Plant RNA-dependent RNA polymerase 1(RDR1) is a key component of antiviral RNA silencing pathway contributing to the biogenesis of the virus-derived small interfering RNAs (vsiRNAs). This enzyme is also responsible for producing virus-activated endogenous siRNAs (vasiRNAs) to stimulate the broad-spectrum antiviral activity through silencing host genes. The expression of RDR1 orthologs in various plants is usually induced by virus infection. However, the molecular mechanisms of activation of RDR1 expression in response to virus infection remain unknown. Here, we show that a monocot-specific miRNA, miR444, is a key factor to relay the antiviral signaling from virus infection to OsRDR1 expression. Expression of miR444 is enhanced by infection of Rice stripe virus (RSV) and overexpression of miR444 improves rice resistance against RSV infection accompanied by upregulation of OsRDR1 expression. We further show that three miR444 targets, the MIKCC-type MADS-box proteins OsMADS23, OsMADS27a and OsMADS57, form homodimers and heterodimers between them to repress the expression of OsRDR1 by directly binding to the CArG motifs of its promoter. Consequently, increased level of miR444 diminishes the repressive roles of OsMADS23, OsMADS27a and OsMADS57 on OsRDR1 transcription thus to activate OsRDR1-dependent antiviral RNA silencing pathway. We also show that overexpression of miR444-resistant OsMADS57 reduced OsRDR1 expression and rice resistance against RSV infection and knockout of OsRDR1 reduced rice resistance against RSV infection. In conclusion, our results unveil a molecular cascade in the rice antiviral pathway in which miR444 and its MADS-box targets directly control OsRDR1 transcription.

MiR444 promotes phytohormone brassinosteroid biosynthesis, conferring ammonium-triggered root elongation inhibition in rice.