Application of molecular chaperone protein TaBiP1 in wheat resistance to Fusarium head blight

Abstract

This invention relates to the field of biotechnology, and more particularly to the application of the wheat molecular chaperone protein TaBiP1 in the control of wheat scab. This invention is the first to discover that overexpression of the wheat molecular chaperone protein TaBiP1 can significantly reduce the infection severity of wheat scab. Compared with the transgenic recipient parent Fielder, TaBiP1-overexpressing plants significantly reduce wheat scab infection and can be used to create new scab-resistant germplasm. In this invention, the TaBiP1 gene in recipient wheat is edited using CRISPR / Cas9 gene editing technology to obtain transgenic plants. bip1 The resistance of wheat to Fusarium head blight was weaker than that of Fielder, which confirms the positive significance of TaBiP1 in enhancing wheat resistance to Fusarium head blight.

Description

Technical Field

[0001] This invention relates to the field of plant breeding, and more particularly to the application of the wheat TaBiP1 gene in wheat scab resistance. Background Technology

[0002] Wheat (Triticum aestivum L.) is the world's most widely planted, highest-yielding, and most distributed grain crop. Global wheat production has increased significantly over the past few decades. However, wheat cultivation and production worldwide still face major challenges and serious threats, with widespread outbreaks of various diseases severely impacting wheat yield and quality. Fusarium head blight (FHB) is a highly destructive and difficult-to-control fungal disease caused by Fusarium graminearum, one of the three major wheat diseases, often referred to as the "cancer" of wheat (Chen Y, Kistler HC, Ma Z. Fusarium graminearum trichothecenemycotoxins: biosynthesis, regulation, and management[J]. Annual Review of Phytopathology, 2019, 57(1): 15-39.).

[0003] Although the combined use of tolerant wheat varieties, fungicides, and specific management practices (such as tillage and crop rotation) can reduce some of the losses caused by the disease, no effective strategy has yet been found to completely control the spread of FHB (Bai G, Su Z, Cai J. Wheat resistance to Fusarium head blight[J]. Canadian Journal of Plant Pathology, 2018, 40(3): 336-346.). Discovering disease-resistant genes and breeding FHB-resistant varieties are the most effective and environmentally friendly measures for controlling wheat FHB. However, only nine genetic loci (Fhb1–Fhb9) for wheat resistance to Fusarium head blight have been identified so far, and only Fhb1 (Su Z, Bernardo A, Tian B, et al. A deletion mutation in TaHRC confers Fhb1 resistance to Fusarium head blight in wheat[J]. Nature genetics, 2019, 51(7): 1099-1105.) and Fhb7 (Wang H, Sun S, Ge W, et al. Horizontal gene transfer of Fhb7 from fungus underlies Fusarium head blight resistance in wheat. Science 368: eaba5435[EB / OL].(2020)) have been cloned. Therefore, the discovery and utilization of efficient wheat Fusarium head blight resistance genes are of great significance for promoting the breeding process of wheat Fusarium head blight resistance.

[0004] Heat shock proteins (HSPs) play a role in plant development and responses to abiotic and biotic stresses, including drought, salt stress, pathogens, and insect infestations. Increasing evidence suggests that HSP70-mediated defense mechanisms play a crucial role in plant disease resistance (Usman MG, Rafii MY, Martini MY, et al. Molecular analysis of Hsp70 mechanisms in plants and their function in response to stress[J]. Biotechnology and Genetic Engineering Reviews, 2017,33(1): 26-39.). Plant HSP70 is associated with endoplasmic reticulum-mediated immunity, and recent studies indicate that plant pathogens can utilize their effectors to regulate host endoplasmic reticulum stress, thereby promoting their infection. The endoplasmic reticulum-resident HSP70 protein BiP, as a key sensor for endoplasmic reticulum stress, participates in plant immunity. Studies have reported that the effector PsAvh262se produced by Phytophthora sojae stabilizes BiP and inhibits endoplasmic reticulum stress-mediated immunity by stabilizing plant binding immunoglobulin proteins [J]. Nature Communications, 2016, 7(1): 11685.. This background suggests that BiP has potential for development in the field of plant disease resistance. Summary of the Invention

[0005] This invention identifies TaBIP1, a key target protein in wheat's response to Fusarium head blight, through genetic engineering and a biochemical system of interaction between Fusarium head blight pathogens and wheat. Therefore, the application of TaBIP1 in wheat resistance to Fusarium head blight is determined by overexpressing and knocking out TaBIP1.

[0006] Based on the inventors' research, it was discovered for the first time that overexpression of TaBiP1 in wheat can reduce the infection degree of wheat scab; it was also discovered for the first time that knockout expression of TaBiP1 in wheat can increase the infection degree of wheat scab. This completes the present invention.

[0007] The present invention first provides information on the TaBiP1 gene, the amino acid sequence of which is shown in SEQ ID No.2, No.4 or No.6.

[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] This invention relates to the application of TaBiP1 in disease resistance. Overexpression of the TaBiP1 gene does not affect the growth and yield of wheat plants; however, transgenic plants overexpressing the TaBiP1 gene exhibit significant resistance to wheat scab and can inhibit the spread of Fusarium graminearum within host cells. Simultaneously, it was found that knocking out the TaBiP1 mutant transgenic plants significantly reduces wheat scab resistance. The transgenic plants involved in this invention do not affect normal growth and fruit setting.

[0013] Furthermore, the present invention provides a method for enhancing plant resistance to wheat scab by overexpressing the gene in transgenic plants through a transgenic method;

[0014] This invention is beneficial for the breeding of disease-resistant wheat varieties and provides a basis for the later screening of highly resistant wheat varieties. For example, this invention can provide TaBiP1 overexpressing plants. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly introduced below.

[0016] Figure 1 Overexpression of TaBiP1 in wheat enhances its resistance to Fusarium head blight. Figure A shows the relative expression level of TaBiP1 in the overexpressing wheat lines. Lines #6, #7, and #8 showed relatively high expression levels and were therefore selected for subsequent experimental analysis. Figure B shows representative images of wheat ears 15 days after infection with Fusarium graminearum by the WT (Fielder) and TaBiP1-OE lines under greenhouse conditions. Figure C shows the quantitative analysis of the disease index of the three TaBiP1-OE lines. The results indicate that overexpression of TaBiP1 significantly enhances wheat resistance to Fusarium head blight.

[0017] Figure 2Representative images (A) and lesion area (B) of leaves infected with Fusarium graminearum in the three strains WT, TaBiP1-OE at 5 days. The data show that the leaves of the three strains TaBiP1-OE have stronger resistance to Fusarium graminearum infection.

[0018] Figure 3 The DNA sequence editing details of the target region for the TaBiP1 gene-edited plants are shown; "." indicates base deletion; red fields indicate base mutations, and three different types of knockout expression lines, #2, #3, and #5, were obtained.

[0019] Figure 4 The image shows the phenotypic resistance to Fusarium head blight in T2 generation homozygous transgenic wheat plants with TaBiP1 gene editing. Figure A shows representative images of wheat ears 15 days after infection with Fusarium graminearum in the WT (Fielder) and bip1 lines. Figure B shows the quantitative analysis of the disease index in the three bip1 lines. The results indicate that knocking out TaBiP1 significantly reduces wheat resistance to Fusarium head blight.

[0020] Figure 5 The study investigated the growth of three transgenic lines, TaBiP1, OE, and TaBiP1, in the field. The results showed that overexpression of TaBiP1 did not affect the normal growth of the plants.

[0021] In summary, TaBiP1 is a protein that plays a positive role in wheat scab resistance. It can significantly improve wheat resistance to scab without affecting normal wheat growth. Detailed Implementation

[0022] The present invention will be described below through specific embodiments to provide a better understanding of it, but these embodiments do not constitute a limitation thereof. The specific details are as follows:

[0023] Example 1: Overexpression of TaBiP1 in wheat enhances its resistance to wheat scab.

[0024] In this invention, the application preferably includes the following steps:

[0025] (1) The full-length TaBiP1 fragment was inserted into the pUbiGW vector with the Ubiquitin promoter via the BamHI site using In-Fusion cloning technology (Clontech, catalog number 638910);

[0026] The nucleotide sequence of the full-length TaBiP1CDS fragment is shown in SEQ ID No. 1;

[0027] (2) The ligation vector obtained in step (1) is transformed into Escherichia coli DH5α, and after screening and sequencing, the plasmid with the BiP1 gene ligated is extracted.

[0028] (3) Transform the plasmid obtained in step (2) into Agrobacterium EHA105 to obtain Agrobacterium carrying the TaBiP1 plasmid;

[0029] (4) The transformant obtained in step (3) is transformed into the Fielder wheat strain, and after gene identification, wheat overexpressing the TaBiP1 gene is obtained;

[0030] Wheat transformation was performed using the Agrobacterium-mediated transformation method (Goetz H., Cornelia M., and Jochen K. (2021). Agrobacterium-Mediated Transformation of Wheat Using Immature Embryos. Rom. Agric. Res. 38, 99-107 (2021)). Genotyping of transgenic plants was evaluated using the Enviologix QuickStix Kit (Envirologix, catalog number AS013). qRT-PCR was used to further confirm whether the T1 and T2 generations overexpressed the gene. Figure 1 (A)

[0031] Subsequently, the resistance of wheat scab to the transgenic material overexpressing TaBiP1 (TaBiP1-OE) (SEQ ID NO: 1) was identified. Under greenhouse conditions, inoculation with Fusarium graminearum resulted in significantly lower disease incidence in TaBiP1-OE compared to the wild-type Fielder. Figure 1 (B, C). We also tested the disease symptoms induced by *Fusarium graminearum* in the leaves of the overexpression materials. Notably, TaBiP1-OE showed a significant reduction in lesion area on leaves compared to the wild type. Figure 2 ).

[0032] The processing method for detached blades is as follows:

[0033] Wild-type and transgenic wheat plants were grown in a growth chamber at 22°C with a photoperiod of 16 hours light and 8 hours dark. Secondary leaves from 2-week-old plants were collected and transferred to square petri dishes containing 1% water agar. A 10µL suspension of conidia (approximately 5 × 10⁻⁶) was cultured in the center of each wheat leaf. 4 Conidia / mL). Infection symptoms were recorded using ImageJ (https: / / www.computerbild.de / download / ImageJ-422527.html) 5-6 days post-infection to assess necrotic lesions.

[0034] Finally, overexpression of TaBiP1 did not affect plant growth and yield. Figure 5 ).

[0035] Example 2: Knocking out TaBiP1 expression in wheat weakens its resistance to wheat scab.

[0036] Wheat knockout expression constructs were created by inserting the intermediate vector pMETaU6.1 fragment into the gene editing backbone vector pLGYE-3 via the Bsa1 site using In-Fusion cloning technology (Clontech, catalog number 638910). All constructs were transformed into Agrobacterium EHA105 strain. Wheat transformation was performed according to the above method. Genotypic evaluation of transgenic plants was performed using nucleotide sequencing, and three different homozygous lines #2, #3, and #5 were obtained through subculture. Figure 3 ).

[0037] Subsequently, the wheat transgenic materials expressing TaBiP1 were subjected to Fusarium head blight resistance identification. Fusarium graminearum was inoculated under greenhouse conditions, and the number of diseased ears was counted. The results showed that the disease severity of the three bip1 lines was significantly higher than that of the wild-type Fielder. Figure 4 ).

Claims

1. Application of wheat TaBiP1 protein or its encoding gene in improving plant disease resistance; The plant in question is wheat; the disease resistance refers to Fusarium head blight caused by Fusarium graminearum. The amino acid sequence of the wheat TabiP1 protein is shown in SEQ ID No.

2.

2. Application of wheat TaBiP1 protein or its encoding gene in the preparation of transgenic plants with enhanced disease resistance; The plant in question is wheat; the disease resistance refers to Fusarium head blight caused by Fusarium graminearum. The amino acid sequence of the wheat TabiP1 protein is shown in SEQ ID No.

2.

3. A method for preparing transgenic plants with enhanced disease resistance, characterized in that, This includes the steps of overexpressing the gene encoding the wheat TaBiP1 protein in plants through transgenic methods, and screening for transgenic plants that enhance resistance to wheat scab. The plant in question is wheat; the disease resistance refers to Fusarium head blight caused by Fusarium graminearum. The amino acid sequence of the protein encoded by the wheat TabiP1 protein gene is shown in SEQ ID No. 2.

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