Twinstar is a chitin synthase interacting protein with an essential role in insect cuticle biosynthesis

doi:10.1016/j.jia.2024.05.027

Journal of Integrative Agriculture

2025, Vol. 24

Issue (1): 209-219 DOI: 10.1016/j.jia.2024.05.027

Plant Protection

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Twinstar is a chitin synthase interacting protein with an essential role in insect cuticle biosynthesis

Xu Zou¹, Jiqiang Chen¹, Yanwei Duan², Weixing Zhu², Qing Yang^{1, 2#}

¹State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China

²Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, China

Highlights
● Twinstar has been shown to directly interact with chitin synthase through various assays.
● Twinstar is essential for chitin synthesis in insect cuticles.
● Twinstar is highly conserved across diverse insect species, suggesting it may play a conserved role in chitin synthesis.

Abstract
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摘要几丁质是一种丰富的天然生物聚合物，是昆虫外骨骼的主要结构组分，对于昆虫的生长和发育至关重要。几丁质合成酶（Chitin synthase，CHS）是一种膜整合的糖基转移酶，被认为是参与几丁质生物合成过程的关键酶。然而，在几丁质生物合成过程中，一些额外的蛋白在促进几丁质合成酶发挥功能方面的具体作用仍然没有被完全阐明。在本研究中，利用分裂泛素膜酵母双杂交（Split-ubiquitin membrane yeast two-hybrid，MYTH）和Pull-down实验，证明了黑腹果蝇（Drosophila melanogaster）Twinstar（Tsr）与几丁质合成酶（Krotzkopf verkehrt，Kkv）在体外存在物理相互作用。Tsr 是一种小分子蛋白，属于肌动蛋白解聚因子ADF/Cofilin家族。利用RNA干扰（RNA interference，RNAi）技术在果蝇全身、表皮、气管和翅中对Tsr基因进行干扰，结果显示，降低果蝇全身、表皮和气管中的Tsr基因均会导致果蝇幼虫死亡；而降低果蝇翅中Tsr基因的表达导致翅褶皱表型。进一步研究表明，异常翅的第一纵脉几丁质含量降低，且几丁质片层结构缺失。为了验证Tsr在其他昆虫目中的功能保守性，对鳞翅目害虫亚洲玉米螟（Ostrinia furnacalis）和鞘翅目害虫赤拟谷盗（Tribolium castaneum）进行了研究。分别对亚洲玉米螟和赤拟谷盗中果蝇Tsr的同源基因OfTsr和TcTsr进行干扰，结果表明，降低亚洲玉米螟OfTsr的表达导致幼虫表皮合成受阻和化蛹异常；降低赤拟谷盗TcTsr的表达引起幼虫致死表型。本研究结果不仅增加了我们对昆虫表皮几丁质生物合成机制的认识，还为农业害虫防治提供了新的潜在靶点。

Abstract

Chitin is an abundant natural biopolymer that plays a crucial role in insect growth and development as a fundamental structural component of the exoskeleton. The membrane-integral β-glycosyltransferase, chitin synthase, has been identified as the central component in chitin biosynthesis. However, the precise roles of other proteins in facilitating chitin synthase in chitin biosynthesis remain unclear. In this study, we employed split-ubiquitin membrane yeast two-hybrid (MYTH) and pull-down assays to demonstrate the physical interaction between Twinstar (Tsr), a small molecular protein in the actin-depolymerizing factor ADF/Cofilin protein family, and chitin synthase Krotzkopf verkehrt (Kkv) in Drosophila melanogaster in vitro. The RNA interference (RNAi)-mediated global knockdown of Tsr in D. melanogaster resulted in larval lethality. Furthermore, targeted suppression of Tsr in the tracheal and epidermal tissues also led to larval mortality, while knocking down Tsr in the wing tissues led to wrinkled wings. Additionally, silencing Tsr not only reduced the chitin content in the first longitudinal vein of the wings but also led to the absence of the chitin lamellar structure. To validate the functional conservation of Tsr in other insect orders, the two agricultural pests Ostrinia furnacalis and Tribolium castaneum, representing lepidoptera and coleoptera insects, respectively, were investigated. Knockdown experiments targeting the Drosophila Tsr orthologues OfTsr in O. furnacalis and TcTsr in T. castaneum produced abnormal larvae during molting or pupation in O. furnacalis and lethality in T. castaneum. Our findings not only improve our knowledge of the chitin biosynthesis machinery in insect cuticles but also provide new potential targets for the control of major agricultural pests.

Keywords: chitin chitin synthase Twinstar insect cuticle pest control

Received: 25 January 2024 Accepted: 17 April 2024

Fund:

This work was supported by the National Natural Science Foundation of China (32161133010), the National Key Research and Development Program of China (2022YFD1700200), the Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-CSCB-202302), the Shenzhen Science and Technology Program, China (KQTD20180411143628272), and the Special Funds for Science Technology Innovation and Industrial Development of Shenzhen Dapeng New District, China (PT202101-02).

About author: Xu Zou, E-mail: xuxuzou@163.com; #Correspondence Qing Yang, E-mail: qingyang@caas.cn

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