Differential Protection of Cry1Fa Toxin against Spodoptera frugiperda Larval Gut Proteases by Cadherin Orthologs Correlates with Increased Synergism

Abstract

The Cry proteins produced by Bacillus thuringiensis (Bt) are the most widely used biopesticides effective against a range of crop pests and disease vectors. Like chemical pesticides, development of resistance is the primary threat to the long-term efficacy of Bt toxins. Recently discovered cadherin-based Bt Cry synergists showed the potential to augment resistance management by improving efficacy of Cry toxins. However, the mode of action of Bt Cry synergists is thus far unclear. Here we elucidate the mechanism of cadherin-based Cry toxin synergism utilizing two cadherin peptides, Spodoptera frugiperda Cad (SfCad) and Manduca sexta Cad (MsCad), which differentially enhance Cry1Fa toxicity to Spodoptera frugiperda neonates. We show that differential SfCad-and MsCad-mediated protection of Cry1Fa toxin in the Spodoptera frugiperda midgut correlates with differential Cry1Fa toxicity enhancement. Both peptides exhibited high affinity for Cry1Fa toxin and an increased rate of Cry1Fa-induced pore formation in S. frugiperda. However, only SfCad bound the S. frugiperda brush border membrane vesicle and more effectively prolonged the stability of Cry1Fa toxin in the gut, explaining higher Cry1Fa enhancement by this peptide. This study shows that cadherin fragments may enhance B. thuringiensis toxicity by at least two different mechanisms or a combination thereof: (i) protection of Cry toxin from protease degradation in the insect midgut and (ii) enhancement of pore-forming ability of Cry toxin. Bacillus thuringiensis (Bt) Cry toxins are a family of bacterial pore-forming proteins that are highly toxic to a range of crop pests and disease vectors. Cry proteins are produced as crystals during the sporulation phase. Crystals are ingested, solubilized in the gut lumen to protoxin, and activated by host gut proteases. Activated toxin crosses the peritrophic matrix (PM) and binds cadherin, which is the primary high-affinity receptor for Cry1 toxins on the apical border of midgut microvilli. A current model postulates that toxin interaction with cadherin causes a conformational change in toxin allowing a specific proteolytic cleavage and formation of a prepore toxin oligomer. Evidence suggests that the prepore oligomer has increased affinity for secondary glycosylphosphatidylinositol (GPI)-anchored receptors, such as aminopeptidases (APNs) or alkaline phosphatases (ALPs) localized in lipid rafts. Oligomers insert into the membrane and disrupt membrane integrity by forming lytic pores, which lead directly to insect mortality or indirectly to mortality due to septicemia (5,7,21,45). A recent modification to the pore formation model described above proposes that activated toxin monomers first bind to abundant low-affinity APN receptors before binding to high-affinity cadherin receptors, which results in toxin oligomerization (32). In contrast to the pore formation model, the cell-signaling model (54) proposes that binding of activated toxin monomers to cadherin activates an intracellular signaling pathway, which ultimately results ...

A Spodoptera exigua Cadherin Serves as a Putative Receptor for Bacillus thuringiensis Cry1Ca Toxin and Shows Differential Enhancement of Cry1Ca and Cry1Ac Toxicity

“…Similarly, the SfCad fragment from S. frugiperda increased Cry1Fa toxicity to S. frugiperda larvae more than did the MsBtR1 fragment from M. sexta (57). Our data support the hypothesis proposed previously by Chen et al (22) and Peng et al (54) that the binding of cadherin peptide to Cry toxin is a prerequisite for the peptide's enhancement effect.…”

“…However, the soluble HaBtR fragment reduces Cry1Ac insecticidal toxicity (27), whereas the HaBtR fragment produced in the inclusion body enhances Cry1Ac insecticidal toxicity (54) to H. armigera larvae. These results suggested that other synergistic mechanisms (for example, soluble versus inclusion body) rather than the cadherin binding affinity may also influence the synergistic ability of cadherin peptides (57). Our enhancement results of rSeCad1bp and rHaBtRp to different Cry toxins in S. exigua and H. armigera provide an excellent system to comparatively address the mechanisms.…”

“…Our data support the hypothesis proposed previously by Chen et al (22) and Peng et al (54) that the binding of cadherin peptide to Cry toxin is a prerequisite for the peptide's enhancement effect. However, both SfCad and MsBtR1 fragments bound Cry1Fa with high affinity and shared a common binding site on Cry1Fa toxin, but the former cadherin fragment exhibited a greater enhancement effect than did the latter in S. frugiperda larvae (57). Similarly, both a soluble HaBtR fragment from H. armigera (27) and an HaBtR fragment produced in an inclusion body (54) showed high affinity to Cry1Ac.…”

Genetically modified soybean expressing insecticidal protein (Cry1Ac): Management risk and perspectives

“…This was demonstrated for native populations of S. frugiperda in Mexico, Brazil, and Colombia, which showed differences in susceptibility to Bt strains due to high genetic variability among populations; a fact that results in groups lacking receptors for certain Bt proteins and, thus, are insensitive to insecticidal proteins (Monnerat et al 2006). Aranda et al (1996) demonstrated in trials with S. frugiperda that insufficient and (or) a weak link of the insecticidal protein in the midgut binding sites can also affect the susceptibility to Cry1Ac, which can also be related to the inactivation of the insecticidal protein by proteases (Rahman et al 2012). …”

Avaliação do risco de resistência de lepidópteros-praga (Lepidoptera: Noctuidae) à proteína Cry1Ac expressa em soja MON 87701 x MON 89788 no Brasil

“…Adicionalmente, uma quantidade insuficiente e/ou fraca ligação da proteína inseticida nos sítios de ligação do intestino médio também pode afetar a suscetibilidade a Cry1Ac, isso foi demonstrado para S. frugiperda a Cry1Ab (ARANDA et al, 1996) e S. exigua a Cry1C (de MAAGD et al, 2000), também pode estar relacionada com a inativação da proteína inseticida por proteases (MIRANDA; ZAMUDIO; BRAVO, 2001;RAHMAN et al, 2012 …”