Wheat powdery mildew avirulence gene AvrPmL180 and application thereof

Abstract

The invention discloses a wheat powdery mildew avirulence gene AvrPmL180 and application thereof, and belongs to the technical field of gene engineering. According to the invention, a novel wheat powdery mildew avirulence gene AvrPmL180 is cloned. Signal prediction is carried out on the protein of the gene, and it is found that the protein coded by the gene contains signal peptide, which indicates that the gene has the potential of being secreted from wheat powdery mildew to host cells and identified by corresponding anti-disease genes. A phenotype identification result shows that a powdery mildew strain containing the gene cannot grow on resistant wheat PI170911; a powdery mildew strain which does not contain the gene can normally grow on resistant wheat PI170911. Therefore, the AvrPmL180 gene can be widely applied to wheat powdery mildew physiological race monitoring and wheat powdery mildew molecular interaction theory research; and a molecular marker detection system for monitoring the pathogenic variation of the natural population of the wheat powdery mildew can be established according to the gene sequence.

Description

Technical Field

[0001] The 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 graminearum, a special type of wheat ( Blumeria graminis f.sp .tritici, Bgt ) is a disease caused by fungi. After powdery mildew occurs, visible round or oval spots may form on wheat stems, leaves, leaf sheaths or ears. In severe cases, a white powdery mold layer may be seen. The pathogen causes damage by robbing host cells of nutrients, affecting photosynthesis; it also affects the accumulation of nutrients, leading to yield losses.

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

[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). Among them, Pm3-AvrPm3 does not conform to the simple gene-to-gene theory, but there is a suppressor of avirulent gene, Supressorr-of-recognition SvrPm3a1 / f1, that is, the recognition mode of Avr-R-Svr. The suppressor can not only inhibit the recognition of avirulent gene, but also greatly enhance the survival adaptability of powdery mildew (Bourras et al., 2016). Therefore, it is very necessary to develop a new avirulent gene for wheat powdery mildew and use it for the prevention and control of wheat powdery mildew 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 object 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 problem is as follows: a wheat powdery mildew avirulence gene AvrPmL180 is provided, and the nucleotide sequence of the wheat powdery mildew avirulence gene AvrPmL180 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 natural populations of wheat powdery mildew.

[0011] The present invention has the following beneficial effects: a new wheat powdery mildew avirulence gene AvrPmL180 is cloned. By predicting the signal of the protein of the gene, it is 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 the corresponding disease resistance 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 subspecies and the theoretical study of the molecular interaction of wheat powdery mildew; a molecular marker detection system for monitoring the 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; Figure 2 This is the signal peptide prediction map of AvrPmL180 gene; Figure 3 The present invention carried out phenotypic identification by inoculating 24 purified strains into the disease-resistant wheat variety PI170911. DETAILED DESCRIPTION

[0013] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0014] Example 1: Cloning of the wheat powdery mildew avirulence gene AvrPmL180 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 the gene was significantly associated with the resistance-susceptibility phenotype. The gene was named AvrPmL180, and the gene was obtained by gene cloning. The gene sequence is shown in SEQ ID NO.1. The specific cloning process is as follows: ① Enzyme system: TaKaRa Tks Gflex DNA Polymerase R060A.

[0015] ② Amplification system: Using the cDNA of the wheat powdery mildew strain as a 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.

[0016] 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; and final extension at 68°C for 5 min.

[0017] Among them, the nucleotide sequences of the upstream and downstream primers (5 μm / L) for cloning the AvrPmL180 gene are: AvrPmL180-F: atgagatctgctaatcttgcttcattg (SEQ ID NO.3) AvrPmL180-R: ttaacgctttatacaagatgacatttgg (SEQ ID NO. 4).

[0018] 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.

[0019] 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.

[0020] Example 2: Phenotypic Identification of the Avirulence Gene AvrPmL180 of Powdery Mildew 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 disease-resistant wheat variety PI170911 was selected as the test wheat, and the susceptible variety 9504 (denoted as ZZ9504) was used as the positive control. After growing for 7 days, healthy leaves were selected and inoculated with the 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, the 24 strains could grow normally in the positive control ZZ9504, and some strains could normally infect the disease-resistant wheat PI170911 after inoculation, such as B1, B7, B8, B11, B12, B17, B19 and B22, and PI170911 showed a susceptible phenotype; the remaining strains could not normally infect the disease-resistant wheat PI170911 after inoculation, such as B2, B3, B4, B5, B6, B9, B10, B13, B14, B15, B16, B18, B20, B21, B23 and B24, and PI170911 showed a resistant phenotype.

[0021] 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. Wheat without this gene had a susceptible phenotype.

[0022] The nucleotide sequence of the CDS region of the wheat powdery mildew avirulence gene AvrPmL180 in the present invention is as follows: CCAAATCTGCTAATCTTGCTTCATTGTCCTTACTTTTTGGCTTTTTGATTCTCGAAAGTGCTGCAGACTATGTATGCTCGGGTGGAACTCGAATACCGGATCATGAAGTCGAGACAAGAGCAAATGAGATTTACTCAAAAGGATTATCCCTCAAAGCTAGTCGGACTCCTGGTCAACAACAAATAGAAGATATCTATTTTGATGATGATGAAGATGATGCAGAAATGTCCTTTTCCAGCGACTTCTATCCTCGGATCAAATCTTCTGGAACATATACAATAACAGTCGATTATCCATCAAAAAATATATTGGTTATAGAGAAAATTGAATATAACGGCAGATACCAAATGTCATCTTGTATAAAGCGTTAA (SEQ ID NO.1); The amino acid sequence of the wheat powdery mildew avirulence gene AvrPmL180 in the present invention is as follows: MRSANLASLSLLFGFLILESAADYVCSGGTRIPDHEVETRANEIYSKGLSLKASRTPGQQQIEDIYFDDDEDDAEMSFSSDFYPRIKSSGTYTITVDYPSKNILVIEKIEYNGRYQMSSCIKR (SEQ ID NO.2).

[0023] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A wheat powdery mildew avirulence gene AvrPmL180, characterized in that: The nucleotide sequence of the wheat powdery mildew avirulence gene AvrPmL180 is shown in SEQ ID NO.

1.

2. Use of the wheat powdery mildew avirulence gene AvrPmL180 described in claim 1 in breeding new disease-resistant wheat varieties.

3. The use of the wheat powdery mildew avirulence gene AvrPmL180 according to claim 1 in detecting the toxicity of powdery mildew, 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 disease-susceptible.

4. Use of the wheat powdery mildew avirulence gene AvrPmL180 described in claim 1 in establishing a molecular marker detection system for monitoring pathogenic variation of natural populations of wheat powdery mildew.

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