Jizhen Wei

Dual mode of action of Bt proteins: protoxin efficacy against resistant insects

Transgenic crops that produce Bacillus thuringiensis (Bt) proteins for pest control are grown extensively, but insect adaptation can reduce their effectiveness. Established mode of action models assert that Bt proteins Cry1Ab and Cry1Ac are produced as inactive protoxins that require conversion to a smaller activated form to exert toxicity. However, contrary to this widely accepted paradigm, we report evidence from seven resistant strains of three major crop pests showing that Cry1Ab and Cry1Ac protoxins were generally more potent than the corresponding activated toxins. Moreover, resistance was higher to activated toxins than protoxins in eight of nine cases evaluated in this study. These data and previously reported results support a new model in which protoxins and activated toxins kill insects via different pathways. Recognizing that protoxins can be more potent than activated toxins against resistant insects may help to enhance and sustain the efficacy of transgenic Bt crops.

New insights on the role of alkaline phosphatase 2 from Spodoptera exigua (Hübner) in the action mechanism of Bt toxin Cry2Aa

Cry1A and Cry2A toxins, which are widely used in Bt transgenic crops, can specifically bind to insect midguts and exert their insecticidal effects. There are interactions between insect midgut-binding proteins and Cry1A toxins; however, little is known about the insect protein that specifically binds to Cry2A. Midgut membrane-bound alkaline phosphatases (ALPs), which are important for the binding of proteins to Cry1A, play dominant roles in Cry1A-mediated toxicity in some lepidopteran larvae. In this study, we cloned and expressed one partial ALP2 peptide from susceptible Spodoptera exigua larvae and studied the binding characteristics of SeALP2 with Cry2Aa. The ALPs proteins was expressed at all larval stages and highly expressed in the first and second instar larvae. The heterologously expressed SeALP2 peptide bound specifically to Cry2Aa with a high affinity. Knocking down ALP2 in vivo revealed that it plays an important role in the susceptibility of S. exigua to Cry2Aa. Based on these findings, we propose that ALP2 in S. exigua serves as a functional receptor for Cry2Aa.

APN1 is a functional receptor of Cry1Ac but not Cry2Ab in Helicoverpa zea.

Lepidopteran midgut aminopeptidases N (APNs) are phylogenetically divided into eight clusters, designated as APN1–8. Although APN1 has been implicated as one of the receptors for Cry1Ac in several species, its potential role in the mode of action of Cry2Ab has not been functionally determined so far. To test whether APN1 also acts as one of the receptors for Cry1Ac in Helicoverpa zea and even for Cry2Ab in this species, we conducted a gain of function analysis by heterologously expressing H. zea APN1 (HzAPN1) in the midgut and fat body cell lines of H. zea and the ovarian cell line of Spodoptera frugiperda (Sf9) and a loss of function analysis by RNAi (RNA interference) silencing of the endogenous APN1 in the three cell lines using the HzAPN1 double strand RNA (dsRNA). Heterologous expression of HzAPN1 significantly increased the susceptibility of the three cell lines to Cry1Ac, but had no effects on their susceptibility to Cry2Ab. Knocking down of the endogenous APN1 made the three cell lines resistant to Cry1Ac, but didn’t change cell lines susceptibility to Cry2Ab. The findings from this study demonstrate that HzAPN1 is a functional receptor of Cry1Ac, but not Cry2Ab.

Functional roles of cadherin, aminopeptidase-N and alkaline phosphatase from Helicoverpa armigera (Hübner) in the action mechanism of Bacillus thuringiensis Cry2Aa

A pyramid strategy combining the Cry1A and Cry2A toxins in Bt crops has been widely used throughout the world to delay pest adaption to transgenic crops and broaden the insecticidal spectrum. Midgut membrane-bound cadherin (CAD), aminopeptidase-N (APN) and alkaline phosphatase (ALP) are important for Cry1A toxicity in some lepidopteran larvae, but the proteins that bind Cry2A in the midgut of target insects and their role in the Cry2A mechanism of action are still unclear. In this study, we found that heterologously expressed CAD, APN4 and ALP2 peptides from the midgut of Helicoverpa armigera could bind to the Cry2Aa toxin with a high affinity. Additionally, the efficiency of Cry2Aa insecticidal activity against H. armigera larvae was obviously reduced after the genes encoding these proteins were silenced with specific siRNAs: CAD- and ALP2-silenced larvae showed significantly similar reductions in mortality due to the Cry2Aa toxin (41.67% and 43.06%, respectively), whereas a larger reduction in mortality was observed in APN4-silenced larvae (61.11%) than in controls. These results suggest that CAD, APN4 and ALP2 are involved in the mechanism of action of Cry2Aa in H. armigera and may play important functional roles in the toxicity of the Cry2Aa toxin.

Activation of Bt Protoxin Cry1Ac in Resistant and Susceptible Cotton Bollworm

Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50) for a laboratory-selected resistant strain (LF120) divided by the LC50 for its susceptible parent strain (LF), the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins.

Decreased Cry1Ac activation by midgut proteases associated with Cry1Ac resistance in Helicoverpa zea : Decreased Cry1Ac activation in H. zea

BACKGROUND Field-evolved resistance of Helicoverpa zea to Bacillus thuringiensis (Bt) toxin Cry1Ac was first reported more than a decade ago, yet the underlying mechanisms remain elusive. Towards understanding the mechanisms of resistance to Cry1Ac, we analyzed a susceptible (LAB-S) and two resistant (GA and GA-R) strains of H. zea. The GA strain was derived from Georgia and exposed to Bt toxins only in the field. The GA-R strain was derived from the GA strain and selected for increased resistance to Cry1Ac in the laboratory. RESULTS Resistance to MVPII, a liquid formulation containing a hybrid protoxin similar to Cry1Ac, was 110-fold for GA-R and 7.8-fold for GA relative to LAB-S. In midgut brush border membrane vesicles, activity of alkaline phosphatase and aminopeptidase N did not vary significantly among strains. The activity of total proteases, trypsin-like proteases and chymotrypsin-like proteases was significantly lower for GA-R and GA than LAB-S, but did not differ between GA-R and GA. When H. zea midgut cells were exposed to Cry1Ac protoxin that had been digested with midgut extracts, toxicity was significantly lower for extracts from GA-R and GA relative to extracts from LAB-S, but did not differ between GA-R and GA. Transcriptional analysis showed that none of the five protease genes examined was associated with the decline in Cry1Ac activation in GA-R and GA relative to LAB-S. CONCLUSION The results suggest that decreased Cry1Ac activation is a contributing field-selected mechanism of resistance that helps explain the reduced susceptibility of the GA-R and GA strains. Relative to the LAB-S strain, the two Cry1Ac-resistant strains had lower total protease, trypsin and chymotrypsin activities, a lower Cry1Ac activation rate, and Cry1Ac protoxin incubated with their midgut extracts was less toxic to H. zea midgut cells.

Cytotoxicity and binding profiles of activated Cry1Ac and Cry2Ab to three insect cell lines

While Cry1Ac has been known to bind with larval midgut proteins cadherin, APN (amino peptidase N), ALP (alkaline phosphatase) and ABCC2 (adenosine triphosphate‐binding cassette transporter subfamily C2), little is known about the receptors of Cry2Ab. To provide a clue to the receptors of Cry2Ab, we tested the baseline cytotoxicity of activated Cry1Ac and Cry2Ab against the midgut and fat body cell lines of Helicoverpa zea and the ovary cell line of Spodoptera frugiperda (SF9). As expected, the descending order of cytotoxicity of Cry1Ac against the three cell lines in terms of 50% lethal concetration (LC50) was midgut (31.0 μg/mL) > fat body (59.0 μg/mL) and SF9 cell (99.6 μg/mL). By contrast, the fat body cell line (LC50 = 7.55 μg/mL) was about twice more susceptible to Cry2Ab than the midgut cell line (16.0 μg/mL), the susceptibility of which was not significantly greater than that of SF9 cells (27.0 μg/mL). Further, ligand blot showed the binding differences between Cry1Ac and Cry2Ab in the three cell lines. These results indicated that the receptors of Cry2Ab were enriched in fat body cells and thus largely different from the receptors of Cry1Ac, which were enriched in midgut cells.

Specific Binding Protein ABCC1 Is Associated With Cry2Ab Toxicity in Helicoverpa armigera

A pyramid strategy combining the crystal (Cry) 1A and 2A toxins in Bacillus thuringiensis (Bt) crops are active against many species of insects and nematode larvae. It has been widely used to delay pest adaption to genetically modified plants and broaden the insecticidal spectrum in many countries. Unfortunately, Cry2A can also bind with the specific receptor proteins of Cry1A. ATP-binding cassette (ABC) transporters can interact with Cry1A toxins as receptors in the insect midgut, and ABC transporter mutations result in resistance to Bt proteins. However, there is limited knowledge of the ABC transporters that specifically bind to Cry2Ab. Here, we cloned the ABCC1 gene in Helicoverpa armigera, which expressed at all larval stages and in nine different tissues. Expression levels were particularly high in fifth-instar larvae and Malpighian tubules. The two heterologously expressed HaABCC1 transmembrane domain peptides could specifically bind to Cry2Ab with high affinity levels. Moreover, transfecting HaABCC1 into the Spodoptera frugiperda nine insect cell significantly increased its mortality when exposed to Cry2Ab in vitro, and silencing HaABCC1 in H. armigera by RNA interference significantly reduced the mortality of larvae exposed to Cry2Ab in vivo. Altogether current results suggest that HaABCC1 serves as a functional receptor for Cry2Ab.

Effects of antibiotics on biological activity of Cry1Ac in Bt-susceptible and Bt-resistant Helicoverpa armigera strains

In this study, the results showed that the population of midgut bacteria and larval mortality due to Cry1Ac are significantly reduced in antibiotic-treated larvae from Bt-susceptible, -resistant and field-collected strains (96S, BtR, FS respectively) of Helicoverpa armigera. The percentage reduction of larval mortality with increasing concentrations of antibiotics was significantly different among strains with the smallest effect observed in FS. It has been suggested that antibiotics could influence the toxicity of Cry1Ac, possibly by eliminating gut bacteria, hence gut bacteria might be playing essential roles in Bt-induced killing of H. armigera. But elimination of midgut microflora with antibiotics had no effect on resistance level.