Wheat-sourced puccinia striiformis effect protein Pst8724, coding gene and application

Abstract

The invention provides a wheat-derived stripe rust effect protein Pst8724, a coding gene and application, and belongs to the technical field of gene engineering. The invention provides a stripe rust effect protein Pst8724 from wheat, the amino acid sequence of the stripe rust effect protein Pst8724 is as shown in SEQ ID No.1, and the nucleotide sequence of the coding gene of the stripe rust effect protein Pst8724 is as shown in SEQ ID No.2. According to the invention, RNA interference (RNAi) and overexpression wheat plants of Pst8724 are created through a genetic transformation technology. Disease resistance identification results show that Pst8724 positively regulates pathogenicity of puccinia striiformis to wheat, histological and cytological observation shows that accumulation of H2O2 and expression of PR genes can be promoted by reduction of expression quantity of Pst8724 in interaction of puccinia striiformis and wheat, and resistance of wheat to puccinia striiformis is enhanced. The invention provides a new material for the cultivation of the stripe rust resistant wheat variety.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a wheat-derived stripe rust effector protein Pst_8724, its encoding gene, and its applications. Background Technology

[0002] Wheat is one of the world's three major food crops and an important food source upon which human life depends. Approximately 35-40% of the global population relies on wheat as their staple food, making increased wheat production crucial for stabilizing global food security. Wheat stripe rust, caused by the pathogenic fungus *Puccinia striiformis* f. sp. *Tritici*, has become one of the most significant diseases threatening global wheat production. Planting resistant varieties is the most economical, effective, and environmentally friendly measure for controlling wheat stripe rust. However, due to the rapid mutation of the pathogenicity of wheat stripe rust, resistant varieties quickly lose their resistance, leading to frequent and severe outbreaks of the disease. Therefore, there is an urgent need to develop new disease control methods to provide theoretical basis and technical support for the green and sustainable control of wheat stripe rust.

[0003] In the long-standing competition between pathogens and plants, pathogens have evolved effector molecules that are secreted into the host to disrupt plant cellular responses—a process known as the effector-targeted pathway (ETP). In recent years, ETP reserves have been steadily increasing, and mounting evidence suggests that the proteasome and autophagy pathways are central hubs for microbial effectors. Both of these degradation pathways involve a wide range of cellular responses, thus constituting an attractive target for effector proteins to exert a broader impact on the host.

[0004] Therefore, systematically elucidating the interaction mechanism between stripe rust fungi and host plants will provide important theoretical support for developing new and persistent control strategies. Summary of the Invention

[0005] This invention provides a wheat-derived stripe rust effector protein Pst_8724, its encoding gene, and its application. The stripe rust effector protein and its encoding gene can be used to improve and breed new wheat varieties resistant to stripe rust, thus broadening the research scope of new wheat materials resistant to stripe rust.

[0006] This invention provides a wheat-derived stripe rust effector protein Pst_8724, the amino acid sequence of which is shown in SEQ ID No. 1.

[0007] The present invention also provides the encoding gene of the stripe rust effector protein Pst_8724.

[0008] In a preferred embodiment of the present invention, the nucleotide sequence of the encoding gene is shown in SEQ ID No. 2.

[0009] This invention also provides the application of the above-mentioned stripe rust effector protein Pst_8724 or the above-mentioned encoding gene in regulating wheat resistance to stripe rust.

[0010] In a preferred embodiment of the present invention, reducing the content of the stripe rust effector protein Pst_8724 or reducing the expression level of the encoding gene improves the resistance of wheat to stripe rust. Increasing the content of the stripe rust effector protein Pst_8724 or overexpressing the encoding gene can reduce wheat resistance to stripe rust.

[0011] The present invention also provides the application of a reagent for reducing the expression level of the above-mentioned coding gene in improving wheat resistance to stripe rust.

[0012] In a preferred embodiment of the present invention, the reagent comprises RNAi.

[0013] The present invention also provides an RNAi vector that reduces the expression level of the above-mentioned coding gene, wherein the backbone vector of the RNAi vector includes a PC336 vector, and a specific fragment is inserted into the backbone vector; The specific fragment was amplified using the coding gene as a template and with the upstream primer shown in SEQ ID No. 3 and the downstream primer shown in SEQ ID No. 4.

[0014] The present invention also provides a method for improving wheat resistance to stripe rust, including reducing the expression level of the above-mentioned coding gene in the wheat genome, or silencing the above-mentioned coding gene.

[0015] This invention also provides the application of the above-mentioned stripe rust effector protein Pst_8724, the above-mentioned encoding gene, or the above-mentioned RNAi vector in the cultivation of stripe rust-resistant wheat.

[0016] Beneficial effects: This invention provides a wheat-derived stripe rust effector protein, Pst_8724, whose amino acid sequence is shown in SEQ ID No. 1. This invention utilizes HIGS technology to obtain... Pst_ 8724 Transgenic plants derived from RNAi were subjected to stripe rust resistance assessment. Results showed that, compared to the control Fielder, they exhibited superior resistance. Pst_ 8724 On RNAi plants, the pathogenicity of stripe rust fungus was reduced, and the interaction between stripe rust fungus and wheat was also found to be... Pst_8724 Decreased expression levels promote the accumulation of H2O2 and the expression of the PR gene. This invention also utilizes the Gateway method to create... Pst_ 8724 Overexpression of the material was performed, and resistance to stripe rust was identified. The results showed... Pst_8724 Overexpression of this substance in wheat plants weakened their disease resistance. Furthermore, it was found that… Pst_8724 The regulation of stripe rust pathogenicity is negatively correlated with H2O2 accumulation and PR gene expression in wheat, i.e., the aforementioned Pst_8724 It positively regulates the pathogenicity of stripe rust fungus to wheat and negatively regulates the resistance of wheat to stripe rust fungus. Attached Figure Description

[0017] Figure 1 for Pst_8724 - RNAi plant stripe rust resistance identification (A) and biomass statistics 7 days after inoculation (B), CYR31 is the physiological race CYR31 of stripe rust; in the figure , P <0.05; , P <0.01; Figure 2 for Pst_8724 - Histological observation (A) and statistical results (B) of H2O2 accumulation in RNAi plants after inoculation with CYR31; , P <0.05; Figure 3 for Pst_8724 -Graphs (A) showing the hyphal length (B), colony area (C), and statistical results of stripe rust fungus at 24 h and 48 h after RNAi plant inoculation with CYR31. In the graphs, SV represents the substomatal sac, HMC represents the haustoria mother cell, IH represents the infected hyphae, and H represents the haustoria. , P <0.05; Figure 4 for Pst_8724 Overexpression plants ( Pst_8724 -OE) stripe rust disease resistance identification (A) and biomass statistics 7 days after inoculation (B), CYR23 is the physiological race CYR23 of stripe rust; in the figure , P <0.05; Figure 5 for Pst_8724 Histological observation (A) and statistical results (B) of H2O2 accumulation in OE plants after inoculation with CYR23; , P <0.05; Figure 6 for Pst_8724-Figure A shows the mycelial length (B), colony area (C), and statistical results of stripe rust fungus at 24 h and 48 h after inoculation of OE plants with CYR23. In the figure, SV represents the substomatal sac, HMC represents the haustoria mother cell, IH represents the infecting hyphae, and H represents the haustoria. , P <0.05; , P <0.01; Figure 7 for Pst_8724 -RNAi and Pst_8724 -OE plants were inoculated with CYR31 and CYR23 at 24 hpi. TaPR1 Gene expression levels (A and C) and at 48 hpi TaPR2 Gene expression levels (Figures B and D) , P <0.05; , P <0.01; , P <0.001. Detailed Implementation

[0018] This invention provides a wheat-derived stripe rust effector protein, Pst_8724, the amino acid sequence of which is shown in SEQ ID No. 1: MIFTYDLIAVCLLVLRAIAPHLPPEDLLSHDEIARGIVEQLQPVPERVQFGPLVGGTTPAGGSIARRRDFTRPSEDTHGAFPLHTGQVGNVQSAIDGQQSQRRVLDARLSEIGTPNERWCPLVPFFRRISERVQRLFWRNGRRRIQIEMQVHQQFNAFLQTLTTPTREMIGPLVYLGTSGNTMEFRFNIFLHRLSPRSSYWVTFPIHIGLM GHHPDAYILRGRFDRFMQRFLRDLHRYNKHNLQAGDTIPNTKKLGYMTMPTDYQQGTSGANRRVKVPLDQAIGMLESTSNNELESVHKDLDIITNEQGVNTNRKNTEDE THKDLITSSKSIHDDTGDGTRLTSSLQDISEVLLGDPLHGSCVICQCEFCQPEKNSENVWMFEKICNIDGCDHFFHPHCLEEWIIKNQQNSCPECRAQVFPLTLVPEP.

[0019] In one embodiment of the present invention, using the wheat variety Fielder as the receptor, HIGS technology was used to obtain... Pst_ 8724 -RNAi transgenic plants. At the two-leaf stage of the T3 generation, seedlings were inoculated with the physiological race CYR31 of stripe rust. Tissue samples and RNA were collected at 24 hpi and 48 hpi. DNA was collected on day 7 for biomass assays, and sporulation phenotype was photographed and recorded 14 days later. Results showed that wild-type Fielder exhibited abundant spore accumulation on leaves, while... Pst_8724 -Only a few spore masses were formed on the leaves of RNAi plants, and obvious necrosis was observed near the spore masses, indicating that... Pst_8724 The pathogenicity of stripe rust fungus was reduced in RNAi plants, proving that... Pst_8724 It is an important pathogenic factor in the process of stripe rust infecting wheat, and the above-mentioned Pst_8724 After silencing, the length of the hyphae and the area of ​​hyphal infection decreased significantly.

[0020] In one embodiment of the present invention, a receptor was constructed using the wheat variety Fielder. Pst_8724 overexpression of wheat Pst_8724-OE In the T3 generation Pst_8724-OE The fungus was inoculated with stripe rust race CYR23, and tissue samples and RNA were collected at 24 hpi and 48 hpi. DNA was collected on day 7 for biomass assay, and sporulation phenotype was photographed and recorded 14 days later. Results showed that, compared with Fielder, Pst_8724-OE Numerous stripe rust spores are produced on the plant's leaves, and Pst_8724- OE The increased hyphal length and infection area on plant leaves indicate that the mycelium is overexpressed in wheat. Pst_8724 It can weaken the disease resistance of wheat.

[0021] The present invention also provides the encoding gene of the stripe rust effector protein Pst_8724.

[0022]

[0023] This invention also provides the application of the above-mentioned stripe rust effector protein Pst_8724 or the above-mentioned encoding gene in regulating wheat resistance to stripe rust.

[0024] This invention reduces the content of the stripe rust effector protein Pst_8724 or reduces the expression level of the encoding gene to improve wheat resistance to stripe rust; it also increases the content of the stripe rust effector protein Pst_8724 or overexpresses the encoding gene to reduce wheat resistance to stripe rust.

[0025] In the embodiments of the present invention, it was found that the Pst_8724 Regulation of wheat stripe rust resistance is associated with H2O2 accumulation and PR gene expression. RNAi technology can be used to silence the PR gene in the wheat genome. Pst_8724 Following expression, after inoculation with stripe rust, wheat plants showed... Pst_8724 The expression levels of TaPR1 and TaPR2 genes were reduced, while the expression levels of TaPR1 and TaPR2 genes were significantly increased, promoting the accumulation of H2O2 and leading to enhanced resistance of wheat to stripe rust. When overexpression occurred in wheat... Pst_8724 Afterwards, following inoculation with stripe rust fungus, the wheat plants... Pst_8724 Increased expression levels and significantly reduced expression levels of TaPR1 and TaPR2 genes, while inhibiting H2O2 accumulation, led to decreased wheat resistance to stripe rust.

[0026] The present invention also provides the application of a reagent for reducing the expression level of the above-mentioned coding gene in improving wheat resistance to stripe rust.

[0027] This invention reduces the Pst_8724 There are no particular limitations on the method for measuring gene expression; conventional methods in the field can be used. Gene knockout can be performed using gene editing methods, or gene suppression can be performed using shRNA. In one embodiment of the present invention, gene silencing is performed using RNAi, but this should not be considered as the entire scope of protection of the present invention.

[0028] The present invention also provides an RNAi vector that reduces the expression level of the above-mentioned coding gene, wherein the backbone vector of the RNAi vector includes a PC336 vector, and a specific fragment is inserted into the backbone vector; The specific fragment was amplified using the coding gene as a template and with the upstream primer shown in SEQ ID No. 3 and the downstream primer shown in SEQ ID No. 4.

[0029] This embodiment is for the purpose of creating Pst_8724 -RNAi plants, selected Pst_8724A specific 300 bp fragment was selected as the target sequence for RNAi. The target sequence was amplified using the following primers and procedure: Pst_8724 -PC336-F (SEQ ID No.3): TTTAGCCCTGCCTTTCATACGCGTGACTTTACAAGGCCA; Pst_8724 -PC336-R (SEQ ID No. 4): CACGCAAGTCCGCATCTTCATCTGGATCTCTATCCTCC; Amplification system: 1 μL cDNA template, 1 μL each of the above primers, 20 μL 2X Rapid Taq Master Mix, 17 μL ddH2O. Amplification program: Pre-denaturation 95℃ for 5 min, 95℃ for 30 sec, 65℃ for 30 sec, 72℃ for 15 sec, 35 cycles, 72℃ for 10 min, 16℃ forever. The primer pairs described in this invention... Pst_8724 -PC336-F and Pst_8724 -PC336-R can also be used for subsequent colony PCR verification.

[0030] Then use BP The enzyme performs a BP reaction, catalyzing the amplification product containing the attB site and the Donor221 vector containing the attP site to generate an intermediate vector. After correct sequencing, it is used... Nru The linearization of enzyme I, the specific reaction system and reaction procedure are as follows: BP reaction mixture: 2.0 μL 1×TE Buffer, 0.5 μL BP Enzyme, 1.0 μL pDONR221 vector, 1.5 μL gene fragment. BP reaction program: overnight incubation at 25°C for 18 h. Linearization reaction system: 2.0 μL recombinant vector, 3.0 μL... Nru I enzyme, 2.0 μL 10×Buffer, 33.0 μL ddH2O. Linearization reaction program: 37℃, 30 min.

[0031] use LR The enzyme performs an LR reaction, and the intermediate vector (containing the target gene with the attL site on its flanking side) is recombined in vitro with the final vector PC336 (containing the attR site) to generate an expression clone, thus obtaining the RNAi vector. LR reaction system: 2.0 μL 1×TE Buffer, 0.5 μL LREnzyme, 1.0 μL PC336 vector, 1.5 μL linearized plasmid; LR reaction program: overnight incubation at 25℃ for 18 h. In this invention, the constructed RNAi vector was transformed into EHA105 cells using Agrobacterium-mediated transformation. Colonies were detected by PCR, and single colonies with the correct band size were labeled. Then, a modified Agrobacterium-mediated transformation method was used to transform the RNAi vector into immature embryos of the wheat variety Fielder, constructing gene-silenced plants.

[0032] The present invention also provides a method for improving wheat resistance to stripe rust, including reducing the expression level of the above-mentioned coding gene in the wheat genome, or silencing the above-mentioned coding gene.

[0033] In one embodiment of the present invention, the method of silencing the [unclear] is employed. Pst_8724 The specific methods for genes are the same as above, and will not be repeated here.

[0034] This invention also provides the application of the above-mentioned stripe rust effector protein Pst_8724, the above-mentioned encoding gene, or the above-mentioned RNAi vector in the cultivation of stripe rust-resistant wheat.

[0035] The RNAi method described in this invention can be used to construct wheat varieties with high resistance to stripe rust, and the trait can be stably propagated.

[0036] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of the wheat-derived stripe rust effector protein Pst_8724, its encoding gene, and its applications, should not be construed as limiting the scope of protection of the present invention.

[0037] Unless otherwise specified, the materials and methods used in the embodiments of this invention are all from conventional sources and methods in the art. BP The enzyme is from Thermo Fisher Scientific, product number: 11789020. LR The enzyme was obtained from Thermo Fisher Scientific, catalog number 11791043. The intermediate vector Donor 221 was obtained from Thermo Fisher Scientific, catalog number 12536017. The PC336 and pANIC-6E vectors were preserved in our laboratory and have been published in the article (Shu Weixue. Study on the mechanism of wheat immunity regulated by stripe rust effector Pst_215 [D]. Northwest A&F University, 2023. DOI:10.27409 / d.cnki.gxbnu.2023.000238.).

[0038] Example 1: Construction of RNAi plants and overexpression plants Using the above Pst_8724 -PC336-F and Pst_8724 PC336-R primer amplification Pst_8724 A specific 300 bp fragment is used as the target sequence for RNAi. BP The enzyme performs a BP reaction to construct the target sequence containing the attB site into the intermediate vector Donor 221 containing the attP site. After correct sequencing, it is used... Nru I enzyme linearization, utilizing LR The enzyme performs an LR reaction, and the intermediate vector (containing the target gene with the attL site on its flank) and the final vector PC336 (containing the attR site) undergo in vitro recombination to produce an expression clone.

[0039] use Pst_8724 -6E-F and Pst_8724 -6E-R amplification Pst_8724 Genes, will be amplified Pst_8724 Gene utilization BP The enzyme performs a BP reaction, catalyzing the amplification product containing the attB site and the Donor 221 vector containing the attP site to generate an intermediate vector. The correctly sequenced intermediate vector is then used... Nru Linearization of enzyme I was performed, utilizing... LR The enzyme performs an LR reaction, and the intermediate vector (containing the target gene with the attL site flanking it) undergoes in vitro recombination with the final vector pANIC-6E (containing the attR site) to generate an expression clone. Pst_8724 The C-end integrates the HA tag.

[0040] Pst_8724 -6E-F (SEQ ID No. 5): GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTACCCATACGACGTCCCAGACTACGCTCCACATTTACCACCAGAGGA; Pst_8724 -6E-R (SEQ ID No. 6): GGGGACCACTTTGTACAAGAAAGCTGGGTCTCATGGTTCAGGCACTAGAGT. The primer pair described in this invention... Pst_8724 -6E-F and Pst_8724 -6E-R can also be used for subsequent colony PCR verification.

[0041] Finally, the constructed vector was transformed into EHA105 using Agrobacterium-mediated transformation. PCR detection was performed on the colonies, and single colonies with the correct band size were labeled. Then, a modified Agrobacterium-mediated transformation method (He Yi. Optimization of Agrobacterium-mediated transformation system for wheat [D]. Huazhong University of Science and Technology, 2016.) was used to transform the colonies into immature embryos of the wheat variety Fielder.

[0042] T0 generation wheat seedlings were transplanted into a flowering substrate and allowed to acclimate for two weeks in a high-humidity incubator. Leaves were then collected and genomic DNA was extracted. Based on the sequence information of the vector and the inserted fragment, primer pairs were used to... Pst_8724 -6E-F、 Pst_ 8724 -PC336-R and Pst_8724 -PC336-F, Pst_8724 -PC336-R was tested separately Pst_8724 of OE Strains and RNAi strains.

[0043] Example 2 Seeds from correctly identified T0 generation plants (T1 generation) were sown into a flower substrate. Genomic DNA was extracted as previously described to detect gene editing, and seeds from these correctly identified T1 generation plants were obtained (T2 generation). These were then sown again to form the T3 generation. In the T3 generation... Pst_8724 -RNAi and Pst_8724-OE At the two-leaf stage, the plants were inoculated with stripe rust physiological races CYR31 (compatible) and CYR23 (incompatible) (both published in the article: Li S, Li T, Zhang P, et al. The E3 ubiquitinligase TaGW2 facilitates TaSnRK1γ and TaVPS24 degradation to enhance striperust susceptibility in wheat[J]. Plant Biotechnology Journal, 2025, 23(3):750-765.). Tissue samples were collected and RNA was extracted at 24 hpi and 48 hpi. DNA was collected on day 7 for biomass detection. Sporulation phenotype was photographed and recorded 14 days later.

[0044] The tissue sample processing method is as follows: DAB staining and reactive oxygen species accumulation area statistics: After collecting leaves, they were cut into three sections evenly with scissors. The morphological lower end of the leaves was immersed in a 2 mL centrifuge tube containing prepared DAB staining solution (0.1 mg / mL) and placed under strong light for 3-6 h until black spots were visible at the top of the leaves, indicating that the DAB staining solution had completely penetrated to the top of the leaves. The DAB staining solution was discarded, and the leaves were decolorized in a fume hood with a decolorizing solution (equal volumes of anhydrous ethanol and glacial acetic acid mixed). The decolorizing solution was changed once a day until the leaf color completely faded. Saturated chloral hydrate solution was added to the centrifuge tube to make the leaves transparent. After treatment for 3 days, the leaves were stored in 50% glycerol for subsequent experiments. The transparent leaves were removed, and the chloral hydrate on the surface of the leaves was washed off with ddH2O. After slide preparation, the tissue samples were observed using a fluorescence microscope (Olympus BX-51). Fifty infection points were randomly selected under bright field to count the reactive oxygen species area.

[0045] Wheat stripe rust fungus WGA staining and observation statistics: After the area of ​​reactive oxygen species accumulation was counted, wheat tissue samples were washed twice with 20% ethanol solution by blowing and rinsing, followed by washing the leaves three times with ddH2O. Centrifuge tubes were filled with 1M KOH solution, the caps were perforated or sealed with tape, and autoclaved at 121℃ for 5 min to soften the tissue samples. Leaves were soaked twice with 2mL ddH2O for 10 min each time to thoroughly remove the KOH solution. The tubes were then washed with Tris-HCl (50mM, pH 7.4) buffer, repeated three times. The liquid in the tubes was discarded, and the prepared WGA-Alexa448 staining solution was added. The tubes were covered with aluminum foil to protect them from light and stained overnight at 4℃. The stained leaves were then stored in 50% glycerol for subsequent experiments. The WGA-Alexa448 staining solution was filtered and recovered using a 0.45μm filter. The stained leaves were removed, and the glycerol on the leaf surface was washed off with ddH2O. After slide preparation, the samples were examined using a fluorescence microscope (Olympus). BX-51) Observe the tissue sample and randomly select 50 infection points to count the hyphal length and hyphal area.

[0046] The method for detecting stripe rust biomass in wheat leaves is as follows: First, plot the standard curve, then amplify... PstEF Genes and TaEF Genes are measured at concentrations and serially diluted, typically at 100 ng / μL × 10⁻⁶. -5The copy number can be obtained by inputting the DNA sample concentration and base pair length on the tool website (https: / / agbio.com.cn / soft / tools / copy / ). Using diluted DNA at different concentrations as templates, amplification is performed using quantitative primers to obtain the Ct values ​​for each group. The logarithm of the DNA copy number is plotted on the x-axis, and the Ct values ​​are plotted on the y-axis to create a regression curve. A regression curve with a determination coefficient greater than 0.99 is acceptable. After phenotypic scanning, leaf DNA is extracted, its concentration is measured, and then diluted to 200 ng / μL. This concentration is used as a template for qPCR experiments to obtain the Ct values. The Ct values ​​are then substituted into the plotted standard curve to calculate the Ct values ​​in the template. PsEF Genes and TaEF The ratio of the logarithm of the gene copy number to the logarithm of the gene copy number is the biomass of wheat stripe rust in the sample.

[0047] TaEF -F (SEQ ID No.7): TGGTGTCATCAAGCCTGGTATGGT; TaEF -R (SEQ ID No.8):ACTCATGGTGCATCTCAACGGACT; PstEF -F (SEQ ID No.9): TTCGCCGTCCGTGATATGAGACAA; PstEF -R (SEQ ID No. 10): ATGCGTATCATGGTGGTGGAGTGA.

[0048] The results are shown in the figure. Pst_8724 -Inoculation of RNAi plants with the physiological race CYR31 (affinity) of stripe rust fungus revealed a large accumulation of spore masses on the leaves of wild-type Fielder, while Pst_8724 -RNAi plants only produce a small number of spore masses on their leaves, and obvious necrosis is observed near the spore masses. Figure 1 (A) indicates that in Pst_8724 In RNAi plants, the pathogenicity of stripe rust fungus is reduced, and its biomass is significantly decreased. Figure 1 (B) To explore silence Pst_8724 The effect of H2O2 accumulation during the interaction between wheat and stripe rust fungus was investigated using DAB staining to observe H2O2 accumulation during stripe rust fungus infection. Figure 2 (A), compared to the control Fielder, Pst_8724 -RNAi plants showed increased H2O2 accumulation 24 hpi and 48 hpi after inoculation with stripe rust ( Figure 2(Middle B). To further understand the effect of silencing-induced siRNA on the hyphal development of wheat stripe rust, the histological characteristics after inoculation were observed using the WGA staining method for wheat stripe rust. Figure 3 (A) It was found that the length of hyphae and the area of ​​hyphal infection were significantly reduced after silencing. Figure 3 (B and C in the middle). Pst_8724-OE Inoculation of plants with the physiological race CYR23 (incompatible) of stripe rust fungus revealed that, compared to Fielder, Pst_8724 -OE-1、 Pst_8724 -OE-2 and Pst_8724 -OE-3 plants have a large number of stripe rust spores on their leaves ( Figure 4 In the middle (A), the biomass of stripe rust fungi increased significantly ( Figure 4 (Middle B). Histological staining results showed that, compared with the control Fielder, Pst_8724 -OE plants showed reduced H2O2 accumulation 24 hpi and 48 hpi after inoculation with stripe rust. Figure 5 The hyphal length and hyphal infection area increased significantly. Figure 6 This indicates that overexpression in wheat Pst_8724 It can weaken the disease resistance of wheat.

[0049] Example 4 verify Pst_8724 -RNAi and Pst_8724-OE Disease resistance of plants after inoculation with stripe rust fungus, and determination of disease resistance-related genes. TaPR1 and TaPR2 The expression level. The primers involved include TaEF-F / TaEF-R as mentioned above, as well as the following primers: TaPR1 -F (SEQ ID No.11):GAGAATGCAGACGCCCAAGC; TaPR1 -R (SEQ ID No.12):CTGGAGCTTGCAGTCGTTGATC; TaPR2 -F (SEQ ID No.13):AGGATGTTGCTTCCATGTTTGCCG; TaPR2 -R (SEQ ID No. 14): AAGTAGATGCGCATGCCGTTGATG.

[0050] The results are as follows Figure 7 As shown, compared to the control Fielder, Pst_8724 -RNAi plants showed a significant increase in the expression levels of TaPR1 and TaPR2 genes at 24 hpi and 48 hpi after inoculation with stripe rust fungus CYR31. Figure 7(A and B in the middle). Pst_8724 -OE plants showed significantly reduced expression levels of TaPR1 and TaPR2 genes at 24 hpi and 48 hpi after inoculation with stripe rust fungus CYR23. Figure 7 (C, D)

[0051] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A wheat-derived stripe rust effector protein, Pst_8724, characterized in that, The amino acid sequence of the stripe rust effector protein Pst_8724 is shown in SEQ ID No.

1.

2. The gene encoding the stripe rust effector protein Pst_8724 as described in claim 1.

3. The encoding gene according to claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID No.

2.

4. The application of the stripe rust effector protein Pst_8724 as described in claim 1 or the encoding gene as described in claim 2 or 3 in regulating wheat resistance to stripe rust.

5. The application according to claim 4, characterized in that, Reducing the content of the stripe rust effector protein Pst_8724 or reducing the expression level of the encoding gene can improve wheat resistance to stripe rust. Increasing the content of the stripe rust effector protein Pst_8724 or overexpressing the encoding gene can reduce wheat resistance to stripe rust.

6. The use of an agent that reduces the expression level of the encoding gene as described in claim 2 or 3 in improving wheat resistance to stripe rust.

7. The application according to claim 6, characterized in that, The reagents include RNAi.

8. An RNAi vector for reducing the expression level of the encoding gene as described in claim 2 or 3, characterized in that, The backbone vector of the RNAi vector includes the PC336 vector, and a specific fragment is inserted into the backbone vector; The specific fragment was amplified using the coding gene as a template and with the upstream primer shown in SEQ ID No. 3 and the downstream primer shown in SEQ ID No.

4.

9. A method for improving wheat resistance to stripe rust, characterized in that, This includes reducing the expression level of the coding gene of claim 2 or 3 in the wheat genome, or silencing the coding gene of claim 2 or 3.

10. The application of the stripe rust effector protein Pst_8724 of claim 1, the encoding gene of claim 2 or 3, or the RNAi vector of claim 8 in the cultivation of stripe rust-resistant wheat.

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