Arylphorin is a mitogen in the Heliothis virescens midgut cell secretome upon Cry1Ac intoxication

Authors: CASTAGNOLA, A. - University Of Tennessee; JACKSON, J. - University Of Tennessee; Perera, Omaththage; OPPERT, C. - University Of Tennessee; EDA, S. - University Of Tennessee; JURAT-FUENTES, J.L. - University Of Tennessee

Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/19/2017
Publication Date: 10/3/2017
Citation: Castagnola, A., Jackson, J., Perera, O.P., Oppert, C., Eda, S., Jurat-Fuentes, J. 2017. Arylphorin is a mitogen in the Heliothis virescens midgut cell secretome upon Cry1Ac intoxication. Insect Biochemistry and Molecular Biology. 5:e3886. https://doi.org/10.7287/peerj.preprints.2878v1.
DOI: https://doi.org/10.7287/peerj.preprints.2878v1

Interpretive Summary: Crystal (Cry) proteins produced by the soil bacterium Bacillus thuringiensis (Bt) cause species-specific lethality by damaging midgut in susceptible larvae. Cry1Ac protein is toxic to lepidopteran larvae, but Cry3Aa is non-toxic. In order to understand the midgut response to intoxication with Cry1Ac toxin, biological activity of proteins secreted by tobacco budworm midgut cells intoxicated with Cry1Ac toxin were compared with the proteins secreted by midgut cells treated with Cry3Aa or buffer solution. Proteins secreted by Cry1Ac treated cells induced cell division and differentiation and reduced mortality levels in Cry1Ac treated cells compared to secreted proteins from controls. Comparative analysis of secreted proteins in Cry1Ac treated and control samples identified seven differentially expressed proteins. One of the proteins, arylphorin, was only detected in secreted proteins from Cry1Ac treated cells. Feeding purified arylphorin to tobacco budworm larvae significantly reduced susceptibility to Cry1Ac. The data suggest that arylphorin plays a role in midgut regeneration process in response to Cry1Ac intoxication.

Technical Abstract: Insecticidal crystal (Cry) proteins produced by the bacterium Bacillus thuringiensis (Bt) target cells in the midgut epithelium of susceptible larvae. While the mode of action of Cry toxins has been extensively investigated, the midgut response to Cry intoxication and its regulation are not well characterized. In this work, we compared the secreted proteome (secretome) of primary mature midgut cell cultures from Heliothis virescens larvae after exposure to Cry1Ac toxin compared to Cry3Aa (non-active toxin) or buffer treatment. Biological activity of Cry1Ac-induced secretomes was monitored as higher proliferation and differentiation and an overall reduction in total cell mortality in primary H. virescens midgut stem cell cultures when compared to buffer secretome. Using differential proteomics, we identified 7 proteins with significant differences in abundance in Cry1Ac-treated compared to control secretomes. The most significant difference was detected for an arylphorin, which was only detected in the Cry1Ac-induced secretome. The effect of arylphorin on the midgut response to Cry1Ac intoxication was explored by feeding purified arylphorin and detecting midgut hyperplasia that led to significantly reduced susceptibility to Cry1Ac in H. virescens larvae. Taken together, our data identify arylphorin as a protein with a relevant role in the midgut regeneration process in response to Cry1Ac intoxication.