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

Authors: LIANG, CHAOQIONG - China Agricultural University; HAO, JIANJUN - University Of Maine; LI, JIANQIANG - China Agricultural University; Baker, Barbara; LUO, LAIXIN - Huazhong Agricultural University

Submitted to: Planta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/6/2019
Publication Date: 8/6/2019
Citation: Liang, C., Hao, J., Li, J., Baker, B.J., Luo, L. 2019. Artificial microRNA-mediated resistance to cucumber green mottle mosaic virus in Nicotiana benthamiana. Planta. 250:1591–1601. https://doi.org/10.1007/s00425-019-03252-w.
DOI: https://doi.org/10.1007/s00425-019-03252-w

Interpretive Summary: Cucumber green mottle mosaic virus (CGMMV) infects cucurbit plants and causes severe economic losses to crop production. RNA silencing has been employed successfully to control several viral diseases but has not yet been applied for management of CGMMV disease. We developed CGMMV-Nicotiana benthamiana as a model virus-host pathosystem to test if artificial microRNAs (amiRNAs) targeting CGMMV genes can protect plants from CGMMV disease. We used this pathosystem to test amiRNAs directed against conserved sequences of three CGMMV genes offered protection against viral disease. Our results showed that amiRNAs targeting viral genes correlated with viral “resistance” with reduced viral replication and reduced incidence of CGMMV disease compared to controls and demonstrates the successful application of amiRNA silencing for protection against CGMMV disease. Our model system for evaluating artificial microRNA-mediated resistance to plant viruses will streamline the process of generating amiRNA virus resistant crops and can be applied to identify active antiviral amiRNAs against a broad spectrum of viruses to control viral diseases in diverse crops.

Technical Abstract: Artificial microRNA (amiRNA) technology has been used to control viral diseases, however an efficient screening process to identify amiRNAs active in protecting crops from disease has not been described. ARS scientists in Albany, California in collaboration with scientists at China Agricultural University developed an N. benthamiana system for rapid identification of artificial microRNAs to control virus disease. Cucumber green mottle mosaic virus (CGMMV) infects cucurbit plants, and can cause severe economic losses to crop production. RNA silencing has been employed successfully to control several viral diseases but has not yet been applied for management of CGMMV disease. We developed a CGMMV-Nicotiana benthamiana pathosystem to test if artificial microRNAs (amiRNAs) targeting CGMMV genes can protect plants from CGMMV disease. In this study we constructed six amiRNAs (amiR1-CP, amiR2-CP, amiR3-MP, amiR4-MP, amiR5-Rep and amiR6-Rep) targeting conserved sequences of CGMMV genes for coat protein (CP), movement protein (MP) or replicase (Rep) using amiRNA expression vectors based on Arabidopsis thaliana miRNA precursor backbones. The CGMMV-amiRNA vectors were introduced into N. benthamiana using Agrobacterium tumefaciens infiltration and protection from viral disease was assessed by subsequent viral infection of infiltrated plants. Our results showed that N. benthamiana expressing high levels of amiR1-CP, amiR4-MP or amiR6-Rep showed viral “resistance” or “tolerance” with reduced viral replication and reduced incidence of CGMMV disease compared to N. benthamiana expressing “empty” amiRNA vector controls. This work streamlines the process of generating amiR 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.