Figure 2

Relative RNA and coat protein accumulation levels for CMVin II and CMVar II variants having wt or modified RNA 4 leader sequences. After 6 days post-infiltration, infiltrated leaves were harvested and total RNAs and proteins were extracted as described in Methods. For CMVinII variants (see Table 2), estradiol was applied 18 hrs after infiltration using a cotton swab. For CMVinII variants, two leaves each from 3 plants for each treatment (n = 6) were used for 1^st, 2^nd biological trials, and three leaves each from 2 plants for 3^rd, 4^th trials (n = 6) yielding four different biological trials (total n = 24). For CMVarII variants (see Table 2), two leaves each from 3 plants for each treatment (n = 6) were used for analysis. These experiments were replicated three times (total n = 18). For immunoblot analysis, each lane was loaded with 15 μg of total protein. Real-time PCR reactions were performed using an ABI 7500 with gene specific primer sets. Relative Ct values were calculated to the respective 18S rRNA relative concentration. Relative folds were calculated to the repective wildtype CMVinII and CMVarII value, respectively. Group superscript letters next to the numbers represent different statistical groups, means with the same letter are not significantly different by the Bonferroni (Dunn) t test using the SAS 9.1 program. Panel (A) CMVinII variants; H , healthy uninfiltrated leaf; pR1R2, pCMVinII RNA 1 and RNA 2 only; CMVinII wt , wt leader; CMVinII 2 , #2 leader; CMVinII 6, #6 leader; CMVinII 8 , #8 leader; CMViva, pCMV (see Methods). Panel (B) CMVarII variants; H, healthy, uninfiltrated leaf; R1 + R2, pCMVarII RNA 1 and RNA 2 only; CMVarII wt , wild type leader; CMVarII 2 , #2 leader; CMVarII 6 , #6 leader; CMVarII 8, #8 leader; pQ123, pCassQ123 (see Methods).