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Dimeric artificial microRNAs mediate highly efficient RSV and RBSDV resistance in transgenic rice plants

Lin Sun;Chao Lin;Yunzhi Song;Jinwen Du;Fujiang Wen;Changxiang Zhu

  Infection of Rice stripe virus(RSV) and Rice black streaked dwarf virus(RBSDV) on rice results in severe economic losses on crop production.As the natural resistance resources against these viruses are limited,it is imperative to elaborate a biotechnological approach that will provide effective and safe immunity to RSV and RBSDV.In this study,we generated three dimeric amiRNA precursor expression vectors(pamiR-M,pamiR-3 and pamiR-U) that simultaneously targeting RSV and RBSDV based on the structure of rice osa-MIR528 precursor.The transgenic rice plants were obtained by Agrobacterium tumefaciens-mediated transformation and proved to express amiRNAs successfully.The viral challenge assays revealed that these transgenic plants could be conferred highly specific resistance against RSV and RBSDV infection simultaneously.The amiR-RSVU and the amiR-RBSDVU induced the highest virus resistance against RSV and RBSDV,respectively.The target viral RNAs could be cleavaged by the amiRNAs and there was a relationship between the resistance level and the accumulation of amiRNA expression.A Northern blot assay verified that silencing was induced by the original amiRNAs and could be bilaterally extended by the siRNA pathway.The amiRNA together with the secondary siRNAs mediated the degradation of viral RNAs.Genetic stability assay showed that transgene and amiRNAmediated virus resistance can be stably inherited toT2.……