Expressing a fusion protein with protease and chitinase activities increases the virulence of the insect pathogen Beauveria bassiana
Expressing a fusion protein with protease and chitinase activities increases the virulence of the insect pathogen Beauveria bassiana
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DOI：
10.1016/j.jip.2009.07.013
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发表时间：
2009-10-01
影响因子：
3.4
通讯作者：
Pei, Yan
中科院分区：
文献类型：
--
作者：
Fang, Weiguo;Feng, Jin;Pei, Yan
昆虫病原真菌，如白僵菌和金龟子绿僵菌，正在开发作为化学杀虫剂的替代品。它们利用物理压力和细胞外水解酶（例如蛋白酶和几丁质酶）的组合，通过直接穿透角质层来感染昆虫。之前我们发现枯草杆菌蛋白酶样蛋白酶 (Pr1A) 或几丁质酶 (Bbchit1) 的过度表达分别导致 M. anisopliae 和 B. bassiana 的毒力增加。在这项研究中，我们发现白僵菌 Pr1A 同源物 (CDEP1) 和 Bbchit1 的混合物在体外比单独使用 CDEP1 或 Bbchit1 更有效地降解昆虫角质层。在此基础上，我们产生了三种质粒构建体； (1) Bbchit1、(2) CDEP1 和 (3) 与 CDEP1 连接的 Bbchit1 融合基因，各自受构巢曲霉组成型 gpd 启动子的控制。分泌融合蛋白（CDEP1：Bbchit1）的白僵菌转化体穿透角质层的速度明显快于野生型或过表达 Bbchit1 或 CDEP1 的转化体。与野生型相比，过表达CDEP1的转化体的LT(50)降低了12.5%，而LC(50)没有降低。表达CDEP1:Bbchit1的转化体的LT(50)降低了24.9%。引人注目的是，CDEP1: Bbchit1 的表达导致 LC(50) 降低 60.5%，是 Bbchit1 过表达 (28.5%) 降低的两倍多。这项工作代表了开发高毒力昆虫病原体以有效控制害虫的重要一步。 (C) 2009 Elsevier Inc. 保留所有权利。
Entomopathogenic fungi, such as Beauveria bassiana and Metarhizium anisopliae are being developed as alternatives to chemical insecticides. They infect insects by direct penetration of the cuticle using a combination of physical pressure and extracellular hydrolytic enzymes such as proteases and chitinases. Previously we found that overexpression of a subtilisin-like protease (Pr1A) or a chitinase (Bbchit1) resulted in increased virulence of M. anisopliae and B. bassiana, respectively. In this study, we found that a mixture of the B. bassiana Pr1A homolog (CDEP1) and Bbchit1 degraded insect cuticle in vitro more efficiently than either CDEP1 or Bbchit1 alone. Based on this we produced three plasmid constructs; (1) Bbchit1, (2) CDEP1, and ( 3) a fusion gene of Bbchit1 linked to CDEP1 each under the control of the constitutive gpd promoter from Aspergillus nidulans. B. bassiana transformants secreting the fusion protein (CDEP1: Bbchit1) penetrated the cuticle significantly faster than the wild type or transformants overexpressing either Bbchit1 or CDEP1. Compared to the wild type, the transformant overexpressing CDEP1 showed a 12.5% reduction in LT(50), without a reduction in LC(50). The LT(50) of the transformant expressing CDEP1: Bbchit1 was reduced by 24.9%. Strikingly, expression of CDEP1: Bbchit1 resulted in a 60.5% reduction in LC(50), more than twice the reduction obtained by overexpression of Bbchit1 (28.5%). This work represents a significant step towards the development of hypervirulent insect pathogens for effective pest control. (C) 2009 Elsevier Inc. All rights reserved.