FIGURE 1.

Controlled expression of Rep and RepA in Nicotiana benthamiana leaves. (A) Generalized schematic representation of the BeYDV vectors used in this study. RB and LB, the right and left borders of the T-DNA region from Agrobacterium; NOS 3′, the nopaline synthase terminator from Agrobacterium; P19, the RNA silencing suppressor from tomato bushy stunt virus; 35S, the 35S promoter from cauliflower mosaic virus; LIR, the long intergenic region from BeYDV; 5′ UTR, the 5′ untranslated region as described in each experiment; GOI, the gene of interest, as described in each experiment; Ext 3′, the 3′ region from the tobacco extensin gene; SIR, the short intergenic region from BeYDV; Rep/RepA, the replication proteins from BeYDV, which are either present in wild-type form, or are deleted or mutated as described in each experiment. (B) Generalized schematic representation of the T-DNA region of the separated Rep/RepA vectors used in this study. NPTII, kanamycin resistance cassette; VspB 3′, vegetative storage protein B gene terminator from soybean; Promoter, various promoters as described with 5′ UTR from tobacco etch virus; NOS, the nopaline synthase promoter from Agrobacterium; VspB, the vegetative storage protein B promoter from soybean; Ubi, the ubiquitin-3 promoter from potato; UbiF, Ubi with ubiquitin fusion (C) Agrobacterium carrying the indicated T-DNA vectors mixed to a final OD of 0.2 for each construct and were infiltrated into the leaves of N. benthamiana. After 4 days post-infiltration (DPI), leaf tissue samples were harvested, and protein extracts were analyzed by reducing or non-reducing western blot. In the “Reduced” gel, the lane “35S Rep/35S RepA” was pasted from a different gel than the other lanes (two representative gels of Rep/RepA expression shown in Supplementary Figures S1, S2 were combined into a single panel). For RT-PCR, RNA was extracted from leaf samples and 50 ng of converted cDNA were PCR amplified with Rep-specific primers.