Exploiting plant resistance-proteins for crop protection

进化见证了植物和入侵病原体之间的军备竞赛。病原体向植物细胞注入蛋白质，以阻止植物细胞的保护反应。植物细胞通过抗性蛋白的进化做出反应，这些抗性蛋白检测到这些细菌蛋白并对抗它们的活性。抗性蛋白的一个重要功能是允许植物细胞自杀，从而限制病原体的进一步传播。了解抗性蛋白的功能，并重要的是，操纵这一功能来提高植物对病原体的防御能力，可以帮助养活全球数百万人。尽管它们在保护植物免受病原体入侵方面发挥了关键作用，但人们对抗性蛋白如何发挥作用的细节知之甚少，部分原因是难以产生抗性蛋白在试管中进行分析。在这里，我们描述了用于试管分析的抗性蛋白的关键部分的产生。我们观察到了一种独特而令人兴奋的生化活动，腺嘌呤核苷酸生成腺苷(一种核苷酸酶活性)。更令人惊讶的是，在试管中观察到了这种抗性蛋白与DNA结合，这一结果很能说明问题，因为许多抗性蛋白被假设在细胞核中发挥作用。因此，我们基于试管的实验为研究抗性蛋白的功能提供了新的视角。在这里，我们建议开发我们的新方法来产生用于试管分析的抗性蛋白片段。首先，我们将研究试管中抗性蛋白核苷酸酶活性的特异性。我们将询问(I)抗性蛋白能够靶向的核苷酸类分子的范围，(Ii)抗性蛋白如何以及在哪里攻击其目标分子，以及(Iii)抗性蛋白的哪些部分对这种活性是重要的。这些重要的研究将揭示抗性蛋白如何在植物中发挥作用，并为我们进一步的研究提供保护植物的工具。其次，我们将在试管中研究抗性蛋白与DNA结合的分子基础。我们将询问(I)抗性蛋白针对和结合的DNA结构，以及(Ii)抗性蛋白的哪些部分对于结合DNA是重要的。在试管中调查核苷酸酶和DNA结合活性相互依赖的程度也将是有趣的，因为这可以揭示对统一抗性蛋白功能的更深层次的了解。总之，这些实验将提供对细胞内抗性蛋白功能的关键洞察，并为即将到来的植物保护实验提供进一步的工具。第三，我们将利用我们的试管实验作为作物保护战略的起点。我们将允许烟草叶片中产生抗性蛋白片段，从而导致细胞死亡。我们将调查在试管中被确认为核苷酸酶或DNA结合活性所需的那些抗性蛋白部分是否也需要指导烟草叶片的细胞死亡。这项重要的实验将把我们的试管实验与工厂中实际发生的情况统一起来。下一步，我们将改造植物，在这些植物中，只有当植物检测到入侵的病原体时，才会产生抗性蛋白片段。我们预计，这些植物中的特定细胞只有在接触病原体时才会死亡。这将阻止病原体的进一步传播，从而保护植物的其余部分。这种保护植物免受病原体侵袭的策略有两个明显的好处：1)它使用不产生有毒产物的植物蛋白质；2)我们可以利用我们基于试管的实验所获得的知识来产生抗性蛋白质，在这些蛋白质中，活性已经进行了微调，以补偿否则会损害植物适应性的影响。重要的是，这一植物保护战略应该广泛适用于各种作物。

Evolution has seen an arms race between plants and invading pathogens. Pathogens inject plant cells with proteins to block plant cell protective responses. Plant cells have responded through the evolution of resistance proteins that detect these bacterial proteins and counter their activity. An important function of the resistance protein is to permit plant cells to suicide thus restricting further spread of the pathogen. Understanding resistance protein function and, importantly, manipulating this function to improve plant defences to pathogens can help to feed millions of people globally. Despite their key role in protecting plants from invading pathogens, relatively little is known of the specifics of how resistance proteins function, in part due to difficulties in generating resistance proteins for analysis in the test tube. Here we describe the generation of a critical portion of a resistance protein for test tube based analysis. We observe a distinct and exciting biochemical activity, the generation of adenosine from adenine nucleotides (a nucleotidase activity). In a further surprise, the resistance protein was observed to bind DNA in the test tube, a telling result given that many resistance proteins are hypothesized to function in the nucleus. Our test tube based experiments therefore provide a new insight into resistance protein function. Here we propose to exploit our new method for generating resistance protein fragments for test tube analysis First, we will investigate the specifics of the resistance protein nucleotidase activity in the test tube. We will ask (i) what range of nucleotide like molecules is the resistance protein able to target, (ii) how and where does the resistance protein attack its target molecules, and (iii) which portions of the resistance protein are important for this activity. These important studies will reveal how the resistance protein is able to function in the plant and provide tools for protecting plants in our further studies. Second, we will investigate the molecular basis of DNA binding by resistance proteins in the test tube. We will ask (i) what DNA structures are targeted and bound by resistance proteins, and (ii) what portions of the resistance protein are important for binding DNA. It will also be interesting to investigate the extent to which nucleotidase and DNA binding activities are mutually dependent in the test tube as this can reveal a deeper insight into a unified resistance protein function. Together these experiments will provide key insight into resistance protein function within the cell and further tools for the plant protection experiments to come. Third, we will utilize our test tube experiments as a starting point for a crop protection strategy. We will permit the production of resistance protein fragments in tobacco leaves, thus causing cell death. We will investigate whether those portions of the resistance protein identified as being required for nucleotidase or DNA binding activity in the test tube are also required to direct cell death in tobacco leaves. This important experiment will unify our test tube experiments with what actually occurs in the plant. Next we will engineer plants in which a resistance protein fragment is only produced when the plant detects an invading pathogen. We anticipate that specific cells in these plants will only die when exposed to the pathogen. This will block any further spread of the pathogen and therefore protect the remainder of the plant. This strategy for protecting plants from pathogens has two clear benefits 1) It uses plant proteins that do not generate toxic products, 2) We can utilize knowledge from our test tube based experiments to generate resistance proteins in which the activity has been fine tuned to compensate for effects that would otherwise compromise plant fitness. Importantly, this strategy for plant protection should be broadly applicable across a broad spectrum of crops.