Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects

Abstract

Evolving levels of resistance in insects to the bioinsecticide Bacillus thuringiensis (Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants. When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330- to 393-fold) tobacco budworm Heliothis virescens, cotton bollworm Helicoverpa zea, and the beet armyworm Spodoptera exigua, 100% mortality was observed against all insect species and strains. Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross-resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa). Southern blot analysis confirmed stable integration of cry2Aa2 into all of the chloroplast genomes (5, 000-10,000 copies per cell) of transgenic plants. Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20- to 30-fold higher levels than current commercial nuclear transgenic plants. These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field.

Figure 1

(A) Structure of the chloroplast genome with the site of foreign integration represented by a shaded dotted line. (B) PCR analysis to screen for chloroplast transformants. Primers used: rbcL 5′ region and aadA. Lanes 1 and 8, 1-kb ladder; template DNA from lane 2, nontransformed tobacco, lanes 3–6, clones 2, 5, 7 and 8, respectively; and lane 7, plasmid pZS-KM-cry2A.

Figure 2

(A) Southern blot analysis to confirm homoplasmy. Hybridization of EcoRV-digested total genomic DNA, with EcoRV/SstI-digested chloroplast border flanking sequences. DNA from lane 1, nontransformed tobacco and lanes 2–4, DNA from clones 2, 5, and 7, respectively. (B) Southern blot analysis to confirm chloroplast integration of cry2Aa2. Hybridization of EcoRV-digested total genomic DNA, with BglII/HindIII-digested cry2Aa2 probe. Lanes 1 and 7, 1-kb ladder; DNA from lane 2, plasmid pZS-KM-cry2A; lane 3, nontransformed tobacco, and lanes 4–6, clones 2, 5, and 7, respectively.

Figure 3

Immunoblot analysis. Lane A, molecular weight marker; lanes B and C, 10 μg of total soluble leaf proteins from clone 2 and nontransformed tobacco, respectively; lanes D-H, dilutions (0.0002, 0.002, 0.02, 0.2, and 2.0 μg, respectively) of solubilized Cry2Aa2 protoxin.

Figure 4

Leaf bioassay of control (Left) and Cry2Aa2 chloroplast transgenic tobacco leaves (Right) assayed against various H. virescens strains. YDK, susceptible (Top), YHD₂ 1000MVP, Cry1Ac-resistant (Middle), and CxC 1000IIA, Cry2Aa2-resistant (Bottom). Photographs were taken on day 4 of the assay.

Figure 5

Leaf bioassay of control (Left) and Cry2Aa2 chloroplast transgenic tobacco leaves (Right) assayed against H. zea (Upper) and S. exigua (Lower). Photographs were taken on day 4 of the assay.