The yeast [PSI+] element represents a new type of genetic inheritance, in which changes in phenotype are transmitted by a 'protein only' mechanism reminiscent of the 'protein-only' transmission of mammalian prion diseases. The underlying molecular mechanisms for both are poorly understood and it is not clear how similar they might be. Sup35, the [PSI+] protein determinant, and PrP, the mammalian prion determinant, have different functions, different cellular locations and no sequence similarity; however, each contains five imperfect oligopeptide repeats-PQGGYQQYN in Sup35 and PHGGGWGQ in PrP. Repeat expansions in PrP produce spontaneous prion diseases. Here we show that replacing the wild-type SUP35 gene with a repeat-expansion mutation induces new [PSI+] elements, the first mutation of its type among these newly described elements of inheritance. In vitro, fully denatured repeat-expansion peptides can adopt conformations rich in beta-sheets and form higher-order structures much more rapidly than wild-type peptides. Our results provide insight into the nature of the conformational changes underlying protein-based mechanisms of inheritance and suggest a link between this process and those producing neurodegenerative prion diseases in mammals.

Laboratory selection with Cry1Ac, the Bacillus thuringiensis (Bt) toxin in transgenic cotton, initially produced 300-fold resistance in a field-derived strain of pink bollworm, Pectinophora gossypiella (Saunders), a major cotton pest. After additional selection increased resistance to 3,100-fold, we tested the offspring of various crosses to determine the mode of inheritance of resistance to Cry1Ac. The progeny of reciprocal F1 crosses (resistant male x susceptible female and vice versa) responded alike in bioassays, indicating autosomal inheritance. Consistent with earlier findings, resistance was recessive at a high concentration of Cry1Ac. However, the dominance of resistance increased as the concentration of Cry1Ac decreased. Analysis of survival and growth of progeny from backcrosses (F1 x resistant strain) suggest that resistance was controlled primarily by one or a few major loci. The progression of resistance from 300- to 3,100-fold rules out the simplest model with one locus and two alleles. Overall the patterns observed can be explained by either a single resistance gene with three or more alleles or by more than one resistance gene. The pink bollworm resistance to Cry1Ac described here fits "mode 1" resistance, the most common type of resistance to Cry1A toxins in Lepidoptera.

The mode of action of Bacillus thuringiensis insecticidal proteins is not well understood. Based on analogies with other bacterial toxins and ion channels, we hypothesized that charged amino acids in helix 4 of the Cry1Aa toxin are critical for toxicity and ion channel function. Using Plutella xylostella as a model target, we analyzed responses to Cry1Aa and eight proteins with altered helix 4 residues. Toxicity was abolished in five charged residue mutants (E129K, R131Q, R131D, D136N, D136C), however, two charged (R127E and R127N) and one polar (N138C) residue mutant retained wild-type toxicity. Compared with Cry1Aa and toxic mutants, nontoxic mutants did not show greatly reduced binding to brush border membrane vesicles, but their ion channel conductance was greatly reduced in planar lipid bilayers. Substituted cysteine accessibility tests showed that in situ restoration of the negative charge of D136C restored conductance to wild-type levels. The results imply that charged amino acids on the Asp-136 side of helix 4 are essential for toxicity and passage of ions through the channel. These results also support a refined version of the umbrella model of membrane integration in which the side of helix 4 containing Asp-136 faces the aqueous lumen of the ion channel.During sporulation, the Gram-positive bacterium Bacillus thuringiensis (Bt) 1 produces insecticidal crystal (Cry) proteins. Each of these proteins has a unique spectrum of toxicity (1). Because of rapidly expanding use of Bt toxins in pest control, knowledge of the mode of action of Bt toxins is becoming increasingly important for proper deployment of this valuable biopesticide and avoidance of insect resistance (2). In particular, millions of hectares of transgenic crops that produce Bt toxins from the Cry1 family are being grown in the United States and elsewhere to control lepidopteran pests (3). After lepidopteran larvae ingest Cry1 proteins, protoxins are solubilized in the alkaline midgut and activated by proteases. The activated toxins bind to specific target sites in the midgut epithelium, creating pores that disrupt the ionic balance of the cell and kill the insect (4).A major goal in understanding how Bt toxins work is to elucidate the functions of their three domains (5, 6). Domains II and III are involved in binding specificity and structural integrity (7, 8). Post-binding events thought to be associated with domain I remain poorly understood (8), but progress in this area may be facilitated by exploiting knowledge of other bacterial toxins and ion channels. Domain I is composed of a central helix (␣5) encased by six other helices (6). The helical nature of domain I and the ability of Cry proteins to form ion channels (6, 9, 10) places them in the general class of globular bacterial toxins, like diphtheria toxin and colicin A. After binding, diphtheria toxin and colicin A undergo conformational changes enabling one or more helices to insert into the target cell membrane and form ion channels (11). A general working hypothesis is that simil...

There is a great demand for safe and effective alternative fumigants to replace methyl bromide and other toxic fumigants for postharvest pest control. Nitric oxide, a common signal molecule in biological systems, was found to be effective and safe to control insects under ultralow oxygen conditions. Four insect species including western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera Thripidae); aphid, Nasonovia ribisnigri (Mosley) (Homoptera: Aphididae); confused flour beetle, Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae); and rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae), at various life stages were fumigated with 0.1-2.0% nitric oxide under ultralow oxygen levels of < or = 50 ppm in 1.9-liter glass jars at 2-25 degrees C depending on insect species. Fumigations were effective against all four insect species. Efficacy of nitric oxide fumigation increased with nitric oxide concentration, treatment time, and temperature. There were also considerable variations among insect species as well as life stages in susceptibility to nitric oxide fumigation. Complete control of thrips was achieved in 2 and 8 h with 2.0 and 0.2% nitric oxide, respectively, at 2 degrees C. At the same temperature, complete control of the aphid was achieved in 3, 9, and 12 h with 1.0, 0.5, and 0.2% nitric oxide, respectively. Larvae, pupae, and adults of confused flour beetle were effectively controlled in 24 h with 0.5% nitric oxide at 20 degrees C. Complete mortality of confused flour beetle eggs was achieved in 24 h with 2.0% nitric oxide at 10 degrees C. Rice weevil adults and eggs were effectively controlled with 1.0% nitric oxide in 24 and 48 h, respectively, at 25 degrees C. These results indicate that nitric oxide has potential as a fumigant for postharvest pest control.

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.

U.S. exported lettuce, broccoli, asparagus, and strawberries often harbor western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), a quarantined pest in Taiwan, and therefore require quarantine treatment. Fumigation with diluted pure phosphine at a low temperature of 2 degrees C was studied to control western flower thrips and to determine effects on the quality of the treated products. Total thrips control was achieved in > or = 18-h fumigation treatments with > or = 250 ppm phosphine. One day fumigation treatment with 1,000 ppm phosphine was tested on lettuce and broccoli. One-day fumigation treatments with 500 ppm and 1,000 ppm phosphine were tested on asparagus and strawberry. Visual quality of lettuce, broccoli, and asparagus was evaluated after 2-wk posttreatment storage. Strawberry quality was evaluated immediately after fumigation and after 1-wk posttreatment storage. For all the products, there were no significant differences between the treatments and the controls in postharvest quality, and there were no injuries caused by the fumigation treatments. Therefore, phosphine fumigation at low temperature was promising for postharvest control of western flower thrips on lettuce, broccoli, asparagus, and strawberry.

SUMMARYTheoretical projections suggest that refuges from exposure can delay insect adaptation to environmentally benign insecticides derived from Bacillus thuringiensis, but experimental tests of this approach have been limited. We tested the refuge tactic by selecting two sets of two colonies of diamondback moth (Plutella x lostella) for resistance to B. thuringiensis subsp. ai a ai in the laboratory. In each set, one colony was selected with no refuge and the other with a 10 % refuge from exposure to B. thuringiensis subsp. ai a ai. Bioassays conducted after nine selections were completed show that mortality caused by B. thuringiensis subsp. ai a ai was significantly greater in the refuge colonies than in the no-refuge colonies. These results demonstrate that the refuges delayed the evolution of resistance. Relative to a susceptible colony, final resistance ratios were 19 and eight for the two no-refuge colonies compared to six and five for the refuge colonies. The mean realized heritability of resistance to B. thuringiensis subsp. ai a ai was 0.046 for colonies without refuges, and k0.002 for colonies with refuges. Selection with B. thuringiensis subsp. ai a ai decreased susceptibility to B. thuringiensis toxin Cry1Ab, but not to Cry1C or B. thuringiensis subsp. kurstaki. Although the ultimate test of refuges will occur in the field, the experimental evidence reported here confirms modelling results indicating that refuges can slow the evolution of insect resistance to B. thuringiensis.

Classical and molecular genetic analyses show that two independently derived resistant strains of pink bollworm, Pectinophora gossypiella (Saunders), share a genetic locus at which three mutant alleles confer resistance to Bacillus thuringiensis (Bt) toxin Cry1Ac. One laboratory-selected resistant strain (AZP-R) was derived from individuals collected in 1997 from 10 Arizona cotton fields, whereas the other (APHIS-98R) was derived from a long-term susceptible laboratory strain. Both strains were previously reported to show traits of "mode 1" resistance, the most common type of lepidopteran resistance to Cry1A toxins. Inheritance of resistance to a diagnostic concentration of Cry1Ac (10 microg per gram of diet) was recessive in both strains. In interstrain complementation tests for allelism, F1 progeny from crosses between the two strains were resistant to the diagnostic concentration of Cry1Ac. These results indicate that a major resistance locus is shared by the two strains. Analysis of DNA from the pink bollworm cadherin gene (BtR) using allele-specific polymerase chain reaction (PCR) tests showed that the previously identified resistance alleles (r1, r2, and r3) occurred in both strains, but their frequencies differed between strains. In conjunction with previous findings, the results reported here suggest that PCR-based detection of the three known cadherin resistance alleles might be useful for monitoring resistance to Cry1Ac-producing Bt cotton in field populations of pink bollworm.