Untitled

104

Bacillus thuringiensis (Bt) crystal protein genes encode insecticidal ␦-endotoxins that are widely used for the development of insectresistant crops. In this article, we describe an alternative transgenic strategy that has the potential to generate broader and more sustainable levels of resistance against insect pests. Our strategy involves engineering plants with a fusion protein combining the ␦-endotoxin Cry1Ac with the galactose-binding domain of the nontoxic ricin B-chain (RB). This fusion, designated BtRB, provides the toxin with additional, binding domains, thus increasing the potential number of interactions at the molecular level in target insects. Transgenic rice and maize plants engineered to express the fusion protein were significantly more toxic in insect bioassays than those containing the Bt gene alone. They were also resistant to a wider range of insects, including important pests that are not normally susceptible to Bt toxins. The potential impact of fusion genes such as BtRB in terms of crop improvement, resistance sustainability, and biosafety is discussed.Bt genes ͉ transgenic plants ͉ transgenic maize ͉ transgenic rice T ransgenic plants expressing Bacillus thuringiensis (Bt) toxins have been used successfully to provide resistance against selected insect pests for several years. Indeed, insect resistance is the second most widely used trait in transgenic crops (after herbicide tolerance) in world agriculture (1-5). One potential problem with Bt genes is that Bt insecticides are very widely used, with up to 90% of microbiological insect control products based on topically applied Bt toxins. For this reason, there is concern that insects might evolve resistance to Bt toxins (6, 7). The diamondback moth (Plutella xylostella) has evolved resistance in some open field populations in response to repeated exposure to foliar sprays containing Bt proteins (8), whereas laboratory selection experiments with other insect pests have shown that recessive mutant alleles can confer resistance to multiple Bt toxins (7-10). However, note that the evolution of resistance in insects against transgenic plants expressing Bt toxins has yet to be seen in the field.Recent strategies to address potential limitations in conventional transgenic insect pest control include the stacking or pyramiding of multiple transgenes in the same transgenic plant (11) and the use of hybrid toxins (e.g., fusions between a synthetic truncated Cry1Ba and domain II from Cry1Ia; ref. 12). We have devised an alternative strategy in which the Bt toxin Cry1Ac is fused to the nontoxic ricin B-chain (RB). The recognition of toxin-binding sites in the insect midgut is an important factor determining the spectrum of Bt toxin activity and the severity of toxemia (13). Several groups investigating the mechanism of toxin recognition have identified N-acetyl galactosamine residues as an important component of Bt toxinbinding receptors (14-16). Therefore, we selected the ricin B subunit as a fusion partner for the Bt toxin because RB is a galactose...

Section: Resultscontrasting

confidence: 78%

An alternative strategy for sustainable pest resistance in genetically enhanced crops

Mehlo

Gahakwa²,

Nghia³

et al. 2005

104

“…In Ϸ10% of these plants, the toxin levels were as high as 3% of the total soluble proteins. This is 10 to Ͼ100 times higher than the CryIA(b) and CryIA(c) contents in the previously reported transgenic rice plants (4)(5)(6)(7). This is a significant advance because such high levels have been proposed as a necessary component of an effective integrated pest management program limiting build-up of insect resistance in transgenic crops (33).…”

Section: Discussionmentioning

confidence: 81%

“…Insecticidal activity of the transgenic plants toward two major rice insects SSB and YSB was assayed by using laboratory culture dishes, similar to described methods (6,7). At the flowering stage, stem cuttings with sheath tissue were taken from R 1 plants of three primary transformants.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Agrobacterium-transformed rice plants expressing syntheticcryIA(b)andcryIA(c)genes are highly toxic to striped stem borer and yellow stem borer

Cheng

Sardana

Kaplan

et al. 1998

334

137

Edited by Marc C. E. Van Montagu, University of Ghent, Ghent, Belgium, and approved December 17, 1997 (received for review September 15, 1997) ABSTRACTOver 2,600 transgenic rice plants in nine strains were regenerated from >500 independently selected hygromycin-resistant calli after Agrobacterium-mediated transformation. The plants were transformed with fully modified (plant codon optimized) versions of two synthetic cryIA(b) and cryIA(c) coding sequences from Bacillus thuringiensis as well as the hph and gus genes, coding for hygromycin phosphotransferase and ␤-glucuronidase, respectively. These sequences were placed under control of the maize ubiquitin promoter, the CaMV35S promoter, and the Brassica Bp10 gene promoter to achieve high and tissue-specific expression of the lepidopteran-specific ␦-endotoxins. The integration, expression, and inheritance of these genes were demonstrated in R 0 and R 1 generations by Southern, Northern, and Western analyses and by other techniques. Accumulation of high levels (up to 3% of soluble proteins) of CryIA(b) and CryIA(c) proteins was detected in R 0 plants. Bioassays with R 1 transgenic plants indicated that the transgenic plants were highly toxic to two major rice insect pests, striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas), with mortalities of 97-100% within 5 days after infestation, thus offering a potential for effective insect resistance in transgenic rice plants.Rice is one of the world's most important food crops, and intense efforts, including use of genetic engineering technologies, must be engaged to increase its yield if the impending global rice shortage is to be avoided (1). Engineering rice for pest resistance is a major challenge, one strategy being the introduction of Bacillus thuringiensis (Bt) crystal insecticidal protein (␦-endotoxin) genes (cry genes). These proteins (Bt toxins) are highly toxic to lepidopteran, dipteran, and coleopteran insects (2), among which are important pests of rice such as striped stem borer (SSB), yellow stem borer (YSB), and leaffolder (Cnaphalocrocis medinalis and Marasmia patnalis) that cause annual losses of an estimated 10 million tonnes (3).Rice plants containing cryIA(b) or cryIA(c) have been obtained by using protoplast (4) or particle bombardment methods (5-7). However, the numbers of plants obtained and levels of the toxin proteins in these studies were unfortunately still very low from a breeder's point of view. In contrast, Ͼ2,600 transgenic plants were produced with the modified cry genes in nine rice strains by using a modified Agrobacteriumbased rice transformation procedure (8). Herein we report that high levels of CryIA(b) and CryIA(c) were detected among these transgenic plants, indicating that many candidate transgenics in this large screen may be the result of optimal position effects. Insect feeding assays with R 1 plant tissues indicated that the transgenic plants were highly toxic to two major rice insects, SSB and YSB, with near 100% mortality within 5 days....

“…The most commonly used Bt genes in transgenic crops including rice are Cry1Ab, Cry1Ac, and a fusion gene of Cry1Ac/Cry1Ab or Cry1Ab/c (19)(20)(21)(22)(23)(24)(25)(26)(27)(28). However, binding tests of midgut brush border membrane vesicles showed that Cry1Aa, Cry1Ab, and Cry1Ac toxins share a common binding site (29)(30)(31); thus, a mutant that is able to overcome one of the Cry1A genes is also likely to be resistant to other Cry1A genes as well.…”

Section: Progresses In Gene Identification and Development Of Gsrmentioning

confidence: 99%

Strategies for developing Green Super Rice

Zhang

2007

729

438

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on May 1, 2007.From a global viewpoint, a number of challenges need to be met for sustainable rice production: (i) increasingly severe occurrence of insects and diseases and indiscriminate pesticide applications; (ii) high pressure for yield increase and overuse of fertilizers; (iii) water shortage and increasingly frequent occurrence of drought; and (iv) extensive cultivation in marginal lands. A combination of approaches based on the recent advances in genomic research has been formulated to address these challenges, with the long-term goal to develop rice cultivars referred to as Green Super Rice. On the premise of continued yield increase and quality improvement, Green Super Rice should possess resistances to multiple insects and diseases, high nutrient efficiency, and drought resistance, promising to greatly reduce the consumption of pesticides, chemical fertilizers, and water. Large efforts have been focused on identifying germplasms and discovering genes for resistance to diseases and insects, N-and P-use efficiency, drought resistance, grain quality, and yield. The approaches adopted include screening of germplasm collections and mutant libraries, gene discovery and identification, microarray analysis of differentially regulated genes under stressed conditions, and functional test of candidate genes by transgenic analysis. Genes for almost all of the traits have now been isolated in a global perspective and are gradually incorporated into genetic backgrounds of elite cultivars by molecular marker-assisted selection or transformation. It is anticipated that such strategies and efforts would eventually lead to the development of Green Super Rice.genomics ͉ rice genetic improvement ͉ sustainable agriculture