Several publications and patent documents are cited throughout this application in order to better describe the state of the art to which this invention pertains. Each of these citations is incorporated by reference herein.
Potato tuber moth (Phthorimaea operculella, Lepidoptera, Gelechiidae) is one of the most destructive insect pests of potato with a pandemic distribution. In the field, the moths lay their eggs on the potato foliage and the larvae mine the foliage and the stems. Larvae attack the tubers through infected stems or may enter the tubers directly. Development of cultivars resistant to potato tuber moth through conventional breeding has not been successful because of lack of reliable resistance sources in potato germplasm. However, considerable degree of protection has been achieved by using insecticidal crystal proteins of the soil bacterium Bacillus thuringiensis (B.t.). Insecticidal crystal proteins are susceptible to UV damage necessitating frequent sprays on the standing crop. To overcome this, transgenic potato lines with tuber moth resistance have been developed by engineering cry1 class B.t. genes. Transgenic potato lines with variable level of PTM resistance have been obtained by expressing the native cry1Aa (Chan et al., 1996), cry1Ab (Jansens et al., 1995) and cry1Ac (Ebora et al., 1994) genes. Nuclear transformation with native cry genes in plants, however, results in very low levels of B.t. protein expression due to instability of prokaryotic transcripts in plant systems (Murray et al., 1991). Relatively high level of B.t. protein expression with better PTM control could be achieved by using codon modified and truncated cry1Ac9 (Beuning et al., 2001; Davidson et al., 2002), cry 1Ia1 (Mohammed et al., 2000; Douches et al., 2002), and a hybrid Bt toxin (SN19) gene consisting of domain I and III of cry1Ba and domain II of cry1Ia (Naimov et al., 2003). Gleave et al. cloned and sequenced a B.t. gene, later named cry9Aa2, from Bacillus thuringiensis var. galleriae (strain DSIR517) that showed strong insecticidal activity to P. operculella (LC50 80 ng/ml) (Gleave et al., 1992). The amino acid sequence of this new B.t. protein was significantly different from those belonging to Cry1 class and, therefore, it was placed under the new class of Cry9 (Crickmore et al., 1998). In their later work, Gleave et al. transformed tobacco with the native and the modified versions of the cry9Aa2 gene and found significant improvement in B.t., expression as well as PTM resistance in those expressing the truncated and codon modified versions (Gleave et al., 1998). It is apparent from the published work that sequence modification of cry nuclear genes is an essential requirement for achieving satisfactory levels of toxin expression and PTM control in transgenic plants.