Double-stranded rna molecules targeting the oxidative sterol binding protein 1 of phytophthora capsici and uses thereof

Abstract

The application discloses a double-stranded RNA molecule capable of efficiently inhibiting expression of an oxidized sterol binding protein 1 gene of Phytophthora capsici, and belongs to the field of agricultural biotechnology.The double-stranded RNA molecule is composed of nucleic acid sequences shown in SEQ ID No.1 and SEQ ID No.2.The application also discloses application of the double-stranded RNA molecule in preventing and treating plant diseases caused by the Phytophthora capsici.The double-stranded RNA molecule can inhibit the invasion of the Phytophthora capsici by directly treating the Phytophthora capsici or spraying the Phytophthora capsici on plants.Meanwhile, the double-stranded RNA molecule is specific to essential genes of the Phytophthora capsici, and does not have the problem of drug resistance, so that the double-stranded RNA molecule can be used for preparing biological pesticides, and provides an effective way for preventing and treating diseases caused by the Phytophthora capsici.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a double-stranded RNA molecule that can inhibit the expression of the oxidosterol-binding protein 1 gene of Phytophthora capsici, and the application of this molecule in the prevention and control of plant diseases caused by Phytophthora capsici. Background Technology

[0002] Phytophthora capsici is an important plant pathogenic oomycete distributed worldwide. It can infect more than 70 species of plants in the Solanaceae, Fabaceae, and most Cucurbitaceae families, causing damping-off, wilting, and root, stem, and fruit rot. The disease can occur from the seedling stage to the fruiting stage, causing severe economic losses. Phytophthora capsici-induced pepper blight is a devastating disease that can be spread through rainwater, soil, and air currents, causing symptoms such as leaf wilting, fruit rot, and even the death of the entire plant. It has a significant impact on crop yield and quality, and in severe cases, can lead to yield reductions of more than 50%.

[0003] Control of Phytophthora capsici mainly relies on chemical control. However, due to the limited variety of omphalosporins and the long-term use of chemical pesticides alone, Phytophthora capsici has developed resistance to many chemical pesticides. Furthermore, the increasing dosage of chemical pesticides has caused serious environmental pollution. In addition, plant varieties resistant to Phytophthora capsici are scarce, necessitating the development of new methods to control plant diseases caused by Phytophthora capsici.

[0004] RNA interference (RNAi) is a gene silencing phenomenon induced by double-stranded RNA, which can inhibit gene expression by blocking the transcription or translation of specific genes. RNAi is ubiquitous in organisms. The double-stranded or hairpin-structured RNA sequence formed by DNA transcription can be processed into small RNAs of 20-30 bp. These small RNAs mediate the gene silencing complex to specifically recognize the complementary target gene DNA or mRNA sequence, causing mRNA cleavage, translational repression, or DNA methylation, ultimately inhibiting normal gene expression (Fire, A. et al. 1998. Nature. 391(6669):806). RNAi technology has achieved significant results in plant disease control in recent years. By directly spraying double-stranded RNA molecules, key genes of pathogens can be interfered with, leading to a decrease in pathogen fitness and reduced pathogenicity, a phenomenon known as Spray-Induced Gene Silencing (SIGS).

[0005] SIGS (Synthetic Infection Sequences) have been a hot topic in RNAi (RNAi) in recent years for controlling crop diseases. They are generally achieved by directly interfering with the expression of important genes in pathogens using double-stranded RNA. Spraying double-stranded RNA targeting the CYP51 gene, essential for sterol synthesis in Fusarium graminearum, and the DCL and AGO genes, important RNAi elements, on barley leaves significantly reduced the pathogenicity of Fusarium graminearum on barley leaves (Koch, A. et al. 2016. PLoS pathogens. 12(10):e1005901; Werner, BT et al. 2020. Frontiers in plant science. 11:476). Using double-stranded RNA targeting different cotton leaf curl virus (CLCuV) genes on cotton significantly reduced the expression of these genes, successfully controlling the severity of CLCuV transmission by whiteflies (Verma, P. et al. 2018. Journal of entomology and zoology studies. 6(4):1055-1060). These studies demonstrate that cross-species gene silencing based on SIGS can be effectively used for plant disease control.

[0006] Oxidosterone-binding protein 1 (ORP1) is the target of the highly effective fungicide fluthiazopyrone. Amino acid mutations in PcORP1 can lead to high levels of resistance to the fungicide by *Phytophthora capsici*. A search revealed no literature on the control of plant diseases caused by *Phytophthora capsici* based on RNA silencing technology. Summary of the Invention

[0007] The purpose of this invention is to provide a double-stranded RNA molecule that can inhibit the expression of the oxidized sterol-binding protein 1 gene in Phytophthora capsici.

[0008] A second objective of this invention is to provide the use of the above-mentioned double-stranded RNA molecule in inhibiting the expression of the oxidizing sterol-binding protein 1 gene in Phytophthora capsici.

[0009] A third objective of this invention is to provide the use of the above-mentioned double-stranded RNA molecule in the prevention and control of plant diseases caused by Phytophthora capsici.

[0010] A fourth object of the present invention is to provide a pesticide containing the above-mentioned double-stranded RNA molecule.

[0011] The fifth object of the present invention is to provide the application of pesticides containing the above-mentioned double-stranded RNA molecules in the prevention and control of plant diseases caused by Phytophthora capsici.

[0012] The sixth object of the present invention is to provide a method for controlling plant diseases caused by Phytophthora capsici using the above-mentioned double-stranded RNA molecules.

[0013] The technical solution for implementing the present invention is as follows:

[0014] A double-stranded RNA molecule that can inhibit the expression of the oxidosterol-binding protein 1 gene from Phytophthora capsici, the sequence of one strand of which is shown in SEQ ID No. 1 and the sequence of the other strand of which is shown in SEQ ID No. 2.

[0015] The application of the above-mentioned double-stranded RNA molecules in inhibiting the expression of the oxidosterol-binding protein 1 gene in Phytophthora capsici.

[0016] Application of the above-mentioned double-stranded RNA molecules in the prevention and control of plant diseases caused by Phytophthora capsici.

[0017] Pesticides containing the aforementioned double-stranded RNA molecules.

[0018] Application of pesticides containing the above-mentioned double-stranded RNA molecules in the control of plant diseases caused by Phytophthora capsici.

[0019] Methods for controlling plant diseases caused by Phytophthora capsici using the above-mentioned double-stranded RNA molecules include directly spraying solutions or preparations containing the above-mentioned double-stranded RNA molecule carriers onto the surface of plant tissues or treating the soil (such as root irrigation).

[0020] The double-stranded RNA molecule involved in this invention can be produced via prokaryotic expression.

[0021] The method of using the double-stranded RNA molecule involved in this invention is as follows: spraying an aqueous solution of double-stranded RNA molecules of a suitable concentration (e.g., about 100 nM) onto the surface of plant leaves or performing root irrigation treatment on the plant rhizosphere soil can effectively inhibit the infection and spread of Phytophthora capsici in plant leaves or rhizomes.

[0022] The preferred plants are tobacco or chili peppers.

[0023] The advantages or beneficial effects of this invention are as follows: (1) The double-stranded RNA molecule involved in this invention has a significant inhibitory effect on the expression of oxidosterol-binding protein 1 of Phytophthora capsici, which can effectively inhibit the infection of plants by Phytophthora capsici and provide a new and effective way to control plant diseases caused by Phytophthora capsici. (2) The double-stranded RNA molecule involved in this invention has strong specificity for the oxidosterol-binding protein 1 gene of Phytophthora capsici, and theoretically can specifically inhibit a variety of plant pathogens including Phytophthora capsici, without the problem of drug resistance. (3) The target gene involved in this invention is also the target of fluoxetine or similar ultra-high-efficiency agents, and can be used to control the drug resistance of Phytophthora populations that have developed resistance to such agents due to amino acid mutations, or to delay the development of resistance to such high-risk drugs when used in combination with fungicides. (4) The double-stranded RNA molecule involved in this invention is safe for humans or animals, and can effectively reduce the amount of chemical pesticides used, without environmental pollution problems, which is beneficial to environmental protection. Attached Figure Description

[0024] Figure 1 This is a gel electrophoresis image of double-stranded RNA molecules according to the present invention. Caption: From left to right, the bands in the gel electrophoresis image represent the double-stranded RNA molecule dsORP3, the blank vector control L4440, the negative control double-stranded RNA molecule dsGFP, and the Mark.

[0025] Figure 2 Photographs showing the infection effect of *Phytophthora capsici* on detached leaves after the application of the double-stranded RNA molecule involved in this invention. A: Protective effect of double-stranded RNA on tobacco leaves; B: Protective effect of double-stranded RNA on pepper leaves. Figure 2 In section A: After spraying dsRNA crude extract onto detached tobacco leaves for 1 day, inoculate with *Phytophthora capsici* zoospores (10... 4 Tobacco leaves that developed disease 3 days after (from left to right: water control, blank vector control L4440, negative control double-stranded RNA molecule dsGFP and double-stranded RNA molecule dsORP3). Figure 2 In section B: After spraying detached pepper leaves with crude dsRNA extract for 1 day, inoculate with *Phytophthora capsici* zoospores (10... 4 Tobacco leaves that developed disease 3 days later (from left to right: water control, blank vector control L4440, and double-stranded RNA molecule dsORP3).

[0026] Figure 3 This is a bar graph showing the area of ​​lesions formed by inoculation with *Phytophthora capsici* after the application of the double-stranded RNA molecule of this invention to detached leaves. A shows the protective effect of double-stranded RNA on tobacco leaves; B shows the protective effect of double-stranded RNA on pepper leaves. Figure 3 In A: The diameter of tobacco lesions that developed after spraying crude dsRNA extract for 1 day and inoculating with Phytophthora capsici for 3 days (from left to right: water control, blank vector control L4440, negative control double-stranded RNA molecule dsGFP, and double-stranded RNA molecule dsORP3). Figure 3 B: Diameter of pepper lesions that developed after spraying crude dsRNA extract for 1 day and inoculating with Phytophthora capsulatum cake for 3 days (from left to right: water control, blank vector control L4440, and double-stranded RNA molecule dsORP3).

[0027] Figure 4 This invention relates to the efficacy of double-stranded RNA molecules applied to living plants against *Phytophthora capsici*. Caption: One day after root drenching pepper plants with crude dsRNA extract, 3 mL of *Phytophthora capsici* zoospore suspension (10...) was applied to the roots. 4 (number / mL), disease status of peppers after 7 days (from left to right: water control, blank vector control L4440, negative control double-stranded RNA molecule dsGFP and double-stranded RNA molecule dsORP3).

[0028] Figure 5This is a bar chart showing the disease index resulting from the application of the double-stranded RNA molecule involved in this invention to living plants followed by inoculation with Phytophthora capsici.

[0029] Caption: One day after root drenching pepper plants with crude dsRNA extract, 3 mL of Phytophthora capsici zoospore suspension (10) was applied to the roots. 4 (number / mL), the disease index of pepper plants after 7 days (from left to right: water control, blank vector control L4440, negative control double-stranded RNA molecule dsGFP and double-stranded RNA molecule dsORP3). Detailed Implementation

[0030] Unless otherwise specified, the reagents used in the following embodiments are all commercially available reagents, and the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0031] Example 1: Source of the double-stranded RNA molecule that inhibits the expression of the oxidized sterol protein 1 gene in Phytophthora capsici, as involved in this invention.

[0032] (1) The sequence of the double-stranded RNA molecule of the present invention is derived from the cDNA of PcORP1. The cDNA sequence of PcORP1 is derived from the gene numbered PHYCAscaffold_14:545241-548188 in the JGI database of Phytophthora capsici.

[0033] (2) The 556 bases from 924 to 2479 of the PcORP1 cDNA sequence were selected as the sequence of the double-stranded RNA molecule involved in this invention. Its sense strand sequence is shown in Sequence 1 of the sequence listing, and its antisense strand sequence is shown in Sequence 2 of the sequence listing.

[0034] (3) The above sequences were compared with the genome sequences (NCBI) of common hosts of Phytophthora capsici, such as tobacco, tomato, and pepper, to detect the possibility of non-specific silencing of plant genes. The comparison results showed that the maximum consecutive match between the sequences involved in this invention and the plant genome sequences was less than 10 bases, and the siRNA prediction by the potential generation of the sequences showed that the siRNAs generated by the above sequences could not reach the effective length for gene silencing, thus ruling out the possibility of silencing host plant genes.

[0035] Example 2: Preparation of the double-stranded RNA molecule of the present invention

[0036] (1) The double-stranded RNA expression plasmid L4440 contains two bidirectional T7 promoters, which can be transcribed into double-stranded RNA in RNase III-deficient Escherichia coli. The target fragment was amplified by PCR, and the amplification primers are shown in Table 1. Using the cDNA of the standard strain of Phytophthora capsici LT1534 (doi:10.1094 / MPMI-02-12-0028-R) as a template, the amplified fragment was inserted into the space between the two T7 promoters of the L4440 plasmid (between the Sac II and Xba I enzyme recognition sites) by enzyme digestion and ligation. After verification by PCR and sequencing, the plasmid capable of expressing double-stranded RNA molecules was obtained.

[0037] Table 1 Primer sequences for constructing double-stranded RNA molecules

[0038]

[0039] (2) The plasmid that can express double-stranded RNA molecules obtained in step (1) is transformed into RNase III-deficient Escherichia coli strain HT115 to induce the expression of dsRNA.

[0040] (3) Select single colonies of the double-stranded RNA molecular expression strain HT115 that has been verified by PCR and successfully sequenced, and incubate them overnight in LB liquid medium containing ampicillin and then store them in a -80℃ freezer.

[0041] (4) Streak the double-stranded RNA expression strain on LB plates with resistance markers, dip a single colony into 5 mL of liquid LB medium, and incubate overnight at 37°C and 200 rpm.

[0042] (5) Take 500 μL of the shaken bacterial culture and add it to 10 mL of LB liquid medium. Shake at 37℃ and 230 rpm for about 2.5 h, until the bacterial culture OD reaches 0.05. 600 Between 0.5 and 0.8.

[0043] (6) Add IPTG to the bacterial culture to a final concentration of 2mM and incubate at 37℃ and 230rpm for 8 hours.

[0044] (7) Take 2 mL from the cultured bacterial solution after shaking and transfer it to an enzyme-free centrifuge tube. Extract total RNA using the Triozol method.

[0045] (8) The extracted RNA was subjected to agarose gel electrophoresis, and the size of the double-stranded RNA bands was determined based on the electrophoresis results to determine whether they met expectations. Figure 1 As shown, the double-stranded RNA molecules prepared in this invention exhibit clear electrophoretic bands of the expected size. Sequencing results indicate that the sense strand sequence of the obtained double-stranded RNA molecules is shown in Sequence 1 of the sequence listing, and the antisense strand sequence is shown in Sequence 2 of the sequence listing.

[0046] Example 3: Evaluation of the resistance of plants treated with the double-stranded RNA molecule involved in this invention to Phytophthora capsici.

[0047] This patent utilizes two methods to clarify the effects of double-stranded RNA on the growth, development, and pathogenicity of *Phytophthora capsici*: a) double-stranded RNA molecules are directly sprayed onto the surface of host plants (tobacco and pepper detached leaves), followed by inoculation with *Phytophthora capsici* zoospores 1 day later, and leaf lesion diameter is observed and measured 3 days later; b) double-stranded RNA molecules are first applied to the roots of plants, followed by zoospore inoculation of pepper seedlings in a simulated field environment, and the disease index of the pepper seedlings is measured.

[0048] The preparation of zoospores of *Phytophthora capsici* strain BYA5 (isolated from pepper plants infected with blight in Baiyun, Guangdong Province, by the Fungicide Pharmacology and Pathogen Resistance Laboratory of China Agricultural University, and confirmed as *Phytophthora capsici* by morphological and molecular biological identification) was as described in Example 3. The zoospore concentration was adjusted to 10 after counting with a hemocytometer. 5 Units / mL are available for use.

[0049] Crude extraction of double-stranded RNA: Following the induction conditions described in Example 1, *E. coli* prokaryotic cells were induced to express the target dsRNA in a 100 mL system. After induction, the bacterial culture was centrifuged at 4000 rpm for 30 min at 4°C to collect the bacterial sediment. The sediment was resuspended in 50 mL of TE buffer, and the OD value of the resuspended bacterial culture was determined. 600 Adjust to around 1.5. Use a high-pressure cell disruptor to disrupt the bacterial suspension at a pressure of 0.5 kPa for 2.5 minutes. The disrupted bacterial suspension should be clear, transparent, and highly fluid.

[0050] a. Double-stranded RNA molecules were directly sprayed onto the surface of host plants (exposed leaves of Nicotiana sambac and pepper) and inoculated with Phytophthora capsici zoospores 1 day later. The diameter of leaf lesions was observed and measured 3 days later.

[0051] Leaves of 6-8 weeks old *Nicotiana benthamiana* with similar growth stages were collected. Using a 20 mL autoclaved spray bottle, 5 mL of crude double-stranded RNA (dsRNA) extract was sprayed onto both the upper and lower surfaces of isolated *Nicotiana benthamiana* and pepper leaves. After the leaves were allowed to air dry, they were placed in humidified culture dishes. Ten leaves were treated with each crude dsRNA extract. One day after dsRNA spraying, the leaves were inoculated with 10 μL of *Phytophthora capsici* zoospore suspension (10 μL / 1000 μL). 4 The lesion diameter was measured after 3 days using a cross-sectional method (number of lesions per mL) on the underside of the leaf and placed in a humidified culture dish. The activity of the average double-stranded RNA against *Phytophthora capsici* was determined based on the average diameter of the lesions on the detached leaf.

[0052] like Figure 2 As shown, compared with the control, the lesions formed by *Phytophthora capsici* infection on leaves sprayed with double-stranded RNA molecules targeting the *Phytophthora capsici* oxidosterol-binding protein 1 gene were significantly reduced.

[0053] Statistical results are shown below Figure 3 The double-stranded RNA of this invention significantly reduced the infectivity of Phytophthora capsici after treatment of the leaves, indicating that the double-stranded RNA molecule of this invention has a significant inhibitory effect on the growth and pathogenicity of Phytophthora capsici.

[0054] b. First, the plant roots were irrigated with double-stranded RNA molecules, and then the chili seedlings were inoculated with zoospores in a simulated field environment to determine the disease index of the chili seedlings.

[0055] Rhizosphere soil was drenched in tobacco plants of similar growth stages (6-8 weeks old), with approximately 3 mL of crude double-stranded RNA extract per plant. One day later, 3 mL of Phytophthora capsici zoospore suspension (10) was applied to the rhizosphere soil. 4 (number of plants / mL). Disease index was assessed 7 days post-inoculation, and the disease index grading criteria were referenced in Table 2. Approximately 10 biological replicates were used for each treatment, and the average disease index was calculated.

[0056] Table 2 Grading Criteria for Phytophthora capsici disease

[0057]

[0058]

[0059] See results Figure 4 As shown, compared with control plants sprayed with sterile water or E. coli bacterial sediment lysate, plants sprayed with the double-stranded RNA molecules of this invention were significantly healthier, showing no obvious constriction, necrosis, or lodging at the stem base. Statistical results are shown in […]. Figure 5 The average disease index of plants sprayed with the double-stranded RNA molecules of the present invention was significantly lower than that of control plants, indicating that spraying with the double-stranded RNA molecules of the present invention significantly inhibited the infection of Phytophthora capsici on the base of the plant stem.

[0060] Example 4: Determination of the efficacy of plants treated with the double-stranded RNA molecule involved in this invention against Phytophthora capsici.

[0061] In a greenhouse, pepper seedlings were planted in 80-cell trays. After 1.5 months of growth, 3 mL of crude double-stranded RNA molecule extract obtained through induction was directly sprayed onto the rootstock of the plants. One day later, inoculation was performed by drenching the roots with a suspension of Phytophthora capsici zoospores, using a pipette to extract a concentration of 10... 4 Three mL of a spore suspension of 1 spore / mL was directly applied to the soil near the base of the pepper plants. Ten days after inoculation, the disease incidence was assessed, and the disease severity levels (0, 1, 2, 3, 4, 5) were recorded according to Table 3. The disease index and the control efficiency of the crude dsRNA extract were then calculated.

[0062] Prevention and control efficiency = (Disease index of control group - Disease index of treatment group) / Disease index of control group × 100%

[0063] As shown in Table 3, spraying the double-stranded RNA molecule of the present invention showed a control efficacy of 46.57% against Phytophthora capsici. The above results indicate that the double-stranded RNA molecule of the present invention can be used as a green and safe new pesticide for the prevention and control of plant diseases caused by Phytophthora capsici.

[0064] Table 3. Statistical analysis of the greenhouse efficacy of the double-stranded RNA molecule involved in this invention against pepper blight.

[0065] sequence list <110> China Agricultural University <120> Double-stranded RNA molecules targeting and silencing capsaicin oxidosterol-binding protein 1 and their applications <130> WHOI210026 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 556 <212> RNA <213> Artificial sequence <400> 1 ucgucuucac uccgcaauca cgucguguuu cggccuauug gcgcggugaa ugguguuuua 60 cgcagcauug ggacagauuc gagcucuggg aagcaguacg cacgggcuag uguggugcuc 120 ccggugucgu cagaaguagc ggcgauucua cuggcugacc augcgcgcag agcugaaugg 180 gacgugcauu uuccucaauc ggcacauguu gcuacauucg augacgcgac ggaucucgug 240 cauuugucaa guggaagcuu ugcccagauc caacagacca agccaauugu ggcuccucau 300 guggcugcug cugcgugugc cuugugugca gcacucuucu cuggugcuuc aucuugggag 360 gcgcugcuga uugcgaugau cuacgcugca gccguaggug gcaucgugag uagcaucgac 420 uauagcacuu ugaccgcacc ucgcgaccuc guaguucuuc gucaugugcg ugagucugcu 480 acgccugauu cacaggauuc aagcgacgac aagucagcgg acgagauggg ccagucagug 540 gugcuuaucc uggaga 556 <210> 2 <211> 556 <212> RNA <213> Artificial sequence <400> 2 ucuccaggau aagcaccacu gacuggccca ucucguccgc ugacuugucg ucgcuugaau 60 ccugugaauc aggcguagca gacucacgca caugacgaag aacuacgagg ucgcgaggug 120 cggucaaagu gcuauagucg augcuacuca cgaugccacc uacggcugca gcguagauca 180 ucgcaaucag cagcgccucc caagaugaag caccagagaa gagugcugca cacaaggcac 240 acgcagcagc agccacauga ggagccacaa uuggcuuggu cuguuggauc ugggcaaagc 300 uuccacuuga caaaugcacg agauccgucg cgucaucgaa uguagcaaca ugugccgauu 360 gaggaaaaug cacgucccau ucagcucugc gcgcaugguc agccaguaga aucgccgcua 420 cuucugacga caccgggagc accacacuag cccgugcgua cugcuuccca gagcucgaau 480 cugucccaau gcugcguaaa acaccauuca ccgcgccaau aggccgaaac acgacgugau 540 ugcggaguga agacga 556

Claims

1. A double-stranded RNA molecule capable of targeting and silencing the *Phytophthora capsici* oxidosterol-binding protein 1 gene, characterized in that, The sequence of one of its chains is shown in SEQ ID No. 1, and the sequence of the other chain is shown in SEQ ID No.

2.

2. The application of the double-stranded RNA molecule of claim 1 in interfering with the expression of the oxidizing sterol-binding protein 1 gene in Phytophthora capsici.

3. The application of the double-stranded RNA molecule according to claim 1 in the prevention and control of plant diseases caused by Phytophthora capsici.

4. A pesticide containing the double-stranded RNA molecule of claim 1.

5. The application of the pesticide according to claim 4 in the prevention and control of plant diseases caused by Phytophthora capsici.

6. A method for controlling plant diseases caused by Phytophthora capsici using the double-stranded RNA molecule as described in claim 1, characterized in that, The solution or preparation containing the double-stranded RNA molecule as described in claim 1 can be directly sprayed onto the surface of plant tissues or used for soil treatment.

Images