] The present application will be further described in conjunction with the accompanying drawings and specific examples, which are only used to explain the present application and are not used to limit the scope of the present application. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0047] The wild-type sugarcane smut fungus used in the following examples MAT-1 and MAT-2 Isolated, identified and preserved by the Fungus Room of South China Agricultural University, disclosed in the prior art “Transcriptome analysis of Sporisorium scitamineum reveals critical environmental signals for fungal sexual mating and filamentous growth ”.

[0048] The medium formula used in the following examples is as follows:

[0049] YePS liquid medium: take yeast extract 10 g, proteose peptone 20 g, sucrose 20 g, add deionized water to 1 L, sterilize at 121 ℃ for 20 min; YePS solid medium: add 2 % agar powder to the YePS liquid medium, sterilize at 121 ℃ for 20 min.

[0050] The pEASY-COM vector is constructed and preserved by the laboratory of the inventor, which is a DNA fragment of GPA promoter and anti-blasticidin gene fusion inserted at the Hind III enzyme cutting site of the pEASY-Blunt vector. The public can obtain the vector from the applicant; the pDNA-HPT vector carries the anti-hygromycin gene (HPT), which is constructed and preserved by the laboratory of the inventor, and the vector map is shown in Figure 1 . Disclosed in the document “Establishment of gene knockout method of DNA double fragment of sugarcane smut fungus protoplast transformation” (Cai EP, Mei D, Zhang XM, et al. Mycosystema, 2020, 39(12): 14. DOI: 10.13346 / j.mycosystema.200273), the public can obtain the vector from the applicant.

[0051] Example 1 SsCYP64 Obtained

[0052] Transcriptomic analysis of the sexual reproduction stage of *Ustilago maydis* revealed a gene associated with the sexual mating stage. Its nucleotide sequence, as shown in SEQ ID NO:1, is 1563 bp in length, and the amino acid sequence of the protein it encodes, as shown in SEQ ID NO:2, is 394 amino acids in length. This gene was submitted to the SMART online analysis platform (http: / / smart.embl-heidelberg.de / ) for protein domain prediction.

[0053] The results showed that it possesses one LCR region (Low Complexity Region) and two conserved cytochrome p450 domains (see...). Figure 2 This indicates that SsCyp64 may be a conserved cytochrome p450 enzyme, and it was named... SsCYP64 .

[0054] Example 2 SsCYP64 Changes in expression levels during sexual mating phase

[0055] I. Experimental Methods

[0056] Wild-type cells were cultured overnight in YePS liquid medium. MAT-1 and MAT-2 Wait for its OD 600 When the bacterial cells were between 1.5 and 2.0 and in a basically uniform growth state, they were collected by centrifugation, washed once with sterile deionized water, and resuspended to achieve an OD of 1.5-2.0. 600 It is 1.0.

[0057] Then the two resuspended bacterial solutions were mixed in equal volumes. MAT-1 × MAT-2 The sample was spread onto YePS solid medium, and total RNA was extracted at 0, 12, 24, 36, 48, and 60 h after spreading.

[0058] RT-qPCR detection of different time points during sexual mating of *Smuts spp.* SsCYP86 Gene transcription and expression status, in order to ACTIN The gene is used as an internal reference. Primers are shown in Table 1.

[0059] Table 1: RT-qPCR Primer Information

[0060]

[0061] RT-qPCR was performed using ChamQ Universal SYBR qPCR Master Mix. The reaction system was as follows: 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 was added to bring the volume to 20 μL. The reaction procedure is shown in Table 2.

[0062] Table 2:

[0063]

[0064] II. Experimental Results

[0065] See results Figure 3 The results showed SsCYP86 Genes in wild type MAT-1 × MAT-2 Expression significantly increased 24 h after mixing, peaked at 48 h, and then decreased, indicating that... SsCYP86 Genes are involved in the sexual mating and mycelial growth of *Ustilago maydis*.

[0066] Example 3 SsCYP64 Construction of knockout and complement strains

[0067] I. Experimental Methods

[0068] Constructed using homologous recombination technology SsCYP64 Knockout and reintroduction of strains.

[0069] 1. SsCYP64 Construction of knockout mutants

[0070] (1) Amplification of the right homologous arm and the left homologous arm

[0071] wild-type strain of sugarcane smut MAT-1 Using genomic DNA as a template, a left homologous arm forward primer was used. SsCYP64 -LB-F and 5' end introduction HPT -Reverse primer for the upstream end of the 15-25 bp sequence. SsCYP64 PCR was performed using LB-R to amplify the left homologous arm; using the genomic DNA of wild-type sugarcane smut fungus strain MAT-1 as a template, the right homologous arm reverse primer was used. SsCYP64 -RB-R and 5' end introduction HPT -The forward primer for the downstream 15-25 bp sequence. SsCYP64 PCR was performed using RB-F primers to amplify the right homologous arm; specific primers are shown in Table 3.

[0072] Table 3: SsCYP64 Left and right homologous arm primer information

[0073]

[0074] The PCR amplification reaction system was as follows: 2x Phanta® Max Buffer 12.5 μL, dNTP (10 mM) 0.5 μL, Forward primers (5 μM) 1 μL, Reverse primers (5 μM) 1 μL, DNA template (10 ng / μL) 1 μL, Phanta Max Super-Fidelity DNA Polymerase 0.5 μL, and deionized water supplemented to 25 μL.

[0075] The PCR amplification reaction program is shown in Table 4,

[0076] Table 4:

[0077]

[0078] (2) Amplification of resistance hygromycin gene fragments HPT-up and HPT-down

[0079] The pDNA-HPT vector was used as a template, and the forward primer pDNA-LB-F and the reverse primer LB-226-R were used for PCR amplification to obtain the DNA sequence of the resistance hygromycin gene upstream fragment (HPT-up). The pDNA-HPT vector was used as a template, and the forward primer RB-225-F and the reverse primer pDNA-RB-R were used for PCR amplification to obtain the DNA sequence of the resistance hygromycin gene downstream fragment (HPT-down). The PCR amplification reaction system and reaction program were the same as above, and the related primers are shown in Table 5.

[0080] Table 5: Left and right homologous arm primer information of resistance hygromycin gene

[0081]

[0082] (3) Perform fusion PCR amplification

[0083] After the above amplification, the PCR products were identified by gel electrophoresis, and after confirmation of no error, each product was diluted 10 times.

[0084] After dilution, 1 μL of LB and HPT -up were mixed as a template, and the left homologous arm forward primer SsCYP64 -LB-F and HPT-up reverse primer LB-226-R is a PCR primer, and PCR amplification is carried out to obtain LB and HPT -up into a linear DNA fragment LB- HPT -up (nucleotide sequence as shown in SEQ ID NO: 15);

[0085] diluted RB and HPT -down each 1 μL, mixed as a template, right homologous arm reverse primer SsCYP64 -RB-R and HPT -down forward primer RB-225-F is a PCR primer, and PCR amplification is carried out to obtain RB and HPT -down into a linear DNA fragment RB- HPT -down (nucleotide sequence as shown in SEQ ID NO: 16);

[0086] The PCR amplification reaction system and the reaction procedure are the same as above. After PCR amplification, the PCR products are identified by gel electrophoresis, and LB- HPT -up and RB- HPT -down are recovered by gel electrophoresis and stored in a refrigerator at -20°C for standby use.

[0087] (4) Construction of knockout mutants

[0088] The wild type MAT-1 and MAT-2 strains are taken as backgrounds, LB- HPT -up and RB- HPT -down are co-transformed into protoplasts by PEG-mediated protoplast transformation method, and SsCYP64 are homologously knocked out, respectively, to obtain knockout mutants sscyp64 Δ -1 and sscyp64 Δ -2 , and Hyg B is used for resistance screening.

[0089] (II) SsCYP64 Construction of complementation strains

[0090] SsCYP64 The principle of in situ complementation is similar to that of two-fragment homologous recombination knockout. There is a DNA insertion fragment of about 3.0 kb in each correct homologous recombination knockout mutant (including the HPT sequence of the anti-hygromycin gene HPT Therefore, the anti-hygromycin gene ZEO R) For resistance screening gene, then PEG-mediated protoplast transformation will introduce two homologous recombination DNA fragments for back complementation into knockout mutant protoplast, then homologous knockout mutant HPT partial sequence of the gene.

[0091] (1) SsCYP64 Cloned into pEASY-COM vector, the specific method is as follows:

[0092] Using wild type genomic DNA as template, using 5' end of linearization vector end sequence of back complementation forward primer reverse primer SsCYP64 -COM-F and SsCYP64 -COM-R for PCR amplification SsCYP64 Back complementation DNA fragment (including SsCYP64 gene itself promoter, gene full length and terminator), PCR amplification reaction system and reaction program are the same as above, see Table 6 for related primers.

[0093] Table 6: SsCYP64 Back complementation primer information

[0094]

[0095] Then, using Hind III, the pEASY-COM vector is cut into linearization;

[0096] Finally, the linearized vector and the above SsCYP64 back complementation DNA fragment, according to the molar ratio of vector to insert fragment is 1:2, mixed, under the catalysis of recombinase ClonExpress® II (Vazyme), incubated at 37℃ for 30 min to complete the cloning, construct in situ back complementation vector pEASY-COM- SsCYP64 .

[0097] (2) Amplification of the fused DNA fragment

[0098] Using the correct pEASY-COM- SsCYP64 vector as template, using universal primer COM-LB-F and COM-LB for PCR amplification, obtaining a DNA fragment 1 fused by left homologous arm sequence, complete SsCYP64 and partial resistance to bleomycin gene (nucleotide sequence as shown in SEQ ID NO: 19).

[0099] Using the correct pEASY-COM- SsCYP64The vector is a template, and a DNA fragment 2 (nucleotide sequence is shown as SEQ ID NO: 20) fused by part of the anti-blastic gene and the right homologous arm sequence is obtained by PCR amplification using the universal primer COM-RB-F and COM-RB-R. The PCR amplification reaction system and reaction procedure are the same as above, and the related primers are shown in Table 7.

[0100] Table 7: Information of universal backfill primers

[0101]

[0102] (3) PEG-mediated protoplast transformation

[0103] The DNA fragment 1 and the DNA fragment 2 are jointly transformed into sscyp64 Δ -1 and sscyp64 Δ -2 The corresponding backfill strains are obtained in the mutant cells sscyp64 Δ / CYP64-1 and sscyp64 Δ / CYP64-2 .

[0104] The PCR amplification reaction system and reaction procedure are the same as above.

[0105] (Three) SsCYP64 Identification of knockout and backfill strains

[0106] (1) PCR detection

[0107] The genomic DNA of the wild type of the sugarcane smut fungus, SsCYP64 knockout mutant sscyp64 Δ -1 , SsCYP64 knockout mutant sscyp64 Δ -2 , backfill strain sscyp64 Δ / CYP64-1 and backfill strain sscyp64 Δ / CYP64-2 is used as a template to perform internal detection PCR amplification and external detection PCR amplification, respectively, and electrophoresis is performed.

[0108] The primers for internal detection PCR amplification are SsCYP64 -inside-F and SsCYP64 -inside-R (specific primers designed according to the SsCYP64 knocked-out DNA sequence); the primers for external detection PCR amplification are SsCYP64 -outside-F and SsCYP64 -outside-R (specific primers designed according to the SsCYP64DNA sequence upstream of the left homologous arm and downstream of the right homologous arm of the sequence was designed to be specific primers). The primers are shown in Table 8.

[0109] Table 8: SsCYP64 Endogenous and peripheral primer detection primer information

[0110]

[0111] PCR reaction system: 12.5 μL of 2x Green Taq Mix (Vazyme), 1 μL of DNA, 1 μL of Forward primers (5 μM) and 1 μL of Reverse primers (5 μM), and deionized water was added to 25 μL. The reaction program is shown in Table 9.

[0112] Table 9:

[0113]

[0114] (2) RT-qPCR detection

[0115] RT-qPCR analysis was performed using ChamQ Universal SYBR qPCR Master Mix to detect the expression of MAT-1 and MAT-2 ), SsCYP64 knockout mutant sscyp64 Δ -1 , SsCYP64 knockout mutant sscyp64 Δ -2 , complemented strain sscyp64 Δ / CYP64-1 and complemented strain sscyp64 Δ / CYP64-2 , and the specific detection method is as follows: SsCYP64

[0116] , MAT-1 , MAT-2 , sscyp64 Δ -1 , sscyp64 Δ -2 , sscyp64 Δ / CYP64-1 and sscyp64 Δ / CYP64-2 were grown in YePS solid medium for 24 h, and the total RNA of the mixed bacteria was extracted according to the method of Example 2. The transcriptional expression level of SsCYP64 was detected by RT-qPCR; and ACTIN gene was used as an internal reference gene.

[0117] II. Experimental results ​

[0118] See Figure 4 A in the text refers to wild-type and complement strains. sscyp64 Δ / CYP64-1 and sscyp64 Δ / CYP64-2 The target DNA fragment can be amplified by internal detection primers, while the knockout mutant... sscyp64 Δ -1 and sscyp64 Δ -2 The target DNA fragment cannot be amplified by internal detection primers; simultaneously, wild-type and knockout mutants... sscyp64 Δ -1 and knockout mutants sscyp64 Δ -2 Both can be amplified to produce target DNA fragments of different sizes.

[0119] RT-qPCR analysis results showed (see) Figure 4 (B in the middle) SsCYP64 Knockout mutants sscyp64 Δ -1 and knockout mutants sscyp64 Δ -2 No signal was detected in the supplemented strain. sscyp64 Δ / CYP64-1 and sscyp64 Δ / CYP64-2 The signal was detected in the medium-sized cells and was indistinguishable from that of the wild-type cells.

[0120] The above results indicate that the system was successfully built. SsCYP64 Knockout and reintroduction of strains.

[0121] Example 4 SsCYP64 Influence on sexual mating and mycelial growth

[0122] I. Experimental Methods

[0123] Wild-type cells were cultured overnight in YePS liquid medium. MAT-1 , wild type MAT-2 Knockout mutants sscyp64 Δ -1 Knockout mutants sscyp64 Δ -2 , replenishing strains sscyp64 Δ / CYP64-1 and replenishment strains sscyp64 Δ / CYP64-2 strain, waiting for its OD 600 When the bacterial cells were between 1.5 and 2.0 and in a basically uniform growth state, they were collected by centrifugation, washed once with sterile deionized water, and resuspended to achieve an OD of 1.5-2.0. 600 For 1.0, use the following combination MAT-1 × MAT-2 , sscyp64Δ -1 × sscyp64 Δ -2 and sscyp64 Δ / CYP64-1 × sscyp64 Δ / CYP64-2 Mix equal volume of different mating type strains;

[0124] Then take 1.5 μL mixed bacteria solution and spot on YePSA solid medium, dry, and place at 28℃ for 1-2 days, then take photos to record the sexual mating and mycelial growth of S. sacchari.

[0125] II. Experimental results

[0126] The results are shown in Figure 5 , and the results show MAT-1 × MAT-2 , and sscyp64 Δ / CYP64-1 × sscyp64 Δ / CYP64-2 can form soft and white mycelium, while sscyp64 Δ -1 × sscyp64 Δ -2 The ability to form white mycelium is significantly weakened, indicating that SsCyp64 regulates sexual mating and mycelial growth of S. sacchari.

[0127] Example 5 SsCYP64 Effect on H2O2 and sodium dodecyl sulfate (SDS) stress

[0128] I. Experimental method

[0129] Use YePS liquid medium to culture wild type MAT-1 , knock-out mutant sscyp64 Δ -1 and complemented strain sscyp64 Δ / CYP64-1 overnight. When the OD 600 is between 1.5-2.0 and the growth state is basically the same, centrifuge to collect the bacteria, wash once with sterile deionized water, and resuspend the bacteria with sterile deionized water to make the OD 600 of the resuspended bacteria solution 1.0.

[0130] Dilute the resuspended bacteria solution 10-fold to 10 0 , 10 -1 , 10 -2 , 10 -3 , then take 1.5 μL of resuspended bacteria solution of different dilution concentrations and spot on YePSA medium (freshly prepared) containing or not containing 1.8 mM H2O2 or 0.011% SDS, dry, and culture at 28℃ for 3-4 days.

[0131] Finally, the growth of S. sacchari under H2O2 and SDS stress was recorded by taking pictures.

[0132] II. Experimental results

[0133] The results are shown in Figure 6 , which showed that, relative to the wild type and SsCYP64 the complemented strain, SsCYP64 the growth of the knockout mutant on YePSA medium containing 1.8 mM H2O2 was almost inhibited, indicating that SsCyp64 is an antioxidant factor essential for S. sacchari to participate in antioxidant stress; and, SsCYP64 the knockout mutant had significantly increased tolerance to SDS.

[0134] This indicates that SsCyp64 may affect membrane stability by negatively regulating membrane repair-related pathways (such as lipid metabolism, membrane protein synthesis, or vesicle transport).

[0135] Example 6 SsCYP64 Effect on pathogenicity

[0136] I. Experimental methods

[0137] The wild type MAT-1 , wild type MAT-2 , knockout mutant sscyp64 Δ -1 , knockout mutant sscyp64 Δ -2 , complemented strain sscyp64 Δ / CYP64-1 and complemented strain sscyp64 Δ / CYP64-2 strains were cultured overnight using YePS liquid medium, and the OD 600 was between 1.5 and 2.0 and the growth state was basically consistent. The bacterial cells were centrifuged, washed once with sterile deionized water, and resuspended to make the resuspension liquid OD 600 1.0, and equal volumes of strains of different mating types were mixed according to the following combinations MAT-1 x MAT-2 (Wild Type) sscyp64Δ-1 x sscyp64Δ-2 ( sscyp64Δ ) and sscyp64Δ / CYP64-1 x sscyp64Δ / CYP64-2 sscyp64Δ / CYP64 ( Figure 7 ) to obtain a mixed bacterial solution.

[0138] The bacterial solution was injected near the growth point of the stems of the susceptible sugarcane variety XTT22 seedlings (five-leaf stage) at a dose of 0.5 mL per injection, with 12 sugarcane plants per injection and three biological replicates. The sugarcane was then placed in a greenhouse for cultivation for 3-6 months, and the black whip symptoms were observed and the incidence results were counted.

[0139] II. Experimental Results

[0140] The results show that ( SsCYP64 ), inoculated with wild type and sscyp64Δ / CYP64 Replacement strains ( Figure 7 Sugarcane seedlings exhibited typical black whip symptoms (see...) Figure 7 In the A group, the incidence rate was higher than 70% (see A). SsCYP6 In contrast, vaccination (B) SsCYP6 Knockout mutant ( SsCYP6 The incidence of typical whip-like symptoms in sugarcane seedlings was significantly reduced, with a morbidity rate of 33% (see...). SsCYP6 A and ​ B) indicates that SsCyp64 is essential for the pathogenicity of sugarcane smut.

[0141] Example 7 Using ​ Gene and SsCyp64 protein were used as targets for screening and evaluation of drugs against sugarcane smut.

[0142] 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. ​ The expression status of the SsCyp64 protein was detected using Western blot.

[0143] If the candidate drug inhibits ​ The expression of the gene or SsCyp64 protein within the bacteria... ​ The expression levels of the gene and SsCyp64 protein were significantly reduced, or even absent.

[0144] 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 anti-sugarcane smut effect.

Claims

1. The use of a knock-out or silencing of the product of a pathogenic protein or pathogenic gene of Smut of sugarcane, characterized by, The pathogenic protein is a protein with an amino acid sequence as shown in SEQ ID NO: 2, and the pathogenic gene is a gene encoding a protein with an amino acid sequence as shown in SEQ ID NO: 2; and the application is to reduce the pathogenicity of the sugarcane smut fungus.

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