An Engineered Citrus Tristeza Virus (T36CA)-Based Vector Induces Gene-Specific RNA Silencing and Is Graft Transmissible to Commercial Citrus Varieties.

A protein-expressing citrus tristeza virus-based vector construct, pT36CA-V1.3, obtained from a California isolate of the T36 strain (T36CA), was retooled into a virus-induced gene silencing system intended for use with studies of California citrus. Virus-induced gene silencing constructs engineered with a truncated () gene sequence in the sense or antisense orientation worked equally well to silence the endogenous gene. In a parallel effort to optimize vector performance, two nonsynonymous nucleotides in open reading frame 1a of pT36CA-V1.3 were replaced with those conserved in the reference sequences from the T36CA cDNA library. The resulting viruses, T36CA-V1.4 (with one amino acid modification: D760N) and T36CA-V1.5 (with two amino acid modifications: D760N and P1174L), along with T36CA-V1.3, were individually propagated in and plants. Enzyme-linked immunosorbent assay (ELISA) measurements of extracts of the newly emerged leaves suggested that all three viruses accumulated to similar levels in plants at 5 weeks postinoculation. ELISA values of T36CA-V1.4- and -V1.5-infected samples were significantly higher than that of T36CA-V1.3-infected samples within an 8- to 12-month postinoculation window, suggesting a higher accumulation of T36CA-V1.4 and -V1.5 than T36CA-V1.3. However, at 36 months postinoculation, the ELISA values suggested that all three viruses accumulated to similar levels. When plants infected with each of the three viruses were grafted to commercial citrus varieties, a limited number of receptor plants became infected, demonstrating a weak but nonetheless (the first) successful delivery of T36CA to California-grown commercial citrus.