A small secreted peptide and its use in plant immune priming

Abstract

The application belongs to the technical field of plant immunity, and particularly relates to a small secretion peptide and application thereof in plant immunity stimulation. A small secretion peptide SlSolP12 (SEQ ID NO. 1) is first discovered in solanaceae plants, and when the small secretion peptide is used alone or in combination with other small secretion peptides, the small secretion peptide can activate the immune response of the plant, thereby triggering the systemic resistance of the plant to pathogens. Therefore, the SlSolP12 provided by the application can be used as a widely effective plant immunity stimulant, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant immunity, and particularly relates to a small secreted peptide and application thereof in plant immune stimulation. BACKGROUND

[0002] Small secreted peptides (SSPs) refer to a class of short-chain polypeptide molecules synthesized by organisms and secreted to the extracellular by a classical signal peptide mechanism. Such polypeptides are usually composed of less than 250 amino acid residues, have a relatively small molecular weight, and have various biological functions. Plant genomes encode thousands of small secreted peptides. As short- and long-distance signaling molecules, plant small secreted peptides are usually perceived by resident receptor kinases on the cell surface, and regulate plant development, reproduction, immunity, and environmental adaptation.

[0003] Studies have shown that in order to cope with the continuous invasion of pathogens, plants have developed a complex perception and defense system. In this system, plant small secreted peptides play a crucial role in development and cell communication (including ligand-receptor interaction) through direct or indirect interaction with pathogens. In the past few decades, a large number of SSPs have been identified in various plants. In Arabidopsis, small plant cell factors (SCREWs) that regulate defense and water loss have been shown to be involved in responding to abscisic acid (ABA) and microbe-associated molecular pattern (MAMP) signals to regulate stomatal closure; sulfonated peptide phytosulfochrome (PSK) and plant peptide containing tyrosine sulfonation 1 (PSY1) were initially identified as promoters of cell proliferation and tissue growth, and were recently shown to weaken the PAMP-induced immune (PTI) response and enhance susceptibility to biotrophic and resistance to necrotrophic pathogens; PAMP-induced peptide 1 (PIP1) enhances the activation of PTI response through receptor-like kinase 7 (RLK7) to enhance plant immunity. Similarly, the serine-rich endogenous peptide (SCOOP12) enhances plant immunity by utilizing the MIK2 receptor. In rice, immune response peptide (IRP) enhances the expression of defense gene phenylalanine ammonia lyase 1 (PAL1) and is involved in regulating the immune process; drought tolerance 11 peptide (OsDT11) enhances plant drought resistance in an ABA signaling pathway-dependent manner; five-amino acid peptide 1 (PEP1) regulates rice root development.

[0004] Solanaceae is one of the most important plant families in horticulture. It includes many important crops and ornamental plants such as tobacco, tomato, pepper, and eggplant. These plants play an important role in the food industry and horticulture, and have a huge impact on human life and economy. Small secreted peptides play a crucial role in protecting Solanaceae plants from pathogen invasion and are a key factor for their survival and reproduction. Therefore, it is essential to understand how Solanaceae plants adapt and survive under adverse environmental conditions through small secreted peptides. The first small secreted peptide found in tomato is systemin (Sys), which mediates long-distance immune responses through the receptor RECEPTOR1 (SYR1) (Pearce et al., 1991, Science, 253, 895-897.). Interestingly, Sys appears to be specific to Solanaceae, and has only been identified in certain species of the family, such as eggplant and pepper. While the initial discovery of small peptides originated from Solanaceae plants, subsequent exploration and functional studies of SSPs have mainly focused on model plants such as Arabidopsis and rice. Therefore, research specifically targeting Solanaceae plants remains relatively limited, with only a few peptides, such as Sys, CAPE, and NbPPI1, being first identified and extensively studied within the Solanaceae family. With advancements in genomics and sequencing assembly technologies, significant progress has been made in Solanaceae genomics research in recent years. Chromosome-level whole-genome sequencing has been performed and deciphered for several species, including tomato, pepper, and tobacco, providing great convenience for the study of small secreted peptides in Solanaceae. Therefore, Solanaceae plants have great potential for discovering new small peptides.

[0005] In summary, discovering more new small secreted peptides has high value in fields such as the food industry and horticulture, and therefore it remains an important topic in the field. SUMMARY

[0006] To address the problems of the prior art, the present application provides a small secreted peptide and its use in plant immune stimulation.

[0007] A small secreted peptide is a polypeptide A having at least 90% sequence similarity to the amino acid sequence set forth in SEQ ID NO. 1.

[0008] Preferably, the amino acid sequence of the polypeptide A is set forth in SEQ ID NO. 1.

[0009] The present application also provides a small secreted peptide that is a peptide capable of activating the receptor of the above-mentioned small secreted peptide (polypeptide A).

[0010] The present application also provides the use of the above-mentioned small secreted peptide as a plant immune stimulant.

[0011] Preferably, the plant immune stimulant is used to trigger the basal immune response of the plant.

[0012] The present application also provides a plant immune elicitor, which is prepared by using the above-mentioned small secreted peptide as an active ingredient and adding an acceptable agricultural adjuvant.

[0013] The present application also provides a use of the polypeptide B in combination with the above-mentioned small secreted peptide as a plant immune elicitor, wherein the polypeptide B has an amino acid sequence with at least 90% sequence similarity to the amino acid sequence described in SEQ ID NO. 2 or SEQ ID NO. 3.

[0014] Preferably, the polypeptide B has an amino acid sequence as described in SEQ ID NO. 2 or SEQ ID NO. 3.

[0015] Preferably, the above-mentioned small secreted peptide and the polypeptide B are used simultaneously or sequentially.

[0016] Preferably, the method for using the plant immune elicitor is to use the above-mentioned small secreted peptide first, and then use the polypeptide B after 1 hour.

[0017] The present application also provides a plant immune elicitor, which is prepared by using the polypeptide B and the above-mentioned small secreted peptide as active ingredients, wherein the polypeptide B has an amino acid sequence with at least 90% sequence similarity to the amino acid sequence described in SEQ ID NO. 2 or SEQ ID NO. 3.

[0018] Preferably, the polypeptide B has an amino acid sequence as described in SEQ ID NO. 2 or SEQ ID NO. 3.

[0019] The present application also provides a method for constructing a disease-resistant or pest-resistant crop, which comprises inserting a gene for expressing the above-mentioned small secreted peptide into the genome of the crop.

[0020] In the present application, the "sequence similarity" is an index to measure the degree of similarity in structure and evolution of two biological sequences (such as protein or nucleic acid sequence). It is not only based on the number of completely identical amino acid residues or nucleotides in two sequences, but also considers the possibility of partial matching, that is, different but biologically conservative substitution relationship of residues can also contribute to a certain similarity score. For protein (or polypeptide) sequence, scoring matrix (such as PAM or BLOSUM matrix) is usually used to calculate similarity, which assigns a score to each pair of possible amino acid substitutions, reflecting their probability of replacement in the evolution process and the expected impact on function. By aligning two sequences and accumulating scores, a global or local similarity value can be obtained. A large number of previous studies have shown that small peptides with homologous sequences and activating the same receptor have the same or similar biological functions. For example, AtPIP1 (RLASGPSPRGRGH) and AtPIP2 (VKHSGPSPSGPGH) of PIP family in Arabidopsis thaliana both activate plant immune function through receptor RLK7 (Hou et al., 2014, PLoS Pathogens, 183, 399-413.). In addition, Arabidopsis CLE40 (RQVPTGSDPLHH) and CLV3 (RTVPSGPDPLHH) as homologous sequences have similar functions, which promote stem cell differentiation by binding to receptor kinase ACR4 ARABIDOPSIS CRINKLY 4) / CLV1 (Stahl et al., 2013, Current Biology, 23, 362-371; Stahl et al., 2009, Current Biology, 19, 909-914); In addition, rice FON4 (FLORAL ORGAN NUMBER 4) is a homologous gene of Arabidopsis CLV3, which can regulate rice meristem development and affect its yield traits (Ren et al., 2019, Plant Biotechnology Journal, 17, 1007-1009). Similarly, CLV3 homologous sequences in corn, tomato and other plants also play an important role in yield trait regulation (Liu et al., 2021, Nature Plants, 7, 287-294; Wang et al., 2021, Nature Plants, 7, 419-427). Therefore, sequences of the same family or activating the same receptor across species should be considered as equivalent sequences and should be within the protection scope of the present patent.

[0021] In the present application, the "polypeptide A" or "polypeptide B" refers to a polypeptide, wherein "A" or "B" is used to number different kinds of polypeptides, and the purpose is to distinguish different kinds of polypeptides, and has no limiting effect on the technical features (such as amino acid sequence and spatial structure, etc.) of the polypeptide.

[0022] The present application discovers a new small secreted peptide SlSolP12 in Solanaceae plants, which can trigger basal immune responses such as ROS burst, callose deposition, stomatal closure, MAPK activation, and up-regulation of defense genes PR1 and NPR1. Exogenous application of the same has the potential to confer broad-spectrum disease resistance to crops. Therefore, the small secreted peptide and its variants provided by the present application have application prospects as plant immune elicitors, and in addition, they also have the potential to be used for the development of genetically engineered or biological pesticides, and to cultivate crops with improved broad-spectrum and systemic properties of disease resistance or insect resistance.

[0023] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, substitutions or changes can be made without departing from the above basic technical idea of the present application.

[0024] The above content of the present application will be further described in detail through the specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present application to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Analysis of SSPs in Solanaceae plants (tomato, tobacco and pepper). Among them, (A) is a schematic diagram of the identification process of SSPs in Solanaceae plants; (B) is the distribution of SSP content in Solanaceae plants (tomato, tobacco and pepper); (C) is the proportion of SSPs in the proteome of Solanaceae plants (tomato, tobacco and pepper); (D) is a comparison of known and unknown SSPs in Solanaceae plants (tomato, tobacco and pepper); (E) is an evolutionary tree of unknown SSPs in Solanaceae plants (tomato, tobacco and pepper).

[0026] Figure 2 Sequence alignment of SolP family members in tomato, tobacco and pepper.

[0027] Figure 3Tomato plants were immunized by SlSolP12. Among them, (A) SlSolP12 induced ROS burst. Leaves of 6-week-old soil-borne plants were treated with 10 μM SlSolP12 or without 10 μM SlSolP12, and the relative light units (RLU) of ROS were measured within 60 minutes using a luminometer; error bars represent the SE of the mean (n > 3); (B) MAPK activation by SolP in tomato leaves. 6-week-old soil-borne plants were exposed to 1 μM polypeptides for 15 minutes; Western blotting analysis was performed using phospho-p44 / 42 MAPK antibodies; (C) Fluorescence microscope imaging and (D) quantification of stomatal closure induced by SlSolP12; (E) Fluorescence microscope imaging and (F) quantification of callose deposition in tomato leaves induced by SlSolP12. Error bars represent the SE of the mean (n > 5); (G) Gray mold symptoms in SlSolP12-treated tomato leaves; (H) Measurement of lesion area in tomato leaves 6 days after inoculation.

[0028] Figure 4 SlSolP12 interfered with flg22 and Sys. Among them, (A) ROS burst in tomato after co-treatment with 1 μM flg22 and SlSolP12; (B) 1 μM flg22 pretreatment for 1 hour before 1 μM SlSolP12 elicitation; (C) 1 μM SlSolP12 pretreatment for 1 hour before 1 μM flg22 elicitation; (D) Expression levels of SlSolP12 precursor genes after flg22 pretreatment for 1 hour and 6 hours; (E) ROS burst in WT after co-elicitation with 1 μM Sys and SlSolP12; (F) 1 μM Sys pretreatment for 1 hour before 1 μM SlSolP12 elicitation; (G) 1 μM SlSolP12 pretreatment for 1 hour before 1 μM Sys elicitation; (H) Expression levels of SlSolP12 precursor genes after Sys pretreatment for 1 hour and 6 hours; (I) Expression levels of Sys precursor genes after SlSolP12 pretreatment for 1 hour and 6 hours.

[0029] Figure 5 Expression of SlSolP12 gene and its regulation on defense genes.

[0030] Figure 6 SlSolP12 induced systemic resistance in tomato leaves. Among them, (A) SlSolP12 treatment induced both local and systemic resistance in tomato leaves; (B) Measurement of lesion area in tomato leaves 6 days after inoculation. Error bars represent the SE of the mean (n > 3). Differences were statistically significant (p < 0.01) and were indicated by different letters; (C) Imaging and (D) quantification of superoxide anion in tomato leaves induced by SlSolP12.

[0031] Figure 7 Activity oxygen burst caused by SlSolP12 in different species. Among them, (A) activity oxygen burst caused by SlSolP12 in pepper; (B) activity oxygen burst caused by SlSolP12 in tobacco; (C) activity oxygen burst caused by SlSolP12 in Arabidopsis; (D) activity oxygen burst caused by SlSolP12 in rice; (E) activity oxygen burst caused by SlSolP12 in soybean. DETAILED DESCRIPTION

[0032] In the following examples and experimental examples, the reagents and raw materials not specifically explained are commercially available.

[0033] Example 1 Discovery of a novel small secreted peptide SlSolP12 and its use

[0034] I. Experimental methods and materials

[0035] 1. Plant materials

[0036] Tomato was grown in a mixture of perlite and vermiculite 1:1, and the growth chamber conditions were: 25°C, light intensity 400 μmol m -2 s -1 , 16 hours light / 8 hours dark cycle, relative humidity 80%.

[0037] 2. Synthetic peptides

[0038] All polypeptides with 95% purity were synthesized by GenScript Company (Nanjing, China), including:

[0039] SlSolP12 (SEQ ID NO. 1: VTSNALALVNRFAD),

[0040] Flg22 (SEQ ID NO. 2: QRLSTGSRINSAKDDAAGLQIA),

[0041] Sys (SEQ ID NO. 3: AVQSKPPSKRDPPKMQTD).

[0042] The "polypeptide" described in the following experimental methods is any one of the above three polypeptides.

[0043] 3. Activity oxygen burst

[0044] The activity oxygen in plant leaves was quantified by a luminol-based analysis method. Specifically, plant leaf segments of about 3 mm cut in a 96-well plate were incubated in 30 mL water for 12 hours, and then 99 μL of reaction solution (200 μM luminol, 2 μg mL -1peroxidase) and 1 μΐ, of 1 μΜ polypeptide. Measurements were taken every minute for a total of 60 minutes per well.

[0045] 4. Callose deposition

[0046] The method for callose deposition detection was slightly modified according to the literature (Clay et al., 2009, Science, 323, 95-101). Specifically, plant leaves were injected with 0.3 mL of 1 μΜ polypeptide (water as control) and treated overnight (approximately 12 hours). Leaves were fixed in FAA solution (10% formaldehyde, 5% acetic acid, 50% ethanol) for 24 hours. Leaves were then transferred to absolute ethanol for decolorization for 6 hours, followed by incubation in 50% ethanol for 30 minutes. Ethanol was removed, 67 mM K2HP04(pH 12) was added and incubated for 30 minutes. The K2HP04solution was removed, and staining solution (0.01% aniline blue in 67 mM K2HP04, pH 12) was added and incubated for 1 hour. Callose deposition was observed by epifluorescence microscopy and photographed by light microscope Olympus BX-53 (Olympus, Tokyo, Japan).

[0047] 5. MAPK experiment

[0048] Leaves of 6-week-old soil-grown plants were immersed in sterile water overnight and then induced with 1 μΜ polypeptide for 15 minutes. Next, seedlings were quickly frozen in liquid nitrogen and ground into fine powder, and total proteins were extracted therefrom using protein extraction solution (Tris-HCl (pH 6.8), 25% (V / V) glycerol, 2% (V / V) SDS, 0.001% (W / V) bromophenol blue, 5% (V / V) mercaptoethanol). Active MPK6 and MPK3 were detected by immunoblotting using anti-phospho p44 / p42 MAPK antibody (Huabio, China). Ponceau staining was used to compare whether the total protein amount loaded during electrophoresis reached a consistent level for different samples.

[0049] 6. Stomata observation

[0050] Leaves of 6-week-old soil-grown plants were treated with or without 5 mL of 1 μΜ polypeptide for 4 hours, and then epidermis was peeled off. Leaf stomata were observed by light microscope Olympus BX-53 (Olympus, Tokyo, Japan).

[0051] 7. Quantitative RT-PCR analysis

[0052] Total RNA was extracted from plant tissues by FastPure Plant Total RNA Isolation Kit (Vazyme, Nanjing, China) according to the manufacturer’s method. Reverse transcription was performed on a total of 1 pg total RNA preparation using HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The resulting cDNA was amplified using Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) and gene-specific primers (as shown in Table 1). SlActin was used as a reference sequence.

[0053] Table 1 Sequence of specific primers

[0054]

[0055]

[0056] 8. Fungal infection experiment

[0057] Tomato leaves were taken from the middle-aged leaves at the end of the vegetative and reproductive stages. The leaves were placed on wet filter paper in a 9 cm diameter Petri dish to prevent drying. Subsequently, the leaves were inoculated with 10 6 spores / ml of Botrytis cinerea spore suspension. Photos were taken after 6 days.

[0058] In the above method, there were three treatments each time, and three plants for each treatment.

[0059] 9. Bioinformatics analysis

[0060] Signal peptide prediction was performed by Signal 6.0. Transmembrane domains were predicted using DeepTMHMM. Phylogenetic analysis of SSPs protein sequences was performed by MAFFT and phylogenetic analysis of SolP sequences was performed by MUSCLE. The phylogenetic tree file was visualized by iTOL 5. Sequence alignment was performed by Jalview2.11.1.0. Amino acid conservation analysis was performed by Weblogo.

[0061] 11. Statistical analysis

[0062] Statistical analysis of the data obtained was performed using the ANOVA procedure using IBM SPSS Statistics version 26 to test the significant effect of all the study variables. Duncan’s multiple range test (DMRT) was used as a significant test at p<0.05 to separate the means.

[0063] II. Experimental results

[0064] 1. Identification of SSPs in Solanaceae

[0065] To identify small peptides in Solanaceae, the experimental example uses protein sequences obtained from Solanaceae Genomics Network (solgenomics.net SGN) and uses the pipeline shown in Figure A to construct a library of small proteins (ranging from 25 to 250 amino acids). SignalP 6.0 predicts small proteins with N-terminal signal sequences of the secretory pathway. Proteins lacking transmembrane domains predicted by DeepTMHMM and potential endoplasmic reticulum lumen retention sequences at the C-terminus. The study analyzes identified a total of 1056 small secreted peptides in pepper, 1555 in tobacco, and 721 in tomato (B), accounting for 2.07-3.04% (C) of the respective genomes. Plant SSPs are generally classified into different families according to their conserved peptide domains at the C-terminus. To identify members of known plant SSP families in Solanaceae, the sequence of potential SSPs was searched against the Medicago truncatula small secreted peptide database (Boschiero et al., 2020, Plant Physiology, 183, 399-413.). The results show that 407 SSPs in tomato, 557 SSPs in tobacco, and 295 SSPs in pepper belong to known SSP families, accounting for 38.54%, 35.82%, and 40.92% of the total number of SSP families in these species, respectively. These identified members belong to 43 different SSP families (D), respectively. Unknown SSPs were clustered and multiple aligned, using MAFFT to construct a phylogenetic tree to find new SSP families (E). Figure 1 A. Constructing a library of small proteins (ranging from 25 to 250 amino acids) using the pipeline shown in Figure A. SignalP 6.0 predicts small proteins with N-terminal signal sequences of the secretory pathway. Proteins lacking transmembrane domains predicted by DeepTMHMM and potential endoplasmic reticulum lumen retention sequences at the C-terminus. Figure 1 B. Total number of small secreted peptides identified in pepper, tobacco, and tomato. Figure 1 C. Percentage of small secreted peptides in the respective genomes. Figure 1 D. Number of identified members of known plant SSP families in Solanaceae. Figure 1 E. Phylogenetic tree of unknown SSPs in Solanaceae.

[0066] 2. Identification of SolP family and activation of plant immune response

[0067] After manual screening, it was found that there was a cluster of sequences in the unknown SSP group, whose C-terminal sequence was very conservative, named Solanaceae Peptide (SolP). There are a total of 45 SolP family members in tomato, tobacco, and pepper ( Figure 2 ).

[0068] An amazing discovery was found in the SolP family of the three plant species. It was found that these species share a common peptide sequence, SlSolP12, NtSolP15, or CaSolP1 ( Figure 2). Therefore, in the subsequent experiments, this small peptide was selected for functional studies. It is well known that PTI plays a crucial role in preventing the entry of various pathogens into cells. This is achieved by initiating active oxygen burst, callose deposition, stomatal aperture regulation, and mitogen-activated protein kinase (MAPK) activation. Among them, active oxygen burst refers to the massive production of active oxygen in plants under the stimulation of small peptides, which in turn activates a series of signaling pathways to resist the invasion of pathogenic bacteria; MAPK activation refers to the phosphorylation and activation of MAPK kinase in plants under the stimulation of small peptides, which in turn resists pathogenic bacteria. In tomato plants, SlSolP12 can induce the massive production of ROS, while also activating MAPK Figure 3 A and 3B). In addition, SlSolP12 is also involved in callose deposition and stomatal closure in tomato plants ( Figure 3 C to 3F). Among them, callose is a glucan with β-1, 3 bonds, and its increased content can resist the invasion of pathogenic bacteria; similarly, the closure of stomata also resists the invasion of pathogenic bacteria. Pre-treatment of tomato plants with SlSolP12 reduces the susceptibility of Botrytis cinerea pathogen infection ( Figure 3 G and 3H).

[0069] From the above experimental results, it can be seen that SlSolP12 is a small secreted peptide that induces PTI response.

[0070] 3. SlSolP12 can be mixed with other small peptides to enhance its immune effect

[0071] In order to further enhance the immune function effect of SlSolP12 small peptide, we selected the classic small peptides Flg22 and Sys, and then explored the effect of their mixed use. In this experiment, SlSolP12 and Flg22 were applied alone as the control group, and the two were mixed and used as the experimental group. The results showed that the active oxygen content produced by the combination of the two was between the two ( Figure 4 A), that is, there was no mutual enhancement or antagonistic effect between the two. However, pre-treatment with Flg22 for 1 hour before applying SlSolP12 reduced the active oxygen burst of SlSolP12 ( Figure 4 B). On the contrary, pre-treatment with SlSolP12 for 1 hour before applying Flg22 reduced the active oxygen burst of Flg22 ( Figure 4 C). In addition, this study further explained the possible reasons for the expression level of SlSolP12 precursor. Pre-treatment with Flg22 caused the expression level of SlSolP12 precursor gene to decrease ( Figure 4 D). Similarly, in this experiment, SlSolP12 and Sys were applied alone as the control group, and the two were mixed and used as the experimental group. The results showed that the active oxygen content produced by the combination of the two was higher than that of the two alone (Figure 4 E), indicating that SlSolP12 and Sys have a synergistic effect, and the combination of the two can enhance the immune effect. However, the active oxygen burst of SlSolP12 was weakened after pre-treatment with Sys for 1 hour ( Figure 4 F), and on the contrary, the active oxygen burst of Sys was enhanced after pre-treatment with SlSolP12 for 1 hour ( Figure 4 G). Subsequently, the present study further explained the possible reasons through the expression levels of SlSolP12 and Sys precursors. The experimental results showed that SlSolP12 treatment increased the expression level of Sys precursor ( Figure 4 H), while Sys treatment led to a decrease in the expression level of SlSolP12 precursor ( Figure 5 I).

[0072] In summary, there is an interaction between SlSolP12 and Flg22 and Sys, and in actual application or production, they can be compounded or used together according to their effects. The preferred use is: (1) SlSolP12 pretreatment of crops for 1 hour before applying Flg22 or Sys; (2) mixing SlSolP12 and Sys and applying them to crops at the same time.

[0073] 4. SlSolP12 participates in the SA signaling pathway and defense gene expression

[0074] To determine the immune pathway activated by SlSolP12 and identify the responsive genes, several classical disease resistance genes were selected for analysis using qRT-PCR. The results showed that the exogenous application of SlSolP12 significantly increased the expression of marker genes PR1 and NPR1 in the salicylic acid signaling pathway, as well as the expression of classical PTI reporter genes GRAS, FRK1, and PTI4 ( Figure 6 ). Therefore, SlSolP12 is very likely to induce plant immune response through the salicylic acid signaling pathway.

[0075] 5. SlSolP12 induces systemic and broad-spectrum resistance in plants

[0076] Further experiments were conducted to verify whether the application of SlSolP enhanced the systemic resistance of tomato plants to pathogens (whole plant resistance). In this study, plants not sprayed with SlSolP12 were used as the control group, and one side of the plants was sprayed with SlSolP12 (local application of SlSolP12), and the sprayed side (Local group) and the other side (System group) were detected as the experimental group. It is worth noting that according to the size of the lesion ( Figure 6A and 6B), it was found that the local and systemic tomato plants treated with SlSolP12 were more resistant to fungal pathogens (Botrytis cinerea) than the control plants. In addition, the results of superoxide anion (a reactive oxygen species) staining showed that SlSolP12 can activate the local and systemic PTI response Figure 7 C and 6D). These results indicate that exogenous application of SlSolP12 coordinates the local and systemic immune response in tomato plants. In addition, in order to study whether the application of SlSolP12 can effectively stimulate the immune response in different plant species. Pepper (Solanaceae), tobacco (Solanaceae), Arabidopsis (Brassicaceae), soybean (Leguminosae) and rice (Poaceae) were selected for reactive oxygen species burst experiments. The results showed that SlSolP12 treatment can also activate ROS burst in other plant species ​ A-E).

[0077] The above experimental results show that SlSolP12 is a widely effective plant immune elicitor.

[0078] As can be seen from the above examples, the present application first discovers a small secreted peptide SlSolP12 in Solanaceae plants, which can activate the immune response of plants when used alone, and can further enhance the effect of activating the immune response of plants when used in combination with other polypeptides (such as flg22 or Sys). Further experiments have verified that SlSolP12 can trigger systemic resistance of plants to pathogens, therefore, the SlSolP12 polypeptide provided by the present application can be used as a widely effective plant immune elicitor, which has good application prospect.

Claims

1. A small secretory peptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. Use of the small secretory peptide according to claim 1 for preparing a plant immune stimulant.

3. The use according to claim 2, characterized in that: The plant immune stimulant is used to trigger the basic immune response of the plant.

4. A plant immune stimulant, characterized in that: The invention is prepared by using the small secretory peptide as claimed in claim 1 as an active ingredient and adding auxiliary materials acceptable to pesticides.

5. Use of polypeptide B in combination with the small secretory peptide according to claim 1 for preparing a plant immune stimulant, wherein the amino acid sequence of polypeptide B is as shown in SEQ ID NO. 2 or SEQ ID NO.

3.

6. The use according to claim 5, characterized in that: The small secretory peptide of claim 1 and the polypeptide B are used simultaneously; or, the method of using the plant immune stimulant is: first use the small secretory peptide of claim 1, and then use the polypeptide B with the amino acid sequence as SEQ ID NO. 3 at least 1 hour later.

7. A plant immune stimulant, characterized in that: The invention is prepared by using polypeptide B and the small secretory peptide according to claim 1 as active ingredients. The amino acid sequence of polypeptide B is as described in SEQ ID NO. 2 or SEQ ID NO.

3.

8. A method for constructing disease-resistant or insect-resistant crops, characterized in that: The method comprises inserting a gene for expressing the small secretory peptide according to claim 1 into the genome of a crop.

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