Use of dithio-ursine for the control of plant viruses

Abstract

The application discloses application of dithiouridine in prevention and treatment of plant viruses and belongs to the technical field of plant virus prevention and control. The application finds that dithiouridine has broad-spectrum resistance to tobacco mosaic virus, tomato mosaic virus, tomato spotted wilt virus and various plant viruses, and can be used for development of a new broad-spectrum anti-plant virus drug, which has important significance for prevention and treatment of plant virus diseases.

Description

Technical Field

[0001] This invention relates to the field of plant virus control technology, specifically to the application of dithiouridine in the prevention and control of plant viruses. Background Technology

[0002] Plant viruses are a significant threat to agricultural production, widely present in various crops and severely impacting crop yield and quality. Plant viruses can enter plants through insects, mechanical transmission, or seed dispersal. Once infected, plant viruses cause symptoms such as stunted growth, yellowing leaves, and deformed fruits, ultimately leading to crop yield loss. In the field environment, viruses spread rapidly and widely, posing a substantial threat to agriculture.

[0003] Common plant viruses include tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), tomato mottle mosaic virus (ToMMV), and tomato brown rugose fruit virus (ToBRFV). TMV is one of the earliest discovered plant viruses and belongs to the genus *Tobacco Mosaic Virus*. TMV can infect a variety of plants, including tomatoes, peppers, and tobacco. ToMV, ToMMV, and ToBRFV also belong to the *Tobacco Mosaic Virus* genus and primarily affect tomatoes and peppers; among them, ToBRFV has become the most serious tomato virus.

[0004] Although certain methods for controlling plant viruses exist, including the breeding of virus-resistant varieties and chemical control, the lack of broad-spectrum antiviral agents remains a significant challenge. Therefore, finding new and effective antiviral strategies has become an urgent task in agricultural research.

[0005] 2-Thiouridine (s2U; CAS No.: 20235-78-3) is a naturally occurring modified nucleoside compound whose structure consists of 2-thiouracil linked to a pentose sugar (D-ribose) via a β-N1-glycosidic bond. This compound is widely found in transfer RNA (tRNA) of various organisms, especially at the "wobbling base" sites (such as position 34) of the anticodon loop. The introduction of the 2-thio group enhances the specificity and thermal stability of base pairing, playing a crucial role in maintaining translation accuracy. 2-Thiouridine is generated in vivo through a specific enzymatic modification process. However, research on the control of plant viruses using 2-thiouridine has not yet been reported. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide the application of dithiouridine in the prevention and control of plant viruses. An unexpected discovery of this invention is that dithiouridine exhibits broad-spectrum resistance to various plant viruses, including tobacco mosaic virus, tomato mosaic virus, tomato mottle mosaic virus, and tomato brown wrinkled fruit virus, and can be used for the prevention and control of plant viral diseases.

[0007] In a first aspect, the invention provides the use of dithiouridine in either (1) or (2) below:

[0008] (1) Prevention and control of plant viral diseases;

[0009] (2) Prepare drugs for the prevention and treatment of plant viral diseases.

[0010] In the above applications, the plant viral diseases are caused by viruses of the genera *Tobacco Mosaic Virus*, *Geminivirus*, *Tomato Spotted Wilt Virus*, *Cucumber Mosaic Virus*, and *Potato Virus X*. Plant viruses include, but are not limited to: tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), tomato mottle mosaic virus (ToMMV), tomato brown rugose fruit virus (ToBRFV), tomato yellow leafcurl virus (TYLCV), tomato spotted wilt tospovirus (TSWV), cucumber mosaic virus (CMV), and potato virus X (PVX).

[0011] Preferably, the plant viral disease is caused by a virus of the Tobacco Mosaic Virus genus; more specifically, the plant viral disease is caused by Tobacco Mosaic Virus, Tomato Mosaic Virus, Tomato Mottled Mosaic Virus and / or Tomato Brown Wrinkled Fruit Virus.

[0012] A second aspect of the present invention provides a method for preventing and controlling plant viral diseases, comprising the following steps:

[0013] Dithiouridine is applied to plant leaves or rhizomes.

[0014] In the above method, preferably, the concentration of dithiouridine is 40μM-160μM.

[0015] In the above method, preferably, the plant is tomato, pepper, or tobacco.

[0016] The beneficial effects of this invention are:

[0017] This invention is the first to discover that dithiouridine has broad-spectrum resistance to a variety of plant viruses, including tobacco mosaic virus, tomato mosaic virus, tomato mottle mosaic virus, and tomato brown wrinkled fruit virus. It can be used to develop new broad-spectrum antiviral drugs for plant viruses, which is of great significance for the prevention and control of plant viral diseases. Attached Figure Description

[0018] Figure 1 Screening of effective concentrations of dithiouridine; In the figure, A shows the fluorescence of *Tobacco Benedictine* leaves after co-infiltration with different concentrations of dithiouridine and ToBRFV-GFP; B shows the relative accumulation of ToBRFV RNA in *Tobacco Benedictine* leaves after co-infiltration with different concentrations of dithiouridine and ToBRFV-GFP.

[0019] Figure 2 Effect of dithiouridine spraying on tobacco resistance to ToBRFV-GFP infection; In the figure, A shows the fluorescence of Tobacco Benedictine leaves after treatment with different concentrations of dithiouridine; B shows the accumulation of ToBRFV CP protein in Tobacco Benedictine leaves after treatment with different concentrations of dithiouridine.

[0020] Figure 3 Effects of dithiouridine spraying on the resistance of tomatoes to ToBRFV infection; In the figure, A represents the susceptibility of tomato leaves after treatment with different concentrations of dithiouridine; B represents the relative accumulation of ToBRFV RNA in tomato leaves after treatment with different concentrations of dithiouridine.

[0021] Figure 4 The resistance of dithiouridine to TMV-GFP, ToMV-GFP, and ToMMV-GFP; In the figure, A shows the fluorescence of *Tobacco Benedictine* leaves after co-infiltration with dithiouridine and the three viruses; B shows the accumulation of CP in *Tobacco Benedictine* leaves after co-infiltration with dithiouridine and the three viruses. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0023] As mentioned earlier, there are many types of plant viruses that are extremely harmful, and there is a lack of broad-spectrum and specific drugs that can prevent and control plant viruses in the current technology.

[0024] 2-Thiouridine, a naturally occurring modified nucleoside compound, has been the subject of much research, primarily focusing on its natural functions in organisms, particularly its roles in tRNA modification, protein synthesis, and translational precision. The inventors, having dedicated years to research on the prevention and control of plant viral diseases, unexpectedly discovered during their research that 2-thiouridine exhibits resistance to multiple plant viruses, suggesting its potential for development into novel antiviral drugs. This led to the development of this invention.

[0025] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0026] Unless otherwise specified, the test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Among them:

[0027] The ToBRFV-SD isolate used has the NCBI accession number MT018320; the TMV has the NCBI accession number MH595920; the ToMV has the GenBank accession number MZ388458; and the ToMMV has the NCBI accession number MW37351.

[0028] 2-thiouridine (s2U) CAS No.: 20235-78-3.

[0029] Example 1: Screening of dithiouridine treatment concentration

[0030] 1. Test method:

[0031] A 40 mM stock solution of dithiouridine was prepared using ddH2O and then added to the Agrobacterium resuspension containing the ToBRFV infectious clone, resulting in six concentrations: 0.625 μM, 2.5 μM, 10 μM, 40 μM, 160 μM, and 640 μM. Using ddH2O as a control, the solution was co-infiltrated with ToBRFV-GFP-transformed Agrobacterium using the half-leaf method in *Nicotiana benthamiana*. Fluorescence expression was observed under UV light four days after infiltration, and ToBRFV CP accumulation was detected by Western blot.

[0032] 2. Test Results:

[0033] The results are as follows Figure 1As shown, 40 μM s²U exhibited significant resistance to ToBRFV-GFP infection, with 160 μM s²U showing a more pronounced effect, and almost no ToBRFV-GFP green fluorescence was observed under 640 μM s²U treatment. qPCR analysis further confirmed that 40 μM s²U was sufficient to resist ToBRFV-GFP infection, and that ToBRFV RNA accumulation gradually decreased under both 160 μM and 640 μM s²U treatments.

[0034] Example 2: Effect of dithiouridine spraying on tobacco resistance to ToBRFV-GFP infection

[0035] 1. Test method:

[0036] Dithiouridine was prepared into solutions of 40 μM, 160 μM, and 640 μM using ddH2O. Using ddH2O as a control, spraying experiments were conducted on *Nicotiana benthamiana*. First, *Agrobacterium benthamiana* transformed with ToBRFV-GFP was infiltrated, and then different concentrations of s2U (1 mL of diluted s2U per plant) were sprayed. Spraying was repeated 2, 4, and 6 days after inoculation. Seven days after inoculation, the fluorescence of *Nicotiana benthamiana* leaves treated with different concentrations of s2U was observed under UV light; simultaneously, the accumulation of ToBRFV CP in *Nicotiana benthamiana* leaves was detected using Western blot.

[0037] 2. Test Results:

[0038] The results are as follows Figure 2 As shown, the GFP fluorescence observed under UV light after continuous spraying with 40 μM s2U was significantly weaker than that of the control group. Western blot results showed that the accumulation of ToBRFV CP after treatment with 40 μM s2U was also significantly lower than that of the control group. No GFP fluorescence was observed on tobacco after continuous spraying with 160 μM and 640 μM s2U, and no accumulation of ToBRFV CP was detected by Western blot. However, continuous spraying with 640 μM s2U led to stunted growth and yellowing of tobacco plants.

[0039] Example 3: Effect of dithiouridine spraying on resistance of tomatoes to ToBRFV infection

[0040] 1. Test method:

[0041] Dithiouridine was prepared into three concentrations (40 μM, 160 μM, and 640 μM) using ddH2O. Using ddH2O as a control, spraying experiments were conducted on tomatoes. First, ToBRFV Agrobacterium tumefaciens was infiltrated into tomato cotyledons, and then different concentrations of s2U (500 μL of diluted s2U per plant) were sprayed. Spraying was repeated 2, 4, and 6 days after inoculation. Seven days after inoculation, the susceptibility of tomato leaves treated with different concentrations of s2U was observed; simultaneously, the relative accumulation of ToBRFV CP in tomato leaves was detected using RT-qPCR.

[0042] 2. Test Results:

[0043] The results are as follows Figure 3 As shown, no obvious ToBRFV symptoms were observed in tomatoes sprayed with 40 μM, 160 μM, and 640 μM s2U for 7 consecutive days. RT-qPCR results revealed that the ToBRFV RNA accumulation in tomato plants treated with 40 μM s2U was only about 50% of that in the control, the ToBRFV RNA accumulation in tomato plants treated with 160 μM s2U was only 22% of that in the control, and almost no ToBRFV RNA accumulation was detected in tomato plants treated with 640 μM s2U.

[0044] Example 4: Antiviral activity of dithiouridine against other viruses in the Tobacco Mosaic Virus genus

[0045] 1. Test method:

[0046] A 40 mM stock solution of dithiouridine was prepared using ddH2O, and then added to the Agrobacterium resuspension containing TMV, ToMV, and ToMMV infectious clones, respectively, and diluted to 160 μM. Using ddH2O as a control (Mock), the solutions were co-infiltrated with Agrobacterium transformed with TMV-GFP, ToMV-GFP, and ToMMV-GFP using the half-leaf method in Nicotiana benthamiana. After 4 days of co-infiltration, the fluorescence of the leaves was observed under UV light; simultaneously, the CP accumulation of TMV, ToMV, and ToMMV in Nicotiana benthamiana leaves was detected using Western blot.

[0047] 2. Test Results:

[0048] The results are as follows Figure 4 As shown, the results revealed that 160 μM s2U effectively inhibited infection by TMV, ToMV, and ToMMV viruses. Therefore, s2U exhibits broad-spectrum resistance to Tobacco Mosaic Virus (TMV) viruses.

[0049] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Application of dithiouridine in the following (1) or (2): (1) Prevention and control of plant viral diseases; (2) Prepare drugs for the prevention and treatment of plant viral diseases.

2. The application according to claim 1, characterized in that, The plant viral diseases mentioned are caused by viruses of the genera *Tobacco Mosaic Virus*, *Geminivirus*, *Tomato Mosaic Virus*, *Cucumber Mosaic Virus*, *Potato Virus X*, *Potato Virus Y*, *Potato Leaf Roll Virus*, or *Hymenovirus*.

3. The application according to claim 2, characterized in that, The plant viral disease is caused by viruses of the Tobacco Mosaic Virus genus.

4. The application according to claim 3, characterized in that, The plant viral diseases mentioned are caused by tobacco mosaic virus, tomato mosaic virus, tomato mottle mosaic virus, or tomato brown wrinkled fruit virus.

5. A method for preventing and controlling plant viral diseases, characterized in that, Includes the following steps: Dithiouridine is applied to plant leaves or rhizomes.

6. The method according to claim 5, characterized in that, The concentration of dithiouridine used is 40μM-160μM.

7. The method according to claim 5, characterized in that, The plant in question is either tobacco or tomato.

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