Application of wheat taalix protein in reducing accumulation of vomitoxin and wheat resistance to scab

Abstract

The application relates to the fields of biotechnology and plant protection, and discloses application of an ESCRT-related protein in prevention and treatment of wheat scab. It is found for the first time that by increasing the expression amount of a wheat ESCRT III protein TaALIX, the resistance of wheat to scab is enhanced, and the content of vomitoxin (DON) is reduced, so that the application has practical significance in plant disease prevention and treatment.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of TaALIX, an ESCRT system-related protein, in reducing the accumulation of vomitoxin in wheat grains and enhancing resistance to wheat scab. Background Technology

[0002] Fusarium head blight (FBB) is one of the most destructive fungal diseases in wheat-producing regions worldwide, causing widespread crop yield reductions and economic losses. More than 20 species of Fusarium fungi can cause FBB, with Fusarium graminearum being the most prevalent in my country. Fusarium graminearum *Fusarium graminearum* is the dominant species. During infection, *Fusarium graminearum* produces various mycotoxins, among which deoxynivalenol (DON) is one of the most significant. DON can produce neurotoxicity, reproductive toxicity, and immunotoxicity. Ingestion of contaminated food by humans and animals can cause adverse reactions such as anorexia and vomiting, commonly known as vomitoxin. Studies have shown that DON participates in cell differentiation, apoptosis, and other related signal transduction processes, leading to the destruction of normal plant cells. It also acts as a pathogenic factor, accelerating crop tissue necrosis and contaminating grain grains. In recent years, due to various environmental factors, DON contamination has become increasingly serious worldwide. Large amounts of DON toxins have been detected in grains such as wheat, barley, and corn, with levels far exceeding the limits, posing a significant threat to human and animal health. Controlling the accumulation of DON toxins in wheat could greatly reduce the damage caused by wheat scab (Mawcha, KT, Zhang, N., Wang, Y., and Yang, W. Advances in wheat breeding for resistance to Fusarium headblight. Czech J. Genet. Plant. 2022). 58 (4), 167-188).

[0003] In recent years, the synthesis and regulatory mechanisms of wheat DON toxin have been extensively studied, such as the regulation of wheat scab protein FgRab7 by the wheat scab fungus. TriDeletion of FgSec2A and FgSec2B, two Sec2 homologs in *Fusarium graminearum*, located at the apical regions of hyphae and conidia, plays a crucial role in wheat DON biosynthesis and secretion. Double deletion of these homologs results in a significant reduction in DON content. (Zheng H, Li L, Miao P, et al., FgSec2A, a guanine nucleotide exchange factor of FgRab8, is important for polarized growth, pathogenicity, and deoxynivalenol production in wheat.) Fusarium graminearum Environ Microbiol. 2018, 20(9):3378-3392). Meanwhile, plants have evolved protective mechanisms against DON. Reports indicate that plant UDP-glucosyltransferase (UGT) can reduce DON toxicity and improve resistance to Fusarium head blight. Furthermore, a novel gene highly expressed by Fusarium head blight has been successfully cloned and identified in Sumai 3. TaUGT6 Enzyme activity assay revealed TaUGT6 DON can be glycosylated into D3G, which has lower toxicity, thereby improving the plant's tolerance to DON and resistance to Fusarium head blight (He Y, Wu L, Liu X, et al.). TaUGT6 A novel UDP-Glycosyltransferase gene enhances the resistance to FHB and DON accumulation in wheat. Front Plant Sci. 2020;11:574775. Furthermore, a cotton-specific glyoxalase (SPG) can isomerize DON-modified products to reduce DON toxicity (Huang J, Fang X, Tian X, et al. Aromatization of natural products by a specialized detoxification enzyme. Nat Chem Biol, 2020, 16(3):250-256.).

[0004] Besides the two main antitoxin accumulation mechanisms of inhibiting toxin synthesis and chemical modification of toxins, a 2021 study reported a novel mechanism by which the fungal toxin DON is transported into vacuoles via the P4-ATPase-mediated vesicle transport pathway in plant cells, thereby enhancing the disease resistance of Arabidopsis thaliana plants. This study identified a detoxification-related protein, AtALA1, in the Arabidopsis genome using DON as a screening agent and found that DON targets vacuoles via the AtALA1-mediated vesicle transport pathway. Overexpression of this gene increased the efficiency of DON targeting vacuoles, and significantly enhanced the resistance of Arabidopsis thaliana plants to Fusarium wilt. More importantly, the vomitoxin content in transgenic maize and Arabidopsis seeds was significantly reduced (Wang, F., Li, X., Li, Y. et al.). Arabidops is P4 ATPase-mediated cell detoxification confers resistance to Fusarium graminearum and Verticillium dahliae (Nat Commun 12, 6426 (2021)). This study utilized this vesicle transport-related cellular detoxification strategy, which significantly improved plant resistance to toxin-related diseases and provided new insights into how to reduce fungal toxins. The modification and transport processes of DON toxins are closely related to the occurrence of wheat scab and grain health, and are therefore a key focus in the fields of crop disease resistance, food safety, and human health. Summary of the Invention

[0005] Based on the inventors' research, it was discovered for the first time that overexpression of the ESCRT-related protein TaALIX can reduce the accumulation of vomitoxin during the course of wheat scab disease and enhance resistance to scab. This invention thus completes the present invention.

[0006] The present invention first provides a key vesicle transport protein TaALIX in the ESCRT system, the amino acid sequence of which is shown in SEQ ID No.2 or its homologous protein sequence, such as the protein shown in SEQ ID No.4 and SEQ ID No.6.

[0007] The present invention also provides a gene encoding the TaALIX, specifically, the nucleic acid sequence of which is such as SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5.

[0008] Furthermore, the present invention provides an expression element containing the aforementioned gene, a recombinant vector, and a host cell.

[0009] Preferably, the gene is overexpressed in the plant via a transgenic method.

[0010] The present invention also provides the application of the gene in the creation of disease-resistant transgenic plants, wherein the gene is overexpressed in the transgenic plants by a transgenic method.

[0011] Preferably, the plant is a monocotyledonous plant, and more preferably, the plant is wheat. More preferably, the disease resistance refers to resistance to Fusarium head blight caused by Fusarium.

[0012] The screening of disease-resistant transgenic plants involves measuring the DON content in the transgenic plants. Specifically, this includes sampling and measuring the DON content of Fusarium graminearum at different times after inoculation.

[0013] The inventors have discovered that overexpression TaALIX The gene-expressing wheat exhibits resistance to Fusarium graminearum with significantly reduced toxin content, and overexpression can be widely applied in practical applications. This invention provides TaALIX overexpressing plants and homozygous overexpressing plants. TaALIX Mutant plants are beneficial for the breeding of disease-resistant wheat varieties and provide a basis for the later screening of highly resistant wheat varieties. Attached Figure Description

[0014] Figure 1 wheat overexpression TaALIX Genotyping of strains.

[0015] Figure 2 Genotyping of wheat TaALIX knockout lines is shown.

[0016] Figure 3 Representative images of wheat ears WI (Fielder), TaALIX-OE, and TaALIX mutants 15 days after infection with Fusarium graminearum under greenhouse conditions.

[0017] Figure 4 for TaALIX Quantitative analysis of the incidence index of -OE.

[0018] Figure 5 For the determination and analysis of DON content. Detailed Implementation

[0019] The present invention will be described below through specific embodiments to provide a better understanding of the invention, but these embodiments do not constitute a limitation thereof.

[0020] Example 1: Wheat overexpression TaALIX Genotyping of strains

[0021] For the wheat overexpression construct, In-Fusion cloning technology (Clontech, catalog number 638910) was used to... TaALIX(SEQ ID NO: 1, encoding the amino acid sequence shown in SEQ ID NO: 2, is a toxin target gene identified in previous laboratory one-to-one interaction screening, and is from Arabidopsis thaliana in wheat.) AtALIX The full-length CDS fragment of the homologous gene (since wheat is an allohexaploid with an ABD chromosome, SEQ ID NO: 3 and SEQ ID NO: 5 are the ABD chromosome homologous gene sequences of this gene) was inserted into the pUbiGW vector with a Ubiquitin promoter via the BamHI site. All constructs were transformed into Agrobacterium EHA105 strain. Wheat transformation was performed according to the previously described method (Goetz H., Cornelia M., and Jochen K. Agrobacterium-Mediated Transformation of Wheat Using Immature Embryos. Rom. Agric. Res. 2021). 38 , 99-107). A total of 13 positive plants were identified by qPCR ( Figure 1 ).

[0022] Example 2: Wheat knockout TaALIX Genotyping of strains

[0023] Wheat contains three sets of chromosomes (A, B, and D) and is a typical allohexaploid (BBAADD), formed by hybridization and genome polyploidization of cultivated tetraploid wheat (BBAA) and diploid Aegilops davidii (DD). This was achieved using a modified CRISPR-Cas9 system in wheat. TaALIX The mutant was designed with two targets (T1 and T2) on the sgRNA. TaALIX Gene editing was performed. The target sequence was evaluated using CRISPRdirect (https: / / crispr.dbcls.jp) to confirm its target specificity in the wheat genome, and a knockout mutant was constructed. Genotyping of the mutant plants was performed using gene-specific primer sequencing, and PCR technology was used to further analyze the results. TaALIX The knockout mutant was identified, and a total of one homozygous mutant was obtained, with all chromosomes ABD covered. TaALIX mutant strains ( Figure 2 ).

[0024] Example 3: Overexpression in wheat TaALIX It can enhance resistance to wheat scab and reduce DON content.

[0025] overexpression TaALIX ( TaALIX Positive plants and homozygous plants obtained in Example 1 of (-OE) TaALIXThe resistance of mutant wheat transgenic materials to Fusarium head blight was identified.

[0026] Under greenhouse conditions, the FHB resistance of transgenic materials was assessed using the single-flower drip method (Zhang, X., Zhou, M., Ren, L., Bai, G., Ma, H., Scholten, OE, Guo, P., and Lu, W. Molecular characterization of Fusarium head blight resistance from wheat variety Wangshuibai. Euphytica, 2004). 139, 59–64.

[0027] TaALIX After the onset of -OE disease, an average of 9 florets on the spikelet were affected, while the control group (wild type) averaged 12. The severity of the disease was significantly lower in the wild type than in the wild type. TaALIX The mutant strain had 14 cases of disease, and the severity of the disease was significantly higher than that of the wild-type Fielder. Figure 3 , Figure 4 ).

[0028] Subsequently, the diseased seeds of the genetically modified material were ground into flour, and the DON content was determined using a vomitoxin rapid detection kit (Huaan Maike, catalog number: HEM1896). TaALIX The DON content in the diseased grains of the -OE strain was 6.7 × 10⁻⁶. 3 μg / kg was significantly lower than that of the wild type (6.98×10). 3 μg / kg, and TaALIX The mutant strain showed a significant increase in DON content in the viral particles, specifically 7.35 × 10⁻⁶. 3 μg / kg ( Figure 5 ).

Claims

1. Key vesicle transport proteins in the ESCRT system TaALIX The application of its encoding gene, expression element containing the encoding gene, recombinant vector, or recombinant host cell in improving wheat disease resistance; The disease resistance refers to wheat scab; the key vesicle transport protein TaALIX The amino acid sequence is shown in SEQ ID No.

2.

2. The application as described in claim 1, characterized in that, The nucleic acid sequence of the encoding gene is shown in SEQ ID No.

1.

3. A method for preparing transgenic plants with enhanced disease resistance, characterized in that, This includes overexpressing key vesicle transport proteins in the ESCRT system in plants through transgenic methods. TaALIX The coding gene, and the steps for screening to obtain transgenic plants with enhanced disease resistance; Key vesicle transport proteins in the ESCRT system TaALIX The amino acid sequence is shown in SEQ ID No. 2; the plant is wheat, and the disease resistance refers to resistance to wheat scab caused by Fusarium graminearum.

4. The method as described in claim 3, characterized in that, The nucleic acid sequence of the encoding gene is shown in SEQ ID No.

1.

5. The method as described in claim 3 or 4, characterized in that, The screening process involved inoculating the transgenic plants with Fusarium graminearum and taking samples at different times to determine the DON content.

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