Application of endogenous plant peptides in regulating resistance to fusarium head blight and foot rot of wheat

Abstract

The application discloses application of three endogenous plant peptides in prevention and treatment of wheat scab and foot rot. The application first finds that three endogenous Pep precursors (TaPROPEP) can be cut to produce endogenous plant peptides TaPep, the endogenous plant peptides can act as immunostimulants, cause plant immune response, reduce infection degree of wheat scab and foot rot, and significantly improve wheat resistance to the wheat scab and foot rot in a greenhouse and in a field.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of three endogenous plant peptides in resistance to wheat scab and stem rot. Background Technology

[0002] Crops are constantly subjected to various biological stresses during their growth, leading to significant yield losses (Savary, S., Willocquet, L., Pethybridge, SJ, Esker, P., McRoberts, N., and Nelson, A. (2019). The global burden of pathogens and pests on major food crops. Nat. Ecol. & Evol. 3(3), 430-439). Fungal pathogens can cause many devastating diseases, resulting in losses exceeding US$200 billion annually, accounting for 10%-20% of global crop losses (www.ars.usda.gov). Wheat is one of the most important staple crops widely grown globally and is particularly susceptible to pathogen infection. Fusarium head blight (FHB) is mainly caused by the Fusarium graminearum (F. graminearum) complex and is one of the top ten crop diseases globally, prevalent in wheat-growing areas. This disease not only leads to a decrease in yield but also produces mycotoxins, mainly deoxynivalenol (DON), which endanger human and animal health. The widespread use of fungicides such as tebuconazole can prevent the spread of food toxins and reduce yield losses, but it will cause environmental and health problems (Mawcha, KT, Zhang, N., Wang, Y., and Yang, W. (2022). Advances in wheat breeding for resistance to Fusarium head blight. Czech J. Genet. Plant. 58(4), 167-188).

[0003] Over the past thirty years, significant progress has been made in research on plant resistance to Fusarium head blight using genetic and other methods. Hundreds of resistance QTLs have been identified, but only a few, such as Fhb1, have shown stable effects. Therefore, it is necessary to find and utilize more effective methods to control wheat Fusarium head blight. However, unlike other wheat diseases, such as immune-related proteins directly involved in wheat resistance to powdery mildew and rust, no immune target proteins directly involved in wheat Fusarium head blight resistance have been reported. For example, Fhb1 and Fhb7 encode histidine-rich calcium-binding proteins and glutathione S-transferases, respectively, but both are involved in wheat scab resistance and toxin mitigation through unknown mechanisms (Su, Z., Bernardo, A., Tian, ​​B., Chen, H., Wang, S., Ma, H., Cai, S., Liu, D., Zhang, D., Li, T., et al. (2019). A deletion mutation in TaHRC confers Fhb1 resistance to Fusarium head blight in wheat. Nat. Genet. 51(7), 1099-1105). Although utilizing innate plant defense mechanisms is a potentially effective strategy for improving crop disease resistance, our understanding of how to utilize these mechanisms to enhance wheat resistance to scab remains limited.

[0004] In plant innate immune responses, conserved pathogen-associated molecular patterns (PAMPs) produced by pathogenic microorganisms can be recognized by anchored pattern recognition receptors (PRRs) on the plant cell surface, thereby triggering PTI immunity. Many PAMPs act as immune inducers, inducing defense signals and conferring disease resistance to crops. For example, the 22-amino acid peptide flg22 conserved by bacterial flagellin can induce PTI and enhance Arabidopsis resistance to bacterial diseases. In addition to microbial elicitors, plants also secrete and release endogenous immune factors or peptides into the apoplast space during interactions with pathogens. These are recognized by PRRs located on the plasma membrane, activating the PTI response and promoting plant survival (Hou, S., Liu, D., and He, P. (2021). Phytocytokines function as immunological modulators of plant immunity. Stress Biol. 1(1), 8).

[0005] In plants, only a few endogenous peptides have been identified as plant cytokines involved in the regulation of plant immunity, such as systemin-like peptides in Solanaceae, which have anti-herbivorous defense functions. Notably, Pep1 (plant activating peptide) is a plant endogenous peptide composed of 23 amino acids and was the first Arabidopsis immune endogenous polypeptide to be discovered. It triggers the signature PTI response through the cell surface-anchored Pep receptor (PEPR), promoting resistance to various stresses (Huffaker, A., Pearce, G., and Ryan, CA (2006). An endogenous peptide signal in Arabidopsisactivates components of the innate immune response. P. Natl. Acad. Sci. 103(26), 10098-10103).

[0006] Pep endogenous peptides are produced by the cleavage of its precursor protein PROPEP by cysteine ​​proteases, ultimately functioning as the N-terminal mature form. PROPEP proteins are found in many angiosperms such as maize, rice, and potato. However, the sequences of these precursor proteins are not conserved, and the resulting mature Peps are often not recognized across species; therefore, the activity of the Peps elicitor is species-specific. It has been reported that Arabidopsis Pep1 enhances plant responses to pathogens, pests, and damage stress (Yamaguchi, Y., Huffaker, A., Bryan, AC, Tax, FE, and Ryan, and C.A. (2010). PEPR2 is a second receptor for the Pep1 and Pep2 peptides and contributes to defense responses in Arabidopsis. Plant Cell, 22(2), 508-522). Maize homologous proteins ZmPep1 and ZmPep3 regulate maize's resistance to pathogens and pests, respectively (Huffaker, A., Pearce, G., Veyrat, N., Erb, M., Turlings, Ted C., Sartor, R., Shen, Z., Briggs, SP, Vaughan, MM, Alborn, HT, et al. (2013). Plant elicitor peptides are conserved signals regulating direct and indirect antiherbivore defense. P. Natl. Acad. of Sci., 110(14), 5707-5712). The rice homolog OsPep3 can enhance crop resistance to pests and diseases (Shen, W., Zhang, X., Liu, J., Tao, K., Li, C., Xiao, S., Zhang, W., Li, JF (2022). Plant elicitor peptide signallingconfers rice resistance to piercing-sucking insect herbivores and pathogens. Plant Biotechnol. J. 20(5), 991-1005). Utilizing endogenous Peps to enhance crop disease resistance is an economical and effective approach. Although most Peps achieve plant protection and defense through immune responses, the application and development of Peps immune inducers still face significant challenges. Summary of the Invention

[0007] Based on the inventors' research, it was discovered for the first time that overexpression of endogenous Pep precursors (TaPROPEP) can reduce the infection severity of wheat scab and stem rot. These four endogenous Pep precursors can be cleaved by cysteine ​​proteases to form their respective mature TaPeps plant peptide elicitors (three in total, with mature sequences identical to SEQ ID NO: 3 and SEQ ID NO: 9, as shown), thereby regulating wheat resistance to scab and stem rot. Overexpression of the TaPROPEP gene in wheat significantly increases resistance to scab and stem rot. This invention thus completes the present invention.

[0008] This invention first provides four endogenous Pep precursors (TaPROPEP), whose amino acid sequences are shown in SEQ ID No. 2, 5, 8, and 11.

[0009] Furthermore, the present invention provides an expression element containing the aforementioned gene, a recombinant vector, and a host cell.

[0010] Preferably, the gene is overexpressed in the plant via a transgenic method.

[0011] 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.

[0012] 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 or stem rot caused by Fusarium.

[0013] This invention relates to the application of four endogenous Pep precursors (TaPROPEP) in disease resistance. Overexpression of the TaPROPEP gene does not affect wheat plant growth or yield; however, transgenic plants overexpressing the TaPROPEP gene exhibit significant resistance to wheat scab and stem rot, inhibiting the spread of Fusarium graminearum within host cells. During Fusarium graminearum infection, TaPROPEP can be cleaved by wheat cysteine ​​protease to form three mature TaPep plant peptides as elicitors, thereby significantly enhancing resistance to wheat scab and stem rot. The transgenic plants involved in this invention do not affect normal growth and fruit setting.

[0014] Furthermore, the present invention provides a method for enhancing plant resistance to wheat scab and stem rot, which involves overexpressing the aforementioned gene in transgenic plants via transgenic methods; or directly treating plants with three synthetic TaPep mature plant peptides, such as the endogenous Pep precursor TaPROPEP prepared by genetic engineering methods, or the TaPeps mature synthesized by chemical methods.

[0015] Furthermore, the present invention also provides an agent for enhancing plant disease resistance, characterized in that it contains the mature TaPep polypeptide. This agent can be used against Fusarium head blight or stem rot of wheat crops.

[0016] The inventors have discovered that wheat treated with exogenous spraying of three mature TaPep variants or overexpression of Pep precursor genes (TaPROPEP7 and TaPROPEP10) exhibits resistance to Fusarium graminearum, which can be widely applied in practice. This invention is beneficial for the breeding of disease-resistant wheat varieties and provides a basis for later screening of highly resistant wheat varieties. For example, this invention can provide TaPROPEP overexpressing plants; furthermore, this invention can provide three synthetically produced mature TaPep plant peptides as elicitors to treat plants, thereby improving plant resistance to Fusarium head blight and stem rot. Attached Figure Description

[0017] Figure 1 This study investigates the induction of wheat resistance to Fusarium head blight by exogenous TaPep application under greenhouse conditions. Figure A shows the TaPep pretreatment before Fusarium graminearum infection of wheat spikelets. Figure B shows a representative image of a Fielder spike 17 days after Fusarium graminearum infection with 20 μM TaPep pretreatment under greenhouse conditions. Figure C quantifies the severity of disease after Fusarium graminearum infection 10-20 days prior, as mentioned in Figure B.

[0018] Figure 2 This study demonstrates the induction of wheat resistance to Fusarium head blight by exogenous application of TaPep under field conditions. Figure A shows a representative image of wheat ears infected with Fusarium graminearum 15 days after Fielder variety pretreatment under field conditions. Figure B quantifies the severity of Fusarium graminearum infection mentioned in Figure A.

[0019] Figure 3 To induce resistance to stem rot in wheat through exogenous application of TaPeps. Figure A shows the disease incidence on wheat stems 14 days after inoculation with Fusarium graminearum following pretreatment with 20 μM TaPep-e and TaPep-g. Figure B shows the quantitative measurement of stem length affected by wheat stem rot.

[0020] Figure 4 Overexpression of the TaPep precursor in wheat enhances its resistance to wheat scab. Figure A shows the relative expression levels of TaPROPEP7 and TaPROPEP10 in the overexpressing wheat lines. Figure B shows representative images of wheat ears 15 days after infection with Fusarium graminearum by WT (Fielder), TaPROPEP7-OE, and TaPROPEP10-OE under greenhouse conditions. Figure C shows the quantitative analysis of the disease index of TaPROPEP7-OE and TaPROPEP10-OE.

[0021] Figure 5 Representative images (top) and lesion areas (bottom) of leaf infection and Fusarium graminearum in WT, TaPROPEP7-OE and TaPROPEP10-OE at 5 days.

[0022] Figure 6 Overexpression of the TaPep precursor in wheat enhances its resistance to wheat stem rot. Figure A shows three wheat plants (WT (Fielder), TaPROPEP7-OE, and TaPROPEP10-OE) inoculated with Fusarium graminearum conidia for 14 days. Figure B shows the quantitative determination of stem length in wheat seedlings with stem rot in WT and TaPROPEP-OE. Detailed Implementation

[0023] The present invention will be described below through specific embodiments to better understand the invention, but these embodiments do not constitute a limitation thereof.

[0024] Example 1: Induction of wheat resistance to wheat scab by exogenous application of TaPep

[0025] Considering that several Pep molecules in Arabidopsis and maize act as plant cytokines to regulate immunity and biological responses, we speculate that wheat homologs may also act as disease resistance inducers.

[0026] The formation of four endogenous wheat Pep precursors (TaPROPEP7-10) resulted in three mature forms: TaPep-e (SEQ ID NO: 3, 9), TaPep-f (SEQ ID NO: 6), and TaPep-g (SEQ ID NO: 12). The precursors TaPROPEP7 (SEQ ID NO: 2) and TaPROPEP9 (SEQ ID NO: 8) formed TaPep-e, the precursor TaPROPEP8 (SEQ ID NO: 5) formed TaPep-f, and the precursor TaPROPEP10 (SEQ ID NO: 11) formed TaPep-g.

[0027] To verify this hypothesis, we artificially synthesized mature TaPep-e, TaPep-f, and TaPep-g (Sangon Biotech, China) (Table 1) and applied them exogenously to wheat spikelets (Fielder), then observed the symptoms of wheat scab. Figure 1 (A). Under greenhouse conditions, the severity of wheat scab disease 9-21 days after pretreatment with TaPep-e, TaPep-f, and TaPep-g was significantly lower than that in the control group. Observation and disease index statistics showed that the number of diseased spikelets after treatment with TaPep-e, TaPep-f, and TaPep-g was significantly less than that in the control group. Figure 1(B, C) indicates that external application of TaPep-e, TaPep-f, and TaPep-g improved wheat resistance to wheat scab.

[0028] Furthermore, under field conditions, application of three groups of exogenous Tapeps to wheat spikelets revealed that TaPep-e, TaPep-f, and TaPep-g, under natural conditions, also reduced the number of diseased spikelets. This indicates that TaPep-e, TaPep-f, and TaPep-g treatments enhanced resistance to wheat scab. Figure 2 (A, B)

[0029] Table 1

[0030]

[0031] The peptide processing methods used in the above experiments are as follows:

[0032] Dissolve the peptide powder in distilled water to a working concentration of 0.5–50 μM. For phenotypic analysis, treat the florets of three central spikelets on an ear with 20 μL of TaPep-e, TaPep-f, or TaPep-g one day before Fusarium graminearum treatment.

[0033] The methods for evaluating the wheat Fusarium head blight phenotype in the above experiments are as follows:

[0034] Fusarium graminearum isolate Fg1312 from Jiangsu, China, was used as inoculum for infection with Fusarium graminearum. The pre-flowering spikelet stage is the optimal time for wheat scab infection. A 10 μL suspension of Fg1312 conidia (approximately 1 × 10⁻⁶) was used. 5 Infect a single floret of a central spikelet with 1 spore / mL of fungus, cover with a plastic bag, and maintain humidity for 2 days (greenhouse) or 3 days (field) to ensure normal infection by Fusarium graminearum. Assess the spread of wheat scab between spikelets (type II resistance) by counting symptomatic spikelets at 7, 9, 11, 13, 15, 17, and 21 days post-inoculation.

[0035] Example 2: Exogenous application of TaPep to induce resistance in wheat to wheat stem rot

[0036] To investigate the role of TaPeps in wheat stem rot caused by Fusarium graminearum, wheat seedlings were pretreated with TaPeps, and the incidence of wheat stem rot (FCR) was detected. Compared with the control group, the length of the brown stem affected by TaPep-e,g was significantly smaller in the pretreated group. TaPep-e and TaPep-g significantly reduced the size of brown necrotic lesions in the basal stem region. Figure 3(A, B) indicates that Group III TaPeps also confers resistance to wheat stem base rot.

[0037] The peptide treatment method involves pretreating wheat seedlings with TaPep-e and TaPep-g one day before Fusarium graminearum spore infection.

[0038] The assessment methods for symptoms of wheat stem base rot are as follows:

[0039] Wheat seeds were disinfected with 70% ethanol for 2 minutes, then rinsed 3-4 times with sterile water and germinated in petri dishes on three layers of water-soaked filter paper. When the seedlings reached a length of 0.5-1 cm, they were germinated in Fg1312 (containing 1×10⁻⁶ ppm). 6 Soak seedlings in a spore suspension (spores / mL) for 1-2 minutes. Ten seedlings inoculated with *Fusarium graminearum* were wrapped in a damp paper towel and grown in a plastic container at 22°C and 60% humidity for 16 hours in a growth chamber. Resistance was assessed by measuring the length of the dark brown lesion at the base of the stem.

[0040] Example 3: Overexpression of TaPep precursor in wheat enhances its resistance to wheat scab.

[0041] For the wheat overexpression constructs, the full-length CDS fragments of TaPROPEP7 (SEQ ID NO: 1) and TaPROPEP10 (SEQ ID NO: 10) were inserted into the pUbiGW vector with a Ubiquitin promoter via the BamH I 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 previously described method (Goetz H., Cornelia M., and Jochen K. (2021). Agrobacterium-Mediated Transformation of Wheat Using Immature Embryos. Rom. Agric. Res. 38, 99-107 (2021)). The genotypes of the transgenic plants were evaluated using the Enviologix QuickStix Kit (Envirologix, catalog number AS013). qRT-PCR was used to further confirm whether the T1 and T2 generations were overexpressing, or gene-specific primer sequencing was used to identify homozygous mutations. Figure 4 (A)

[0042] Subsequently, the scab resistance of wheat transgenic materials overexpressing the precursors TaPROPEP7 (TaPROPEP7-OE) (SEQ ID NO: 1) and TaPROPEP10 (TaPROPEP10-OE) (SEQ ID NO: 10) was identified. Under greenhouse conditions, the severity of spikelet disease after infection with TaPROPEP7-OE and TaPROPEP10-OE was significantly lower than that with wild-type Fielder (…). Figure 4 (B, C). We also tested the disease symptoms induced by *Fusarium graminearum* in the leaves of the overexpression materials. Notably, compared with the wild type, TaPROPEP7-OE and TaPROPEP10-OE showed a significant reduction in lesion area on the leaves ( Figure 5 ).

[0043] The processing method for detached blades is as follows:

[0044] Wild-type (Fielder) and transgenic wheat plants were grown in a growing chamber at 22°C with a photoperiod of 16 hours light to 8 hours dark. Secondary leaves from 2-week-old plants were collected and transferred to square petri dishes containing 1% water agar. A 10 μL 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.

[0045] Example 4: Overexpression of TaPep precursor in wheat enhances its resistance to wheat stem rot.

[0046] To understand the role of endogenous TaPep in response to wheat stem rot, we also tested the disease symptoms induced by *Fusarium graminearum* in the stems of overexpressing materials. Notably, compared to the wild type, TaPROPEP7-OE and TaPROPEP10-OE showed a significant reduction in the lesion area at the base of the stem. Figure 6 (A, B)

Claims

1. Application of an endogenous Pep precursor TaPROPEP or its encoding gene in improving plant disease resistance; The plant in question is wheat; the disease resistance refers to wheat scab or stem rot caused by Fusarium wilt. The amino acid sequence of the endogenous Pep precursor TaPROPEP is shown in SEQ ID No. 2 or 11.

2. The application of an endogenous Pep precursor TaPROPEP or its encoding gene, an expression element containing the encoding gene, and a recombinant vector containing the encoding gene in the preparation of transgenic plants with enhanced disease resistance; The plant in question is wheat; the disease resistance refers to wheat scab or stem rot caused by Fusarium wilt. The amino acid sequence of the endogenous Pep precursor TaPROPEP is shown in SEQ ID No. 2 or 11.

3. A method of making a transgenic plant with enhanced disease resistance, comprising, This includes the steps of overexpressing the gene encoding the endogenous Pep precursor TaPROPEP in transgenic 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 wheat scab or stem rot caused by Fusarium wilt. The amino acid sequence of the endogenous Pep precursor TaPROPEP is shown in SEQ ID No. 2 or 11.

4. The application of an endogenous TaPep mature polypeptide in enhancing plant disease resistance, wherein the plant is wheat; the disease resistance refers to wheat scab or stem rot caused by Fusarium wilt; The amino acid sequence of the endogenous TaPep mature polypeptide is shown in SEQ ID No. 3, 6, 9 or 12.

5. A method of enhancing disease resistance in a plant, comprising, Plants can be treated directly with endogenous mature TaPep peptides to increase their disease resistance. The plant in question is wheat; the disease resistance refers to wheat scab or stem rot caused by Fusarium wilt. The amino acid sequence of the endogenous TaPep mature polypeptide is shown in SEQ ID No. 3, 6, 9 or 12.

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