, Volume 250, Issue 5, pp 1591–1601 | Cite as

Artificial microRNA-mediated resistance to cucumber green mottle mosaic virus in Nicotiana benthamiana

  • Chaoqiong Liang
  • Jianjun Hao
  • Jianqiang Li
  • Barbara BakerEmail author
  • Laixin LuoEmail author
Original Article


Main conclusion

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.


Cucumber 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.

Supplementary material

425_2019_3252_MOESM1_ESM.png (882 kb)
Fig. S1 The positions of amiRNAs target conserved regions of genes for coat protein (CP), movement protein (MP) and replicase (Rep) in CGMMV genome. The purple boxes denote the positions of the target sites of amiRNAs (PNG 881 kb)
425_2019_3252_MOESM2_ESM.pdf (479 kb)
Fig. S2 The construction strategy of the amiRNA plant expression vectors (PDF 479 kb)
425_2019_3252_MOESM3_ESM.tif (185 kb)
Fig. S3 Products of overlapping PCR for six amiRNA precursors. I-12 was synthesized using Primers 1 and 2 in PCR reaction ①. I-345 was synthesized using Primers 3, 4 and 5 in PCR reaction ②. I-12 and I-345 were used as template and Primers 1 and 5 were used to synthesize amiRNA precursor sequence in PCR reaction ③ (TIFF 185 kb)
425_2019_3252_MOESM4_ESM.pdf (42 kb)
Fig. S4 Secondary structure of amiRNA precursors by bioinformatic analysis using MFOLD program (PDF 41 kb)
425_2019_3252_MOESM5_ESM.tif (65 kb)
Fig. S5 The relative amiRNA band intensities with respect to the corresponding miR172 bands, estimated using Quantity One Basic, are shown as a column graph (TIFF 64 kb)
425_2019_3252_MOESM6_ESM.xlsx (20 kb)
Table S1. Primers used to synthesize amiRNA precursors by overlapping PCR. Table S2. Detail of repeat setting of the experiment. Table S3. Probes used to detect amiRNA expression by northern blot hybridization analysis. Table S4. Primers used for qRT-PCR in the detection of amiRNA overexpression. Table S5. Primers used for qRT-PCR in the detection of CGMMV. Table S6. Prediction of secondary structure of amiRNA (XLSX 20 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.College of Plant Protection/Beijing Key Laboratory of Seed Disease Testing and ControlChina Agricultural UniversityBeijingChina
  2. 2.College of HorticultureChina Agricultural UniversityBeijingChina
  3. 3.School of Food and AgricultureThe University of MaineOronoUSA
  4. 4.Department of Plant and Microbial BiologyUniversity of California, BerkeleyBerkeleyUSA
  5. 5.United States Department of Agriculture, Plant Gene Expression CenterAgricultural Research ServiceAlbanyUSA

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