Integrative Cloning, Expression, and Stability of the cryIA(c) Gene from Bacillus thuringiensis subsp. kurstaki in a Recombinant Strain of Clavibacter xyli subsp. cynodontis

Abstract

A bacterial endophyte was engineered for insecticidal activity against the European corn borer. The crylA(c) gene from Bacillus thuringiensis subsp. kurstaki was introduced into the chromosome of Clavibacter xyli subsp. cynodontis by using an integrative plasmid vector. The integration vectors pCG740 and pCG741 included the replicon pGEM5Zf(+), which is maintained in Escherichia coli but not in C. xyli subsp. cynodontis; tetM as a marker for selection in C. xyli subsp. cynodontis; and a chromosomal fragment of C. xyli subsp. cynodontis to allow for homologous recombination between the vector and the bacterial chromosome. Insertion of vector DNA into the chromosome was demonstrated by DNA hybridization. Recombinant strains MDR1.583 and MDR1.586 containing the cryL4(c) gene were shown to produce the 133,000-kDa protoxin and several smaller immunoreactive proteins. Both strains were equally toxic to insect larvae in bioassays. Significant insecticidal activity was demonstrated in planta. The cryL4(c) gene and the tetM gene introduced into strain MDR1.586 were shown to be deleted from some cells, thereby giving rise to a noninsecticidal segregant population. In DNA hybridization experiments and insect bioassays, these segregants were indistinguishable from the wild-type strain. Overall, these results demonstrate the plausibility of genetically engineered bacterial endophytes for insect control. Clavibacter xyli subsp. cynodontis is a fastidious gram-positive coryneform bacterium that naturally inhabits the xylem of Bermuda grass (Cynodon dactylon L.) (6). C. xyli subsp. cynodontis also colonizes the vascular system of corn (Zea mays L.) when artificially inoculated (16). High populations of C. xyli subsp. cynodontis are distributed in the xylem elements following the introduction of inoculum into the tissues of the whorl or stem of corn seedlings or after germination of corn seed infused with the bacterium. C. xyli subsp. cynodontis does not enter developing kernels and is therefore not present in the progeny seed of inoculated plants (5). Additionally, a DNA transformation system and the development of plasmid cloning vectors have been recently reported (13, 19).

Bacillus thuringiensis: a century of research, development and commercial applications

“…Crop Genetics International developed a similar strategy using Clavibacter xyli var. cynodontis , an endophytic bacterium that can penetrate the vascular system of plants, and this confers resistance to the European corn borer (Lampel et al. , 1994).…”

Insect Pathogens as Biological Control Agents: Do They Have a Future?

“…Transgenic plants are developed by incorporation of the genes responsible for production of the toxin into the plant genome. In addition to traditional formulations (such as suspensions, wettable powders, tablets, microencapsulation), various alternative means of delivery, including endophytic bacteria such as Clavibacter xyli Davis et al (Lampel et al, 1994) or Bacillus cereus Frankland and Frankland (Mahaffee et al, 1994), to properly deliver the toxins to the target insects have been investigated. Plant-colonizing bacteria including Pseudomonas fluorescens Migula, P. cepacia (Burkholder) Palleroni and Holmes, Rhizobium leguminosarum Jordan, and Azosporillium spp.…”

Integrative vector for stable transformation and expression of a β-1,3-glucanase gene in Clavibacter xyli subsp. cynodontis

“…cynodontis, there are no plasmid vectors that are stable during growth in the plant xylem. Insertion transformants of this bacterium have been obtained before [5], but those constructs were unstable, gradually losing the heterologous gene. Moreover, the recombinant strain grew more slowly than the wild-type strain.…”