Simultaneous silencing of two different Arabidopsis genes with a novel virus-induced gene silencing vector
Tóm tắt
Virus-induced gene silencing (VIGS) is a useful tool for functional characterizations of plant genes. However, the penetrance of VIGS varies depending on the genes to be silenced, and has to be evaluated by examining the transcript levels of target genes. In this report, we report the development of a novel VIGS vector that permits a preliminary assessment of the silencing penetrance. This new vector is based on an attenuated variant of Turnip crinkle virus (TCV) known as CPB that can be readily used in Arabidopsis thaliana to interrogate genes of this model plant. A CPB derivative, designated CPB1B, was produced by inserting a 46 nucleotide section of the Arabidopsis PHYTOENE DESATURASE (PDS) gene into CPB, in antisense orientation. CPB1B induced robust PDS silencing, causing easily visible photobleaching in systemically infected Arabidopsis leaves. More importantly, CPB1B can accommodate additional inserts, derived from other Arabidopsis genes, causing the silencing of two or more genes simultaneously. With photobleaching as a visual marker, we adopted the CPB1B vector to validate the involvement of DICER-LIKE 4 (DCL4) in antiviral defense against TCV. We further revealed the involvement of ARGONAUTE 2 (AGO2) in PDS silencing and antiviral defense against TCV in dcl2drb4 double mutant plants. These results demonstrated that DOUBLE-STRANDED RNA-BINDING PROTEIN 4 (DRB4), whose protein product (DRB4) commonly partners with DCL4 in the antiviral silencing pathway, was dispensable for PDS silencing induced by CPB1B derivative in dcl2drb4 double mutant plants. The CPB1B-based vector developed in this work is a valuable tool with visualizable indicator of the silencing penetrance for interrogating Arabidopsis genes, especially those involved in the RNA silencing pathways.
Tài liệu tham khảo
Ding SW. RNA-based antiviral immunity. Nat Rev Immunol. 2010;10:632–44.
Wang M, Weiberg A, Jin H. Pathogen small RNAs: a new class of effectors for pathogen attacks. Mol Plant Pathol. 2015;16:219–23.
Hiraguri A, Itoh R, Kondo N, Nomura Y, Aizawa D, Murai Y, Koiwa H, Seki M, Shinozaki K, Fukuhara T. Specific interactions between Dicer-like proteins and HYL1/DRB-family dsRNA-binding proteins in Arabidopsis thaliana. Plant Mol Biol. 2005;57:173–88.
Baulcombe DC. Fast forward genetics based on virus-induced gene silencing. Curr Opin Plant Biol. 1999;2:109–13.
Becker A, Lange M. VIGS—genomics goes functional. Trends Plant Sci. 2010;15:1–4.
Burch-Smith TM, Anderson JC, Martin GB, Dinesh-Kumar SP. Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J. 2004;39:734–46.
Dommes AB, Gross T, Herbert DB, Kivivirta KI, Becker A. Virus-induced gene silencing: empowering genetics in non-model organisms. J Exp Bot. 2019;70:757–70.
Kant R, Dasgupta I. Gene silencing approaches through virus-based vectors: speeding up functional genomics in monocots. Plant Mol Biol. 2019;100:3–18.
Liu Y, Schiff M, Marathe R, Dinesh-Kumar SP. Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J. 2002;30:415–29.
Gedling CR, Ali EM, Gunadi A, Finer JJ, Xie K, Liu Y, Yoshikawa N, Qu F, Dorrance AE. Improved apple latent spherical virus-induced gene silencing in multiple soybean genotypes through direct inoculation of agro-infiltrated Nicotiana benthamiana extract. Plant Methods. 2018;14:19.
Igarashi A, Yamagata K, Sugai T, Takahashi Y, Sugawara E, Tamura A, Yaegashi H, Yamagishi N, Takahashi T, Isogai M, Takahashi H, Yoshikawa N. Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes. Virology. 2009;386:407–16.
Lentz EM, Kuon JE, Alder A, Mangel N, Zainuddin IM, McCallum EJ, Anjanappa RB, Gruissem W, Vanderschuren H. Cassava geminivirus agroclones for virus-induced gene silencing in cassava leaves and roots. Plant Methods. 2018;14:73.
Tzean Y, Lee MC, Jan HH, Chiu YS, Tu TC, Hou BH, Chen HM, Chou CN, Yeh HH. Cucumber mosaic virus-induced gene silencing in banana. Sci Rep. 2019;9:11553.
Cao M, Ye X, Willie K, Lin J, Zhang X, Redinbaugh MG, Simon AE, Morris TJ, Qu F. The capsid protein of Turnip crinkle virus overcomes two separate defense barriers to facilitate systemic movement of the virus in Arabidopsis. J Virol. 2010;84:7793–802.
Zhang X, Zhang X, Singh J, Li D, Qu F. Temperature-dependent survival of Turnip crinkle virus-infected Arabidopsis plants relies on an RNA silencing-based defense that requires dcl2, AGO2, and HEN1. J Virol. 2012;86:6847–54.
Zhang X, Zhang X, Wu K, Liu Z, Li D, Qu F. Incomplete DRB4-dependence of the DCL4-mediated antiviral defense. Sci Rep. 2016;6:39244.
Carrington JC, Heaton LA, Zuidema D, Hillman BI, Morris TJ. The genome structure of Turnip crinkle virus. Virology. 1989;170:219–26.
Qu F, Ye X, Morris TJ. Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1. Proc Natl Acad Sci USA. 2008;105:14732–7.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 2001;25:402–8.
Cui H, Wang A. An efficient viral vector for functional genomic studies of Prunus fruit trees and its induced resistance to Plum pox virus via silencing of a host factor gene. Plant Biotechnol J. 2017;15:344–56.
Singh DK, Lee HK, Dweikat I, Mysore KS. An efficient and improved method for virus-induced gene silencing in sorghum. BMC Plant Biol. 2018;18:123.
Bruun-Rasmussen M, Madsen CT, Jessing S, Albrechtsen M. Stability of Barley stripe mosaic virus-induced gene silencing in barley. Mol Plant Microbe Interact. 2007;20:1323–31.
Scofield SR, Huang L, Brandt AS, Gill BS. Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway. Plant Physiol. 2005;138:2165–73.
Deleris A, Gallego-Bartolome J, Bao J, Kasschau KD, Carrington JC, Voinnet O. Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. Science. 2006;313:68–71.
Carbonell A, Carrington JC. Antiviral roles of plant ARGONAUTES. Curr Opin Plant Biol. 2015;27:111–7.
Adhikary D, Khatri-Chhetri U, Tymm FJM, Murch SJ, Deyholos MK. A virus-induced gene-silencing system for functional genetics in a betalainic species, Amaranthus tricolor (Amaranthaceae). Appl Plant Sci. 2019;7:01221.
Bedoya LC, Martínez F, Orzáez D, Daròs JA. Visual tracking of plant virus infection and movement using a reporter MYB transcription factor that activates anthocyanin biosynthesis. Plant Physiol. 2012;158:1130–8.
Tian J, Pei H, Zhang S, Chen J, Chen W, Yang R, Meng Y, You J, Gao J, Ma N. TRV-GFP: a modified Tobacco rattle virus vector for efficient and visualizable analysis of gene function. J Exp Bot. 2014;65:311–22.
Gilbertson RL, Sudarshana M, Jiang H, Rojas MR, Lucas WJ. Limitations on geminivirus genome size imposed by plasmodesmata and virus-encoded movement protein: insights into DNA trafficking. Plant Cell. 2003;15:2578–91.
Qu F, Morris TJ. Encapsidation of Turnip crinkle virus is defined by a specific packaging signal and RNA size. J Virol. 1997;71:1428–35.
Rao AL. Genome packaging by spherical plant RNA viruses. Annu Rev Phytopathol. 2006;44:61–87.