Yuan Zhiming, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China (e-mail: email@example.com).
Aims: To investigate the interaction between two crystal proteins, Cry1Aa and Cry1C, for future development of biopesticides based on Bacillus thuringiensis, toxicities of the two individual proteins and in combinations have been determined against Spodoptera exigua and Helicoverpa armigera larvae, and synergism between the proteins has been evaluated using synergistic factor.
Methods and Results: SDS-PAGE showed that Cry1Aa and Cry1C proteins could be expressed in acrystalliferous B. thuringiensis 4Q7 strain, with molecular weights of 135 and 130 kDa respectively. The bioassay results indicated a synergistic activity between Cry1Aa and Cry1C against S. exigua and H. armigera, and the highest toxicities could be observed in the combination of Cry1Aa and Cry1C at a ratio of 1 : 1.
Conclusion: The two toxins, Cry1Aa and Cry1C, interact synergistically to exhibit higher toxicity against S. exigua and H. armigera.
Significance and Impact of the Study: This is the investigation on the synergistic activity between two B. thuringiensis Cry1 toxins. It can be applied to the rational design of new generations of B. thuringiensis biopesticides and to strategies for management of resistant insects.
Bacillus thuringiensis (Bt), a Gram-positive bacterium, produces two distinct types of crystalline toxins, Cry and Cyt proteins (Crickmore et al. 1995; Schnepf et al. 1998). The genes encoding these proteins are generally located on large plasmids, and the proteins are synthesized and assembled as crystalline inclusions during sporulation. More than 100 different cry genes have been identified and characterized, the significant homologies among the encoded amino acid sequences of these cry genes, in combination with experimental studies, suggest that they have a common mode of action (Crickmore et al. 1998).
Li et al. (1991) unraveled the structure of Cry3A protein from B. thuringiensis subsp. tenebrionis by X-ray crystallography. Grochulski et al. (1995) showed that Cry1Aa and other Cry toxins shared a similar three-domain structure. Domain I consists of seven alpha helices and is thought to be involved in insertion into the epithelial cell membrane. Domain II, consisting of three beta sheets in a so-called Greek key conformation, is assumed to interact with receptors, thereby contributing to toxin specificity and high-affinity binding (Dean et al. 1996; Schnepf et al. 1998). Domain III, also composed of beta sheets, has been implicated in toxin stability and binding specificity in some insects (Burton et al. 1999). Moreover, Maagd et al. (2000) reported that the Domain III of Cry1C was a major determinant of specificity for Spodoptera exigua. However, the function of the three domains of crystal toxins still needs to be clarified.
Cry1C toxin, triggering channel activity in S. exigua cell lines, shows specific activity against S. exigua (Monette et al. 1994), but its activity is still too low to be used for field application, with no toxicity against third or fourth insect larvae. Meanwhile, it shows moderate activity against H. armigera. In contrast, Cry1Aa was proven to have high toxicity to H. armigera, but low toxicity to S. exigua.
In the last decade, the synergism between Cry and Cyt toxins from B. thuringiensis has been studied, and it has been proven that Cyt1Aa can enhance the activity of other Cry toxins against the target insects. Moreover, this protein can also overcome the high-level resistance to Cry3A and Cry4A in resistant populations of Chrysomela scripta and Culex quinquefasciatus respectively (Wirth et al. 1997; Federici and Bauer 1998).
In an earlier report, Tabashnik (1992) evaluated the synergism of Cry1A toxins against two insect species, Orygia leucostigma and Lymantria dispar. This did not reveal any evidence of synergism among Cry1A toxins. Moar et al. (1990) also demonstrated that Cry1A toxins from HD-1 and NRD-12 strains of B. thuringiensis subsp. kurstaki did not show synergistic effects against S. exigua. However, Lee et al. (1996) observed that synergistic activity could be found in a combination of toxins Cry1Aa and Cry1Ac against L. dispar, while a mixture of toxins Cry1Aa and Cry1Ab showed an antagonistic effect. The interaction or synergism between the Cry toxins, such as Cry1C and Cry1Aa, requires further investigation.
In the present study, we investigated possible synergistic effects between Cry1Aa and Cry1C on two main Chinese agricultural pests, H. armigera and S. exigua, for future development of biopesticides based on a rational designed genetically modified B. thuringiensis.
Materials and methods
Bacterial strains and plasmids
Escherichia coli TG-1 [(lac-proAB) SupE thi hsd D F’(ral)36 proA+proB+lacI lacZ M15] was used for cloning. The shuttle vector pBU4 was a gift from Dr Armelle Delécluse (Institut Pasteur, Paris, France), having tetracycline and ampicillin resistance determinants (Bourgouin et al. 1990). An acrystalliferous strain 4Q7 of B. thuringiensis subsp. israelensis was used as a recipient strain in the transformation experiments. Plasmid pMPCV1 and pMPCV2, which contains cry1Aa gene and cry1C gene, respectively, were kindly provided by Dr Luke Masson (Biotechnology Research Institute, Montréal, Quebec, Canada). Plasmids pCX1A and pCX1C were constructed by ligating the target fragment from pMPCV1 and pMPCV2 into SphI-SmaI fragment of vector pBU4 respectively.
Bacillus thuringiensis transformants were grown with shaking at 28°C in G-Tris medium (Shi et al. 2001) containing 12·5 μg ml−1 tetracycline, until spore formation and cell lysis. Spore–crystal mixtures were collected by centrifugation and washed once with deionized water, then restored to original volume as stock suspension for later protein analysis and bioassay.
DNA manipulations and electroporation
Plasmid DNA from B. thuringiensis was prepared by the methods of Bourgouin et al. (1990). Cloning experiments, restriction enzyme analysis, and other DNA manipulations were carried out as described by Sambrook et al. (1989). The recombinant plasmids pCX1A and pCX1C were transferred to a plasmid-cured, crystal-minus B. thuringiensis 4Q7 by electroporation with the method described by Shi et al. (2001).
The spore–crystal suspension were boiled with the loading buffer and the soluble mixtures were resolved in a 0·1% sodium dodecyl sulfate–10% polyacrylamide gel, then stained with Coomassie brilliant blue R250 after electrophoresis.
Insects and bioassay
The toxicities of two sporulated recombinants B-pCX1A and B-pCX1C alone and in combinations against S. exigua and H. armigera were evaluated by bioassays as described by Liu et al. (2002). The target insects were stable susceptible S. exigua, H. armigera colonies maintained for two years in Experimental Insect Center in Wuhan Institute of Virology, Chinese Academy of Sciences.
The bioassays of each sporulated culture and in combination were performed on larvae confined individually to compartments of a 24-well tissue culture plate. The larvae were infested on semi-artificial diet composed of wheat flour, soybean flour, yeast powder, and a mixture of vitamins and agar. Diluted recombinant B. thuringiensis sporulated cultures alone and in combination were well mixed with the melted diet at about 55°C, giving final concentrations ranging from 0·125 to 10·0 mg culture per gram diet, and then transferred into separate cubes. One neonate larva was placed into each cell and a plastic cover was used to confine the larvae. The plates were incubated at 26 ± 1°C, with humidity of 85% and a photoperiod of 12 : 12 (L : D) hours. Duplicate of 24 larvae for each dose and five doses for each dose–response experiment were used. The bioassay was performed two or three times on different days and the mortalities were recorded after 72 h.
Fifty per cent lethal concentration (LC50) with 95% fiducial limits was determined using probit analysis with a probit program (E. Frachon, Institut Pasteur) and the LC50 was expressed in mg sporulated culture per gram artificial diets. Synergism between Cry1Aa and Cry1C was evaluated by the method described by Tabashnik (1992). The synergism factor (SF) is the ratio of the expected LC50 of the toxin mixture to the observed LC50. The expected LC50 was calculated from an equation described by Wirth et al. (1997). When the SF value was >1·0, the toxin interaction was considered synergistic because toxicity exceeded the value expected from the individual additive activity. When the SF value was less than 1·0, the interaction was considered antagonistic, whereas a SF value of 1·0 indicated that the toxicity was additive.
Cloning and expression of cry1Aa and cry1C individually
After plasmid pMPCV1 and pMPCV2, which contain cry1Aa and cry1C, respectively, were digested with SphI and SmaI, the target fragments of 4·7 and 4·2 kb were purified and ligated to SphI–SmaI digested vector pBU4 to get recombinant plasmid pCX1A and pCX1C respectively (Fig. 1). Bacillus thuringiensis recombinants B-pCX1A and B-pCX1C were obtained by transforming pCX1A and pCX1C into strain 4Q7 strain, respectively, by electroporation. In G-Tris medium, the two B. thuringiensis recombinants grew and developed normally, producing crystal bodies during sporulation. SDS-PAGE showed that the two recombinants could express a 135- and 130-kDa protein respectively (Fig. 2).
Toxicity of two recombinants against S. exigua and H. armigera
The bioassay results showed that Cry1Aa exhibited high activity against neonate H. armigera with a LC50 value of 0·338 mg g−1, but only a low level of toxicity against neonate S. exigua, yielding a LC50 value of 3·936 mg g−1 (Table 1). In contrast, Cry1C exhibited relatively higher toxicity to S. exigua larvae, with a LC50 of 1·466 mg g−1, although it showed only a moderate toxicity against H. armigera.
Table 1. Toxicities of Cry1Aa, Cry1C alone and in combination toward Spodoptera exigua and Helicoverpa armigera
Toxicity (mg g−1)‡ (95%FL†)
SF, synergism factor.
Results after 72 h exposure of toxin (s).
*Duplicate of 24 neonate larvae for each dose and five doses for each dose–response experiment were used and bioassays were performed two or three times on different days.
†FL95% fiducial limits were determined by probit analysis.
‡The LC50 was expressed in mg B. thuringiensis culture per gram artificial diets.
Cry1C + Cry1Aa (1 : 1)
Cry1C + Cry1Aa (5 : 1)
Cry1C + Cry1Aa (1 : 5)
When Cry1C and Cry1Aa were mixed with a ratio of 1 : 1, 1 : 5 and 5 : 1, all mixtures were more toxic to S. exigua than the observed activities for Cry1Aa or Cry1C alone, having LC50 values ranging from 0·537 to 1·309 mg g−1. The combination of 1 : 1 ratio of Cry1C and Cry1Aa yielded a LC50 value of 0·537 mg g−1 to S. exigua larvae (Table 1; Fig. 3a). The calculated synergistic factor (SF) to S. exigua larvae were 4·0, 2·0, and 2·3 at the LC50 level at the mixture ratio of 1 : 1, 5 : 1 and 1 : 5 of Cry1C and Cry1Aa, respectively, indicating synergistic effects between the two toxins against S. exigua.
As for the H. armigera, although the SF values between the two toxins were lower than that of the combinations to S. exigua, synergism between the two toxins was still observed. All combinations of Cry1C and Cry1Aa were more toxic than Cry1C alone, with LC50 values of 0·204, 0·633, 0·285 mg g−1 at ratios of 1 : 1, 5 : 1 and 1 : 5 respectively (Table 1; Fig. 3b). However, SF values of different combinations of two toxins to H. armigera varied from 1·4 to 2·7 at the LC50 level. The highest toxicity to H. armigera was observed at the combination of 1 : 1 of Cry1C and Cry1Aa.
Simply by looking at the SF values, we can see clearly that the synergistic effects of different combinations of the two crystal toxins are more evident in S. exigua than in H. armigera.
Previous work demonstrated that synergism might occur between B. thuringiensis Cry toxins and Cyt toxins, as well as Cyt toxins and Bin toxin from B. sphaericus. Cry and Cyt1A toxins from B. thuringiensis subsp. israelensis interact synergistically to produce a high level of activity against the target insects (Crickmore et al. 1995; Wirth et al. 2000). Synergistic capacity of Cyt1A can also be extended by the recent observation that Cyt1A and B. sphaericus were synergistic, being toxic toward highly resistant Culex quinquefasciatus (Wirth et al. 2001).
Our study revealed that Cry1Aa toxin could enhance the toxicity of Cry1C against S. exigua, and that Cry1C toxin could enhance the toxicity of Cry1Aa against H. armigera. A ratio of 1 : 1 of Cry1C and Cry1Aa was most toxic against both S. exigua and H. armigera and this high level activity might result from the synergistic effects between Cry1Aa and Cry1C. Based on the calculated SF values, it is seen that the synergistic effects of Cry1Aa and Cry1C toxins were more evident in S. exigua than in H. armigera.
Two hypotheses might be used for interpreting the synergism between Cry1Aa and Cry1C. One is that the toxins might oligomerize before or after binding to the receptor (Gill et al. 1992), forming a hetero-oligomer (Cry1Aa and Cry1C combined as one oligomer). The formed hetero-oligomer may have better insertion ability than the individual homocomplex, resulting in higher toxicity against the targets. Another explanation is that the two toxins could induce two kinds of pores on larval midgut membrane, and work together to increase the osmotic ability of the membrane by forming hybrid pores, causing higher larval mortality.
Previous binding experiments and the function/structure studies of hybridized crystal proteins constructed by domain swapping have shown that domain III is involved in binding to putative receptors of target insects, suggesting that domain III may exert its role in specificity through receptor recognition (Maagd et al. 2000). Hybridized crystal protein containing Domain III of Cry1C may exhibit toxicity against S. exigua regardless of the origin of domains I and II (Maagd et al. 2000). As the Cry1C domain III can function as a specificity determinant for S. exigua, the synergistic activity between Cry1Aa and Cry1C is more evident against S. exigua than against H. armigera. Our findings revealed that the SF value at a ratio of 1 : 1 of Cry1C and Cry1Aa is 4·0 against S. exigua, but 2·7 against H. armigera, which is consistent with this explanation.
This investigation was partly supported by State 973 project (2003CB114201), a grant (KSCX2-SW-301-10) from the Chinese Academy of Sciences, and a NSFC project (39770170).