Abstract
Simple SummaryRice stripe virus is a disastrous viral disease that causes significant yield losses in rice production in South, Southeast, and East Asian countries. To decrease the use of chemical insecticides, genetic engineering has become a pivotal strategy to combat the virus. In this study, we constructed a dimeric artificial microRNA precursor expression vector that targets the viral MP gene based on the structure of the rice osa-MIR528 precursor. Marker-free transgenic plants successfully expressing the MP amiRNAs were obtained and were highly resistant to RSV infection. The novel rice germplasms generated are promising for RSV control.Rice stripe virus (RSV) causes one of the most serious viral diseases of rice. RNA interference is one of the most efficient ways to control viral disease. In this study, we constructed an amiRNA targeting the RSV MP gene (amiR MP) based on the backbone sequence of the osa-MIR528 precursor, and obtained marker-free transgenic rice plants constitutively expressing amiR MP by Agrobacterium tumefaciens-mediated transformation. A transient expression assay demonstrated that dimeric amiR MP could be effectively recognized and cleaved at the target MP gene in plants. Northern blot of miRNA indicated that amiR MP-mediated viral resistance could be stably inherited. The transgenic rice plants were highly resistant to RSV (73–90%). Our research provides novel rice germplasm for RSV control.
Highlights
Rice stripe disease, caused by rice stripe virus (RSV), is one of the most devastating viral diseases in rice production worldwide
Artificial microRNAs of RSV movement protein (MP) were predicted and chosen on the website (Figure 1A) and osa-MIR528 precursor was used as the backbone for expressing artificial miRNA (amiRNA) targeting the RSV MP gene (amiR MP), as described before [12,21]
The natural miR528/miR528* sequences were replaced by viral amiR MP/amiR MP* sequences using oligonucleotide-directed mutagenesis and a precursor of amiR MP was obtained, as shown in the schematic diagram (Figure 1B)
Summary
Rice stripe disease, caused by rice stripe virus (RSV), is one of the most devastating viral diseases in rice production worldwide. The viral disease first appeared in Japan, and emerged in over 20 provinces in China, including Jiangsu and Zhejiang. In the period 2002 to 2004, an outbreak of RSV caused devastating economic losses and affected about 80% of the rice cultivated in Jiangsu province [1,2]. RSV is classified in the genus Tenuivirus, and its genome has four single-stranded RNA genome segments. RNA1 encodes the RNA-dependent RNA polymerase (RdRp) on the complementary strand, while RNAs 2, 3 and 4 have an ambisense coding strategy [3]. RNA4 encodes a disease-specific protein (SP) in the virion sense and a movement protein (MP) in the complementary sense [4,5]. Traditional breeding for resistance is slow and inefficient and is challenged by the genetic linkage of agronomic traits with disease resistance genes [8]
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