Group selection as a novel tool to screen and improve biological pesticides

在现代农业和园艺业中，越来越重视利用自然发生的生物体，如昆虫的病原体来进行虫害管理。微生物，如苏云金芽孢杆菌(Bt)，目前是针对毛虫、蚊子和甲虫害虫的有机许可应用的主要活性成分。此外，同样的细菌物种也是转基因植物蛋白质的重要来源，因为它产生许多蛋白质，这些蛋白质对少数昆虫具有高度毒性，但对人类和野生动物是安全的。然而，识别新的有毒细菌，并识别对昆虫有毒的新蛋白质，是一项困难和具有挑战性的任务。这一建议的基础是这样一个想法，即如果我们能够理解对毒力因子(微生物产生的有毒蛋白质)的投资如何增加微生物的繁殖和适应能力，那么我们就可以在实验室中应用基于选择的方法来分离出毒力较高的突变体或变种。在这项提议之前，我们在理解维持毒力的选择压力方面取得了重要进展。像Bt这样的病原体必须投入精力和资源来输出毒力因子，帮助它们感染昆虫。因此，投资于毒力对单个细胞来说是昂贵的，但却是必不可少的，因为细菌繁殖需要宿主死亡。个体水平的选择(在宿主内)可能有利于突变体，也就是所谓的“作弊者”，它们依靠邻居产生的蛋白质成功感染。相反，在群体水平上的选择(在感染之间)保持毒力。初步实验表明，群体水平的选择(与突变的供应相结合)可以导致毒力水平提高的突变的出现，即使这些突变对个体来说代价高昂。我们开发的群体选择方法为：探索现有的微生物多样性和试图改进微生物生防剂提供了一种新的工具。此外，对于Bt来说，杀死寄主的关键蛋白质是Cry毒素，而产生新的、更好的Cry毒素可以杀死难以瞄准的害虫，这方面有巨大的应用兴趣。使用基因工程方法，我们可以人工构建一组Bt菌株，表达这些蛋白质的不同集合，所有这些蛋白质的组成都略有不同。在这里，我们建议使用我们的新选择方法从这种不同的混合物中挑选出新的和有趣的蛋白质。这个建议的一个主要问题是更好地理解我们何时以及如何能够应用这些组选择方法。进化论可以帮助我们改进方法，例如，通过预测群体的组成如何影响选择的力量。另一个重要的考虑因素是，对于理解这种方法的应用潜力，我们是否只能利用选择来改进适应度较低的菌株，或者我们是否也可以改进已经非常有效地杀死昆虫宿主的菌株。另一个重要的预测是，选择实验可能有助于我们更好地了解病原体如何杀死昆虫宿主，并识别Bt中负责的蛋白质。当细菌进化出毒力增强时，我们可以利用DNA序列的变化或表达的蛋白质来识别哪些分子对毒力增强负责。鉴于我们对Bt产生的大多数蛋白质的功能缺乏了解，我们预计将获得有关这些细菌杀死宿主和克服免疫系统的手段的重要额外知识。

In modern agriculture and horticulture there is more and importance placed on exploiting naturally occurring organisms, such as the pathogens of insects, for pest management. Microbes such as Bacillus thuringiensis (Bt) are currently the main active ingredients in organically licensed applications targeted at caterpillar, mosquito and beetle pests. In addition, the same bacterial species is an important source of proteins for genetically modified plants, as it produces many proteins that are highly toxic to a narrow range of insects but safe for humans and wildlife.Nevertheless, identifying novel virulent bacteria, and identifying new proteins that are toxic to insects, is a difficult and challenging task. The foundation of this proposal is the idea that if we can understand how investment in virulence factors (toxic proteins produced by microbes) increases the reproduction and fitness of microbes, then we can apply selection-based methods in the laboratory to isolate mutants or variants with high virulence. Prior to this proposal we made an important advance in understanding the selection pressure that maintains virulence. Pathogens such as Bt have to invest energy and resources in exporting virulence factors that help them infect insects. Investing in virulence is therefore costly for individual cells, but essential, as bacterial reproduction requires the death of the host. Individual level selection (within hosts) can favour mutants, known as 'cheaters', which rely on the proteins produced by their neighbours for successful infection. Conversely, selection at the group level (between infections) maintains virulence. Preliminary experiments have shown that group level selection (in combination with a supply of mutations) can lead to the emergence of mutants with increased levels of virulence, even if these mutations are costly to the individual.The group selection methods we have developed offer a new tool for: exploring existing microbial diversity and trying to improve microbial biocontrol agents. Moreover, for Bt, the critical proteins involved in killing host are the Cry toxins, and there is enormous applied interest in generating new and better Cry toxins that can kill hard-to-target pests. Using genetic engineering methods we can artificially construct a collection of Bt strains that expresses a diverse collection of these proteins, all with a subtly different make-up. Here we propose to use our new selection methods to pick out new and interesting proteins from this diverse mix.A major question for this proposal is to better understand when and how we might be able to apply these group selection methods. Evolutionary theory can help us refine our methods, for instance by predicting how the make up of groups affects the power of selection. Another consideration, that is important for understanding the applied potential of this method, is to understand whether we can only use selection to improve strains with low fitness, or whether we can also improve strains that are already quite effective at killing insect hosts.Another important prediction is that selection experiments might help us better understand how pathogens kill insect hosts, and identify the proteins responsible in Bt. When bacteria have evolved increases in virulence, we can use the change in DNA sequence, or the proteins expressed, to identify which molecules are responsible for increased virulence. Given our lack of knowledge on the function of the of the majority of proteins produced by Bt, we expect to gain significant extra knowledge regarding the means by which these bacteria can kill hosts and overcome their immune systems.