Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance

Abstract

Plant microRNAs (miRNAs) regulate the abundance of target mRNAs by guiding their cleavage at the sequence complementary region. We have modified an Arabidopsis thaliana miR159 precursor to express artificial miRNAs (amiRNAs) targeting viral mRNA sequences encoding two gene silencing suppressors, P69 of turnip yellow mosaic virus (TYMV) and HC-Pro of turnip mosaic virus (TuMV). Production of these amiRNAs requires A. thaliana DICER-like protein 1. Transgenic A. thaliana plants expressing amiR-P69¹⁵⁹ and amiR-HC-Pro¹⁵⁹ are specifically resistant to TYMV and TuMV, respectively. Expression of amiR-TuCP¹⁵⁹ targeting TuMV coat protein sequences also confers specific TuMV resistance. However, transgenic plants that express both amiR-P69¹⁵⁹ and amiR-HC-Pro¹⁵⁹ from a dimeric pre-amiR-P69¹⁵⁹/amiR-HC-Pro¹⁵⁹ transgene are resistant to both viruses. The virus resistance trait is displayed at the cell level and is hereditable. More important, the resistance trait is maintained at 15 °C, a temperature that compromises small interfering RNA–mediated gene silencing. The amiRNA-mediated approach should have broad applicability for engineering multiple virus resistance in crop plants.