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Destruxin A induces and binds HSPs in Bombyx mori Bm12 cells HUANHUAN ZHANG, Weina Hu, Miaomiao XIAO, Shiyi Ou, and Hu Qiongbo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03734 • Publication Date (Web): 19 Oct 2017 Downloaded from http://pubs.acs.org on October 20, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry 1

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Destruxin A induces and binds HSPs in Bombyx mori Bm12 cells Huanhuan Zhang1, Weina Hu1, Miaomiao Xiao1, Shiyi Ou2, Qiongbo Hu1*

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1. Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province, College of

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Agriculture, South China Agricultural University, Guangzhou 510642, China.

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2. Department of Food Science and Engineering, Jinan University, Guangzhou 510632, China.

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* Correspondence: [email protected]; Tel.: +86-20-85280308; Fax: +86-20-85280292

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Abstract: Destruxin A (DA) is a cyclodepsipeptidic mycotoxin isolated from the entomopathogenic fungus,

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Metarhizium anisopliae. It has insecticidal activity against host insect’s innate immunity system, but the

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molecular mechanism is not elucidated yet. In our previous experiment, four HSPs (heat shock proteins,

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BmHSP70-3, BmHSP75, BmHSP83 and BmHSCP) were characterized from the specific protein

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electrophoretic bands of Bombyx mori Bm12 cell line treated with DA in the test of drug affinity responsive

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target stability (DARTS), which implied that these HSPs might be kinds of DA-affinity proteins, or DA

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induces them up-regulated expression. Therefore, in current research, the interactions of DA and HSPs were

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explored through analysis of Bio-Layer Interferometry (BLI) employing FortBio OcteteQK. The expression

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levels of HSPs genes were surveyed by quantitative real-time PCR (qPCR). The results indicated that DA

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had no interactions with BmHSP70-3, BmHSP75 and BmHSP83, but had affinity to BmHSCP with a KD

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value of 88.1 µM, in BLI analysis. However, the expression levels of all HSPs genes were significantly

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up-regulated after the Bm12 cells were treated by DA. In conclusion, DA can induce the four HSPs

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expression in Bm12 cells, but DA only binds to BmHSCP. Our research provides new insights on

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understanding of the action mechanisms of destruxins.

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Keywords: destruxin; heat shock protein; molecular interaction; gene expression

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INTRODUCTION Metarhizium anisoplyae is a well-known entomopathogenic fungus distributing worldwide, which plays

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an important role in biological control of insect pests 1. Destruxins, a group of cyclo-hexadepsipeptide and

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bio-synthesized by non-ribosomal peptide synthetase, were firstly isolated from M. anisopliae 2. Chemically,

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destruxin compounds comprise five amino acids and an α-hydroxy acid, forming a cyclic hexadepsipeptid.

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To date, 39 destruxin analogs have been isolated and identified 3. Destruxin A (DA), the most common

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analogue of destruxins, is the key pathogenic factor of M. anisopliae and exhibits substantial insecticidal

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activity including contact toxicity, antifeedant and growth regulating against insects. Thus, DA was

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considered as a new lead compound of insecticides and a potential food pollution source of biological control

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agents 1, 3-4.

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There have been some experiments exploring the mechanisms of DA killing pests. It was reported that

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DA damaged the morphological characters of insect’s cells such as the B. mori Bm12, Drosophila

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melanogaster S2, Ostrinia furnacalis SYSU-Of He-C, Spodoptera exigua Sf9, etc. 5. Meanwhile, the tissues

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of midguts, visceral muscles, renal epithelia and Malpighian tubules were injured after the insects, D.

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melanogaster, Locusta migratoria and Rhodnius prolixus were treated with DA 6. However, more research

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results supported that DA damages the innate immunity of insects. In fact, DA even at a dosage of