Artificial microRNA-mediated resistance to cucumber green mottle mosaic virus in Nicotiana benthamiana
We describe a Nicotiana benthamiana system for rapid identification of artificial microRNA (amiRNA) to control cucumber green mottle mosaic virus (CGMMV) disease.
Although artificial miRNA technology has been used to control other viral diseases, it has not been applied to reduce severe cucumber green mottle mosaic virus (CGMMV) disease and crop loss in the economically important cucurbits. We used our system to identify three amiRNAs targeting CGMMV RNA (amiR1-CP, amiR4-MP and amiR6-Rep) and show that their expression reduces CGMMV replication and disease in virus-infected plants. This work streamlines the process of generating amiRNA virus-resistant crops and can be broadly applied to identify active antiviral amiRNAs against a broad spectrum of viruses to control disease in diverse crops.
KeywordsCucumber green mottle mosaic virus Artificial microRNA RNA silencing Virus resistance Crop protection
This work was partially supported by the National Key Research and Development Program of China (2017YFD0201601), the National Science Foundation of China (NSFC) project (31371910) and China Scholarship Council (CSC) (201606350070).
Compliance with ethical standards
Conflict of interest
The authors declare no competing interests.
- Ainsworth GC (1935) Mosaic diseases of the cucumber. Ann Appl Biol 22:55–67. https://doi.org/10.1111/j.1744-7348.1935.tb07708.x/full CrossRefGoogle Scholar
- Alvarez JP, Pekker I, Goldshmidt A, Blum E, Amsellem Z, Eshed Y (2006) Endogenous and synthetic microRNAs stimulate simultaneous, efficient, and localized regulation of multiple targets in diverse species. Plant Cell 18:1134–1151. https://doi.org/10.1105/tpc.105.040725 CrossRefPubMedPubMedCentralGoogle Scholar
- Baker C (2016) Cucumber green mottle mosaic virus (CGMMV) found in the United States (California) in melon. Pest alert, Florida Department of Agriculture and Consumer Services, Division of Plant Industry (DACS-P-01863)Google Scholar
- Chen X (2009) Small RNAs and their roles in plant development. Annu Rev Cell Dev Biol 25:21–44. https://doi.org/10.1146/annurev.cellbio.042308.113417 CrossRefPubMedPubMedCentralGoogle Scholar
- Dombrovsky A, Tran-Nguyen LTT, Jones RAC (2017) Cucumber green mottle mosaic virus: rapidly increasing global distribution, etiology, epidemiology, and management. Annu Rev Phytopathol 55:231–256. https://doi.org/10.1146/annurev-phyto-080516-035349 CrossRefPubMedGoogle Scholar
- Hollings M, Komuro Y, Tochihara H (1975) Cucumber green mottle mosaic virus. CMI/AAB Descriptions of Plant Viruses No. 154. Kew, United KingdomGoogle Scholar
- Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAS and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53. https://doi.org/10.1146/annurev.arplant.57.032905.105218 CrossRefPubMedGoogle Scholar
- Komuro Y (1971) Cucumber green mottle mosaic virus on cucumber and watermelon and melon necrotic spot virus on muskmelon. J Agric Res Quart 6:41–45Google Scholar
- Kung YJ, Lin SS, Huang YL, Chen TC, Harish SS, Chua NH, Yeh SD (2012) Multiple artificial microRNAs targeting conserved motifs of the replicase gene confer robust transgenic resistance to negative-sense single-stranded RNA plant virus. Mol Plant Pathol 13:303–317. https://doi.org/10.1111/j.1364-3703.2011.00747.x CrossRefPubMedGoogle Scholar
- Lafforgue G, Martínez F, Niu QW, Chua NH, Daròs JA, Elena SF (2013) Improving the effectiveness of artificial microRNA (amiR)-mediated resistance against Turnip Mosaic Virus by combining two amiRs or by targeting highly conserved viral genomic regions. J Virol 87:8254–8256. https://doi.org/10.1128/JVI.00914-13 CrossRefPubMedPubMedCentralGoogle Scholar
- Liang CQ, Yan M, Luo LX, Liu PF, Li JQ (2015) Phylogenetic and bioinformatics analysis of replicase gene sequence of Cucumber green mottle mosaic virus. Chin J Virol 31:620–628. https://doi.org/10.13242/j.cnki.bingduxuebao.002821 (in Chinese) CrossRefGoogle Scholar
- Sablok G, Pérez-Quintero ÁL, Hassan M, Tatarinova TV, López C (2011) Artificial microRNAs (amiRNAs) engineering-on how microRNA-based silencing methods have affected current plant silencing research. Biochem Biophys Res Commun 406:315–319. https://doi.org/10.1016/j.bbrc.2011.02.045 CrossRefPubMedGoogle Scholar
- Shim CK, Han KS, Lee JH, Bae DW, Kim DK, Kim HK (2005) Isolation and characterization of watermelon isolate of Cucumber green mottle mosaic virus (CGMMV-HY1) from watermelon plants with severe mottle mosaic symptoms. J Plant Pathol 21:167–171. https://doi.org/10.5423/PPJ.2005.21.2.167 CrossRefGoogle Scholar
- Song YZ, Han QJ, Jiang F, Sun RZ, Fan ZH, Zhu CX, Wen FJ (2014) Effects of the sequence characteristics of miRNAs on multi-viral resistance mediated by single amiRNAs in transgenic N. benthamiana. Plant Physiol Biochem 77:90–98. https://doi.org/10.1016/j.plaphy.2014.01.008 CrossRefPubMedGoogle Scholar
- Sunkar R, Kapoor A, Zhu J (2006) Posttranscriptional induction of two Cu/Zn superoxide dismutase genes in Arabidopsis in mediated by downregulation of miR398 and important for oxidative stress tolerance. Plant Cell 18:2051–2065. https://doi.org/10.1105/tpc.106.041673 CrossRefPubMedPubMedCentralGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar