Figure 8.

Virus-induced gene silencing in plants infected with LIYV vectors. (A) Silencing of the phytoene desaturase (PDS) gene with LIYV-L2C_tPDS in Hc-Pro transgenic N. benthamiana plants. (a) Phenotypes of LIYV-WT and LIYV-L2C_tPDS infected N. benthamiana plants photographed at 3 wpi; (b) Detection of viral infection and insertion integrity by RT-PCR with total RNA extracted from upper non-inoculated leaves at 3 wpi and 5 wpi. Two primer sets were used to amplify the sequence of LIYV CP (CP, 530 bp) and the truncated PDS cassette (Insert, 1026 bp), LIYV-WT without the insert was amplified as a control (317 bp). The bands in between at 5 wpi indicate partial deletions of the inserted cassette. (B) Silencing of GFP transgene with LIYV-L2C_tGFP in 16c transgenic N. benthamiana plants. (a) Silencing effects of LIYV-L2CtGFP observed under UV light at 3 wpi and 5 wpi. Plants inoculated with buffer (Mock) and LIYV-WT were negative controls; (b) Viral infection and insertion integrity were assessed by RT-PCR with total RNA extracted from upper non-inoculated leaves at 5 wpi. Two primer sets were used to amplify the sequence of LIYV CP (CP, 530 bp) and the truncated GFP cassette (Insert, 907 bp); (c) Immunoblot analysis of LIYV CP and GFP accumulation in upper non-inoculated leaves of LIYV-WT and LIYV-L2C_tGFP infected plants using LIYV CP and GFP specific antibodies. The Ponceau S stained Rubisco large subunit serves as a loading control.