RNA silencing is an innate immune mechanism of plants against invasion by viral pathogens. Artificial microRNA (amiRNA) can be engineered to specifically induce RNA silencing against viruses in transgenic plants and has great potential for disease control. Here, we describe the development and application of amiRNA-based technology to induce resistance to soybean mosaic virus (SMV), a plant virus with a positive-sense single-stranded RNA genome. We have shown that the amiRNA targeting the SMV P1 coding region has the highest antiviral activity than those targeting other SMV genes in a transient amiRNA expression assay. We transformed the gene encoding the P1-targeting amiRNA and obtained stable transgenic Nicotiana benthamiana lines (amiR-P1-3-1-2-1 and amiR-P1-4-1-2-1). Our results have demonstrated the efficient suppression of SMV infection in the P1-targeting amiRNA transgenic plants in an expression level-dependent manner. In particular, the amiR-P1-3-1-2-1 transgenic plant showed high expression of amiR-P1 and low SMV accumulation after being challenged with SMV. Thus, a transgenic approach utilizing the amiRNA technology appears to be effective in generating resistance to SMV.

Jiangsu Key Laboratory for Food Quality and Safety State Key Laboratory Cultivation Base of Ministry of Science and Technology Institute of Plant Protection Jiangsu Academy of Agricultural Sciences Nanjing 210014 China

Jiangsu Key Laboratory for Pathogens and Ecosystems Jiangsu Engineering and Technology Research Center for Microbiology College of Life Sciences Nanjing Normal University Nanjing 210023 China

Laboratory of Virology Centre for Plant Virus Research Institute of Experimental Botany of the Czech Academy of Sciences Rozvojová 263 165 02 Prague Czech Republic

Plant Virus and Vector Interactions Centre for Plant Virus Research Crop Research Institute Drnovská 507 161 06 Prague Czech Republic

Zobrazit více v PubMed

Bassett M, Salemi M, Rife Magalis B (2022) Lessons learned and yet-to-be learned on the importance of RNA structure in SARS-CoV-2 replication. Microbiol Mol Biol Rev 86:e0005721. https://doi.org/10.1128/mmbr.00057-21 DOI

Carbonell A, Lopez C, Daros JA (2019) Fast-forward identification of highly effective artificial small RNAs against different tomato spotted wilt virus isolates. Mol Plant Microbe Interact 32:142–156. https://doi.org/10.1094/MPMI-05-18-0117-TA DOI

Chatzi A, Doody O (2023) The one-way ANOVA test explained. Nurse Res 31:8–14. https://doi.org/10.7748/nr.2023.e1885 DOI

Duan CG, Wang CH, Fang RX, Guo HS (2008) Artificial microRNAs highly accessible to targets confer efficient virus resistance in plants. J Virol 82:11084–11095. https://doi.org/10.1128/JVI.01377-08 DOI

Gallois P, Marinho P (1995) Leaf disk transformation using Agrobacterium tumefaciens-expression of heterologous genes in tobacco. Methods Mol Biol 49:39–48. https://doi.org/10.1385/0-89603-321-X:39 DOI

Gao L, Ding X, Li K, Liao W, Zhong Y, Ren R, Liu Z, Adhimoolam K, Zhi H (2015) Characterization of Soybean mosaic virus resistance derived from inverted repeat-SMV-HC-Pro genes in multiple soybean cultivars. Theor Appl Genet 128:1489–1505. https://doi.org/10.1007/s00122-015-2522-0 DOI

Hajimorad MR, Domier LL, Tolin SA, Whitham SA, Saghai Maroof MA (2018) Soybean mosaic virus: a successful potyvirus with a wide distribution but restricted natural host range. Mol Plant Pathol 19:1563–1579. https://doi.org/10.1111/mpp.12644 DOI

Hong X, Li S, Cheng X, Zhi H, Yin J, Xu K (2024) Searching for plant NLR immune receptors conferring resistance to potyviruses. Crop J 12:28–44. https://doi.org/10.1016/j.cj.2023.11.010 DOI

Ishibashi K, Saruta M, Shimizu T, Shu M, Anai T, Komatsu K, Yamada N, Katayose Y, Ishikawa M, Ishimoto M, Kaga A (2019) Soybean antiviral immunity conferred by dsRNase targets the viral replication complex. Nat Commun 10:4033. https://doi.org/10.1038/s41467-019-12052-5 DOI

Jian C, Han R, Chi Q, Wang S, Ma M, Liu X, Zhao H (2017) Virus-based microRNA silencing and overexpressing in common wheat (Triticum aestivum L.). Front Plant Sci 8:500. https://doi.org/10.3389/fpls.2017.00500 DOI

Kim HJ, Kim M-J, Pak JH, Im HH, Lee DH, Kim K-H, Lee J-H, Kim D-H, Choi HK, Jung HW, Chung Y-S (2016) RNAi-mediated Soybean mosaic virus (SMV) resistance of a Korean soybean cultivar. Plant Biotechnol Rep 10:257–267. https://doi.org/10.1007/s11816-016-0402-y DOI

Kis A, Tholt G, Ivanics M, Varallyay E, Jenes B, Havelda Z (2016) Polycistronic artificial miRNA-mediated resistance to wheat dwarf virus in barley is highly efficient at low temperature. Mol Plant Pathol 17:427–437. https://doi.org/10.1111/mpp.12291 DOI

Mallory AC, Reinhart BJ, Jones-Rhoades MW, Tang G, Zamore PD, Barton MK, Bartel DP (2004) MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5’ region. EMBO J 23:3356–3364. https://doi.org/10.1038/sj.emboj.7600340 DOI

Miao S, Liang C, Li J, Baker B, Luo L (2021) Polycistronic artificial microrna-mediated resistance to cucumber green mottle mosaic virus in cucumber. Int J Mol Sci. https://doi.org/10.3390/ijms222212237 DOI

Mitter N, Dietzgen RG (2012) Use of hairpin RNA constructs for engineering plant virus resistance. Methods Mol Biol 894:191–208. https://doi.org/10.1007/978-1-61779-882-5_13 DOI

Park W, Zhai J, Lee JY (2009) Highly efficient gene silencing using perfect complementary artificial miRNA targeting AP1 or heteromeric artificial miRNA targeting AP1 and CAL genes. Plant Cell Rep 28:469–480. https://doi.org/10.1007/s00299-008-0651-5 DOI

Qu J, Ye J, Fang R (2007) Artificial microRNA-mediated virus resistance in plants. J Virol 81:6690–6699. https://doi.org/10.1128/JVI.02457-06 DOI

Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121–1133. https://doi.org/10.1105/tpc.105.039834 DOI

Simon AE (2015) 3’UTRs of carmoviruses. Virus Res 206:27–36. https://doi.org/10.1016/j.virusres.2015.01.023 DOI

Szittya G, Silhavy D, Molnar A, Havelda Z, Lovas A, Lakatos L, Banfalvi Z, Burgyan J (2003) Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation. EMBO J 22:633–640. https://doi.org/10.1093/emboj/cdg74 DOI

Varkonyi-Gasic E, Wu R, Wood M, Walton EF, Hellens RP (2007) Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3:12. https://doi.org/10.1186/1746-4811-3-12 DOI

Vu TV, Choudhury NR, Mukherjee SK (2013) Transgenic tomato plants expressing artificial microRNAs for silencing the pre-coat and coat proteins of a begomovirus, tomato leaf curl New Delhi virus, show tolerance to virus infection. Virus Res 172:35–45. https://doi.org/10.1016/j.virusres.2012.12.008 DOI

Xu B, Zhu Y, Cao C, Chen H, Jin Q, Li G, Ma J, Yang SL, Zhao J, Zhu J, Ding Y, Fang X, Jin Y, Kwok CK, Ren A, Wan Y, Wang Z, Xue Y, Zhang H, Zhang QC, Zhou Y (2022) Recent advances in RNA structurome. Sci China Life Sci 65:1285–1324. https://doi.org/10.1007/s11427-021-2116-2 DOI

Yin J, Wang L, Jin T, Nie Y, Liu H, Qiu Y, Yang Y, Li B, Zhang J, Wang D, Li K, Xu K, Zhi H (2021) A cell wall-localized NLR confers resistance to Soybean mosaic virus by recognizing viral-encoded cylindrical inclusion protein. Mol Plant 14:1881–1900. https://doi.org/10.1016/j.molp.2021.07.013 DOI