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Abstract Background To investigate the mechanism of RNA silencing suppression, the genetic transformation of viral suppressors of RNA silencing (VSRs) in Arabidopsis integrates ectopic VSR expression at steady state, which overcomes the VSR variations caused by different virus infections or limitations of host range. Moreover, identifying the insertion of the transgenic VSR gene is necessary to establish a model transgenic plant for the functional study of VSR. Methods Developing an endogenous AGO1-based in vitro RNA-inducing silencing complex (RISC) assay prompts further investigation into VSR-mediated suppression. Three P1/HC-Pro plants from turnip mosaic virus (TuMV) (P1/HC-Pro Tu ), zucchini yellow mosaic virus (ZYMV) (P1/HC-Pro Zy ), and tobacco etch virus (TEV) (P1/HC-Pro Te ) were identified by T-DNA Finder and used as materials for investigations of the RISC cleavage efficiency. Results Our results indicated that the P1/HC-Pro Tu plant has slightly lower RISC activity than P1/HC-Pro Zy plants. In addition, the phenomena are consistent with those observed in TuMV-infected Arabidopsis plants, which implies that HC-ProTu could directly interfere with RISC activity. Conclusions In this study, we demonstrated the application of various plant materials in an in vitro RISC assay of VSR-mediated RNA silencing suppression.