Chloroplast Expression Cassettes (CECs) used in this study; Phenotype of primary transformants (cv. 81V9); Confirmation of homoplastomy; Confirmation of XynA expression. A. Four Chloroplast Expression Cassettes (CECs, designated CEC1 through -C4) with varying configuration of the cis-acting regulatory elements are shown. The integration of the CECs into the tobacco plastome was designed to occur in the transcriptionally-active spacer region between the trnI and trnA genes. The wild type (WT) plastome trnI - trnA region is shown at the bottom. The expected sizes of Rsr II-digested fragments are indicated. Thick black lines represent hybridization sites for the probe used in Southern blot analyses. IEE = Intercistronic Expression Element with the Shine-Dalgarno sequence from the 5' UTR of bacteriophage T7 gene 10 fused to the 3' end; aadA = gene encoding aminoglycoside 3' adenylyltransferase; TpsbC and TrbcL = 3' UTRs of psbC and rbcL from the white poplar plastome; PpsbA = promoter and 5’ UTR of tobacco psbA gene; mPrrn – mutated chloroplast rrn operon promoter; XynA::T = gene encoding the XynA protein fused at the C-terminal with c-myc and strepII tags. B. Transformation with different CECs produced different phenotypes in primary transformants (T0). C. Southern blot RFLP analysis and confirmation of homoplastomy of T0 transformants. Total DNA extracted from 2 independent transformants for each CEC and 1 untransformed plant (WT) was digested with Rsr II and analyzed using Southern blotting. All T0 transformants displayed a single band of expected size, confirming homoplastomy. D. Immunoblot confirmation of XynA expression in plants transformed with different CECs. Lanes 1 through 4 – extracts from CEC1 through CEC4, respectively. Lane 5 – cv. 81V9 WT as negative control. Each lane contains equal amounts of extracted leaf tissue (equivalent to 4.0 mg/lane). Known amounts (ng) of a c-myc-tagged control protein are indicated above the standard curve lanes.