Rice tillering-reducing virus and infectious clone vectors and uses thereof

Abstract

The application provides a rice tillering reduction virus RLTV and an infectious clone vector and application thereof. The full-length genome sequence of the RLTV is constructed into a pCass4-RZ vector to obtain an RLTV infectious clone vector, and after agrobacterium containing the RLTV infectious clone vector is mechanically inoculated on rice, the virus can systemically infect the rice. The agrobacterium containing the RLTV infectious clone vector is genetically transformed into Zhonghua 11 to obtain transgenic rice, and in the transgenic rice, the viral genome RNA, subgenomic RNA and coat protein are all expressed at a high level, and meanwhile, the transgenic rice also shows symptoms of reduction of rice seed setting rate, thousand-grain weight and yield per plant. The medium insects for RLTV transmission are screened, and it is found that the Sitobion miscanthi can transmit the virus from the transgenic rice or wild rice with the virus to healthy rice. The application provides a new resource for preventing and treating the new type of rice virus and studying the interaction among the virus, rice and Sitobion miscanthi.

Description

Technical Field

[0001] This invention relates to the fields of plant virology and genetic engineering, and in particular to a rice tillering reduction virus RLTV, its infectious cloning vector, and its applications. Background Technology

[0002] The outbreaks of rice viral diseases have caused enormous damage to rice production. These outbreaks are often caused by known or unknown pathogenic rice viruses in nature. The most prevalent rice viral diseases in my country are mainly negative-strand RNA viruses and double-stranded RNA viruses, including viruses from the genera *Cellaviridae*, *Rhabdoviridae*, and *Reoviridae*. These viruses can be transmitted through various vectors, such as different leafhoppers and rice planthoppers.

[0003] Rice less tiller virus (RLTV) is a newly discovered pathogenic rice virus belonging to the genus *Polerovirus*. This genus of viruses can infect more than ten different families and genera of plants. The virus mainly accumulates in the phloem of plants, typically causing symptoms of phloem underdevelopment. In nature, this genus of viruses is mainly transmitted by different species of aphids in a persistent, non-replicating manner. The aphid vectors are highly specialized, and the transmission mechanism is mainly based on receptor-mediated endocytosis and exocytosis. The genome size of Polerovirus ranges from 5625 nt to 6244 nt, generally containing seven ORFs and a subgenomic sgRNA1. It possesses a frameshift structure and a readthrough structure. The conserved 5'-ACAA(AA)-3' sequence and the 3' stem loop or tRNA-like structure at the 5' end are key to viral replication template recognition.

[0004] Viral infectivity cloning technology involves constructing the full-length genome sequence of a virus into an infectivity cloning vector, followed by transcription in vivo or in vitro to form a systemically infective virus. In vivo transcription is primarily driven by the 35S promoter of Cauliflower mosaic virus (CaMV), while in vitro transcription is initiated by prokaryotic promoters such as T7, SP6, and T3. Viral reverse genetics systems based on infectivity cloning technology have been successfully established for plant single-stranded DNA viruses, plant positive-sense RNA viruses, and plant negative-sense RNA viruses. However, the infectivity clones of rice viruses, which cause serious damage in my country, are currently unable to infect rice, thus delaying biological research on rice viruses. Summary of the Invention

[0005] The purpose of this invention is to provide a rice tillering-reducing virus RLTV, its infectious cloning vector, and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The present invention first provides a rice tillering reduction virus RLTV, which has the accession number CCTCC NO: V202272 at the China Center for Type Culture Collection and the accession date is December 26, 2022.

[0007] The present invention also provides an infectious cloning vector for rice tillering reduction virus RLTV, which is prepared by cloning the full-length genome sequence of rice tillering reduction virus RLTV into the plant binary expression vector pCass4-RZ. The full-length genome sequence of rice tillering reduction virus RLTV is shown in SEQ ID NO.1.

[0008] Furthermore, the construction of the above-mentioned rice tiller-reducing virus RLTV infectious clonal vector includes the following steps: 1) Extract total RNA from wild rice infected with rice tiller reduction virus RLTV, reverse transcribe the total RNA of wild rice with primer RLTV-R to obtain cDNA, and use the reverse transcribed cDNA as a template to amplify the full-length genome sequence of rice tiller reduction virus RLTV using primers RLTV-F1 and RLTV-R1. 2) The plant binary expression vector pCass4-RZ was digested with restriction endonucleases StUI and BamHI to obtain the pCass4-RZ linearized vector. The full-length genome sequence of rice tillering reduction virus RLTV obtained in step 1) was ligated to the pCass4-RZ linearized vector using seamless cloning technology. 3) The ligation product obtained in step 2) was transformed into Escherichia coli JM109 competent cells, and the plasmid was extracted to obtain a cloning vector for reducing the infectivity of rice tillering virus RLTV. The primer sequences are as follows: RLTV-R: 5'-GTAGTCCCTTTTTCGGAACTTTGGT-3', RLTV-F1: 5'-agttcatttcatttggagaggACAAAAGATATCCAGGCAGGTCCTG-3' RLTV-R1: 5'-cggtgacagggtatcggatccACCAAAGTTCCGAAAAAGGGACTAC-3'.

[0009] The present invention also provides an Agrobacterium strain carrying an infectious cloning vector of rice tillering reduction virus RLTV, which is obtained by introducing the above-mentioned infectious cloning vector of rice tillering reduction virus RLTV into Agrobacterium GV3101 by liquid nitrogen freeze-thaw method.

[0010] The present invention also provides the above-mentioned rice tillering reduction virus RLTV infectivity cloning vector, and / or the application of the above-mentioned Agrobacterium strain carrying the rice tillering reduction virus RLTV infecting rice.

[0011] The present invention also provides the above-mentioned rice tillering reduction virus RLTV infectivity cloning vector, and / or the above-mentioned Agrobacterium strain carrying the rice tillering reduction virus RLTV infectivity cloning vector in the preparation of transgenic rice.

[0012] The present invention also provides the above-mentioned rice tillering reduction virus RLTV infectivity cloning vector, and / or the above-mentioned Agrobacterium strain carrying the rice tillering reduction virus RLTV infectivity cloning vector in pathogenicity analysis and viral gene function research of rice tillering reduction virus RLTV.

[0013] This invention also involves infecting the callus tissue of Zhonghua 11 rice with the aforementioned Agrobacterium strain carrying the infectious clonal vector of the rice tiller-reducing virus RLTV, and obtaining positive transgenic rice carrying the rice tiller-reducing virus through differentiation, rooting, and hardening-off processes. Using the stably inherited positive transgenic rice as the virus source, the virus was acquired on the transgenic rice using aphids such as *Aphis graminearum*, *Aphis graminearum*, *Aphis graminearum*, *Aphis graminearum*, *Brassica oleracea*, and *Leymus chinensis* as virus-transmitting vectors. Three days later, the virus-carrying aphids were transferred to healthy rice plants for virus transmission, and all aphids were killed five days later. The results showed that only the rice tiller reduction virus (RLTV) could transmit it to healthy rice plants, causing symptoms of reduced tillering. Fifteen days after transmission by the rice tiller aphid, Western blot analysis of the viral coat protein in rice revealed that up to 80% of the seedlings tested positive. This indicates that the rice tiller aphid is the dominant vector insect for RLTV. Therefore, controlling the population of the rice tiller aphid, using a pest control strategy, may be effective in preventing and controlling RLTV.

[0014] The significant advantages of this invention are: This invention prepared a recombinant Agrobacterium tumefaciens clone with reduced rice tillering infectivity for the rice virus RLTV. This Agrobacterium can infect rice via mechanical inoculation. Using the genetically transformed Zhonghua 11 transgenic rice strain from this Agrobacterium as the virus strain, RLTV virus can also be inoculated into healthy rice through transmission by the rice aphid. The newly discovered infectivity clone and transmission vector of the rice virus RLTV provide promising applications for understanding the relationship between rice, the viral pathogen, and the vector insect, and also provide important resources and theoretical basis for early warning of outbreaks and prevention of this viral disease. Attached Figure Description

[0015] Figure 1A schematic diagram of the genome structure of the virus RLTV, which reduces rice tillering, and the construction of its infectious cloning vector.

[0016] Figure 2 To reduce the symptoms and viral accumulation of *Agrobacterium tumefaciens* and *Zhonghua 11* rice by inducing tillering of *Agrobacterium tumefaciens* infection with the RLTV infectious clone. (A) Rice tillering reduced the accumulation of viral RNA of the infectious clone in wild-type and mutant *Agrobacterium tumefaciens*. (B) RT-PCR detection of viral RdRP gene expression in wild-type and mutant *Agrobacterium tumefaciens*. (C) Northern blot detection of vsiRNA expression in wild-type and mutant *Agrobacterium tumefaciens*. (D) Rice tillering reduced the accumulation of viral capsid protein, genomic RNA, subgenomic RNA, and vsiRNAs of the infectious clone in rice. (E) Rice tillering reduced the symptoms of viral infection in rice. (F) Rice tillering reduced the number of tillers in viral-infected rice.

[0017] Figure 3 Transgenic rice of Zhonghua 11, genetically transformed with the RLTV infectious cloning vector for reducing rice tillering, and its symptoms and virus accumulation in rice transmitted by the rice aphid *Rhizoctonia solani*. (A) Symptoms of mature rice of transgenic rice with reduced tillering virus. (B) Agronomic traits of mature rice of transgenic rice with reduced tillering virus. (C) Panicle symptoms of mature rice of transgenic rice with reduced tillering virus. (D) Seed symptoms of mature rice of transgenic rice with reduced tillering virus. (E) Detection of RLTV coat protein, genomic RNA, subgenomic RNA, and vsiRNAs in three different lines of transgenic rice. (F) Detection of viral coat protein, genomic RNA, subgenomic RNA, and vsiRNAs in Zhonghua 11 rice after *Rhizoctonia solani* acquired the virus from the transgenic rice with reduced tillering virus. Detailed Implementation

[0018] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0019] The rice tillering reduction virus RLTV described in this invention has the accession number CCTCC: V202272 at the China Center for Type Culture Collection, and the accession date is December 26, 2022.

[0020] Example 1: Construction of an infectious cloning vector for reducing the invasiveness of rice tillering virus RLTV Total RNA was extracted from wild rice from Guangxi infected with rice tillering reduction virus (RLTV) using the thermophenol method. Based on the viral sequence assembled from high-throughput sequencing, the 5' and 3' terminal sequences of RLTV were cloned using RACE technology. Primers were designed based on these viral terminal sequences, and cDNA was obtained by reverse transcription of total RNA from diseased leaves using primer RLTV-R. Using the reverse-transcribed cDNA as a template, the full-length genome sequence of RLTV was amplified using primers RLTV-F1 and RLTV-R1. The amplified products were purified and recovered using conventional techniques. The primer sequences used are as follows: RLTV-R: 5'-GTAGTCCCTTTTTCGGAACTTTGGT-3', RLTV-F1: 5'-agttcatttcatttggagaggACAAAAGATATCCAGGCAGGTCCTG-3' RLTV-R1: 5'-cggtgacagggtatcggatccACCAAAGTTCCGAAAAAGGGACTAC-3'.

[0021] like Figure 1As shown, the plant binary expression vector pCass4-RZ was digested with restriction endonucleases StII and BamHI. The digestion system and conditions were as follows: 2 μg pCass4-RZ vector, 4 μL 10X Cutsmart Buffer, 1 μL each of StII and BamHI, and ddH2O to a final volume of 40 μL. Digestion was performed at 37°C in a metal bath for 2 hours. The pCass4-RZ linearized vector was purified and recovered using conventional methods. The purified and recovered full-length genome sequence of rice tillering reduction virus RLTV was ligated into the pCass4-RZ linearized vector using Novizan Biosciences' Seamless Cloning Kit (Catalog No.: C113-01). The reaction system was as follows: 2 μL of the pCass4-RZ double-digested linearized vector, 4 μL of the purified and recovered RLTV amplification product, 2 μL 5×CE MultiS Buffer, and 2 μL Exnase MultiS. The reaction conditions were as follows: ligation at 37°C in a metal bath for 30 minutes. The ligation product was transformed into *E. coli* JM109 competent cells. The recovered transformation product was plated on LB agar containing kanamycin (50 μg / ml). Clones were detected using primers RLTV-F1 and RLTV-R1. Positive clones were screened and subjected to Sanger sequencing to determine the full-length genome sequence of the rice tillering reduction virus (RLTV), as shown in SEQ ID NO.1. Simultaneously, positive clones were selected and cultured in a shake culture for plasmid extraction. Plasmid extraction was performed using standard techniques, and the extracted plasmid became the infectious cloning vector pCass4-RZ-RLTV for RLTV.

[0022] Example 2: Infectious Cloning of Rice Tillering Reduction Virus (RLTV) Inoculation into Nicotiana benthamiana and Rice. The above-mentioned infectious cloning vector pCass4-RZ-RLTV of rice tillering reduction virus (RLTV) was transformed into Agrobacterium GV3101 using the liquid nitrogen freeze-thaw method to obtain the pCass4-RZ-RLTV(GV3101) strain. The revived pCass4-RZ-RLTV(GV3101) competent cells were plated onto LB solid medium containing kanamycin (50 μg / ml) and rifampin (25 μg / ml) and incubated upside down at 28°C for 2 days. Positive clones were screened by colony PCR and expanded in LB liquid medium to OD600 = 0.8. After centrifugation at 4000 rpm at room temperature, the clones were resuspended in Agrobacterium suspension (10 mM MgCl2, 10 mM ES (pH = 5.6), 150 uM acetylsyl syringone). After standing in the dark for 3 hours, the clones were soaked in the solution of healthy Nicotiana benthamiana seedlings with 5 leaves. After 30 days, systematic leaves of Nicotiana benthamiana were collected for northern blot analysis.

[0023] Step OD 600A bacterial suspension of pCass4-RZ-RLTV(GV3101) with a concentration of 0.8 and good activity was spread onto LB solid medium containing kanamycin (50 μg / ml) and rifampin (25 μg / ml). After drying the bacterial suspension in a clean bench, the medium was inverted and cultured at 28°C for several days until a layer of Agrobacterium tumefaciens was visible on the surface of the solid LB medium. The leaves and stems of 30-day-old rice seedlings were gently scratched with the tip of a 1 ml syringe. Then, an appropriate amount of Agrobacterium tumefaciens was scraped horizontally from the surface of the aforementioned solid LB medium and evenly applied to the micro-wounds on the rice leaves and stems. Five inoculation sites were made on each rice plant. The inoculation was repeated once after 2 days. After 20 days, the virus content in the rice was detected by Western blot and Northern blot.

[0024] like Figure 2 As shown, rice tillering reduces the infectivity of the virus clone. After inoculation with Nicotiana benthamiana, genomic RNA, subgenomic RNA, and vsiRNAs of RLTV could be detected in its systemic leaves. Figure 2 A-2C). Following inoculation of Zhonghua 11 rice with RLTV infectious clones, RLTV CP protein, genomic RNA, subgenomic RNA, and vsiRNAs could be detected in the systemic leaves of rice. Figure 2 D), and RLTV infection significantly reduces rice tillering. Figure 2 E, 2F).

[0025] Example 3: Rice aphid transmission reduces virus infection in rice tillers. Transgenic rice with the background of Zhonghua 11 was prepared by infecting rice callus with Agrobacterium tillering virus infectivity cloning vector. Transgenic seedlings were obtained by differentiating, rooting and hardening of the callus. The specific steps were referred to in the master's thesis (Li Haiyang, 2020, Interference between rice stripe virus NS3 and rice OsIAA25 to cultivate antiviral rice). PCR was used to detect DNA replicons in transgenic rice, northern blot was used to detect genomic RNA and subgenomic RNA of the virus in transgenic rice, and western blot was used to detect the capsid protein of the virus in transgenic rice. Positive transgenic rice containing rice tillering virus RLTV was screened out. Symptoms of positive transgenic rice were observed at the rice maturity stage, various agronomic traits of rice were counted and rice seeds were harvested.

[0026] Stable genetically derived transgenic rice seedlings were obtained by planting transgenic rice seeds. Positive seedlings were screened using Western blot analysis to detect the viral capsid protein, which reduces rice tillering. Using positive transgenic seedlings or wild rice carrying RLTV as the virus source, the aphid *Strombus graminearum* was placed on infected plants to acquire the virus. Three days later, all aphids were transferred to healthy *Zhonghua 11* rice plants. Five days after virus transmission, all aphids were killed with pesticides. In this experiment, the *Strombus graminearum* virus transmission experiment was conducted using a single tube per seedling, with five *Strombus graminearum* aphids placed on each rice plant. Fifteen days after virus transmission to healthy rice plants, the genomic RNA and subgenomic RNA of the virus in the rice were detected by Northern blot, and the viral capsid protein was detected by Western blot.

[0027] like Figure 3 As shown, the pCass4-RZ-RLTV transgenic Zhonghua 11 rice exhibited significant agronomic traits such as decreased seed setting rate, reduced thousand-grain weight, reduced yield per plant, smaller panicles, and shorter grain length. Figure 3 A-3D), and CP protein, genomic RNA, subgenomic RNA and vsiRNAs of RLTV virus can also be detected in this transgenic rice. Figure 3 E). Using this transgenic rice as the source of RLTV virus, the cereal aphid acquired the virus from this rice and transmitted it to healthy Zhonghua 11 rice 3 days later. The CP protein, genomic RNA, subgenomic RNA, and vsiRNAs of the RLTV virus could be detected in the transmitted Zhonghua 11 rice. Figure 3 F).

[0028] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for reducing rice tillering virus RLTV, characterized in that: The rice tillering reduction virus RLTV has the accession number CCTCC NO: V202272 at the China Center for Type Culture Collection, and the accession date is December 26, 2022; the dominant vector of the rice tillering reduction virus RLTV is the rice aphid.

2. A clonal vector for reducing the infectivity of rice tillering virus RLTV, characterized in that: The full-length genome sequence of the rice tillering reduction virus RLTV as described in claim 1 was cloned into the plant binary expression vector pCass4-RZ. The full-length genome sequence of the rice tillering reduction virus RLTV is shown in SEQ ID NO.

1.

3. The rice tillering-reducing virus RLTV infectivity clonal vector according to claim 2, characterized in that: The construction of the rice tiller-reducing virus RLTV infectivity clonal vector includes the following steps: 1) Extract total RNA from wild rice infected with rice tiller reduction virus RLTV, reverse transcribe the total RNA of wild rice with primer RLTV-R to obtain cDNA, and use the reverse transcribed cDNA as a template to amplify the full-length genome sequence of rice tiller reduction virus RLTV using primers RLTV-F1 and RLTV-R1. 2) The plant binary expression vector pCass4-RZ was digested with restriction endonucleases StUI and BamHI to obtain the pCass4-RZ linearized vector. The full-length genome sequence of the rice tillering reduction virus RLTV obtained in step 1) was ligated to the pCass4-RZ linearized vector using seamless cloning technology. 3) The ligation product obtained in step 2) was transformed into Escherichia coli JM109 competent cells, and the plasmid was extracted to obtain the rice tillering-reducing virus RLTV infectivity cloning vector. The primer sequences are as follows: RLTV-R: 5'-GTAGTCCCTTTTTCGGAACTTTGGT-3', RLTV-F1: 5'-agttcatttcatttggagaggACAAAAGATATCCAGGCAGGTCCTG-3' RLTV-R1: 5'-cggtgacagggtatcggatccACCAAAGTTCCGAAAAAGGGACTAC-3'.

4. An Agrobacterium strain carrying a clonal vector for reducing the infectivity of rice tillering virus RLTV, characterized in that: The rice tillering-reducing virus RLTV cloning vector described in claim 2 was introduced into Agrobacterium GV3101 via liquid nitrogen freeze-thaw method.

5. The application of the rice tillering reduction virus RLTV infectivity cloning vector as described in claim 2, and / or the Agrobacterium strain carrying the rice tillering reduction virus RLTV infectivity cloning vector as described in claim 4, in the pathogenicity analysis and viral gene function study of rice tillering reduction virus RLTV.

6. A method for inoculating rice tillers to reduce RLTV virus, characterized in that: Includes the following steps: 1) Infect rice callus tissue with the Agrobacterium strain carrying the rice tiller-reducing virus RLTV clonal vector as described in claim 4, and screen to obtain transgenic rice; 2) Feed the transgenic rice with the poison from the rice aphid in step 1), and then place the infected rice aphids onto the rice plants to be inoculated to transmit the virus.

Images