Phenotype of transgenic rice lines accumulating the Ole18-CecA fusion protein.

<p>(A) Phenotypic appearance of transgenic rice plants 60 days after sowing. (B) Images of representative panicles from transgenic plants. (C) Average weight of 100 seeds per line. (D) Percentage of germinated seeds at 1 to 4 days after imbibition from empty vector (EV) and <i>pOle18</i>:<i>Ole18-CecA</i> transgenic plants. All results are representative of two independent assays, in which 2 WT, 3 EV and 5 <i>pOle18</i>:<i>Ole18-CecA</i> independent lines were analyzed. Bars correspond to SD.</p

The Ole18-CecA fusion protein localizes in rice OBs modifying their size and surface properties.

<p>(A) Morphology of OBs purified from seeds carrying the empty vector (EV) or the <i>pOle18</i>:<i>Ole18-CecA</i> transgene. Images correspond to single scanned confocal microscopy slides of Nile red stained OBs. (B) Increased size of <i>pOle</i>:<i>Ole18-CecA</i> OBs. Box plot of the median sizes of 3 different OB preparations per line (n = 25 per preparation). Boxes represent the 2^nd and 3^rd quartiles of the data, whereas whiskers indicate the minimum and maximum measured OB diameters. Asterisks denote statistically significant differences (**p<0.01, ANOVA) (C) Aggregation of OBs isolated from <i>pOle</i>:<i>Ole18-CecA</i> transgenic seeds. Confocal and light (left panel) microscopy images of Nile red stained OBs after incubation for 1 hour at room temperature. (D) Immunolocalization of CecA in <i>pOle18</i>:<i>Ole18-CecA</i> OBs. CecA was immunodetected and visualized in green using an AlexaFluor488-conjugated secondary antibody. (E) Confocal microscopy images of mature embryos stained with Nile red. Scale bars correspond to 2 μm (A-D), or 2, 20 or 100 μm as indicated (E).</p

Accumulation of the Ole18-CecA fusion protein in rice seeds.

<p>(A) <i>In situ</i> immunolocalization of CecA in <i>pOle18</i>:<i>Ole18-CecA</i> transgenic seeds. Immunoreaction was detected using a fluorescent-labeled secondary antibody. (B) Western blot analysis of OB proteins purified from seeds of the indicated Ariete transgenic lines using anti-cecropin A, anti-oleosin18 and anti-caleosin antibodies (from top to bottom, respectively). Lower panel correspond to the Coomassie Blue stained SDS gel. Molecular weight markers are indicated on the left in kDa. (C) Cecropin A accumulation in seeds from the Ariete (2, 3, 4, 6, 9) and Senia (3.2, 8.1, 8.5, 11.2, 12.1, 16.2) transgenic lines (T3 homozygous) as estimated by immunoblot analysis of OB protein extracts in comparison with known amounts of synthetic cecropin A. Values correspond to the mean of at least 3 independent assays, and bars to the SD.</p

Purification of active cecropin A from <i>pOle18</i>:<i>Ole18-CecA</i> plant seeds.

<p>(A) Schematic purification procedure of CecA. (B) Immunoblot analysis of purified fractions from wild-type (WT) and two independent <i>pOle18</i>:<i>Ole18-CecA</i> transgenic lines (10 seeds per line). As a control, synthetic cecropin A (10 and 50 ng of CecA) was added to one WT F3 fraction. The proteins in the F3 fractions were concentrated by acetone precipitation, and together with the F1 and F2 fraction proteins, were resolved in Tricine-SDS-PAGE and immunodetected with anti-CecA antibodies. (C) MRM chromatograms of CecA synthetic peptide, WT and <i>pOle18</i>:<i>Ole18-CecA</i> (lines #2 and #9) F3 fractions. The MS/MS transitions monitored were 740.9/816.4, 740.9/915.5 and 740.9/1085.6. (D) Amount of CecA in F3 fractions as determined by UV absorbance (280 nm) in comparison with known amounts of synthetic CecA added to wild-type F3 fractions. (E) Antimicrobial activity of purified fractions against <i>Dickeya dadantii</i> bacterial cells. Aliquots (3 μl) of ten-fold serial dilutions of bacterial cells (5 x 10⁴) incubated for 2h with synthetic CecA or with purified fractions (5 μl of F1 or F2, or 50 μl of F3 fractions) from WT or from the indicated <i>pOle18</i>:<i>Ole18-CecA</i> transgenic lines were plated on LB-agar media and grown for 2 days. (F) <i>D</i>. <i>dadantii</i> viable cells after 2h incubation with the indicated amounts of F3 fractions from empty vector (EV) or <i>pOle18</i>:<i>Ole-CecA</i> (line #2) compared to synthetic CecA at indicated concentrations. Mean values of two replicates and SD are shown.</p

Chimeric gene used for the production of cecropin A as an oleosin fusion protein in rice seeds.

<p>(A) Schematic diagram of the construct used for rice transformation. Relevant restriction enzyme sites and primers (arrows) used for cloning are indicated. (B) Amino acid sequence of the fusion protein Ole18-CecA containing the rice 18 kDa oleosin (black) and the cecropin A peptide (blue) linked through a TEV protease recognition site (PRS, red). Red arrow indicates TEV protease cleavage site.</p