The barley m6A demethylase HvALKBH1B undergoes phase separation to enhance immunity to a plant rhabdovirus

N6-methyladenosine (m6A) is one of the most abundant RNA modifications and plays important roles in plant development and stress responses. However, how m6A affects the infection cycles of plant viruses, especially plant negative-stranded RNA (NSR) viruses, remains poorly understood. Here, we demonstrate that the accessory gene P6 mRNA of the insect-borne plant rhabdovirus barley yellow striate mosaic virus (BYSMV) harbors a high level of m6A modification. The m6A-mutated P6 mRNA is less stable than the wild-type P6 mRNA, and the corresponding mutant virus exhibits reduced infectivity. Additionally, we identified HvALKBH1B, a m6A demethylase from barley (Hordeum vulgare) that displays strong antiviral activity through m6A removal from viral mRNA. Furthermore, HvALKBH1B undergoes liquid-liquid phase separation (LLPS) and binds P6 mRNA into cytoplasmic condensates. Deletion assays indicated that the intrinsically disordered region 3 (IDR3) of HvALKBH1B is required for its LLPS capacity and antiviral function. Overexpression of a truncated HvALKBH1B variant (HvALKBH1BT) in transgenic barley confers enhanced resistance to BYSMV, whereas CRISPR knockout lines exhibit increased susceptibility. Altogether, our results reveal that plant rhabdoviruses exploit m6A modification to promote viral mRNA stability and infection, which is counteracted by the host demethylase HvALKBH1B in barley to improve antiviral immunity.