Use of endophytic diazotrophic bacteria as a vector to express the cry3A gene from Bacillus thuringiensis

Abstract

The goal of this study was to evaluate the potential of endophytic diazotrophic bacteria as a vector to express a cry gene from Bacillus thuringiensis, envisaging the control of pests that attack sugarcane plants. The endophytic nitrogen-fixing bacteria Gluconacetobacter diazotrophicus strain BR11281 and Herbaspirillum seropedicae strain BR11335 were used as models. The cry3A gene was transferred by conjugation using a suicide plasmid and the recombinant strains were selected by their ability to fix nitrogen in semi-solid N-free medium. The presence of the cry gene was detected by Southern-blot using an internal fragment of 1.0 kb as a probe. The production of δ-endotoxin by the recombinant H. seropedicae strain was detected by dot blot while for G. diazotrophicus the Western-blot technique was used. In both cases, a specific antibody raised against the B. thuringiensis toxin was applied. The δ-endotoxin production showed by the G. diazotrophicus recombinant strain was dependent on the nitrogen fixing conditions since the cry3A gene was fused to a nif promoter. In the case of H. seropedicae the δ-endotoxin expression was not affected by the promoter (rhi) used. These results suggest that endophytic diazotrophic bacteria can be used as vectors to express entomopathogenic genes envisaging control of sugarcane pests.

History on the biological nitrogen fixation research in graminaceous plants: special emphasis on the Brazilian experience

“…Because of its endophytic life style, this bacterium has been used as a vector to express heterologous genes of interest. One example is the expression of cry3A and cry1Ab genes isolated from Bacillus thuringiensis that are responsible for the entomopathogenic activity against coleopteran and lepdopeteran insects that cause damage to sugarcane plants (Salles et al 2000, Baldani et al 2002b). …”

A Brazilian Bacillus thuringiensis strain highly active to sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae)

“…Bioinsecticides based either on a spore and crystal mixture or the heterologous expression of cry genes in microorganisms (18,31) as well as in plants (33) have been efficiently used for controlling important agricultural pests and mosquitoes. Early attempts to control the sugarcane borer with B. thuringiensis subsp.…”

Toxicity to Eldana saccharina of a recombinant Gluconacetobacter diazotrophicus strain carrying a truncated Bacillus thuringiensis cry1Ac gene

“…Although transgenic sugarcane plants expressing Cry proteins have been constructed and tested against some sugarcane pests (Arencibia et al, 1997), a review of the literature revealed no published papers that evaluated Cry-expressing sugarcane for the control of E. saccharina. Both epiphytes (bacteria that live on the surface of plants) and endophytes (bacteria that inhabit, for at least one period of their life cycle, the interior of a plant) have been used as delivery vehicles for Cry proteins (Bora et al, 1994;Lampel et al, 1994;Salles et al, 2000). To target insects that burrow into a plant, endophytic bacteria are likely to be better vehicles for Cry proteins than epiphytic bacteria.…”

“…Primarily due to its ability to fix atmospheric nitrogen, G. diazotrophicus has been the focus of intense study (Dong et al, 1997;Gillis et al, 1989;James et al, 1994;Lee et al, 2000;Saravanan et al, 2008;Sevilla and Kennedy, 2000). Salles et al (2000) evaluated the potential of using G. diazotrophicus strain BR11281 as a vector to express a Bt cry3A gene for the control of coleopteran pests of sugarcane. Although the authors did not evaluate the insecticidal activity of the recombinant G. diazotrophicus strain, the study of Salles et al (2000) suggested that G. diazotrophicus may have potential as a vector to express Bt cry genes for the control of sugarcane pests.…”

“…Salles et al (2000) evaluated the potential of using G. diazotrophicus strain BR11281 as a vector to express a Bt cry3A gene for the control of coleopteran pests of sugarcane. Although the authors did not evaluate the insecticidal activity of the recombinant G. diazotrophicus strain, the study of Salles et al (2000) suggested that G. diazotrophicus may have potential as a vector to express Bt cry genes for the control of sugarcane pests.…”