Functional interactions between members of the REPAT family of insect pathogen-induced proteins.

Studies on the transcriptional response to pathogens in the insect larval gut have shown the regulation of several genes after the infection. Repat (REsponse to PAThogens) genes were first identified in Spodoptera exigua midgut as being up-regulated in response to the exposure to Bacillus thuringiensis toxins and baculovirus. Recently, new members of the REPAT family showed a constitutive up-regulation in a B. thuringiensis-resistant population. Based on a yeast two-hybrid screening, we have detected the interaction of REPAT1 with other members of the REPAT family, leading to the discovery of a new member: REPAT8. The functional role of this interaction was shown by following the changes of the subcellular localization of REPAT1 in the presence of REPAT8. REPAT1 alone was localized exclusively in the cytoplasm, while the presence of REPAT8 led to the migration of REPAT1 to the nucleus. Finally, analysis of the expression pattern of eight REPAT members has shown that B. thuringiensis-related treatments (Cry1Ca toxin, Xentari™ product and an acrystalliferous strain) induced a general up-regulation of repat genes, especially of repat2. In contrast, no significant effect was detected after treatment with Escherichia coli or Enterococcus sp., or by the presence of microbiota in the midgut. The results suggest that the different repat genes play different roles in response to pathogens.

Permeability changes of Manduca sexta midgut brush border membranes induced by oligomeric structures of different cry toxins.

The pore-formation activity of monomeric and oligomeric forms of different Cry1 toxins (from Cry1A to Cry1G) was analyzed by monitoring ionic permeability across Manduca sexta brush border membrane vesicles. The membrane vesicles were isolated from microvilli structures, showing a high enrichment of apical membrane markers and low intrinsic K(+) permeability. A fluorometric assay performed with 3,3'-dipropylthiodicarbocyanine fluorescent probe, sensitive to changes in membrane potential, was used. Previously, it was suggested that fluorescence determinations with this dye could be strongly influenced by the pH, osmolarity and ionic strength of the medium. Therefore, we evaluated these parameters in control experiments using the K(+)-selective ionophore valinomycin. We show here that under specific ionic conditions changes in fluorescence can be correlated with ionic permeability without effects on osmolarity or ionic strength of the medium. It is extremely important to attenuate the background response due to surface membrane potential and the participation of the endogenous permeability of the membrane vesicles. Under these conditions, we analyzed the pore-formation activity induced by monomeric and oligomeric structures of different Cry1 toxins. The Cry1 toxin samples containing oligomeric structures correlated with high pore activity, in contrast to monomeric samples that showed marginal pore-formation activity, supporting the hypothesis that oligomer formation is a necessary step in the mechanism of action of Cry toxins.

Translation of both 5'TOP and non-TOP host mRNAs continues into the late phase of Baculovirus infection.

Complete cDNA sequences were obtained for ribosomal protein (rp) L15 and eukaryotic initiation factor eIF2alpha from the lepidopteran insect Spodoptera frugiperda, and for elongation factor eEF2 from S. exigua. The presence of a 5' terminal oligopyrimidine (TOP) tract classified the lepidopteran rpL15 transcript as a TOP mRNA. For eEF2, two types of transcripts were observed, one of which had a 5'TOP tract. The transcript levels for rpL15, eEF2 and eIF2alpha decreased following baculovirus infection. Polysome analysis showed that the corresponding mRNAs remained to be translated until at least 16 h post-infection for both TOP and non-TOP mRNAs. Baculovirus-induced host shut-off therefore appears to be regulated at the level of RNA abundance rather than at the translational level.

Bacillus thuringiensis delta-endotoxin Cry1 hybrid proteins with increased activity against the Colorado potato beetle.

Cry1 delta-endotoxins of Bacillus thuringiensis are generally active against lepidopteran insects, but Cry1Ba and Cry1Ia have additional, though low, levels of activity against coleopterans such as the Colorado potato beetle. Here we report the construction of Cry1Ba/Cry1Ia hybrid toxins which have increased activities against this insect species.

How Bacillus thuringiensis has evolved specific toxins to colonize the insect world.

Bacillus thuringiensis is a bacterium of great agronomic and scientific interest. Together the subspecies of this bacterium colonize and kill a large variety of host insects and even nematodes, but each strain does so with a high degree of specificity. This is mainly determined by the arsenal of crystal proteins that the bacterium produces during sporulation. Here we describe the properties of these toxin proteins and the current knowledge of the basis for their specificity. Assessment of phylogenetic relationships of the three domains of the active toxin and experimental results indicate how sequence divergence in combination with domain swapping by homologous recombination might have caused this extensive range of specificities.

Cross-resistance of pink bollworm (Pectinophora gossypiella) to Bacillus thuringiensis toxins.

Two strains of pink bollworm (Pectinophora gossypiella) selected in the laboratory for resistance to Bacillus thuringiensis toxin Cry1Ac had substantial cross-resistance to Cry1Aa and Cry1Ab but not to Cry1Bb, Cry1Ca, Cry1Da, Cry1Ea, Cry1Ja, Cry2Aa, Cry9Ca, H04, or H205. The narrow spectrum of resistance and the cross-resistance to activated toxin Cry1Ab suggest that reduced binding of toxin to midgut target sites could be an important mechanism of resistance.

Bacillus thuringiensis delta-endotoxin Cry1C domain III can function as a specificity determinant for Spodoptera exigua in different, but not all, Cry1-Cry1C hybrids.

In order to test our hypothesis that Bacillus thuringiensis delta-endotoxin Cry1Ca domain III functions as a determinant of specificity for Spodoptera exigua, regardless of the origins of domains I and II, we have constructed by cloning and in vivo recombination a collection of hybrid proteins containing domains I and II of various Cry1 toxins combined with domain III of Cry1Ca. Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ea, and Cry1Fa all become more active against S. exigua when their domain III is replaced by (part of) that of Cry1Ca. This result shows that domain III of Cry1Ca is an important and versatile determinant of S. exigua specificity. The toxicity of the hybrids varied by a factor of 40, indicating that domain I and/or II modulate the activity as well. Cry1Da-Cry1Ca hybrids were an exception in that they were not significantly active against S. exigua or Manduca sexta, whereas both parental proteins were highly toxic. Incidentally, in a Cry1Ba-Cry1Ca hybrid, Cry1Ca domain III can also strongly increase toxicity for M. sexta.

Identification of Bacillus thuringiensis delta-endotoxin Cry1C domain III amino acid residues involved in insect specificity.

Cry1C domain III amino acid residues involved in specificity for beet armyworm (Spodoptera exigua) were identified. For this purpose, intradomain III hybrids between Cry1E (nontoxic) and Cry1E-Cry1C hybrid G27 (toxic) were made. Crossover points of these hybrids defined six sequence blocks containing between 1 and 19 of the amino acid differences between Cry1E and G27. Blocks B, C, D, and E of G27 were shown to be required for optimal activity against S. exigua. Block E was also required for optimal activity against the tobacco hornworm (Manduca sexta), whereas block D had a negative effect on toxicity for this insect. The mutagenesis of individual amino acids in block B identified Trp-476 as the only amino acid in this block essential, although not sufficient by itself, for full S. exigua activity. In block D, we identified a seven-amino-acid insertion in G27 that was not in Cry1E. The deletion of either one of two groups of four consecutive amino acids in this insertion completely abolished activity against S. exigua but resulted in higher activity against M. sexta. Alanine substitutions of the first group had little effect on toxicity, whereas alanine substitutions of the second group had the same effect as its deletion. These results identify groups of amino acids as well as some individual residues in Cry1C domain III, which are strongly involved in S. exigua-specific activity as well as sometimes involved in M. sexta-specific activity.

Interaction between functional domains of Bacillus thuringiensis insecticidal crystal proteins.

Interactions among the three structural domains of Bacillus thuringiensis Cry1 toxins were investigated by functional analysis of chimeric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain I or domain III among Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The activity of the purified trypsin-activated chimeric toxins was evaluated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only Cry1C was active against these cells and only chimeras possessing domain II from Cry1C were functional. Combination of domain I from Cry1E with domains II and III from Cry1C, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was from Cry1Ab or Cry1Ac were slightly smaller than those formed by toxins in which domain I was from Cry1C. The properties of the pores formed by the chimeras are therefore likely to result from an interaction between domain I and domain II or III. Domain III appears to modulate the activity of the chimeric toxins: combination of domain III from Cry1Ab with domains I and II of Cry1C gave a protein which was more strongly active than Cry1C.

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.

Toxicity and binding properties of the Bacillus thuringiensis delta-endotoxin Cry1C to cultured insect cells.

A better understanding of the mode of action of Bacillus thuringiensis delta-endotoxins is needed to develop strategies which may prevent or slow down selection for resistance. We studied the effect of Cry1C on several different cultured insect cell lines by means of toxicity assays, ligand blotting, and toxin binding studies. A clear difference in sensitivity toward Cry1C between the insect cell lines was observed. Spodoptera frugiperda cell line Sf9 was most sensitive, whereas Spodoptera exigua cell lines SeUCR and SelZD2109 showed intermediate sensitivity. Mamestra brassicae (Mb0503) and Drosophila melanogaster (Dm1) cells were the least sensitive as compared to Sf9 cells. Ligand blot analysis of SDS-PAGE size-separated proteins showed that Cry1C specifically binds to a 40-kDa protein in Sf9, SeUCR, and SelZD2109 cells. Cry1Ab does not bind to this protein. The Cry1C-binding protein was not observed in Mb0503 and Dm1 cells, suggesting that the presence of the 40-kDa Cry1C-binding protein is correlated with sensitivity toward Cry1C.

Different domains of Bacillus thuringiensis delta-endotoxins can bind to insect midgut membrane proteins on ligand blots.

We investigated the role of the constituent domains of the CryIA(b) and CryIA(c) delta-endotoxins in binding to midgut epithelial cell membrane proteins of Spodoptera exigua and Manduca sexta on ligand blots. A collection of wild-type and CryIC-CryIA hybrid toxins was used for this purpose. As demonstrated elsewhere (R. A. de Maagd, M. S. G. Kwa, H. van der Klei, T. Yamamoto, B. Schipper, J. M. Vlak, W. J. Stiekema, and D. Bosch, Appl. Environ. Microbiol. 62:1537-1543, 1996), CryIA(b) domain III recognized a 205-kDa protein on S. exigua blots, while no specific binding by domain I or II could be detected. In contrast, on ligand blots of M. sexta proteins CryIA(b) domain II recognized a 210-kDa protein and CryIA(b) domain III recognized a 250-kDa protein. Domain III is responsible for the interaction of CryIA(c) with 120-kDa major binding proteins of both S. exigua and M. sexta. In addition, in M. sexta CryIA(c) also reacts with a 210-kDa binding protein through its domain I and/or domain II. These results show that besides domain II, domain III of delta-endotoxins plays a major role in binding to putative receptors on ligand blots. However, for S. exigua there was no clear correlation between binding of toxins on ligand blots and the in vivo toxicity of the toxins. These and previous results suggest that interactions of insect membrane proteins with both domain II and domain III can occur and that detection of these interactions depends on the type of binding assay used.

Cross-resistance of the diamondback moth indicates altered interactions with domain II of Bacillus thuringiensis toxins.

We compared responses to six insecticidal crystal proteins from Bacillus thuringiensis by a Cry1A-resistant strain (NO-QA) and a susceptible strain (LAB-P) of the diamondback moth, Plutella xylostella. The resistant strain showed > 100-fold cross-resistance to Cry1J and to H04, a hybrid with domains I and II of Cry1Ab and domain III or Cry1C. Cross-resistance was sixfold to Cry1Bb and threefold to Cry1D. The potency of Cry1I did not differ significantly between the resistant and susceptible strains. Cry2B did not kill resistant or susceptible larvae. By combining these new data with previously published results, we classified responses to 14 insecticidal crystal proteins by strains NO-QA and LAB-P. NO-QA showed high levels of resistance to Cry1Aa, Cry1Ab, and Cry1Ac and high levels of cross-resistance to Cry1F, Cry1J, and H04. Cross-resistance was low or nil to Cry1Ba, Cry1Bb, Cry1C, Cry1D, Cry1I, and Cry2A. Cry1E and Cry2B showed little or no toxicity to susceptible or resistant larvae. In dendrograms based on levels of amino acid sequence similarity among proteins, Cry1F and Cry1J clustered together with Cry1A proteins for domain II, but not for domain I or III. High levels of cross-resistance to Cry1Ab-Cry1C hybrid H04 show that although Cry1C is toxic to NO-QA, domain III or Cry1C is not sufficient to restore toxicity when it is combined with domains I and II of Cry1Ab. Thus, diamondback moth strain NO-QA cross-resistance extends beyond the Cry1A family of proteins to at least two other families that exhibit high levels of amino sequence similarity with Cry1A in domain II (Cry1F and Cry1J) and to a protein that is identical to Cry1Ab in domain II (H04). The results of this study imply that resistance to Cry1A alters interactions between the insect and domain II.

Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition.

To test our hypothesis that substitution of domain III of Bacillus thuringiensis delta-endotoxin (Cry) proteins might improve toxicity to pest insects, e.g., Spodoptera exigua, in vivo recombination was used to produce a number of cryIA(b)-cryIC hybrid genes. A rapid screening assay was subsequently exploited to select hybrid genes encoding soluble protoxins. Screening of 120 recombinants yielded two different hybrid genes encoding soluble proteins with domains I and II of CryIA(b) and domain III of CryIC. These proteins differed by only one amino acid residue. Both hybrid protoxins gave a protease-resistant toxin upon in vitro activation by trypsin. Bioassays showed that one of these CryIA(b)-CryIC hybrid proteins (H04) was highly toxic to S. exigua compared with the parental CryIA(b) protein and significantly more toxic than CryIC. In semiquantitative binding studies with biotin-labelled toxins and intact brush border membrane vesicles of S. exigua, this domain III substitution appeared not to affect binding-site specificity. However, binding to a 200-kDa protein by CryIA(b) in preparations of solubilized and blotted brush border membrane vesicle proteins was completely abolished by the domain III substitution. A reciprocal hybrid containing domains I and II of CryIC and domain III of CryIA(b) did bind to the 200-kDa protein, confirming that domain III of CryIA(b) was essential for this reaction. These results show that domain III of CryIC protein plays an important role in the level of toxicity to S. exigua, that substitution of domain III may be a powerful tool to increase the repertoire of available active toxins for pest insects, and that domain III is involved in binding to gut epithelium membrane proteins of S. exigua.

Recombinant Bacillus thuringiensis crystal proteins with new properties: possibilities for resistance management.

To obtain Bacillus thuringiensis crystal proteins with new properties and to identify the regions involved in insecticidal activity, we generated hybrid genes composed of cryIC and cryIE by in vivo recombination. Analysis of the hybrid proteins showed that domain III of CryIC is involved in the toxicity towards Spodoptera exigua and Mamestra brassicae. Transfer of this domain to CryIE, which is not active against these insects, resulted in a new protein with a broader activity. This hybrid protein binds to different receptors than CryIC, suggesting its use as an alternative for CryIC in resistance management programs.

Cloning, nucleotide sequencing, and expression in Escherichia coli of a Rhizobium leguminosarum gene encoding a symbiotically repressed outer membrane protein.

We describe the cloning of a gene from Rhizobium leguminosarum biovar viciae strain 248 encoding protein IIIa, the 36-kDa outer membrane protein forming a part of the outer membrane protein antigen group III. The expression of this antigen group is repressed in the bacteroid form during symbiosis (R. A. de Maagd, R. de Rijk, I. H. M. Mulders, and B. J. J. Lugtenberg, J. Bacteriol. 171:1136-1142, 1989). A cosmid clone expressing the strain 248-specific MAb38 epitope of this antigen group in a nonrelated strain was selected by a colony blot assay. Sequencing revealed one large open reading frame encoding a 39-kDa protein. N-terminal amino acid sequencing of the purified 36-kDa outer membrane protein IIIa revealed that the isolated gene, now designated ropA, is the structural gene for this protein and that the mature protein was formed by processing of the 22-residue N-terminal signal sequence. The gene is preceded by a promoter that was active in R. leguminosarum but not in Escherichia coli. This promoter, which showed no homology to known promoter sequences, was located approximately by determination of the transcription start site. The region upstream of the putative promoter was shown to contain two potential binding sites for integration host factor protein. Expression of protein IIIa under control of the inducible lac promoter in E. coli shows that, of its earlier described properties, the peptidoglycan linkage of protein IIIa is specific for R. leguminosarum but that outer membrane localization and calcium-stabilized oligomer formation can to a large extent also occur in E. coli.

Antigenic changes in lipopolysaccharide I of Rhizobium leguminosarum bv. viciae in root nodules of Vicia sativa subsp. nigra occur during release from infection threads.

Three different monoclonal antibodies raised against the O antigen-containing lipopolysaccharide (LPS I) of free-living cells were used in an immunocytochemical study to follow the fate of LPS I on the outer membrane of Rhizobium leguminosarum bv. viciae 248 during the nodulation of Vicia sativa subsp. nigra. After immunogold labeling, the LPS I epitopes were detected on the outer membrane of bacteria present in infection threads throughout the nodule. Epitopes were not detectable on bacteria released from the infection thread. The data show that the LPS I epitopes present on rhizobia in infection droplets disappear shortly before or during endocytosis of the bacteria into the host plant cell cytoplasm. The abruptness of the change suggests an active degradation or modification of LPS I epitopes rather than only a repression of their synthesis.

nodO, a new nod gene of the Rhizobium leguminosarum biovar viciae sym plasmid pRL1JI, encodes a secreted protein.

The region of the Rhizobium leguminosarum biovar viciae Sym plasmid pRL1JI, responsible for the production and secretion of a previously described 50-kilodalton protein (R. A. de Maagd, C. A. Wijffelman, E. Pees, and B. J. J. Lugtenberg, J. Bacteriol. 170:4424-4427, 1988), was cloned and its nucleotide sequence was determined. A new nod gene, nodO, preceded by a poorly conserved nod box, was identified and its transcriptional start site was determined. Comparison of its predicted protein product with the N-terminal amino acid sequence of the isolated secreted protein showed that nodO is the structural gene of this protein, although the nucleotide sequence predicted a protein only 30,002 daltons in size. This comparison also showed that the secreted protein is not the product of N-terminal processing of a larger precursor. A conventional N-terminal signal sequence was not detected in the NodO protein. The NodO protein has significant homology with a part (residues 720 to 920) of the hemolysin protein (HlyA) of Escherichia coli. Analysis of the transcriptional regulation of the nodO gene revealed that, in contrast with other nod promoters in this species, activity of the nodO promoter is greatly enhanced in the presence of multiple copies of the nodD gene.

Evidence for divalent cation (Ca2+)-stabilized oligomeric proteins and covalently bound protein-peptidoglycan complexes in the outer membrane of Rhizobium leguminosarum.

Two unusual characteristics of some outer membrane proteins of Rhizobium leguminosarum are described. First, most of the major outer membrane proteins could only be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after lysozyme treatment of the isolated cell envelopes, suggesting a very strong, possibly covalent, interaction of these proteins with the peptidoglycan. These peptidoglycan-associated outer membrane proteins belonged to two distinct groups of immunologically related proteins, groups II and III, as defined by typing with monoclonal antibodies. As members of both groups of proteins could be radioactively labeled by growing cells in the presence of N-[3H]acetylglucosamine, we propose that variation in the apparent molecular weight of the antigens within each group is caused by varying numbers of peptidoglycan subunit residues on only two or three different outer membrane proteins. Second, group III outer membrane proteins, with masses of 35 to 46 kilodaltons, formed oligomers stabilized by divalent cations which resisted complete denaturation in 2% sodium dodecyl sulfate at 100 degrees C. Reconstitution experiments showed that of the divalent cations tested, Ca2+ and, to a lesser extent, Mn2+ and Sr2+ were the best stabilizers.

Localization and symbiotic function of a region on the Rhizobium leguminosarum Sym plasmid pRL1JI responsible for a secreted, flavonoid-inducible 50-kilodalton protein.

A previously described (R. A. de Maagd, C. A. Wijffelman, E. Pees, and B. J. J. Lugtenberg, J. Bacteriol. 170:4424-4427, 1988) Sym plasmid-dependent, naringenin-inducible 50-kilodalton protein of Rhizobium leguminosarum biovar viciae is further characterized in this paper. The protein was overproduced by constructing a strain containing multiple copies of the R. meliloti nodD gene, which facilitated its purification. An antiserum was used to screen Tn5 insertion mutants located in the pRL1JI region found to be responsible for the production of the 50-kilodalton protein. These inserts define a new nod locus left of the nod genes identified previously. Mutations in this region affect the nodulation ability in a way which is dependent on the bacterial background as well as on the host plant. The mutants nodulate normally in a strain RBL1532 (R. leguminosarum biovar viciae strain 248, cured of its Sym plasmid) background on all three tested host plant species. In contrast, in a strain RBL5045 (R. leguminosarum biovar trifolii strain RCR5, cured of its Sym plasmid) background, nodulation on Vicia sativa is severely impaired, whereas nodulation on Vicia hirsuta and Trifolium subterraneum is apparently unaltered.