Wheat powdery mildew avirulence gene AvrPmL180 and its application

Abstract

The present invention discloses a wheat powdery mildew avirulence gene AvrPmL180 and its application, belonging to the field of genetic engineering technology. The present invention clones a new wheat powdery mildew avirulence gene AvrPmL180. By performing signal prediction on the protein of the gene, it is found that the protein encoded by the gene contains a signal peptide, indicating that the gene has the potential to be secreted from the wheat powdery mildew into the host cell and recognized by the corresponding disease-resistant gene. The phenotypic identification results show that the powdery mildew strain containing this gene cannot grow on the resistant wheat PI170911; the powdery mildew strain without this gene can grow normally on the resistant wheat PI170911. Therefore, the AvrPmL180 gene of the present invention can be widely used in the monitoring of wheat powdery mildew physiological races and the study of the molecular interaction theory of wheat powdery mildew; and a molecular marker detection system for monitoring the pathogenic variation of the natural population of wheat powdery mildew can also be established based on its gene sequence.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a wheat powdery mildew avirulence gene AvrPmL180 and an application thereof. Background Art

[0002] Wheat powdery mildew is caused by Blumeria graminicola wheat-specific type ( Blumeria graminis f.sp .tritici, Bgt Powdery mildew is a fungal disease. When powdery mildew strikes, visible circular or oval lesions may form on wheat stems, leaves, sheaths, or ears. In severe cases, a white powdery layer of mold may appear. The pathogen causes damage by robbing host cells of nutrients, affecting photosynthesis and hindering nutrient accumulation, leading to yield losses.

[0003] Effector proteins are molecules secreted by pathogens into host cells to promote infection. They typically contain a signal peptide at their N-terminus (Selin et al., 2016). Based on their location of action, they can be divided into intracellular and extracellular effectors (Kamoun, 2006, 2007). RALPHs (RNase-like effector proteins associated with haustoria) are a class of effector genes with a protein structure similar to RNases that are highly expressed in the haustoria of B. graminis.

[0004] Nine wheat powdery mildew avirulence genes have been cloned so far, including: AvrPm3a2 / f2 (Bourras et al., 2015), AvrPm2 (Praz et al., 2017), AvrPm3b2 / c2 (Bourras et al., 2019), AvrPm3d3 (Bourras et al., 2019), AvrPm1 (Hewitt et al., 2021), AvrPm17 (Müller et al., 2022), AvrPm1a.2 (Kloppe et al., 2023), AvrPm8 (Kunz et al., 2023), and AvrPm60 (Kunz et al., 2024). The Pm3-AvrPm3 gene does not conform to a simple gene-for-gene model. Instead, it contains a suppressor of the avirulence gene, SvrPm3a1 / f1, forming an Avr-R-Svr recognition pattern. This suppressor not only prevents the recognition of the avirulence gene but also significantly enhances the survival and adaptability of the powdery mildew (Bourras et al., 2016). Therefore, it is crucial to develop a novel avirulence gene for wheat powdery mildew control and the breeding of disease-resistant wheat varieties. Summary of the Invention

[0005] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a wheat powdery mildew avirulence gene AvrPmL180 and its application for the prevention and control of wheat powdery mildew and the breeding of disease-resistant wheat varieties.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: providing a wheat powdery mildew avirulence gene AvrPmL180, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] Furthermore, the amino acid sequence of the wheat powdery mildew avirulence gene AvrPmL180 is shown in SEQ ID NO.2.

[0008] The present invention provides an application of the wheat powdery mildew avirulence gene AvrPmL180 in breeding new disease-resistant wheat varieties.

[0009] The present invention provides an application of the above-mentioned wheat powdery mildew avirulence gene AvrPmL180 in detecting the toxicity of powdery mildew. If the wheat powdery mildew contains the AvrPmL180 gene, it cannot grow on the resistant wheat PI170911, and the wheat phenotype is disease-resistant; if the wheat powdery mildew does not contain the AvrPmL180 gene, it can grow normally on the resistant wheat PI170911, and the wheat phenotype is disease-susceptible.

[0010] The present invention also provides an application of the wheat powdery mildew avirulence gene AvrPmL180 in establishing a molecular marker detection system for monitoring pathogenic variation of wheat powdery mildew natural populations.

[0011] The present invention has the following beneficial effects: the present invention cloned a new wheat powdery mildew avirulence gene AvrPmL180. By performing signal prediction on the protein of this gene, it was found that the protein encoded by the AvrPmL180 gene contains a signal peptide, indicating that the gene has the potential to be secreted from wheat powdery mildew into host cells and recognized by corresponding disease-resistant genes. The results of phenotypic identification showed that powdery mildew strains containing this gene cannot grow on resistant wheat PI170911; powdery mildew strains not containing this gene can grow normally on resistant wheat PI170911. Therefore, the AvrPmL180 gene of the present invention can be widely used in the monitoring of wheat powdery mildew physiological races and the study of the molecular interaction theory of wheat powdery mildew; a molecular marker detection system for monitoring pathogenic variation of natural populations of wheat powdery mildew can also be established based on its gene sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The PCR identification results of 24 purified strains identified by specific primers in the present invention are as follows;

[0013] Figure 2 This is the predicted signal peptide map of the AvrPmL180 gene;

[0014] Figure 3 The present invention carried out phenotypic identification by inoculating 24 purified strains into the disease-resistant wheat variety PI170911. DETAILED DESCRIPTION

[0015] The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, the experiments were performed under conventional conditions or those recommended by the manufacturer. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0016] Example 1: Cloning of the wheat powdery mildew avirulence gene AvrPmL180

[0017] The inventors previously conducted genome-wide association analysis on the resistance-susceptibility phenotype of wheat variety PI170911 inoculated with different strains and the genotypes of different strains, and obtained a candidate gene. The single nucleotide polymorphism on this gene was significantly associated with the resistance-susceptibility phenotype. This gene was named AvrPmL180 and obtained by gene cloning. Its gene sequence is shown in SEQ ID NO. 1. The specific cloning process is as follows:

[0018] ① Enzyme system: TaKaRa Tks Gflex DNA Polymerase R060A.

[0019] ② Amplification system: Using the cDNA of the wheat powdery mildew strain as the template, the total volume of the amplification system is 50 μL, including 1 μL of cDNA, 1.5 μL of upstream and downstream primers, 1 μL of amplification mix, 25 μL of buffer and 20 μL of ddH2O.

[0020] ③PCR amplification reaction program: pre-denaturation at 94°C for 3 min; denaturation at 98°C for 10 s, annealing at 58°C for 15 s, extension at 68°C for 30 s, 30 cycles; final extension at 68°C for 5 min.

[0021] Among them, the nucleotide sequences of the upstream and downstream primers (5 μm / L) for AvrPmL180 gene cloning are:

[0022] AvrPmL180-F: atgagatctgctaatcttgcttcattg (SEQ ID NO.3)

[0023] AvrPmL180-R: ttaacgctttatacaagatgacatttgg (SEQ ID NO. 4).

[0024] The amplified product was sent to a sequencing company for sequencing. The sequencing results showed that the cloned gene sequence was consistent with the gene sequence of the avirulent gene AvrPmL180, and the cloned gene product was used for subsequent experiments.

[0025] Using SignalP-5.0( https: / / services.healthtech.dtu.dk / services / SignalP- 5.0 / ) predicts the protein signal of the gene, and the prediction results are as follows Figure 2 As shown, AvrPmL180 contains a signal peptide with the sequence: MRSANLASLSLLFGFLILESA (SEQ ID NO. 5), indicating that the gene has the potential to be secreted from wheat powdery mildew into host cells and recognized by the corresponding disease resistance genes.

[0026] Example 2: Phenotypic Identification of the Wheat Powdery Mildew Avirulence Gene AvrPmL180

[0027] In order to further study the pathogenicity of wheat powdery mildew, 24 wheat powdery mildew strains (denoted as B1-B24) that had been purified and preserved in the laboratory in the early stage were selected for phenotypic identification. The specific operation is as follows: the wheat variety PI170911 with strong disease resistance was selected as the test wheat, and the susceptible variety Zhongzuo 9504 (denoted as ZZ9504) was used as the positive control. After it grew for 7 days, healthy leaves were selected and inoculated with these 24 purified wheat powdery mildew strains. After 12 days of inoculation, the growth of different wheat powdery mildew strains on the leaves was carefully observed and recorded to evaluate the pathogenicity of different strains. The phenotypic identification results are as follows: Figure 3 As shown, all 24 strains could grow normally in the positive control ZZ9504, and some strains could normally infect the disease-resistant wheat PI170911, such as B1, B7, B8, B11, B12, B17, B19 and B22, after being inoculated into the disease-resistant wheat PI170911, and PI170911 showed a susceptible phenotype; the remaining strains could not normally infect the disease-resistant wheat PI170911, such as B2, B3, B4, B5, B6, B9, B10, B13, B14, B15, B16, B18, B20, B21, B23 and B24, and PI170911 showed a disease-resistant phenotype.

[0028] Combining the cloning results and phenotypic identification results of the AvrPmL180 gene of different strains, it was found that powdery mildew strains containing this gene could not grow on the resistant wheat PI170911, and the wheat phenotype was disease-resistant; powdery mildew strains without this gene could grow normally on the resistant wheat PI170911, and the wheat phenotype was susceptible to disease. Wheat without this gene was susceptible to disease.

[0029] The nucleotide sequence of the CDS region of the wheat powdery mildew avirulence gene AvrPmL180 in the present invention is as follows: ATGAGATCTGTCAATCTTTGCTTCATTGTCCTTACTTTTTGGCTTTTTGATTCTCGAAAGTGCTGCAGACTATGTATGCTCGGGTGGAACTCGAATACCGGATCATGAAGTCGAGACAAGAGCAAATGAGATTTACTCAAAAGGATTATCCCTCAAAGCTAGTCGGACTCCTGGTCAACAACAAATAGAAGATATCTATTTTGATGATGATGAAGATGATGCAGAAATGTCCTTTTCCAGCGACTTCTATCCTCGGATCAAATCTTCTGGAACATATACAATAACAGTCGATTATCCATCAAAAAATATATTGGTTATAGAGAAAATTGAATATAACGGCAGATACCAAATGTCATCTTGTATAAAGCGTTAA (SEQ ID NO. 1);

[0030] The amino acid sequence of the wheat powdery mildew avirulence gene AvrPmL180 in the present invention is as follows: MRSANLASLSLLFGFLILESAADYVCSGGTRIPDHEVETRANEIYSKGLSLKASRTPGQQQIEDIYFDDDEDDAEMSFSSDFYPRIKSSGTYTITVDYPSKNILVIEKIEYNGRYQMSSCIKR (SEQ ID NO. 2).

[0031] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of a detection reagent for the wheat powdery mildew avirulence gene AvrPmL180 in detecting powdery mildew toxicity, characterized in that: If the wheat powdery mildew contains the AvrPmL180 gene, it cannot grow on the resistant wheat PI170911, and the wheat phenotype is disease-resistant; if the wheat powdery mildew does not contain the AvrPmL180 gene, it can grow normally on the resistant wheat PI170911, and the wheat phenotype is susceptible; wherein, the nucleotide sequence of the wheat powdery mildew avirulence gene AvrPmL180 is shown as SEQ ID NO.1.

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