Application of RabE1a protein and coding gene thereof in resisting tobacco mosaic virus and potyvirus

Abstract

The invention discloses application of RabE1a protein and a coding gene thereof in resisting tobacco mosaic virus and potato Y virus, and belongs to the technical field of biology. The research finds that the RabE1a protein can regulate and control the resistance of the plant to the tobacco mosaic virus and the potato Y virus for the first time, and the coding gene of the RabE1a protein is knocked out or silenced, so that the infection of the tobacco mosaic virus and the potato Y virus can be inhibited, the resistance of the plant to the plant virus is improved, and the plant resistance to the plant virus is improved. The method is beneficial to obtaining disease-resistant plants and has important application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of RabE1a protein and its encoding gene in resisting viruses of the genus Tobamovirus and viruses of the genus Potyvirus. Background Art

[0002] Viruses of the genus Tobamovirus mainly include tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), tomato mottle mosaic virus (ToMMV), and tomato brown rugose fruit virus (ToBRFV). Tobamovirus can infect important crops such as Solanaceae, Cucurbitaceae, and Brassicaceae and cause serious harm, and is one of the most economically important viruses.

[0003] Viruses of the genus Potyvirus are one of the virus genera with a wide range of influence and serious harm, which can cause reduction in yield and quality of host plants. Typical virus species included in this genus are potato virus Y (PVY) and tobacco vein banding mosaic virus (TVBMV); the infected species mainly include crops and cash crops such as Solanaceae, Leguminosae, and Chenopodiaceae.

[0004] Plant viruses are the second largest category of pathogens causing plant diseases, affecting the growth of crops and resulting in a decrease in crop yield. How to prevent and control the occurrence and harm of plant virus diseases has always been a research hotspot. Planting disease-resistant varieties is an effective way to control plant virus diseases, but currently, the variety resources resistant to plant virus diseases are relatively scarce, and screening new antiviral targets is of great significance for guiding the cultivation of disease-resistant varieties.

[0005] Small GTP-binding proteins, namely small G proteins, are monomeric GTP-binding proteins with "GTPase" activity and are involved in biological processes such as vesicle trafficking, plant hormone signal regulation, and environmental stress responses. Small GTP-binding proteins are a relatively complex and large superfamily, which can be divided into 5 families, namely Arf GTPase, Rho GTPase, Ras GTPase, Rab GTPase, and Ran GTPase according to their structures and functions. Among them, the Rab GTPase family has the largest number of members and is divided into 8 subfamilies, RabA-RabH, and each subfamily participates in different regulatory processes. The RabE1a protein belongs to one of the members of the RabE subfamily, and there is currently little research on its function. Patent CN 116947991A discloses that the pepper RABE1a protein can inhibit the infection of the pathogenic fungus Phytophthora capsici, but the relationship between the RabE1a protein and plant virus diseases has not been reported yet. Summary of the Invention

[0006] In view of the above-mentioned prior art, the object of the present invention is to provide the application of RabE1a protein and its encoding gene in resisting viruses of the genus Tobamovirus and viruses of the genus Potyvirus.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, there is provided the application of RabE1a protein in regulating the resistance of plants to viruses of the genus Tobamovirus and / or viruses of the genus Potyvirus;

[0009] The RabE1a protein is the protein shown in the following (1) or (2):

[0010] (1) A protein with an amino acid sequence as shown in SEQ ID NO.1;

[0011] (2) A protein with an amino acid sequence as shown in SEQ ID NO.2.

[0012] In the above application, the plant is tomato, tobacco or potato.

[0013] In the above application, the viruses of the genus Tobamovirus are TMV, ToMV, ToMMV or ToBRFV; the viruses of the genus Potyvirus are PVY or TVBMV.

[0014] The present invention has found through research that inactivating the RabE1a protein or inhibiting the expression of the RabE1a protein can improve the resistance of plants to viruses of the genus Tobamovirus and viruses of the genus Potyvirus and inhibit virus infection.

[0015] The second aspect of the present invention provides the use of the coding gene of RabE1a protein in the following (1) or (2):

[0016] (1) Regulating the resistance of plants to viruses of the genus Tobamovirus and / or viruses of the genus Potyvirus;

[0017] (2) Cultivating plant varieties resistant to viruses of the genus Tobamovirus and / or viruses of the genus Potyvirus.

[0018] In the above applications, the coding gene of the RabE1a protein is a nucleic acid molecule shown in any one of the following (i)-(v):

[0019] (i) A nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.3;

[0020] (ii) A nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.4;

[0021] (iii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.1 other than (i) and (ii);

[0022] (iv) A nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.5;

[0023] (v) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 other than (iv).

[0024] In the above applications, the plant is tomato, tobacco or potato.

[0025] In the above applications, the virus of the genus Tobamovirus is TMV, ToMV, ToMMV or ToBRFV; the virus of the genus Potyvirus is PVY or TVBMV.

[0026] Taking the coding gene of RabE1a protein as a target, by knocking out or silencing the coding gene of RabE1a protein, the resistance of plants to viruses of the genus Tobamovirus and Potyvirus can be improved, and thus varieties resistant to plant virus diseases can be cultivated.

[0027] The third aspect of the present invention provides a method for improving the resistance of plants to viruses of the genus Tobamovirus and / or viruses of the genus Potyvirus, comprising the following steps:

[0028] Knocking out or silencing the coding gene of RabE1a protein in the plant.

[0029] Preferably, knocking out the coding gene of RabE1a protein by CRISPR / Cas9 gene editing technology; or silencing the coding gene of RabE1a protein using the TRV vector.

[0030] Preferably, the plant is tomato, tobacco or potato.

[0031] In a fourth aspect of the present invention, there is provided a method for cultivating a plant variety resistant to viruses of the genus Tobamovirus and / or viruses of the genus Potyvirus, comprising the following steps:

[0032] Knock out or silence the coding gene of RabE1a protein in a wild-type plant to obtain a mutant plant; the mutant plant has higher resistance to viruses of the genus Tobamovirus and / or viruses of the genus Potyvirus than the wild-type plant;

[0033] Self-cross the mutant plant or cross it with other virus-resistant plants to cultivate a plant variety resistant to viruses of the genus Tobamovirus and / or viruses of the genus Potyvirus.

[0034] Preferably, the plant is tomato, tobacco or potato.

[0035] Advantages of the present invention:

[0036] The present invention for the first time studies and discovers that the RabE1a protein can regulate the resistance of plants to viruses of the genus Tobamovirus and Potyvirus. By knocking out or silencing the coding gene of the RabE1a protein, the infection of viruses of the genus Tobamovirus and Potyvirus can be inhibited, and the resistance of plants to the above-mentioned plant viruses can be improved, which is beneficial to the obtaining of disease-resistant plants and has important application value. Description of the drawings

[0037] Figure 1 : Knock out SlRabE1a in the tomato cultivar Ailsa Craig using the CRISPR / Cas9 gene editing technology; in the figure, A is the editing strategy and sequencing results; B is the photos of the obtained SlRabE1a knockout plants (Slrabe1a-5, Slrabe1a-13) and wild-type tomato (WT).

[0038] Figure 2 : Knocking out SlRabE1a can inhibit the infection of ToBRFV, TMV, ToMV and ToMMV; in the figure, A is the symptom photos of wild-type tomato (WT) and SlRabE1a knockout plants (Slrabe1a-5, Slrabe1a-13) after inoculation with ToBRFV, TMV, ToMV and ToMMV; B is the Western blotting detection result of virus coat protein (CP).

[0039] Figure 3 : RT-qPCR analysis of the NbRabE1a silencing efficiency.

[0040] Figure 4 : Silencing NbRabE1a can inhibit the infection of TVBMV and PVY; in the figure, A is the photo of the plants with silenced NbRabE1a (TRV-NbRabE1a) and the TRV-mCherry control plants after inoculation with TVBMV-GFP and PVY-GFP; B is the accumulation level of viral CP in the plants. Detailed implementation manners

[0041] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0042] As mentioned above, the small G protein family has many members with different functions. At present, there is little research on the function of the RabE1a protein, and it mainly focuses on the regulatory role played during the plant-fungal infection process. There is no report on its function during the plant virus infection process.

[0043] In view of this, the present invention has conducted in-depth research on the functions of the RabE1a proteins derived from tomato, Nicotiana benthamiana, and potato. Among them, the amino acid sequences of SlRabE1a derived from tomato and StRabE1a derived from potato are both shown in SEQ ID NO.1, which is specifically as follows:

[0044] MAAPPARARADYDYLIKLLLIGDSGVGKSCLLLRFSDGSFTTSFITTIGIDFKIRTIELDSKRIKLQIWDTAGQERFRTITTAYYRGAMGILLVYDVTDESSFNNIRNWIRNIEQHASDNVNKILVGNKADMDESKRAVPTSKGQALADEYGIKFFETSAKTNMNVEEVFFSIARDIKQRLAESDSKAEPQTIRINQPDQGAGSSQGAQKSACCGS.

[0045] The amino acid sequence of NbRabE1a derived from Nicotiana benthamiana is shown in SEQ ID NO.2, which is specifically as follows:

[0046] MAAPPARARADYDYLIKLLLIGDSGVGKSCLLLRFSDGSFTTSFITTIGIDFKIRTIEFDSKRIKLQIWDTAGQERFRTITTAYYRGAMGILLVYDVTDESSFNNIRNWIRNIEQHASDNVNKILVGNKADMDESKRAVPTSKGQALADEYGIKFFETSAKTNMNVEEVFFSIARDIKQRLAESDSKAEPQTIRINQPDQAAGAGQSAQKSACCGS。

[0047] The nucleotide sequence of the coding gene of SlRabE1a derived from tomato is shown in SEQ ID NO.3, specifically as follows:

[0048] ATGGCCGCTCCACCCGCTAGAGCTCGAGCTGATTACGATTACCTCATCAAACTCCTTTTGATCGGCGACAGCGGTGTGGGTAAGAGTTGCCTTCTTTTACGTTTCTCAGATGGCTCCTTTACGACCAGTTTTATCACAACTATTGGTATTGACTTCAAGATAAGGACCATAGAGCTTGACAGTAAGCGCATCAAACTGCAAATCTGGGATACTGCTGGTCAGGAGCGATTCCGAACAATTACAACTGCTTACTACCGTGGAGCCATGGGTATATTGCTGGTGTATGATGTAACTGATGAGTCATCTTTTAACAACATCAGGAACTGGATAAGAAACATTGAACAGCATGCCTCCGACAATGTCAACAAAATACTGGTCGGCAACAAGGCTGACATGGATGAAAGCAAAAGGGCTGTTCCTACATCCAAGGGCCAAGCACTTGCTGATGAATATGGCATTAAATTCTTTGAAACTAGTGCCAAGACAAATATGAATGTTGAGGAGGTTTTCTTTTCCATAGCTCGGGATATAAAACAAAGGCTTGCCGAATCTGACTCAAAGGCTGAGCCTCAGACCATCAGGATAAATCAACCGGACCAGGGAGCAGGATCTTCACAAGGCGCTCAAAAATCAGCTTGCTGTGGTTCTTAA。

[0049] The nucleotide sequence of the coding gene of StRabE1a derived from potato is shown in SEQ ID NO.4, as follows:

[0050] ATGGCCGCTCCACCCGCTAGAGCTCGAGCTGATTACGATTACCTCATCAAACTCCTTTTGATCGGCGACAGCGGTGTGGGTAAGAGTTGCCTTCTTTTACGTTTCTCAGATGGCTCCTTTACGACCAGTTTTATCACAACTATTGGTATTGACTTCAAGATAAGGACCATAGAGCTTGACAGTAAACGAATCAAACTGCAAATCTGGGATACCGCCGGTCAGGAGCGGTTCCGAACAATTACAACTGCTTACTACCGTGGAGCCATGGGTATATTGCTGGTGTACGATGTAACTGATGAGTCATCTTTTAACAACATCAGGAACTGGATAAGAAACATTGAACAGCATGCTTCCGACAATGTCAACAAAATTCTGGTCGGCAACAAGGCTGACATGGATGAAAGCAAAAGGGCTGTTCCTACATCCAAGGGTCAAGCACTTGCTGATGAATATGGCATTAAATTCTTTGAAACTAGTGCCAAGACAAATATGAATGTTGAGGAGGTTTTCTTTTCCATAGCTCGGGATATAAAACAAAGGCTTGCCGAATCTGACTCAAAGGCTGAGCCTCAGACCATCAGGATAAATCAACCGGACCAGGGAGCAGGATCTTCACAAGGCGCCCAAAAATCAGCTTGCTGTGGTTCTTAA。

[0051] The nucleotide sequence of the coding gene of NbRabE1a derived from Nicotiana benthamiana is shown in SEQ ID NO.5, as follows:

[0052] ATGGCCGCTCCACCCGCTAGAGCTCGAGCCGATTACGATTACCTAATCAAGCTCCTCTTGATCGGCGACAGCGGTGTGGGTAAGAGTTGCCTTCTTTTACGTTTCTCAGATGGCTCCTTCACGACCAGTTTTATTACAACTATTGGCATTGACTTCAAGATAAGGACCATTGAGTTTGATAGCAAACGAATCAAACTACAAATCTGGGATACTGCTGGTCAGGAGCGGTTCCGAACAATTACAACTGCTTACTACCGTGGAGCCATGGGTATATTGCTGGTGTATGACGTAACTGATGAGTCATCTTTTAACAACATCAGGAACTGGATAAGAAACATTGAGCAGCATGCTTCCGACAATGTCAACAAAATTCTGGTCGGCAACAAGGCTGACATGGACGAAAGCAAAAGGGCTGTTCCTACATCAAAAGGTCAAGCACTAGCTGACGAATATGGCATTAAATTCTTTGAGACAAGTGCCAAGACAAATATGAATGTGGAGGAGGTTTTCTTTTCCATAGCTCGGGATATAAAGCAAAGACTTGCTGAATCTGACTCAAAGGCTGAGCCTCAGACTATCAGGATAAATCAACCAGACCAGGCAGCAGGAGCTGGTCAAAGCGCTCAAAAATCAGCTTGCTGTGGCTCTTGA。

[0053] To study the function of RabE1a protein during plant virus infection, the present invention uses CRISPR / Cas9 gene editing technology to knockout SlRabE1a in tomato cultivar Ailsa Craig, obtaining SlRabE1a knockout plants, and testing the resistance of SlRabE1a knockout plants to tobamoviruses such as ToBRFV, TMV, ToMV, and ToMMV. It is found that tomato plants with SlRabE1a knockout can inhibit the infection of ToBRFV, TMV, ToMV, and ToMMV.

[0054] Furthermore, the present invention uses the TRV vector to silence the SlRabE1a homologous gene NbRabE1a in Nicotiana benthamiana plants, and inoculates the plants with silenced NbRabE1a with TVBMV-GFP and PVY-GFP respectively. The results show that silencing NbRabE1a can inhibit the infection of TVBMV and PVY.

[0055] This proves that knocking out or silencing the coding gene of RabE1a protein in plants can improve the resistance of plants to viruses of the genus Tobamovirus and viruses of the genus Potyvirus.

[0056] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0057] The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels. The experimental methods without specific conditions are carried out according to conventional test methods or according to the operation manuals recommended by the suppliers. Among them: the accession numbers of the ToBRFV, TMV, ToMV, and ToMMV isolates used in the present invention in NCBI are MT018320, MH595920, KY967221.1, and MW37351 respectively. The TRV vector was a gift from Professor Yule Liu of Tsinghua University. TVBMV-GFP and PVY-GFP are described in the journal literature (The conserved aromatic residue W122 is a determinant of potyviral coat protein stability, replication, and cell-to-cell movement in plants. Molecular Plant Pathology, 2021, 22, 189-203.).

[0058] Example 1: Construction of SlRabE1a knockout plants and study on the resistance to viruses of the genus Tobamovirus

[0059] 1. Construction of SlRabE1a knockout plants:

[0060] Use the CRISPR / Cas9 gene editing technology to knockout SlRabE1a in the tomato variety Ailsa Craig. The editing strategy and sequencing results are as Figure 1 shown in A.

[0061] We obtained two homozygous SlRabE1a knockout mutant lines, Slrabe1a-5 and Slrabe1a-13 ( Figure 1 B).

[0062] 2. Study on the resistance to viruses of the genus Tobamovirus:

[0063] Three-week-old wild-type (WT) tomato plants and knockout mutant lines Slrabe1a-5 and Slrabe1a-13 plants were inoculated with ToBRFV, TMV, ToMV, and ToMMV.

[0064] At 8 dpi after virus inoculation, the phenotypes of wild-type tomato plants and SlRabE1a knockout plants were observed, and the accumulation levels of viral coat protein (CP) in wild-type tomato plants and SlRabE1a knockout plants were detected by Western blotting.

[0065] The results were as Figure 2 shown, and the systemic leaves of WT tomato plants showed more severe symptoms than those of tomato plants inoculated with mutant line Slrabe1a plants ( Figure 2 A). The Western blotting results showed that the accumulation level of viral coat protein (CP) in knockout line Slrabe1a plants was significantly lower than that in control plants ( Figure 2 B). These results indicate that SlRabE1a is involved in the systemic infection of ToBRFV, TMV, ToMV, and ToMMV.

[0066] Example 2: Construction of Nicotiana benthamiana plants with silenced NbRabE1a and study on resistance to Potyvirus

[0067] 1. Construction of Nicotiana benthamiana plants with silenced NbRabE1a:

[0068] NbRabE1a was silenced in Nicotiana benthamiana plants using the TRV vector. The primers for silencing NbRabE1a are shown in SEQ ID NO.6 and SEQ ID NO.7, as follows:

[0069] TRV-NbRabE1a-F:

[0070] TGTCTTCGGGACATGCCCGGGCTGGTGTATGACGTAACTGATG; (SEQ ID NO.6)

[0071] TRV-NbRabE1a-R: gtgagtAAGGTTACCgaattcTGAGCGCTTTGACCAGCTCC. (SEQ ID NO.7)

[0072] The region of silenced NbRabE1a is shown in SEQ ID NO.8, as follows:

[0073] CTGGTGTATGACGTAACTGATGAGTCATCTTTTAACAACATCAGGAACTGGATAAGAAACATTGAGCAGCATGCTTCCGACAATGTCAACAAAATTCTGGTCGGCAACAAGGCTGACATGGACGAAAGCAAAAGGGCTGTTCCTACATCAAAAGGTCAAGCACTAGCTGACGAATATGGCATTAAATTCTTTGAGACAAGTGCCAAGACAAATATGAATGTGGAGGAGGTTTTCTTTTCCATAGCTCGGGATATAAAGCAAAGACTTGCTGAATCTGACTCAAAGGCTGAGCCTCAGACTATCAGGATAAATCAACCAGACCAGGCAGCAGGAGCTGGTCAAAGCGCTCA。

[0074] After 12 days of silencing, qPCR was used to detect the silencing efficiency. The results showed that the silencing efficiency of NbRabE1a could reach 80% ( Figure 3 ).

[0075] 2. Resistance study against potyviruses

[0076] The plants with silenced NbRabE1a were inoculated with TVBMV-GFP and PVY-GFP respectively. At 6 dpi after virus inoculation, the phenotypes of TRV-mCherry control plants and NbRabE1a-silenced plants were observed, and the accumulation levels of viral coat protein (CP) in TRV-mCherry control plants and NbRabE1a-silenced plants were detected by Western blotting.

[0077] The results are as Figure 4 shown. The systemic leaves of plants inoculated with the TRV-mCherry control showed more obvious mosaic than those inoculated with TRV-NbRabE1a ( Figure 4 A). Western blotting showed that the accumulation level of viral CP in NbRabE1a-silenced plants was lower than that in control plants ( Figure 4 B). The results indicate that silencing NbRabE1a can inhibit the infection of TVBMV and PVY.

[0078] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Application of RabE1a protein in regulating plant resistance to tobacco mosaic virus and / or potato Y virus; The RabE1a protein is the protein shown in (1) or (2) below: (1) a protein whose amino acid sequence is shown in SEQ ID NO.1; (2) A protein whose amino acid sequence is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The plant is tomato, tobacco or potato.

3. The use according to claim 1, characterized in that: The tobacco mosaic virus is TMV, ToMV, ToMMV or ToBRFV; the potyvirus is PVY or TVBMV.

4. Use of the gene encoding RabE1a protein in the following (1) or (2): (1) regulating plant resistance to tobacco mosaic virus and / or potyvirus; (2) Breeding plant varieties that are resistant to tobacco mosaic virus and / or potyvirus.

5. The use according to claim 4, characterized in that: The gene encoding the RabE1a protein is a nucleic acid molecule shown in any one of the following (i)-(v): (i) a nucleic acid molecule whose nucleotide sequence is shown in SEQ ID NO.3; (ii) a nucleic acid molecule whose nucleotide sequence is shown in SEQ ID NO.4; (iii) a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.1 other than (i) and (ii); (iv) a nucleic acid molecule whose nucleotide sequence is shown in SEQ ID NO.5; (v) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 2 other than (iv).

6. The use according to claim 4, characterized in that: The tobacco mosaic virus is TMV, ToMV, ToMMV or ToBRFV; the potyvirus is PVY or TVBMV.

7. A method for improving plant resistance to tobacco mosaic virus and / or potyvirus, characterized in that: The following steps are involved: Knock out or silence the gene encoding RabE1a protein in plants.

8. The method according to claim 7, characterized in that The gene encoding RabE1a protein is knocked out through CRISPR / Cas9 gene editing technology; or the gene encoding RabE1a protein is silenced using TRV vector.

9. The method according to claim 7, characterized in that: The plant is tomato, tobacco or potato.

10. A method for breeding plant varieties resistant to tobacco mosaic virus and / or potyvirus, characterized in that: The following steps are involved: knocking out or silencing the gene encoding the RabE1a protein in the wild-type plant to obtain a mutant plant; the mutant plant has higher resistance to tobacco mosaic virus and / or potyvirus than the wild-type plant; The mutant plants are self-pollinated or hybridized with other plant virus-resistant plants to cultivate plant varieties resistant to tobacco mosaic virus and / or potyvirus.

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