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Quantitative Analysis of Efficient Endogenous Gene Silencing in Nicotiana benthamiana Plants Using Tomato bushy stunt virus Vectors That Retain the Capsid Protein Gene

¹Dipartimento di Scienze e Tecnologie Agroambientali, Bologna, Italy; ²Plant Biology Graduate Group, University of California, Davis, U.S.A.; ³Istituto di Virologia Vegetale, CNR-Torino, Italy; ⁴Department of Plant Sciences and ⁵Department of Plant Pathology, University of California, Davis, U.S.A.

Tomato bushy stunt virus (TBSV) coat protein (CP) replacement vectors have been used previously to silence transgenes (e.g., the green fluorescent protein gene) but have not been effective for silencing endogenous plant genes. New TBSV vectors which retained the CP gene were developed by engineering an XhoI restriction site in three positions (3f, CEB, and CEA) of the pTBSV-100 infectious clone. Magnesium chelatase (ChlH) and phytoene desaturase (PDS) were chosen as targets for endogenous gene silencing. Initial experiments using CP replacement vectors with a 230-bp sense or antisense ChlH insert gave a silencing phenotype prominent only in the first new leaves above those inoculated. No silencing phenotype was apparent beyond these leaves whereas, for PDS, no silencing phenotype was observed. When plants were inoculated with the XhoI insert vectors containing ChlH and PDS sequences, plants showed a silencing phenotype extensively throughout the challenged plant, indicating an improved ability for virus movement and silencing in Nicotiana benthamiana host plants. Silencing efficiencies were quantified using real-time reverse-transcription polymerase chain reaction, indicating specific silencing effects of each individual silencing vector. Only one recombinant vector (pPD-3f5), where the XhoI insert was at the 3′ end of the CP gene, failed to give effective silencing. Here, we show that our new CP-retaining TBSV vectors (CEA-CEB) form typical TBSV virions, retain silencing inserts of variable lengths (110 to 260 nucleotides), and can systemically silence endogenous genes in N. benthamiana.