Integrative model for binding of Bacillus thuringiensis toxins in susceptible and resistant larvae of the diamondback moth (Plutella xylostella).

Insecticidal crystal proteins from Bacillus thuringiensis in sprays and transgenic crops are extremely useful for environmentally sound pest management, but their long-term efficacy is threatened by evolution of resistance by target pests. The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to B. thuringiensis in open-field populations. The only known mechanism of resistance to B. thuringiensis in the diamondback moth is reduced binding of toxin to midgut binding sites. In the present work we analyzed competitive binding of B. thuringiensis toxins Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F to brush border membrane vesicles from larval midguts in a susceptible strain and in resistant strains from the Philippines, Hawaii, and Pennsylvania. Based on the results, we propose a model for binding of B. thuringiensis crystal proteins in susceptible larvae with two binding sites for Cry1Aa, one of which is shared with Cry1Ab, Cry1Ac, and Cry1F. Our results show that the common binding site is altered in each of the three resistant strains. In the strain from the Philippines, the alteration reduced binding of Cry1Ab but did not affect binding of the other crystal proteins. In the resistant strains from Hawaii and Pennsylvania, the alteration affected binding of Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F. Previously reported evidence that a single mutation can confer resistance to Cry1Ab, Cry1Ac, and Cry1F corresponds to expectations based on the binding model. However, the following two other observations do not: the mutation in the Philippines strain affected binding of only Cry1Ab, and one mutation was sufficient for resistance to Cry1Aa. The imperfect correspondence between the model and observations suggests that reduced binding is not the only mechanism of resistance in the diamondback moth and that some, but not all, patterns of resistance and cross-resistance can be predicted correctly from the results of competitive binding analyses of susceptible strains.

Global variation in the genetic and biochemical basis of diamondback moth resistance to Bacillus thuringiensis.

Insecticidal proteins from the soil bacterium Bacillus thuringiensis (Bt) are becoming a cornerstone of ecologically sound pest management. However, if pests quickly adapt, the benefits of environmentally benign Bt toxins in sprays and genetically engineered crops will be short-lived. The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to Bt in open-field populations. Here we report that populations from Hawaii and Pennsylvania share a genetic locus at which a recessive mutation associated with reduced toxin binding confers extremely high resistance to four Bt toxins. In contrast, resistance in a population from the Philippines shows multilocus control, a narrower spectrum, and for some Bt toxins, inheritance that is not recessive and not associated with reduced binding. The observed variation in the genetic and biochemical basis of resistance to Bt, which is unlike patterns documented for some synthetic insecticides, profoundly affects the choice of strategies for combating resistance.

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.

Regulation of insecticidal crystal protein production in Bacillus thuringiensis.

The production of insecticidal crystal proteins (ICPs) in Bacillus thuringiensis normally coincides with sporulation, resulting in the appearance of parasporal crystalline inclusions within the mother cell. In most instances, the temporal and spatial regulation of ICP gene expression is determined at the transcriptional level by mother-cell-specific sigma factors that share homology with sigma E and sigma K from Bacillus subtilis. The cryIII ICP genes are a notable exception; these genes are transcribed from sigma A-like promoters during vegetative growth, are induced or derepressed at the onset of stationary phase, and are overexpressed in sporulation mutants of B. thuringiensis blocked in the phosphorylation of Spo0A, a key regulator of sporulation initiation. Transcription alone, however, cannot account for the impressive ability of this bacterium to accumulate insecticidal proteins. A variety of post-transcriptional and post-translational mechanisms also contribute to the efficient production of ICPs in B. thuringiensis, thus making this bacterium a cost-effective biological control agent.

The yeast CCR4 protein is neither regulated by nor associated with the SPT6 and SPT10 proteins and forms a functionally distinct complex from that of the SNF/SWI transcription factors.

The CCR4 protein is specifically required for the increased transcription at the ADH2 locus resulting from mutations in the SPT10 (CRE1) and SPT6 (CRE2) genes and is also required for the expression of ADH2 and other genes under non-fermentative growth conditions. The mechanism by which mutations in CCR4 suppress defects in SPT10 and SPT6 was examined. The SPT10 and SPT6 genes were shown not to control CCR4 mRNA or protein expression nor did SPT10 and SPT6 proteins co-immuneprecipitate with CCR4. CCR4 association with two other proteins, 195 and 185 kDa in size, was unaffected by either spt10 or spt6 mutations. Also, the ability of CCR4 to activate transcription when fused to the LexA DNA binding domain was not specifically enhanced by defects in either SPT10 or SPT6. These results suggest that SPT10 and SPT6, in negatively regulating transcription at ADH2, act through a factor that requires CCR4 function, but do not regulate CCR4 expression, control its activity, physically interact with it, or affect its binding to other factors. The relationship of CCR4 to the group of general transcription factors, SNF2, SNF5, SNF6 and SWI1 and SWI3, which comprise a multisubunit complex required for ADH2 and other genes' expression, was also examined. CCR4 protein expression was not controlled by these factors nor did they co-immuneprecipitate or associate with CCR4. In addition, a ccr4 mutation had little effect on an ADH2 promoter alteration in contrast to the large effects displayed by mutations in SNF2 and SNF5. These data suggest that CCR4 acts by a separate mechanism from that used by the SNF/SWI general transcription factors in affecting gene expression.

Overexpression of Bacillus thuringiensis HknA, a histidine protein kinase homology, bypasses early Spo mutations that result in CryIIIA overproduction.

The Bacillus thuringiensis CryIIIA insecticidal crystal protein (ICP) is a vegetatively expressed protein that is toxic to coleopteran insect larvae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the asporogenous B. thuringiensis subsp. morrisoni strain EG1351, which harbors the native cryIIIA-encoding 88-MDa plasmid, showed a 2.5-fold overproduction of the CryIIIA protein compared with that of an isogenic wild-type strain. Further studies showed that neither CryIIIA protein synthesis nor CryIIIA protein processing was affected in strain EG1351 during vegetative growth. In an attempt to characterize the EG1351 mutation by complementation of function, the hknA gene was identified and cloned from a B. thuringiensis cosmid library. Primer extension analysis of hknA mRNA in wild-type B. thuringiensis demonstrated that the hknA gene is transcribed during vegetative growth from a sigma A-like promoter. Multiple copies of either the hknA gene or the Bacillus subtilis kinA (spoIIJ) gene were shown to bypass the sporulation defect in strain EG1351 as well as a spo0F mutation in B. thuringiensis EG1634. Additional studies showed that the hknA gene was not defective in strain EG1351. The results of this study suggest that hknA encodes a novel histidine protein kinase involved in B. thuringiensis sporulation. We also propose that the CryIIIA-overproducing phenotype of strain EG1351 is most likely due to a defect in the phosphorylation of Spo0A and confirm that CryIIIA production is not dependent on sporulation.

Tn5401 disruption of the spo0F gene, identified by direct chromosomal sequencing, results in CryIIIA overproduction in Bacillus thuringiensis.

The Bacillus thuringiensis spo0F gene was identified by chromosomal DNA sequencing of sporulation mutants derived from a B. thuringiensis transposon insertion library. A spo0F defect in B. thuringiensis, which was suppressed by multicopy hknA or kinA, resulted in the overproduction of the CryIIIA insecticidal crystal protein.

Characterization of two genes encoding Bacillus thuringiensis insecticidal crystal proteins toxic to Coleoptera species.

Bacillus thuringiensis EG2838 and EG4961 are highly toxic to Colorado potato beetle larvae, and only strain EG4961 is toxic to southern corn rootworm larvae. To investigate the cause of the different insecticidal activities of EG2838 and EG4961, cryIII-type genes toxic to coleopterans were cloned from each strain. The cryIIIB gene, cloned as part of an 8.0-kb EcoRI fragment of EG2838 DNA, encoded a crystal protein (CryIIIB) of 74,237 Da. The cryIIIB2 gene, cloned as part of an 8.3-kb PstI-Asp718 fragment of EG4961 DNA, encoded a crystal protein (CryIIIB2) of 74,393 Da that was 94% identical to CryIIIB. Analysis of the transcriptional start sites showed that cryIIIB and cryIIIB2 were initiated from a conserved region located within 130 nucleotides upstream from the translation start sites of both genes. Although the CryIIIB and CryIIIB2 proteins were similar in sequence, they displayed distinct insecticidal activities: CryIIIB was one-third as toxic as CryIIIB2 to Colorado potato beetle larvae, and CryIIIB2, but not CryIIIB, was toxic to southern corn rootworm larvae. Genes encoding crystal proteins of approximately 32 and 31 kDa were located adjacent to the cryIIIB and cryIIIB2 genes, respectively. The 32- and 31-kDa crystal proteins failed to enhance the insecticidal activities of CryIIIB and CryIIIB2.

The CCR4 protein from Saccharomyces cerevisiae contains a leucine-rich repeat region which is required for its control of ADH2 gene expression.

The CCR4 gene from Saccharomyces cerevisiae is required for the transcription of the glucose-repressible alcohol dehydrogenase (ADH2). Mutations in CCR4 also suppress the transcription at the ADH2 and his4-912delta loci caused by defects in the SPT10 (CRE1) and SPT6 (CRE2) genes. The CCR4 gene was mapped to the left arm of chromosome I and cloned by complementation of function using previously isolated segments of chromosome I. DNA sequence analysis of the cloned gene defined CCR4 as a 2511 bp open reading frame that would encode a polypeptide of 837 amino acids. The CCR4 mRNA was found to be 2.8 kb in size and Western analysis identified CCR4 as a 95,000 D protein. Disruption of the CCR4 gene resulted in reduced levels of ADH2 expression under both glucose and ethanol growth conditions and in temperature sensitive growth on nonfermentative medium, phenotypes essentially indistinguishable from previously identified mutations in CCR4. The amino terminus of the CCR4 protein was found to be rich in glutamine residues similar to a number of genes which are required for transcription. More importantly, CCR4 showed similarity to a diverse set of proteins sharing a leucine-rich tandem repeat motif, the presence of which has been implicated in mediating protein-protein interactions. Deletions of several of the five leucine-rich repeats in CCR4 were shown to produce nonfunctional proteins indicating the importance of the repeats to CCR4 activity. This leucine-rich repeat region may mediate the contact CCR4 makes with another factor.

The CCR4 gene from Saccharomyces cerevisiae is required for both nonfermentative and spt-mediated gene expression.

Mutations in the yeast CCR4 gene inhibit expression of the glucose-repressible alcohol dehydrogenase (ADH2), as well as other nonfermentative genes, and suppress increased ADH2 expression caused by the cre1 and cre2 alleles. Both the cre1 and ccr4 alleles were shown to affect ADH II enzyme activity by altering the levels of ADH2 mRNA. Mutations in either CRE1 or CRE2 bypassed the inhibition of ADH2 expression caused by delta insertions at the ADH2 promoter which displace the ADH2 activation sequences 336 bp upstream of the TATA element. These cre1 and cre2 effects were suppressible by the ccr4 allele. The cre1 and ccr4 mutations also affected ADH2 expression when all the ADH2 regulatory sequences upstream of the TATA element were deleted. The relationship of the CRE genes to the SPT genes, which when mutated are capable of bypassing the inhibition of HIS4 expression caused by a delta promoter insertion (his4-912 delta allele), was examined. Both the cre1 and cre2 mutations allowed his4-912 delta expression. ccr4 mutations were able to suppress the ability of the cre alleles to increase his4-912 delta expression. CRE2 was shown to be allelic to the SPT6 gene, and CRE1 was found to be allelic to SPT10. We suggest that the CRE genes comprise a general transcriptional control system in yeast that requires the function of the CCR4 gene.

Hydrolysis of dolichyl esters by oviduct membranes and characterization of endogenous dolichyl esters.

The chick oviduct system has been employed to study whether dolichol esters might serve as a storage form of dolichol to be converted to dolichyl phosphate (Dol-P) during periods when Dol-P levels increase. Chicken oviduct membranes catalyze the hydrolysis of dolichyl-[14C]oleate; the reaction is dependent on detergent (0.04% NP-40 is optimal), is unaffected by divalent cations and EDTA, and exhibits a pH optimum of 6.0. Oviduct membranes also hydrolyze cholesteryl-[14C]oleate, which exhibits similar properties except the pH optimum is 5.0-5.5. Neither Dol-[14C]palmitate nor Chol-[14C]palmitate is hydrolyzed by membranes. Chol-ester hydrolysis is more sensitive to heat-denaturation than is Dol-ester hydrolysis. Esterase activity was assayed in membranes prepared from immature chicks, chicks treated with diethylstilbestrol, chicks withdrawn from diethylstilbestrol, and mature hens. The highest esterase specific activity was observed in membranes obtained from chicks withdrawn from hormone. In order to characterize the fatty acid composition of Dol-esters they were purified from mature hen oviducts by chromatography on DEAE-cellulose and Fractogel ORPVA-6000, reverse-phase HPLC, and TLC. About 15-25% of oviduct dolichol is in the esterified form. Fatty acid analysis revealed that approximately 85% of the dolichol was esterified to oleic acid. The fact that the highest esterase activity is found in membranes from chicks withdrawn from hormone and that only 20% of the dolichol is esterified argues against a role for Dol-esters as a reservoir of dolichol for conversion to Dol-P.

Structure-activity relationships of cytochalasins in the differentiation of cytolytic T lymphocytes.

Four naturally occurring cytochalasins and three synthetic congeners have been studied for their effects on in vitro sensitization of murine lymphocytes to P815 mastocytoma. The relative order of effectiveness of these secondary fungal metabolites in inhibiting cytotoxic T cell development is as follows: cytochalasin D greater than cytochalasin E greater than cytochalasin A greater than cytochalasin B, 21,22- dihydrocytochalasin A greater than 7- acetylcytochalasin D. The 7,20 diacetylcytochalasin B derivative was inactive at the highest level tested (4 X 10(-6) M). Cytochalasin D is the most effective compound, producing at 5 X 10(-8) M a 50% inhibition of 51Cr release in a 4-hr cytolysis assay. This response pattern is in keeping with other test systems that implicate actin involvement, and underscores the contribution of an unsubstituted 7-hydroxyl drug function in receptor recognition. Inhibition produced by the cytochalasins is reversible if the compounds are removed from the tissue culture medium within the first 24 hr of a 4-day culture period. Delayed addition of cytochalasin D inhibits T cell development only within this first 24 hr of culture. These data suggest that the effects of cytochalasins are at an early step in the sensitization process, possibly antigen recognition.

CT demonstration of an ectopic pancreatic tail causing a renal pseudotumor.

Renal pseudotumors may be intrinsic or extrinsic to the renal parenchyma. A case in which an ectopic position of the pancreatic tail simulated a solid left renal mass on nephrotomography and ultrasonography is presented. Computed tomography was important in confirming the etiology of the mass.