Mosquitocidal action of Bacillus thuringiensis toxins

描述（申请人提供）：苏云金芽孢杆菌亚种。在田间使用了二十多年，未对任何目标昆虫产生抗性。与此相反，在许多国家的野外已观察到蚊子对球形芽孢杆菌的抗药性。对两种杀蚊芽孢杆菌菌株产生耐药性的倾向存在显著差异，可能是由于苏云金芽孢杆菌亚种中存在多种毒素。israelensis。然而，蚊子能够迅速对该菌株的个别毒素产生抗药性。蚊子明显不能对苏云金芽孢杆菌产生抗性的一个主要原因。以色列蚊是存在的细胞溶解（Cyt）毒素在这个和其他灭蚊剂菌株。Cyt毒素与杀虫结晶（Cry）毒素相互作用的确切机制尚不清楚，因此形成了这一建议的基础。我们假设Cyt和Cry毒素在膜中相互作用，促进任一蛋白形成毛孔。在本文中，我们重点研究苏云金芽孢杆菌的Cry11Aa和Cyt1Aa毒素。这些毒素协同作用，增强每种毒素的杀蚊活性。缺乏耐药发展也有两个因素：一是Cry和Cyt毒素作用所涉及的分子靶点不同，二是Cyt和Cry毒素之间的协同作用。因此，本研究的目标是首先确定Cry11Aa发挥其毒性作用的机制，其次是了解Cry和Cyt毒素之间协同作用的分子基础。在第一个目标中，我们将识别与受体和毒素有关的结构域，识别受体，最后分离相关的受体。一旦受体被分离，我们将使用dsrna介导的基因沉默来确定其作为Cry11A受体的功能作用。在第二个目标中，我们的重点是毒素的膜结合和随后的两种毒素的孔形成过程。这两个目标对于我们理解为什么没有蚊子对苏云金芽孢杆菌产生抗药性至关重要。israelensis。这个项目也是三个不同的研究者之间的联合提议，以最好地利用每个实验室的专业知识。

DESCRIPTION (provided by applicant): Bacillus thuringiensis subsp. israelensis has been used in the field for over twenty years without resistance development in any target insect. In contrast, mosquito resistance to Bacillus sphaericus has been observed in the field in many countries. This remarkable difference in the propensity to develop resistance to two mosquitocidal Bacillus strains is likely due to the presence of multiple toxins in B. thuringiensis subsp. israelensis. However, mosquitoes are able to rapidly develop resistance to individual toxins from this strain. A major reason for the apparent inability of mosquitoes to develop resistance to B. thuringiensis subsp. israelensis is the presence of cytolytic (Cyt) toxins in this and other mosquitocidal strains. The precise mechanism by which Cyt toxins interact with the insecticidal crystalline (Cry) toxins is not known, and therefore forms the basis for this proposal. We hypothesize that Cyt and Cry toxins interact in the membrane facilitating the formation of pores by either protein. In this proposal, our emphasis is on Cry11Aa and Cyt1Aa toxins of B. thuringiensis subsp. israelensis, which interact synergistically to enhance mosquitocidal activity of each toxin. The lack of resistance development is also due to two factors: the different molecular targets involved in the action of Cry and Cyt toxins, and secondly, the synergism between the Cyt and Cry toxins. Hence the objectives of the proposal are to first characterize the mechanism by which the Cry11Aa exerts its toxic effects, and the second is to understand the molecular basis of synergism between the Cry and Cyt toxins. In the first objective we will identify the domains involved in the receptor and toxin, identify the receptor(s), and finally isolate the receptor involved. Once the receptor is isolated we will use dsRNA-mediated gene silencing to determine its functional role as a Cry11A receptor. In the second objective our focus is on membrane binding of the toxins and the subsequent pore forming processes of both toxins. These two objectives are critical to our understanding of why no mosquito species have developed resistance to Bacillus thuringiensis subsp. israelensis. This project is also a joint proposal between three different investigators to best use the expertise of each laboratory.