INTEGRATIVE CLONING, EXPRESSION, AND STABILITY OF THE CRYIA(C) GENE FROM BACILLUS-THURINGIENSIS SUBSP. KURSTAKI IN A RECOMBINANT STRAIN OF CLAVIBACTER-XYLI SUBSP CYNODONTIS

A bacterial endophyte was engineered for insecticidal activity against the European corn borer. The cryIA(c) gene from Bacillus thuringiensi s subsp. kurstaki was introduced into the chromosome of Clavibacter xy li subsp. cynodontis by using an integrative plasmid vector. The integ ration vectors pCG740 and pCG741 included the replicon pGEM5Zf(+), whi ch is maintained in Escherichia coli but not in C. xyli subsp. cynodon tis; tetM as a marker for selection in C. xyli subsp. cynodontis; and a chromosomal fragment of C. xyli subsp. cynodontis to allow for homol ogous recombination between the vector and the bacterial chromosome. I nsertion of vector DNA into the chromosome was demonstrated by DNA hyb ridization. Recombinant strains MDR1.583 and MDR1.586 containing the c ryIA(c) gene were shown to produce the 133,000-kDa protoxin and severa l smaller immunoreactive proteins. Both strains were equally toxic to insect larvae in bioassays. Significant insecticidal activity was demo nstrated in planta. The cryIA(c) gene and the tetM gene introduced int o strain MDRI.586 were shown to be deleted from some cells, thereby gi ving rise to a noninsecticidal segregant population. In DNA hybridizat ion experiments and insect bioassays, these segregants were indistingu ishable from the wild-type strain. Overall, these results demonstrate the plausibility of genetically engineered bacterial endophytes for in sect control.