Construction and optimization of TRV-mediated VIGS silencing system of Helianthus annuus

Authors

  • Dongqi Liu 1. College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Hohhot 010011, China
  • Xiangjiu Kong 1. College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Hohhot 010011, China
  • Yan Lu 1. College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Hohhot 010011, China
  • Shuang Guo 2. Arun Banner Agricultural Development Center, Naji Town 162750, Inner Mongolia, China
  • Lan Jing 1. College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Hohhot 010011, China

Abstract

To analyze gene function in plants lacking a stable genetic transformation system, virus-induced gene silencing (VIGS) is essential. Sunflower (Helianthus annuus), the world’s fourth most important oil crop and a significant cash crop in China, was selected as the experimental material. Three specific fragments of the H. annuus phytoene desaturase gene (HaPDS) were amplified and cloned into the pTRV vector to construct the recombinant vector pTRV-HaPDS for VIGS. Confectionery sunflower cultivars Huiyuan119, LD5009, and 3936, as well as oilseed sunflower cultivars R5 and GK2002, were used to optimize silencing conditions. Agrobacterium tumefaciens-mediated infiltration was performed, and HaPDS gene silencing was evaluated by considering factors such as the position of the silencing fragment within the gene, vacuum treatment, A. tumefaciens co-cultivation time, A. tumefaciens concentration (OD600), and plant cultivation temperature. Silencing efficiency was statistically analyzed, and HaPDS expression levels were quantified by qPCR. After various trials, it was determined that seed vacuum infiltration followed by 6 h of co-cultivation produced the most effective VIGS results. The optimal conditions involved silencing the HaPDS3 fragment near the 3′ end, an OD600 value of 1.0, and a plant ambient temperature of 22°C, resulting in relatively high silencing efficiency in Huiyuan119 and GK 2002. This study aims to develop an efficient and stable gene silencing system for sunflower, establishing a foundation for the subsequent validation of gene functions in this species.      

Keywords: H. annuus, HaPDSgene, VIGS system, gene silencing

DOI: 10.25165/j.ijabe.20261902.10338

 

Citation: Liu D Q, Kong X J, Lu Y, Guo S, Jing L. Construction and optimization of TRV-mediated VIGS silencing system of Helianthus annuus. Int J Agric & Biol Eng, 2026; 19(2): 78–87.

References

[1] Food and Agriculture Organization of the United Nations (FAO). FAOSTAT. Available: https://www.fao.org/faostat/en/#home. Accessed on [2026-02-09].

[2] Chen X H, Duan X F, Wang S, Wu W Y, Zhang X C. Virus-induced gene silencing (VIGS) for functional analysis of MYB80 gene involved in Solanum lycopersicum cold tolerance. Protoplasma, 2019; 256(2): 409–418.

[3] Li X Y, Na T, Xu B, Xu J Q, Yang Z G, Jiang C Q, et al. Establishment and application of a root wounding-immersion method for efficient virus-induced gene silencing in plants. Front Plant Sci, 2024; 15: 1336726.

[4] Ekengren S K, Liu Y, Schiff M, Dinesh-Kumar S P, Martin G B. Two MAPK cascades, NPR1, and TGA transcription factors play a role in Pto-mediated disease resistance in tomato. Plant J, 2003; 36(6): 905–917.

[5] Gama F, Saavedra T, Dandlen S, de Varennes A, Correia P J, Pestana M, et al. Silencing of the FRO1 gene and its effects on iron partition in Nicotiana benthamiana. Plant Physiol Biochem, 2017; 114: 111–118.

[6] Zhang G F, Li W C, Han T, Huang T Y, Sun L R, Hao F S. GhWRKY207 improves drought tolerance through promoting the expression of GhCSD3 and GhFSD2 in Gossypium hirsutum. Plant Sci, 2025; 352: 112392.

[7] Zhang H, Ye Z, Liu Z X, Sun Y, Li X Y, Wu J, et al. The cassava NBS-LRR genes confer resistance to cassava bacterial blight. Front Plant Sci, 2022; 13: 790140.

[8] Kumagai M H, Donson J, della-Cioppa G, Harvey D, Hanley K, Grill L K. Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. Proc Natl Acad Sci U S A, 1995; 92(5): 1679–1683.

[9] Robertson D. VIGS vectors for gene silencing: many targets, many tools. Annu Rev Plant Biol, 2004; 55: 495–519.

[10] Burch-Smith T M, Schiff M, Liu Y, Dinesh-Kumar S P. Efficient virus-induced gene silencing in Arabidopsis. Plant Physiol, 2006; 142(1): 21–27.

[11] Ratcliff F, Martin-Hernandez A M, Baulcombe D C. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J, 2001; 25(2): 237–245.

[12] Wang X Y, Lv K, Cai C P, Xu J, Guo W Z. Establishment and application of TRV-mediated virus-induced gene silencing in cotton. Acta Agric Sin, 2014; 40(8): 1356–1363. (in Chinese)

[13] Senthil-Kumar M, Mysore K S. Tobacco rattle virus-based virus-induced gene silencing in Nicotiana benthamiana. Nat Protoc, 2014; 9(7): 1549–1562.

[14] Sun T H, Tadmor Y, Li L. Pathways for carotenoid biosynthesis, degradation, and storage. In: Walker J M. (Ed.). Methods in Molecular Biology, 2020; pp.3–23.

[15] Mehboob I, Mughees M, Baig A, Ali S, Sajjad Y, Iqbal S, et al. An efficient virus-induced gene silencing of PDS gene in Solanum lycopersicum (cv. Rio Grande) and its functional analysis. Braz. J. Bot, 2023; 46: 881–892.

[16] Liu H P, Fu D Q, Zhu B Z, Yan H X, Shen X Y, Zuo J H, et al. Virus-induced gene silencing in eggplant (Solanum melongena). J Integr Plant Biol, 2012; 54(6): 422–429.

[17] Yuan C, Li C, Yan L J, Jackson A O, Liu Z Y, Han C G, et al. A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots. PLoS One, 2011; 6(10): e26468.

[18] Liu N, Xie K, Jia Q, Zhao J P, Chen T Y, Li H A, et al. Foxtail mosaic virus-induced gene silencing in monocot plants. Plant Physiol, 2016; 171(3): 1801–1807.

[19] Kalia D, Jose-Santhi J, Sheikh F R, Singh D, Singh R K. Tobacco rattle virus-based virus-induced gene silencing (VIGS) as an aid for functional genomics in Saffron (Crocus sativus L.). Physiol Mol Biol Plants, 2024; 30(5): 749–755.

[20] Mardini M, Kazancev M, Ivoilova E, Utkina V, Vlasova A, Demurin Y, et al. Advancing virus-induced gene silencing in sunflower: key factors of VIGS spreading and a novel simple protocol. Plant Methods, 2024; 20(1): 122.

[21] Fu D Q, Zhu B Z, Zhu H L, Zhang H X, Xie Y H, Jiang W B, et al. Enhancement of virus-induced gene silencing in tomato by low temperature and low humidity. Mol Cells, 2006; 21(1): 153–160.

[22] Guo Y, Liu Z D, Kang L R, Bao T, Yang X, Zhao J, et al. Optimization of efficient silencing system of tomato VIGS based on PDS gene. Crops, 2023(2): 46–50. (in Chinese)

[23] Patil B L, Fauquet C M. Light intensity and temperature affect systemic spread of silencing signal in transient agroinfiltration studies. Mol Plant Pathol, 2015; 16(5): 484–494.

[24] Senthil-Kumar M, Hema R, Anand A, Li K, Udayakumar M, Mysore K S. A systematic study to determine the extent of gene silencing in Nicotiana benthamiana and other solanaceae species when heterologous gene sequences are used for virus-induced gene silencing. New Phytol, 2007; 176(4): 782–791.

[25] Liu E W, Page J E. Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus. Plant Methods, 2008; 4: 5.

[26] Burch-Smith T M, Anderson J C, Martin G B, Dinesh-Kumar S P. Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J, 2004; 39(5): 734–746.

[27] Song Z, Li Z A, Zhou C Y. Research advances of virus-induced gene silencing (VIGS). Acta Horticulturae Sinica, 2014; 41(9): 1885–1894. (in Chinese)

[28] Dong Y X, Wei Q W, Hong H, Huang Y, Zhao Y X, Feng M F, et al. Establishment of ALSV-induced gene silencing in Chinese soybean cultivars. Scientia Agricultura Sinica, 2022; 55(9): 1710–1722. (in Chinese)

[29] Wang J E, Li D W, Gong Z H, Zhang Y L. Optimization of virus-induced gene silencing in pepper (Capsicum annuum L.). Genet Mol Res, 2013; 12(3): 2492–2506.

[30] Faivre-Rampant O, Gilroy E M, Hrubikova K, Hein I, Millam S, Loake G J, et al. Potato virus X-induced gene silencing in leaves and tubers of potato. Plant Physiol, 2004; 134(4): 1308–1316.

[31] Shi G Y, Hao M Y, Tian B M, Cao G Q, Wei F, Xie Z Q. A methodological advance of tobacco rattle virus-induced gene silencing for functional genomics in plants. Front Plant Sci, 2021; 12: 671091.

[32] Zhang J, Yu D S, Zhang Y, Liu K, Xu K D, Zhang F L, et al. Vacuum and co-cultivation agroinfiltration of (germinated) seeds results in tobacco rattle virus (TRV) mediated whole-plant virus-induced gene silencing (VIGS) in wheat and maize. Front Plant Sci, 2017; 8: 393.

[33] Rahman J, Baldwin I T, Gase K. California TRV-based VIGS vectors mediate gene silencing at elevated temperatures but with greater growth stunting. BMC Plant Biol, 2021; 21(1): 553.

[34] Ramegowda V, Mysore K S, Senthil-Kumar M. Virus-induced gene silencing is a versatile tool for unraveling the functional relevance of multiple abiotic-stress-responsive genes in crop plants. Front Plant Sci, 2014; 5: 323.

[35] Li W C, Liu X, Kang Y, Li W, Qi Z, Yu L, et al. Optimization and application of tobacco rattle virus-induced gene silencing system in soybean. Biotechnology Bulletin, 2023; 39(7): 143–150. (in Chinese)

[36] Sheludko Y V, Sindarovska Y R, Gerasymenko I M, Bannikova M A, Kuchuk N V. Comparison of several Nicotiana species as hosts for high-scale Agrobacterium-mediated transient expression. Biotechnol Bioeng, 2007; 96(3): 608–614.

[37] Wang C C, Cai X Z, Wang X M, Zheng Z. Optimisation of tobacco rattle virus-induced gene silencing in Arabidopsis. Funct Plant Biol, 2006; 33(4): 347–355.

[38] Zhang J X, Wang F R, Zhang C Y, Zhang J H, Chen Y, Liu G D, et al. A novel VIGS method by agroinoculation of cotton seeds and application for elucidating functions of GhBI-1 in salt-stress response. Plant Cell Rep, 2018; 37: 1091–1100.

[39] Dobnik D, Lazar A, Stare T, Gruden K, Vleeshouwers V G, Žel J. Solanum venturii, a suitable model system for virus-induced gene silencing studies in potato reveals StMKK6 as an important player in plant immunity. Plant Methods, 2016; 12: 29.

[40] Shi G Y, Hao M Y, Tian B M, Cao G Q, Wei F, Xie Z Q. A newly established virus-induced gene silencing method via seed imbibition for functional genomics at early germination stages in cotton. Industrial Crops & Products, 2022; 172: 114040.

[41] Alter H, Peer R, Dombrovsky A, Flaishman M, Spitzer-Rimon B. Tobacco rattle virus as a tool for rapid reverse-genetics screens and analysis of gene function in Cannabis sativa L. Plants (Basel), 2022; 11(3): 327.

[42] Deng X, Elomaa P, Nguyen C X, Hytönen T, Valkonen J P, Teeri T H. Virus-induced gene silencing for Asteraceae--a reverse genetics approach for functional genomics in Gerbera hybrida. Plant Biotechnol J, 2012; 10(8): 970–978.

[43] Xu Y Y, Cui Y M, Chen H Y, Pu Y, Zhang C Y, Huang H. Development and application of the TRV-induced gene-silencing system in different Rhododendron species. Plant Cell Tiss Organ Cult, 2024; 157: 61.

Downloads

Published

2026-05-21

How to Cite

(1)
Liu, D.; Kong, X.; Lu, Y.; Guo, S.; Jing, L. Construction and Optimization of TRV-Mediated VIGS Silencing System of Helianthus Annuus. Int J Agric & Biol Eng 2026, 19, 78-87.

Issue

Section

Animal, Plant and Facility Systems