Comprehensive analysis of Cry1Ac protoxin activation mediated by midgut proteases in susceptible and resistant Plutella xylostella (L.).

Insecticidal Cry toxins produced by Bacillus thuringiensis (Bt) have been widely used to control agricultural pests in both foliage sprays and transgenic crops. Nevertheless, rapid evolution of insect resistance to Cry toxins requires elucidation of the molecular mechanisms involved in Cry resistance. Two proposed models have been described to explain the toxicity of Cry proteins, the classic model states that Cry protoxin is activated by midgut proteases resulting in activated toxin that binds to receptors and forms a pore in the midgut cells triggering larval death, and the newly proposed dual model of the mode of action of Bt Cry toxins states that protoxin and activated toxins may have different mechanisms of action since several resistant strains to activated Cry toxins are still susceptible to the same Cry-protoxin. Protoxin activation by midgut proteases is a key step in both models. Herein, we evaluated Cry1Ac protoxin activation in a susceptible Plutella xylostella (L.) strain (DBM1Ac-S) and in the near-isogenic strain (NIL-R) with high field-evolved Cry1Ac resistance. Previous work showed that Cry1Ac resistance in NIL-R correlates with reduced binding to midgut receptors due to enhanced MAPK signaling pathway and down regulation of ABCC2 receptor. However, reduced midgut trypsin levels and altered midgut protease gene transcription were also observed in the Cry1Ac-resistant field isolated strain that is parent of the NIL-R strain. Therefore, we analyzed the midgut protease activities in both DBM1Ac-S and NIL-R strains. Detection of enzymatic activities showed that caseinolytic protease, trypsin and chymotrypsin activities were not significantly different between the susceptible and resistant strains. Furthermore, treatment with different trypsin or chymotrypsin inhibitors, such as Nα-tosyl-l-lysine chloromethyl ketone (TLCK) or Np-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not affect the susceptibility to Cry1Ac protoxin of the DBM1Ac-S and NIL-R larvae. Bioassay results indicated that the NIL-R larvae showed similar resistant levels to both Cry1Ac protoxin and trypsin-activated toxin. Taken together, our results demonstrated that high-level field-evolved Cry1Ac resistance in the NIL-R strain is independent of Cry1Ac protoxin activation and the specific protoxin mechanism of action. This discovery will strengthen our comprehensive understanding of the complex mechanistic basis of Bt resistance in different insects.

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