Functional analysis of fungal nonself recognition

真菌非自识别的功能分析

• 批准号: RGPIN-2014-05436
• 负责人: Smith, Myron
• 金额: $ 5.17万
• 依托单位: Carleton University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=665479
• 关键词: Functional; analysis; fungal; nonself; recognition

The proposed research explores a fundamental characteristic of life - the ability of cells to identify and appropriately respond to nonself. Similar to other organisms, fungi use nonself recognition to reduce disease transmission, and to modulate intra- and interspecies interactions. We study a form of nonself recognition referred to as vegetative incompatibility, whereby cell fusion between different fungi results in Programmed Cell Death (PCD). We aim to learn how this process works, how it integrates into cellular networks, and how we can apply this knowledge to control fungi that have negative impacts on human affairs. We have four specific objectives for this grant cycle. Our first is to explore protein interactions that trigger nonself recognition in the filamentous fungi Neurospora crassa and Cryphonectria parasitica. We will use genetic and biochemical techniques to study how incompatibility proteins interact during nonself recognition to trigger PCD. For example, we have identified a small 63 amino acid segment of the `PA' form of UN-24 that causes cell death when it interacts with the OR form of this protein. This is interesting since, in addition to incompatibility function, the un-24 gene encodes the large subunit of ribonucleotide reductase (RNR), an essential enzyme that synthesizes the nucleotides needed for DNA synthesis. RNR is thus a prime chemotherapeutic target and understanding how to specifically inhibit this enzyme has applications in cancer therapy and antimicrobial drug development. Second, we aim to understand the basis of `escape' from self-incompatibility in N. crassa, a process that results in mutagenesis of the `het-6' nonself recognition gene. This mutational process resembles hypermutation of antibody genes in vertebrates - an adaptive response to enhance binding by antibodies to foreign substances. We have developed the protocols that allow us to identify what genes are involved in hypermutation of het-6 in N. crassa and show that the process requires genes that play a role in DNA damage signaling. This study of escape will provide novel information on how nonself recognition systems rapidly evolve. For our third objective we will transfer incompatibility genes from filamentous fungi into brewer's yeast to further study the biochemical pathways affected by these genes. Yeast is ideal for this; it offers a powerful experimental system and does not have an endogenous vegetative incompatibility system. Therefore, with yeast we can more efficiently study how incompatibility proteins interact with each other, and how these interactions perturb biochemical processes to bring about PCD. This provides a platform to study protein-based growth inhibitors using methods that we currently employ to determine mode-of-action of novel, plant-derived antifungals. Finally, we will investigate how C. parasitica responds to nonself recognition using high-throughput sequencing of the `transcriptome', or all the RNA molecules in the cell. We will also investigate how a naturally occurring virus of C. parasitica, CHV1, and a suppressor mutation, dcl2-, are able to attenuate incompatibility-associated PCD. This work will take advantage of our recent completion of the genome sequence of C. parasitica, the causal agent of the blight that decimated our chestnut trees. The study will provide insights into biological control strategies of this and other plant pathogens. Overall, this research will help train 6 PhD, 7 MSc, and over 30 undergraduate students in highly marketable areas of genetics, microbiology and biochemistry. This student-centered research will advance our knowledge in areas of both basic and applied science.

这项拟议中的研究探索了生命的一个基本特征--细胞识别非我并做出适当反应的能力。与其他生物类似，真菌使用非自我识别来减少疾病传播，并调节种内和种间的相互作用。我们研究了一种被称为营养体不相容性的非自我识别形式，即不同真菌之间的细胞融合导致程序性细胞死亡（PCD）。我们的目标是了解这一过程是如何工作的，它如何融入细胞网络，以及我们如何应用这些知识来控制对人类事务有负面影响的真菌。我们在这个赠款周期有四个具体目标。我们的第一个是探索蛋白质的相互作用，触发非自我识别的丝状真菌粗糙脉孢菌和Cryphonectria寄生。我们将使用遗传和生物化学技术来研究不相容蛋白在非自我识别过程中如何相互作用以触发PCD。例如，我们已经鉴定了UN-24的“PA”形式的一个小的63个氨基酸片段，当它与该蛋白质的OR形式相互作用时，该片段导致细胞死亡。这是有趣的，因为除了不相容性功能，un-24基因编码核糖核苷酸还原酶（RNR）的大亚基，RNR是合成DNA合成所需的核苷酸的必需酶。因此，RNR是一个主要的化疗靶点，了解如何特异性抑制这种酶在癌症治疗和抗微生物药物开发中具有应用。第二，我们的目标是了解的基础上'逃避'自交不亲和性N。crassa，这是一种导致“het-6”非自身识别基因突变的过程。这种突变过程类似于脊椎动物中抗体基因的超突变-一种增强抗体与外来物质结合的适应性反应。我们已经开发了一种方法，使我们能够确定哪些基因参与了N. crassa的研究表明，这一过程需要在DNA损伤信号中发挥作用的基因。这项关于逃避的研究将为非自我识别系统如何快速进化提供新的信息。我们的第三个目标是将丝状真菌中的不亲和基因转移到啤酒酵母中，以进一步研究这些基因影响的生化途径。酵母是理想的，它提供了一个强大的实验系统，没有内源性营养不亲和系统。因此，使用酵母，我们可以更有效地研究不相容蛋白质如何相互作用，以及这些相互作用如何干扰生化过程，从而导致PCD。这提供了一个平台来研究基于蛋白质的生长抑制剂，使用我们目前采用的方法来确定新型植物源性抗真菌剂的作用模式。最后，我们将研究如何C。寄生虫响应非自我识别使用高通量测序的“转录组”，或所有的RNA分子在细胞中。我们还将研究自然发生的C.寄生虫CHV 1和抑制突变dcl 2-能够减弱不相容性相关的PCD。这项工作将利用我们最近完成的C.寄生虫，枯萎病的病原体，摧毁了我们的栗子树。该研究将为这种和其他植物病原体的生物控制策略提供见解。总的来说，这项研究将有助于培养6名博士，7名硕士和30多名本科生在遗传学，微生物学和生物化学的高度市场化领域。这种以学生为中心的研究将推进我们在基础和应用科学领域的知识。