Cry1C Protein Research Articles

Silencing of Midgut Aminopeptidase N of Spodoptera litura by Double-stranded RNA Establishes Its Role asBacillus thuringiensis Toxin Receptor

Insecticidal crystal proteins of Bacillus thuringiensis bind to receptors in the midgut of susceptible insects leading to pore formation and death of the insect. The identity of the receptor is not clearly established. Recently a direct interaction between a cloned and heterologously expressed aminopeptidase (slapn) from Spodoptera litura and the Cry1C protein was demonstrated by immunofluorescence and in vitro ligand blot interaction. Here we show that administration of slapn double-stranded RNA to S. litura larvae reduces its expression. As a consequence of the reduced expression, a corresponding decrease in the sensitivity of these larvae to Cry1C toxin was observed. The gene silencing was retained during the insect's moulting and development and transmitted to the subsequent generation albeit with a reduced effect. These results directly implicate larval midgut aminopeptidase N as receptor for Bacillus thuringiensis insecticidal proteins.

The InhA2 metalloprotease of Bacillus thuringiensis strain 407 is required for pathogenicity in insects infected via the oral route.

The entomopathogenic bacterium Bacillus thuringiensis is known to secrete a zinc metalloprotease (InhA) that specifically cleaves antibacterial peptides produced by insect hosts. We identified a second copy of the inhA gene, named inhA2, in B. thuringiensis strain 407 Cry(-). The inhA2 gene encodes a putative polypeptide showing 66.2% overall identity with the InhA protein and harboring the zinc-binding domain (HEXXH), which is characteristic of the zinc-requiring metalloproteases. We used a transcriptional inhA2'-lacZ fusion to show that inhA2 expression is induced at the onset of the stationary phase and is overexpressed in a Spo0A minus background. The presence of a reverse Spo0A box in the promoter region of inhA2 suggests that Spo0A directly regulates the transcription of inhA2. To determine the role of the InhA and InhA2 metalloproteases in pathogenesis, we used allelic exchange to isolate single and double mutant strains for the two genes. Spores and vegetative cells of the mutant strains were as virulent as those of the parental strain in immunized Bombyx mori larvae infected by the intrahemocoelic route. Exponential phase cells of all the strains displayed the same in vitro potential for colonizing the vaccinated hemocoel. We investigated the synergistic effect of the mutant strain spores on the toxicity of Cry1C proteins against Galleria mellonella larvae infected via the oral pathway. The spores of DeltainhA2 mutant strain were ineffective in providing synergism whereas those of the DeltainhA mutant strain were not. These results indicate that the B. thuringiensis InhA2 zinc metalloprotease has a vital role in virulence when the host is infected via the oral route.

Broccoli plants with pyramided cry1Ac and cry1C Bt genes control diamondback moths resistant to Cry1A and Cry1C proteins.

This study was undertaken to determine the effects of pyramiding two Bacillus thuringiensis (Bt) genes in the same plant on the production of Bt proteins and the control of diamondback moths (DBM, Plutella xylostella) resistant to one or the other protein. Broccoli lines carrying both cry1Ac and cry1C Bt genes were produced by sexual crosses of cry1Ac- and cry1C-transgenic plants. Plants containing both genes were selected by tests for resistance to kanamycin and hygromycin, and confirmed by PCR analysis for the Bt genes. Both cry1Ac and cry1C mRNAs were detected in the hybrid lines, and Cry1Ac and Cry1C proteins were stably produced at levels comparable to the parental plants. Plants producing both Cry1Ac and Cry1C proteins caused rapid and complete mortality of DBM larvae resistant to Cry1A or Cry1C, and suffered little or no leaf damage. These plants, in combination with the resistant DBM populations available, will allow greenhouse or field studies of resistance management strategies involving gene pyramiding.

Molecular Genetic Manipulation of Truncated Cry1C Protein Synthesis in Bacillus thuringiensis To Improve Stability and Yield

Cry1 protoxins of Bacillus thuringiensis are insecticidal 135-kDa proteins synthesized and assembled into parasporal crystals during sporulation. After ingestion, these crystals dissolve in the midgut and active toxins with molecular masses of about 65-kDa are released from the N-terminal half of the molecule by midgut proteases. Direct synthesis of the toxin-containing N-terminal half of Cry1 molecules using recombinant DNA techniques results in a low level of unstable truncated proteins that do not crystallize. In the present study, inclusions of truncated Cry1C (Cry1C-t) were obtained by combining genetic elements from other endotoxin genes and operons that enhance Cry protein synthesis and crystallization. Increased levels of Cry1C-t synthesis were achieved by using cyt1A promoters to drive expression of the 5' half of cry1C that included in the construct the 5' cry3A STAB-SD mRNA stabilizing sequence and the 3' stem-loop transcription terminator. RNA dot blot analysis showed that the STAB-SD and 3' transcriptional termination sequences were important for stabilization of truncated cry1C (cry1C-t) mRNA. A low level of cry1C-t mRNA was present when only the cyt1A promoters were used to express cry1C-t, but no accumulation of Cry1C-t was detected in Western blots. The orientation of the transcription terminator was important to enhancing Cry1C-t synthesis. Inclusion of the 20- and 29-kDa helper protein genes in cry1C-t constructs further enhanced synthesis. The Cry1C-t protein was toxic to Spodoptera exigua larvae, though the toxicity (50% lethal concentration [LC(50)] = 13.2 microg/ml) was lower than that of full-length Cry1C (LC(50) = 1.8 microg/ml). However, transformation of the HD1 isolate of B. thuringiensis subsp. kurstaki with the cry1C-t construct enhanced its toxicity to S. exigua as much as fourfold.

Development and characterization of diamondback moth resistance to transgenic broccoli expressing high levels of Cry1C.

A field-collected colony of the diamondback moth, Plutella xylostella, had 31-fold resistance to Cry1C protoxin of Bacillus thuringiensis. After 24 generations of selection with Cry1C protoxin and transgenic broccoli expressing a Cry1C protein, the resistance that developed was high enough that neonates of the resistant strain could complete their entire life cycle on transgenic broccoli expressing high levels of Cry1C. After 26 generations of selection, the resistance ratios of this strain to Cry1C protoxin were 12,400- and 63,100-fold, respectively, for the neonates and second instars by a leaf dip assay. The resistance remained stable until generation 38 (G38) under continuous selection but decreased to 235-fold at G38 when selection ceased at G28. The Cry1C resistance in this strain was seen to be inherited as an autosomal and incompletely recessive factor or factors when evaluated using a leaf dip assay and recessive when evaluated using Cry1C transgenic broccoli. Saturable binding of (125)I-Cry1C was found with brush border membrane vesicles (BBMV) from both susceptible and Cry1C-resistant strains. Significant differences in Cry1C binding to BBMV from the two strains were detected. BBMV from the resistant strain had about sevenfold-lower affinity for Cry1C and threefold-higher binding site concentration than BBMV from the susceptible strain. The overall Cry1C binding affinity was just 2.5-fold higher for BBMV from the susceptible strain than it was for BBMV from the resistant strain. These results suggest that reduced binding is not the major mechanism of resistance to Cry1C.

Transgenic broccoli with high levels of Bacillus thuringiensis Cry1C protein control diamondback moth larvae resistant to Cry1A or Cry1C

A synthetic Bacillus thuringiensis (Bt) cry1C gene was introduced into broccoli (Brassica oleracea ssp. italica) by Agrobacterium-mediated transformation. Twenty-one Cry1C transgenic plants were regenerated from 400 hypocotyl and petiole explants. Variable amounts of stable steady- state cry1C mRNA accumulated in different transgenic plants. Cry1C protein (up to 0.4% of total soluble protein) was produced in correlation with the cry1C mRNA levels. Leaf section and whole-plant bioassays were done using diamondback moth (DBM) larvae from lines susceptible to Bt or resistant to Cry1A or Cry1C proteins (Cry1AR or Cry1CR, respectively). Plants with high levels of Cry1C protein caused rapid and complete mortality of all three types of DBM larvae with no defoliation. Plants with lower levels of Cry1C protein showed an increasing differential between control of susceptible of Cry1AR DBM. This study demonstrated that high production of Cry1C protein can protect transgenic broccoli not only from susceptible or Cry1AR DBM larvae but also from DBM selected for moderate levels of resistance of Cry1C. The Cry1C- transgenic broccoli were also resistant to two other lepidopteran pests of crucifers (cabbage looper and imported cabbage worm). These plants will be useful in studies of resistance management strategies involving multiple transgenes.

Green-tissue-specific expression of a reconstructed cry1C gene encoding the active fragment of Bacillus thuringiensis delta-endotoxin in haploid tobacco plants conferring resistance to Spodoptera litura.

The DNA sequence of a truncated cry1C gene encoding the active fragment of Bacillus thuringiensis (Bt) delta-endotoxin was fully reconstructed by introduction of silent mutations. Each of the truncated wild type and the synthetic genes encoding the active fragment of the protoxin was introduced into haploid tobacco plants under the control of the rbcS promoter. To facilitate selection of transgenic tobacco plants with high insecticidal activity, a fusion gene encoding both rat CYP1A1 cytochrome P450 and yeast NADPH-P450 oxidoreductase was cotransformed with the wild type cry1C gene. The synthetic gene elevated the levels of Cry1C protein and the mRNA in transgenic tobacco plants as well as mortality in Spodoptera litura larvae. The Cry1C protein was accumulated mainly in the leaf tissues of the transgenic tobacco plants. The results reported here imply that the green-tissue-specific expression of the synthetic cry1C gene is useful for the control of S. litura which was rather resistant to the other types of Bt toxins.

Domain III of the Bacillus thuringiensis delta-endotoxin Cry1Ac is involved in binding to Manduca sexta brush border membranes and to its purified aminopeptidase N.

Three types of binding assays were used to study the binding of Bacillus thuringiensis delta-endotoxin Cry1Ac to brush border membrane vesicle (BBMV) membranes and a purified putative receptor of the target insect Manduca sexta. Using hybrid proteins consisting of Cry1Ac and the related Cry1C protein, it was shown that domain III of Cry1Ac is involved in specificity of binding as observed by all three techniques. In ligand blotting experiments using SDS-PAGE-separated BBMV proteins as well as the purified putative receptor aminopeptidase N (APN), the presence of domain III of Cry1Ac in a hybrid with Cry1C was necessary and sufficient for specific binding to APN. Using the surface plasmon resonance (SPR) technique with immobilized APN, it was shown that the presence of domain III of Cry1Ac in a hybrid is sufficient for binding to one of the two previously identified Cry1Ac binding sites, whereas the second site requires the full Cry1Ac toxin for binding. In addition, the role of domain III in the very specific inhibition of Cry1Ac binding by the amino sugar N-acetylgalactosamine (GalNac) was determined. Both in ligand blotting and in surface plasmon resonance experiments, as well as in binding assays using intact BBMVs, it was shown that the presence of domain III of Cry1Ac in a toxin molecule is sufficient for the inhibition of binding by GalNAc. These and other results strongly suggest that domain III of delta-endotoxins play a role in insect specificity through their involvement in specific binding to insect gut epithelial receptors.

Cry1Ac, a bacillus thuringiensis toxin, triggers extracellular Ca2+ influx and Ca2+ release from intracellular stores in Cf1 cells

Intracellular Ca2+ concentration was measured in single Cf1 cells (Choristoneura fumiferana, spruce budworm) loaded with Fura-2, a Ca2+-sensitive fluorescent probe. Cf1 cells displayed Ca2+ surges in response to Cry1Ac and Cry1C proteins, two Cf1-toxic Bacillus thuringiensis products, but not to Cry1Aa and Cry3A, which are not toxic to Cf1 cells. In the presence of extracellular Ca2+, the toxin-induced Ca2+ response was insensitive to methoxyverapamil, a voltage-dependent Ca2+ channel blocker, but was abolished by lanthanum, a general inhibitor of Ca2+ transport. In the absence of external Ca2+, Cry1Ac induced a small intracellular Ca2+ transient which was inhibited by TMB-8, a blocker of Ca2+ release from inositol-1,4,5-trisphosphate-sensitive pools. Under these conditions, thapsigargin, which inhibits intracellular Ca2+-ATPases, elicited a Ca2+ surge when applied alone. However, subsequent addition of Cry1Ac failed to induce a Ca2+ signal, indicating a depletion of intracellular Ca2+ pools. In Cf1 cells, therefore, bioactive B. thuringiensis toxins triggered intracellular Ca2+ surges which were mainly due to the influx of extracellular Ca2+ through toxin-made pores, as confirmed by planar lipid bilayer experiments. Furthermore, TMB-8- and thapsigargin-sensitive Ca2+ stores contributed to the Cry1Ac-induced Ca2+ signal.

Influence of the 20-kDa protein from Bacillus thuringiensis ssp. israelensis on the rate of production of truncated Cry1C proteins.

The potential role of a molecular chaperone on the rate of production of extensively altered Bacillus thuringiensis Cry1C proteins was investigated. Analysis of the proteins produced by the recombinant B. thuringiensis strains showed that the truncated proteins were produced at a low rate. Expression of the 20-kDa protein gene from B. thuringiensis ssp. israelensis in tandem with the truncated-cry1C genes led to the production of a greater amount of proteins. The formation of inclusion bodies, however, did not occur even when the 20-kDa protein gene was expressed.

Influence of the 20-kDa protein from Bacillus thuringiensis ssp. israelensis on the rate of production of truncated Cry1C proteins

The potential role of a molecular chaperone on the rate of production of extensively altered Bacillus thuringiensis Cry 1C proteins was investigated. Analysis of the proteins produced by the recombinant B. thuringiensis strains showed that the truncated proteins were produced at a low rate. Expression of the 20-kDa protein gene from B. thuringiensis ssp. israelensis in tandem with the truncated- cry1C genes led to the production of a greater amount of proteins. The formation of inclusion bodies, however, did not occur even when the 20-kDa protein gene was expressed.

Contribution of Bacillus thuringiensis Spores to Toxicity of Purified Cry Proteins Towards Indianmeal Moth Larvae

The influence of Bacillus thuringiensis subsp. kurstaki HD-1 spores upon the toxicity of purified Cry1Ab and Cry1C crystal proteins toward susceptible and BT-resistant Indianmeal moth (IMM, Plodia interpunctella) larvae was investigated. With susceptible larvae, HD-1 spores were toxic in the absence of crystal protein and highly synergistic (approximately 35- to 50-fold) with either Cry1Ab or Cry1C protein. With BT-resistant IMM larvae, HD-1 spores were synergistic with Cry1Ab and Cry1C protein in all three resistant strains examined. Synergism was highest (approximately 25- to 44-fold) in insects with primary resistance toward Cry1C (IMM larvae with resistance to B. thuringiensis subsp. aizawai or entomocidus). However, HD-1 spores also synergized either Cry1Ab or Cry1C toxicity toward larvae resistant to B. thuringiensis subsp. kurstaki at a lower level (approximately five- to sixfold). With susceptible larvae, the presence of spores reduced the time of death when combined with each of the purified Cry proteins. Without spores, the speed of intoxication and eventual death for larvae treated with Cry1C and Cry1Ab proteins was much slower than for the HD-1 preparation containing both spores and crystals together. Neither spores nor toxin dose affected the mean time of death of resistant larvae treated with either Cry1Ab or Cry1C toxins. Both Cry1Ab and Cry1C toxins appeared to reduce feeding and consequently toxin consumption.