Background: N⁶-methyladenosine (m⁶A) is the most common RNA modification in eukaryotes and has been implicated as a novel epigenetic marker that is involved in various biological processes. The pattern and functional dissection of m⁶A in the regulation of several major human viral diseases have already been reported. However, the patterns and functions of m⁶A distribution in plant disease bursting remain largely unknown.Results: We analyse the high-quality m⁶A methylomes in rice plants infected with two devastating viruses. We find that the m⁶A methylation is mainly associated with genes that are not actively expressed in virus-infected rice plants. We also detect different m⁶A peak distributions on the same gene, which may contribute to different antiviral modes between rice stripe virus or rice black-stripe dwarf virus infection. Interestingly, we observe increased levels of m⁶A methylation in rice plant response to virus infection. Several antiviral pathway-related genes, such as RNA silencing-, resistance-, and fundamental antiviral phytohormone metabolic-related genes, are also m⁶A methylated. The level of m⁶A methylation is tightly associated with its relative expression levels.Conclusions: We revealed the dynamics of m⁶A modification during the interaction between rice and viruses, which may act as a main regulatory strategy in gene expression. Our investigations highlight the significance of m⁶A modifications in interactions between plant and viruses, especially in regulating the expression of genes involved in key pathways.

RNA viruses encode various RNA binding proteins that function in many steps of viral infection cycles. These proteins function as RNA helicases, methyltransferases, RNA-dependent RNA polymerases, RNA silencing suppressors, RNA chaperones, movement proteins, and so on. Although many of the proteins bind the viral RNA genome during different stages of infection, our knowledge about the coordination of their functions is limited. In this study, we describe a novel role for the Barley stripe mosaic virus (BSMV) γb as an enhancer of αa RNA helicase activity, and we show that the γb protein is recruited by the αa viral replication protein to chloroplast membrane sites of BSMV replication. Mutagenesis or deletion of γb from BSMV resulted in reduced positive strand (+) RNAα accumulation, but γb mutations abolishing viral suppressor of RNA silencing (VSR) activity did not completely eliminate genomic RNA replication. In addition, cis- or trans-expression of the Tomato bushy stunt virus p19 VSR protein failed to complement the γb replication functions, indicating that the direct involvement of γb in BSMV RNA replication is independent of VSR functions. These data support a model whereby two BSMV-encoded RNA-binding proteins act coordinately to regulate viral genome replication and provide new insights into strategies whereby double-stranded viral RNA unwinding is regulated, as well as formation of viral replication complexes. In order to maximize reproduction, RNA viruses often encode multifunctional proteins to perform different activities during replication. We report that the Barley stripe mosaic virus γb VSR protein is recruited to viral replication sites through interactions with the αa replicase subunit protein where it enhances αa helicase activity to increase the levels of BSMV replication. Our discovery of the γb RNA helicase enhancement activity provides a novel function associated with a viral suppressor of RNA silencing that enhances understanding of multifunctional strategies whereby virus-encoded proteins contribute to genome replication and counter host defense mechanisms.

Three-dimensional visualization identifies structural remodeling in chloroplasts during barley stripe mosaic virus infection.