Protocol for propagating and inoculating rice grassy stunt virus in rice plants using brown planthoppers

Rice grassy stunt virus (RGSV) causes a severe viral disease in rice and is transmitted by brown planthoppers (BPHs) ( Nilaparvata lugens Stål). Here, we present a protocol for propagating RGSV and establishing controlled infection in rice plants. We describe steps for preparing rice seedlings, maintaining and propagating BPH colonies, generating viruliferous BPHs, and performing genotype-separated inoculation. This protocol provides a standardized framework for BPH-mediated RGSV inoculation and helps reduce vector-associated bias in infection assays across rice genotypes. For complete details on the use and execution of this protocol, please refer to Yang et al. 1