Insecticidal proteins from Bacillus thuringiensis protect corn from corn ...

6 downloads 97 Views 2MB Size Report
Daniel J. Moellenbeck1*, Melvin L. Peters1, James W. Bing2, James R. ..... Fowler, IN. WCRW. 1,000 WCRW eggs/row foot + natural WCRW. Arlington, WI.
© 2001 Nature Publishing Group http://biotech.nature.com

RESEARCH ARTICLE

© 2001 Nature Publishing Group http://biotech.nature.com

Insecticidal proteins from Bacillus thuringiensis protect corn from corn rootworms Daniel J. Moellenbeck1*, Melvin L. Peters1, James W. Bing2, James R. Rouse2, Laura S. Higgins1, Lynne Sims1, Tony Nevshemal1, Lisa Marshall1, R. Tracy Ellis3, Paul G. Bystrak2, Bruce A. Lang2, James L. Stewart2, Kristen Kouba1, Valerie Sondag1, Vicki Gustafson1, Katy Nour1,4, Deping Xu1, Jan Swenson1, Jian Zhang1, Thomas Czapla1, George Schwab3, Susan Jayne1, Brian A. Stockhoff3, Kenneth Narva3, H. Ernest Schnepf3, Steven J. Stelman3, Candace Poutre3,5, Michael Koziel1, and Nicholas Duck1

Field tests of corn co-expressing two new delta-endotoxins from Bacillus thuringiensis (Bt) have demonstrated protection from root damage by western corn rootworm (Diabrotica virgifera virgifera LeConte). The level of protection exceeds that provided by chemical insecticides. In the bacterium, these proteins form crystals during the sporulation phase of the growth cycle, are encoded by a single operon, and have molecular masses of 14 kDa and 44 kDa. Corn rootworm larvae fed on corn roots expressing the proteins showed histopathological symptoms in the midgut epithelium.

biopesticides for agriculture, forestry, and mosquito vector control3. Bt organisms also produce two known classes of vegetatively expressed insecticidal proteins. These include the binary toxins Vip1 and Vip2 with coleopteran specificity and Vip3 with lepidopteran specificity8,9.

Corn rootworm is one of the principal insect pests of corn. When cost of control and yield loss are considered, rootworms cause about $1 billion damage annually in the United States1. Typically organophosphate or pyrethroid soil insecticides are used for control of rootworm on corn, accounting for the second largest crop pesticide use in the United States after cotton. Currently the only alternative to pesticides for controlling rootworms is crop rotation. In the spring, rootworms emerge from eggs deposited in the soil the previous year and feed on the roots of corn seedlings. Feeding continues as the plant grows, weakening the root system. Plants with rootworm damage are more susceptible to drought and root disease, have decreased yield, and can lean or fall over, all of which considerably reduce harvest yields. The rootworms develop through three larval stages and then pupate and emerge as adult beetles. The beetles feed on corn silks and pollen, causing minimal damage, then mate and lay eggs that overwinter in the soil. To interrupt this cycle, growers employ a strategy of crop rotation with soybeans. However, years of annual corn and soybean rotation in Illinois have resulted in selection for a rootworm population that also oviposit eggs in neighboring soybean fields, effectively “anticipating” corn being planted in those fields the next year2. These expanded populations lead to increased rootworm pressure on corn crops and subsequently impose a greater need for alternatives to annual crop rotation for their control. Bt is a common soil microorganism that has been used as a biological pesticide for several decades3. Various strains of Bt are known to produce several different classes of insecticidal proteins, and more than 100 different insecticidal genes have been identified to date. The largest class consists of the delta-endotoxins, which often are produced as a 125–140 kDa protein precursor that is solubilized and proteolytically processed in the insect gut to yield a 55–75 kDa biologically active core protein. Delta-endotoxins have been expressed in many important crop plants including cotton, potatoes, rice, and corn4–7. These plants resist insect attack, and commercial seed is available for planting in the United States for all but rice. Delta-endotoxins also comprise the active ingredient of many commercially available

Results and discussion Discovery and characterization of insecticidal toxins. Upon sporulation, Bt strain PS149B1 produces protein crystals that exhibit oral toxicity to western corn rootworm larvae when tested in vitro. Examination of parasporal inclusion bodies showed they comprised two proteins with molecular masses of approximately 14 kDa and 44 kDa. Some proteolytic processing of the 44 kDa component occurs near the C terminus, but insecticidal activity is retained when combined with the 14 kDa component. Molecular cloning and DNA sequencing of the genes encoding these proteins, detailed elsewhere (R.T. Ellis et al. unpublished results), revealed that they were encoded by a single operon (Fig. 1A). Sequence analysis revealed no homology with any of the previously identified classes of Bt insecticidal proteins, although evidence does exist for evolutionary relatedness between the 44 kDa polypeptide and the 42 kDa and 51 kDa dipteran-active toxins from Bacillus sphaericus10. Bioassays using recombinant proteins expressed in heterologous bacterial systems demonstrated the requirement of both proteins for optimal insecticidal activity against rootworms (Table 1). The positive slopes of the probit lines indicate a dosedependent effect of the test materials. The reason(s) for the differences in the slopes of PS149B1, MR546, and the combination of MR1253 and MR1256 is unknown. One may speculate that the differences in the molar ratio or intermolecular interactions of the 14 kDa and the 44 kDa proteins between the native PS149B1 and the recombinant samples could contribute to the differences in the slopes. Furthermore, the proteins are produced as crystals in the native PS149B1 and in the Bt expression system MR546. However, in the Pseudomonas fluorescens expression system these proteins are expressed in inclusion bodies but not as defined crystals as in Bt.

1Pioneer Hi-Bred International, Inc., 7250 NW 62nd Avenue, P.O. Box 552, Johnston, IA 50131-0552. 2Dow AgroSciences, LLC, 301 Campus Drive, Huxley, IA 50124. 3Dow AgroSciences, LLC, 5501 Oberlin Drive, San Diego, CA 92121. 4Current address: SemBioSys Genetics, Inc., 500, 3605 29th Street N.E., Calgary, AB T1Y-5W4, Canada. 5Current address: 209 Hudson Pond Road, West Greenwich, RI 02817. *Corresponding author ([email protected]).

668

nature biotechnology



VOLUME 19



JULY 2001



http://biotech.nature.com

© 2001 Nature Publishing Group http://biotech.nature.com

RESEARCH ARTICLE

analysis (data not shown) and immunoblot analysis to check for integration of the genes encoding the PS149B1 proteins and expression of both proteins (Fig. 2G). Expression of the 14 kDa protein ranged from 0.03% to 0.2% of the total soluble protein in the roots and from 0.05% to 0.1% in the leaves. Expression of the 44 kDa protein ranged from 0.02% to 0.1% of the total soluble protein in the roots and from 0.4% to 0.9% in the leaves. Corn plants expressing both the 14 kDa and 44 kDa proteins were resistant to corn rootworm feeding. Transformed corn lines with outstanding root ratings and good seed set were backcrossed into commercial inbred lines for field-testing. Field testing for rootworm resistance. To determine if the events expressing the 14 kDa and 44 kDa proteins provide resistance to corn rootworm under field conditions, we planted second-generation backcross seed (T2) derived from 10 independent transformation events in nine locations across the US corn belt. The locations of the tests and the rootworm species present are shown in Table 2. There was a significant location effect (F = 87.11; d.f. = 8, 285; P < 0.001), which, of course, is expected given the varying levels of natural infestation. Averaged across the locations, plants co-expressing the 14 kDa and 44 kDa proteins showed excellent protection from damage by corn rootworms compared to both negative control plants and insecticide-treated plants. Negative controls ranged from 4.6 to 5.0 on the 1–6 root rating scale (Fig. 2A, Table 3) and were significantly more damaged than all other treatments tested. A score of 5 corresponds to two root nodes being completely destroyed, showing that insect pressure was extremely heavy across the locations. The terbufos and tefluthrin insecticide controls averaged 2.3 and 2.7, respectively (Fig. 2B, Table 3). These plants had several roots pruned to within 1.5 inches (3.8 cm) of the stem. By comparison, several of the corn lines co-expressing the 14 kDa and 44 kDa proteins from PS149B1 had significantly less damage than either negative controls or the tefluthrin insecticide controls (F = 87.11; d.f. = 15, 285; P < 0.001) and also averaged less than 2 on the 1–6 root rating scale (Fig. 2C–F, Table 3). Although feeding damage scars were seen, few if any roots were pruned by corn rootworm feeding. Because the proteins from PS149B1 are orally active, some feeding must occur if the rootworm larvae are to be affected. Northern corn rootworm. At two locations (Huron, SD and Schaller, IA), northern corn rootworms (Diabrotica barberi Smith and Lawrence) were present as the predominant naturally occurring rootworm pest. At the Huron location, 117 rootworm eggs were found in soil samples before artificial infestation and 94 were northern corn rootworm eggs (∼80%), as would be expected from the geo-

A

© 2001 Nature Publishing Group http://biotech.nature.com

B

Figure 1. Insecticidal proteins from Bt strain PS149B1. (A) Schematic representation of the PS149B1 toxin operon. (B) SDS–PAGE analysis of purified native and recombinant insecticidal crystal proteins from PS149B1. Some proteolytic processing of the 44 kD protein occurs near the C terminus. Native crystal proteins (lane 1); recombinant Bt MR546 co-expressing both polypeptides from the native toxin operon (lane 2); recombinant P fluorescens MR1253 expressing the 14 kDa polypeptide (lane 3); recombinant P. fluorescens MR1256 expressing the 44 kDa polypeptide (lane 4); combination of MR1254 and MR1256 (lane 5).

Multicomponent toxicity is complex, and investigation into the precise mode of action is underway. Expression of PS149B1 genes in plants. To test whether the PS149B1 proteins would provide resistance to rootworms in plants, synthetic genes encoding the 14 kDa and 44 kDa proteins, under transcriptional control of a constitutive promoter11, were introduced together into corn using particle gun transformation12. Phosphinothricin acetyltransferase was used as a selectable marker conferring resistance to the herbicides glufosinate or bialaphos. After transfer of plants from tissue culture, we immediately screened them for resistance to western corn rootworm in greenhouse infestations. Each transgenic plant was infested with 100 western corn rootworm eggs. After two weeks of rootworm feeding, the amount of damage was evaluated and resistant plants were transplanted for subsequent analysis and seed production. Transformants were analyzed by Southern

Table 1. LC50 estimates for insecticidal proteins from Bt strain PS149B1a Strain

PS149B1 MR546 MR1253 + MR1256e MR1253 MR1256 MR839 (P.f. host)f

n

LC50 µg/cm2

(95% CI)b

Gc

Sloped

X2d

d.f.d

239 300 237 257 196 292

55 17 51 NDg ND ND

(34–102) (5–27) (37–69) ND ND ND

0.2 0.4 0.1

1.3 2.3 2.6 ND ND ND

13 44 13 ND ND ND

10 10 10 ND ND ND

Average % mortality at 100 µg/cm2 62 99 77 6 6 7

aLC (concentration causing 50% mortality of the test population) estimates and statistics of Probit Analysis calculated by POLO-PC for the Bt strain PS149B1, 50 Bt recombinant MR546, and P. fluorescens recombinants MR1253 or MR1256. b90% confidence interval (CI) is presented for MR546. cIndex of significance for potency estimation of confidence interval20. When G ≥ 0.5 for the 95% level of confidence, it exceeds the limit for which the 95% CI may be reliably calculated. As a result the more reliable 90% CI for MR546 is presented. dDefinition of statistical terms: “slope” refers to the rate of change in mortality per unit change in concentration. “X2” is the Chi-square test statistic, commonly used to compare observed data with data we would expect to obtain according to a specific hypothesis. “d.f.” is the degrees of freedom, that is, the number of independent pieces of information that go into the estimate of a parameter. eMixtures applied at a 3:1 molar ratio (14 kDa protein to 44 kDa protein). fThe P. fluorescens host MR839 was applied at similar cell biomass rates. gND, Not determined; indicates that the LC estimate exceeded the dosages tested. 50

http://biotech.nature.com



JULY 2001



VOLUME 19



nature biotechnology

669

© 2001 Nature Publishing Group http://biotech.nature.com

© 2001 Nature Publishing Group http://biotech.nature.com

RESEARCH ARTICLE

A

B

C

D

Figure 2. Field performance of transgenic corn events co-expressing the 14 kDa and 44 kDa proteins from Bt strain PS149B1. (A) Roots of nontransgenic control plants without insecticide protection. (B) Roots of nontransgenic control plants treated with insecticide. (C–F) Roots of different transgenic events coexpressing the 14 kDa and 44 kDa proteins from strain PS149B1. Average root ratings for all plants within those events are shown below each picture for comparison (see Table 3). (G) Immunoblot of event 1 compared with the bacterial proteins.

G

foregut and hindgut is an extension of the exoskeleton, leaving only the midgut accessible for nutrient absorption. A peritrophic membrane, comprising a carbohydrate and protein matrix, lines the entire length of the midgut13. In the rootworm the membrane E F is laid down in continuous layers initiated at the base of the microvilli and then extruded into the lumen. The holes that are left where the microvilli had protruded impart a meshlike or netlike appearance. This membrane is semipermeable, allowing proteins and nutrients to pass through while excluding larger particulate matter. Feeding studies were done to visualize the effects of the PS149B1 proteins on western corn rootworm behavior and histology. Neonate, second-instar, and third-instar western corn rootworms graphical distribution of these pests. Although the artificial infestawere fed roots of transgenic plants co-expressing the PS149B1 protion of western corn rootworm may mask the impact of the natural teins. Insects were removed from the roots, fixed, embedded, and infestation of northern corn rootworm at these locations, the data sectioned. Results from second-instar rootworms are shown (Fig. 3). suggest that the presence of northern corn rootworms did not result For comparison, starved larvae were used to simulate the effects of in additional damage at those locations. For example, event 6 averfeeding avoidance (data not shown). Starved insects often had a colaged 1.5 across all nine locations (Table 3) and was one of the lowest lapsed and empty gut lumen with intact epithelial cells, and did not scoring events tested, with the greatest resistance to rootworms. At resemble intoxicated larvae. Huron and Schaller, the event scored 1.1 and 2.4, respectively. Event The first effects of the toxin become evident 12 h after insects 10, which had the highest average score (2.2) across all nine locations are added to the diet arena with transgenic roots. Cells begin (Table 3), scored 2.0 and 2.5 at Huron and Schaller, respectively. In swelling and numerous vacuoles appear, especially near the apical comparison, the 09B negative control averaged 5.0 at Huron and 4.6 surface of the epithelium. Only the epithelial cells are affected; the at Schaller. Though not definitive proof of efficacy against northern regenerative cells and endocrine cells near the basal surface of the corn rootworms, these results indicate that the presence of northern gut are unchanged. From 12 to 48 h after feeding begins, substancorn rootworm at these two sites did not result in any additional tial numbers of large vacuoles appear and the gut epithelial cells damage on the events. Additional studies conducted at locations swell, protruding into the lumen. The swelling cells bleb off large with northern corn rootworms present (data not shown) gave vesicles into the lumen (Fig. 3B). The vesicles contain dense similar results. cytoplasm, large vacuoles, and, in some instances, nuclei or fragEffects of 149B1 proteins on rootworm larvae. The alimentary mented nuclei. Ultimately, the microvilli on these cells collapse, canal of a corn rootworm comprises a cuticle-lined foregut, a forming a continuous flat membrane at the apical surface. These midgut composed of columnar epithelial cells with protruding results suggest that the midgut epithelium is the primary target microvilli, and a cuticle-lined hindgut. The goblet cells, found in tissue for the PS149B1 proteins, as is also the case with Cry1- and many other insects, are not present. The cuticle that lines the Vip3-type insecticidal proteins3. We have described Bt proteins that confer resistance to western corn rootTable 2. Field testing locations worm when expressed in transgenic corn plants. As with other Bt proteins, a Location Rootworm species Infestation level the histopathological effects of the PS149B1 proteins suggest that the Johnston, IA WCRW 1,000 WCRW eggs/plant midgut epithelium is the York, NB WCRW 1,000 WCRW eggs/plant + natural WCRW Huron, SD WCRW, NCRW 1,000 WCRW eggs/plant + natural NCRW and WCRW primary target tissue affected. The Princeton, IL WCRW 1,000 WCRW eggs/plant proteins, however, have no known Windfall, IN WCRW 1,000 WCRW eggs/plant similarity to other Cry proteins from Slater, IA WCRW 1,000 WCRW eggs/row footb Bt. The protection from rootworms Fowler, IN WCRW 1,000 WCRW eggs/row foot + natural WCRW conferred on transgenic plants Arlington, WI WCRW 1,000 WCRW eggs/row foot + natural WCRW expressing these proteins is superior to Schaller, IA WCRW, NCRW 1,000 WCRW eggs/row foot + natural NCRW and WCRW the control provided by chemical aWCRW, Western corn rootworm (Diabrotica virgifera virgifera LeConte). NCRW, Northern corn rootworm insecticides on nontransgenic plants (Diabrotica barberi Smith & Lawrence). Plots were artificially infested and locations where natural infestations also in field tests. They promise to occurred are noted. bA row foot corresponds to a foot of a row of corn. provide a new and effective alternative 670

nature biotechnology



VOLUME 19



JULY 2001



http://biotech.nature.com

© 2001 Nature Publishing Group http://biotech.nature.com

RESEARCH ARTICLE

Table 3. Average root ratings across all nine trial locations

© 2001 Nature Publishing Group http://biotech.nature.com

Event

09B Negative control DAS1 Negative control P38 Negative control B37 Negative control 09B + Terbufos insecticide 09B + Tefluthrin insecticide Event 1 Event 2 Event 3 Event 4 Event 5 Event 6 Event 7 Event 8 Event 9 Event 10

Total number of plants evalutated 62 52 57 51 35 34 55 43 126 266 236 408 52 49 111 50

Root ratinga

Standard error

4.7 5.0 4.6 4.8 2.3b 2.7b 2.0b 1.5b,c 1.6b,c 1.7b,c 1.5b,c 1.5b,c 1.8b 1.7b,c 1.7b,c 2.2b

Bioassays were held in darkness at 25°C for four days. Four days post infestation, plates were inspected for the number of both dead and surviving larvae. The 50% lethal concentration (LC50) and associated parameters were calculated using POLO PC (LeOra Software, 1987).

0.21 0.22 0.21 0.21 0.24 0.24 0.20 0.20 0.11 0.08 0.09 0.07 0.20 0.20 0.11 0.20

Plant transformation. The maize model genotype Hi-II was used in particle gun transformation experiments15. Ears were harvested at 8–10 days after pollination, and immature embryos approximately 1.0–2.0 mm in length were aseptically excised and cultured on modified N6 medium16 supplemented with 1.0 mg/L 2,4-D, 2.88 g/L proline, and 8.5 mg/L silver nitrate for 3–4 days in the dark at 25°C. Between 4 and 16 h before bombardment, embryos were transferred to medium containing 12% sucrose for an osmotic treatment. Tungsten particles coated with DNA were prepared essentially as described12. The DNA encoded a selectable marker gene for glufosinate resistance and the 45 kDa and 14 kDa genes under the control of a constitutive promoter11. Approximately 0.1 µg of DNA per shot was delivered to each plate of target embryos using the Bio-Rad PDS-1000/He device (Bio-Rad Laboratories, Hercules, CA) with a rupture pressure of 650 p.s.i., a vacuum pressure of 27–28 inches of Hg, and a particle flight distance of 8.5 cm. Embryos were then aRoot ratings18 can be defined briefly as follows: 6, three root nodes destroyed; 5, two root transferred to modified N6 medium supplemented with 2.0 mg/L nodes destroyed; 4, one root node destroyed; 3, several roots pruned to within 1.5 inches (3.8 cm) of the plant; 2, feeding scars and less than three roots pruned to within 1.5 inch- 2,4-D, 0.69 g/L proline, and 0.85 mg/L silver nitrate for 3–7 days after bombardment, and then transferred to selective medium cones (3.8 cm) of the plant; 1, no damage or only minor feeding scars. bStatistically better than negative controls; Tukey’s HSD (ref. 19), P = 0.05. taining 3 mg/L bialaphos. Tissue was subcultured every two weeks b,cStatistically better than negative controls and 09B + tefluthrin; Tukey’s HSD (ref. 19), for approximately 8–10 weeks. Resistant calli were transferred to P = 0.05. modified MS medium17 containing 1.0 mg/L indoleacetic acid (IAA), 0.026 mg/L abscisic acid (ABA), and 6% sucrose for 2–3 weeks to promote somatic embryo maturation. Regeneration was B A induced from the well-developed somatic embryos using hormone-free MS medium in the light at 25°C for 1–2 weeks. A total of 226 events were transferred to soil and placed in a growth chamber (25 ± 2°C, 16 h light) for 4–6 days before being transferred to the greenhouse for insect feeding bioassays. Field testing. Every location was artificially infested with western corn rootworm eggs when plants were at the V2–V3 stage of development (two or three collared leaves). Several locations also had a natural infestation of northern and/or western corn rootworm eggs in the soil. Because the events segregated for the presence of the transgenes including the cotransformed herbicide resistance gene, plants were treated with glufosinate at a rate of 28 ounces per acre to identify positive plants. All negative plants were removed before V4 leaf stage to eliminate larval establishment on negatively segregating plants. Two insecticide treatments were used as positive controls. Terbufos (Counter 20CR, American Cyanamid Co., Parsippany, NJ) and tefluthrin (Force 3G, Zeneca Ag Products, Wilmington, DE) were applied at the time of planting using the maximum rate recommended for corn rootworm control. Four nontransgenic inbred corn lines were used as negative control lines: 09B and P38 are two inbred lines that correspond to the genetic backgrounds for the introgression of the transformation events; B37 and DAS1 represent two other corn rootworm–susceptible inbred lines. Plants were evaluated by examining the washed root system and scoring for rootworm feeding damage. A visual damage rating system18 with a scale of 1–6 was used to score each plant. Roots within a plot were averaged to provide a plot score. Plot scores were then analyzed using SAS-JMP 4.0 (SAS Institute, Inc. Cary, NC). Mean separations were conducted using Tukey’s HSD test at the P = 0.05 level19.

Figure 3. Effects of PS149B1 toxin on midguts of western corn rootworm. (A) Cross section of a second-instar rootworm after 48 h on a control plant. mv, Microvilli; ge, midgut epithelium. (B) Cross section of a secondinstar rootworm after 48 h on PS149B1 transgenic root.

to chemical insecticides for controlling important pests in crop plants.

Experimental protocol Insecticidal activity of PS149B1 crystal proteins. The 14 or 44 kDa toxin genes of the PS149B1 toxin operon (GenBank accession no. AY011120) were engineered separately into plasmid vectors by standard DNA-cloning methods and transformed into P. fluorescens MB214 or Bt MR546 for protein expression. Recombinant strains were grown, and washed culture pellets were pipetted onto the surface of artificial diet based on Marrone et al.14 in 48-well plates at the rate of 156 µl/cm2. Three topical bioassays of four 1–100 µg/cm2 dosages were performed against western corn rootworm using washed culture pellets. The negative control was MR839, a P. fluorescens host strain for gene cloning that lacks insect toxin genes. MR839 was applied at the highest cell mass as was applied with the test materials. Quantitation of the insecticidal proteins expressed in heterologous hosts was achieved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using a Molecular Dynamics densitometer (Molecular Dynamics, Sunnyvale, CA) and BSA as a standard. Treated bioassay plates were surface-dried in a laminar flow hood and infested with approximately six neonate western corn rootworm larvae per well. Larvae were hatched from eggs obtained from French Agricultural Research (Lamberton, MN). Following infestation, plates were sealed with Mylar and holes were punched above each well to provide for gas exchange. http://biotech.nature.com



JULY 2001

Microscopy. Neonate, second-instar, and third-instar western corn rootworms were fed roots of transgenic plants co-expressing the PS149B1 proteins for 12, 24, and 48 h. Insects fed on nontransformed tissues or starved were used as controls. Insects were removed from the roots, fixed in 4% paraformaldehyde, dehydrated through an ethanol series, infiltrated with limonene (Histo-clear, National Diagnostics, Atlanta, GA), embedded in paraffin, and sectioned (10 µm). Sections were stained with toluidine blue for microscopic visualization. Immunoblot analysis. Tissue extracts were prepared for western blots by homogenizing 10–30 mg tissue samples in 500 µl PBST (136 mM NaCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, and 2.7 mM KCl with 0.05% Tween-20). •

VOLUME 19



nature biotechnology

671

© 2001 Nature Publishing Group http://biotech.nature.com

© 2001 Nature Publishing Group http://biotech.nature.com

RESEARCH ARTICLE Tissue proteins (denatured, reduced) were analyzed by SDS–PAGE using 4–20% gradient gels and a modified Laemmli buffer system. Positive controls for the 14 kDa and 44 kDa proteins are included. The Bacillus microbial genes were expressed in Pseudomonas and the 14 kDa and 44 kDa complex were purified as positive controls. Proteins were then transferred to 0.2 µm polyvinyl difluoride (PVDF) membrane for immunoblot analysis (all gels and buffers for SDS–PAGE and transfer were obtained from Novex, San Diego, CA). Resulting blots were developed using reagents in the WesternLight Immunodetection System obtained from Tropix (Bedford, MA). Affinity-purified rabbit polyclonal anti-14 kDa and anti-44 kDa PS149B1

antibodies were used at 1:1,000 dilution. Blots were developed in a Kodak M35A X-OMAT Processor. Acknowledgments We thank Theodore Kahn for helpful discussions and contributions to preparing figures and reviewing this manuscript. We thank Leo Kim, Dave Grothaus, Larry Sernyk, and Roger Kemble for their organizational leadership and helpful discussions. We thank Steve Ritchie for his contribution in corn breeding, and Clara Alarcon and David Hondred for Southern analysis. The authors also wish to acknowledge the excellent technical work of Josh Russell. Received 19 January 2001; accepted 15 May 2001

1. Metcalf, R.L. In Methods for the study of the pest Diabrotica. (eds Krysan, J.L. & Miller, T.A.) vii–xv (Springer-Verlag, New York; 1986). 2. Levine, E. & Oloumi-Sadeghi, H. Western corn rootworm (Coleoptera: Chrysomelidae) larval injury to corn grown for seed production following soybeans grown for seed production. J. Econ. Entomol. 89, 1010–1016 (1996). 3. Schnepf, E. et al. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 775–806 (1998). 4. Perlak, F.J. et al. Insect resistant cotton plants. Bio/Technology 8, 939–943 (1990). 5. Perlak, F.J. et al. Genetically improved potatoes: protection from damage by Colorado potato beetles. Plant Mol. Biol. 22, 313–321 (1993). 6. Fujimoto, H., Itoh, K., Yamamoto, M., Kyozuka, J. & Shimamoto, K. Insect resistant rice generated by introduction of a modified δ-endotoxin gene of Bacillus thuringiensis. Bio/Technology 11, 1151–1155 (1993). 7. Koziel M.G. et al. Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/Technology 11, 194–200 (1993). 8. Warren, G.W. Vegetative insecticidal proteins: novel proteins for control of corn pests. In Advances in insect control. (eds Carozzi, N. & Koziel, M.) 109–121 (Taylor and Francis, London, UK; 1997). 9. Estruch, J.J. et al. Vip3a, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Proc. Natl. Acad. Sci. USA 93, 5389–5394 (1996). 10. Baumann, L., Broadwell, A.H. & Baumann, P. Sequence analysis of the mosquitocidal toxin genes encoding 51.4 and 41.9 kilodalton proteins from Bacillus sphaericus 2362 and 2297. J. Bacteriol. 170, 2045–2050 (1988). 11. Christensen, A.H., Sharrock, R.A. & Quail, P.H. Maize polyubiquitin genes: struc-

672

nature biotechnology



VOLUME 19

12.

13.

14.

15. 16.

17. 18. 19. 20.



ture, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol. Biol. 18, 675–689 (1992). Klein, T.M., Gradziel, T., Fromm, M.E. & Sanford, J.C. Factors influencing gene delivery into Zea mays cells by high velocity microprojectiles. Biotechnology 6, 559–563 (1989). Ryerse, J.S., Purcell, J.P. & Sammoms, R.D. Structure and formation of the peritrophic membrane in the larva of the southern corn rootworm, Diabrotica undecimpunctata. Tissue Cell 26, 431–437 (1994). Marrone, P., Ferri, F.D., Mosley, T.R. & Meinke, L.J. Improvements in laboratory rearing of the southern corn rootworm, Diabrotica undecimpunctata howardi Barber (Coleoptera: Chrysomelidae), on an artificial diet and corn. J. Econ. Entomol. 78, 290–293 (1985). Armstrong, C.E. Development and availability of germplasm with high type II culture formation response. Maize Genet. Cooperative Newslett. 65, 92–93 (1991). Chu, C.C. et al. Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Science Sinica 18, 659–668 (1975). Murashige, T. & Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473–497 (1962). Hills, T.M. & Peters, D.C. A method of evaluating postplanting insecticide treatments for control of western corn rootworm larvae. J. Econ. Entomol. 64, 764–765 (1971). Tukey, J.W. The problem of multiple comparisons. (Princeton University, Princeton. NJ; 1953). Russell, R.M., Robertson, J.L. & Savin, N.E. POLO: a new computer program for Probit Analysis. ESA Bull. 23, 209–213 (1977).

JULY 2001



http://biotech.nature.com