Midgut juice components affect pore formation by the Bacillus thuringiensis insecticidal toxin Cry9Ca

Area of Science:

• Insect toxicology
• Biochemistry
• Molecular biology

Background:

• Bacillus thuringiensis (Bt) toxins are crucial for insect pest control.
• The pore-forming ability of Bt toxins is essential for their insecticidal activity.
• Cry9Ca is a Bt toxin with potential applications in agriculture.

Purpose of the Study:

• To evaluate the pore-forming ability of Cry9Ca and its mutants.
• To investigate the role of M. sexta midgut juice in modulating Cry9Ca activity.
• To identify factors in midgut juice that affect Bt toxin efficacy.

Main Methods:

• Electrophysiological assays were used to measure membrane depolarization.
• Toxin activity was assessed with wild-type Cry9Ca, single-site mutants (R164A, R164K), and a 55-kDa fragment.
• Experiments were conducted with and without M. sexta midgut juice, denatured juice, dialyzed juice, extracted lipids, and protease inhibitors.

Main Results:

• Cry9Ca and its mutants depolarized M. sexta midgut cell membranes.
• The 55-kDa fragment showed higher activity than intact Cry9Ca and mutants.
• Midgut juice significantly enhanced the activity of Cry9Ca and its mutants, an effect mimicked by lipids and protease inhibitors.

Conclusions:

• Further proteolysis of activated Cry9Ca can modulate its activity.
• M. sexta midgut juice contains factors, likely protease inhibitors and lipids, that enhance Bt toxin pore-forming ability.
• These findings suggest a significant role for midgut juice components in the efficacy of B. thuringiensis toxins against insects.