Functional analysis of fungal nonself recognition

这项拟议的研究探索了生命的一个基本特征--细胞识别和适当回应非我的能力。与其他生物类似，真菌利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。我们研究一种被称为营养不亲和性的非自我识别形式，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个过程是如何工作的，它是如何整合到细胞网络中的，以及我们如何应用这一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中有四个具体目标。我们的第一个目标是探索在丝状真菌粗脉孢子菌和栗疫霉菌中触发非我识别的蛋白质相互作用。我们将使用遗传和生化技术来研究不相容蛋白在非我识别过程中如何相互作用来触发PCD。例如，我们已经确定了UN-24‘pa’形式的一个63个氨基酸的小片段，当它与该蛋白的OR形式相互作用时会导致细胞死亡。这很有趣，因为除了不相容功能外，un-24基因还编码核糖核苷酸还原酶(RNR)的大亚基，RNR是合成DNA合成所需核苷酸的关键酶。因此，RnR是一个主要的化疗靶点，了解如何特异性地抑制这种酶在癌症治疗和抗菌药物开发中具有应用价值。第二，我们的目标是了解粗粒拟青霉自交不亲和性“逃逸”的基础，这是一个导致‘het-6’非自我识别基因突变的过程。这种突变过程类似于脊椎动物抗体基因的高度突变--一种增强抗体与外来物质结合的适应性反应。我们已经开发了一些方案，使我们能够确定哪些基因参与了粗糙奈瑟氏菌中het-6的超突变，并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我识别系统如何快速进化提供新的信息。为了我们的第三个目标，我们将丝状真菌的不亲和性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。酵母菌是这方面的理想选择；它提供了一个强大的实验系统，并且没有内源营养不亲和系统。因此，对于酵母，我们可以更有效地研究不相容蛋白如何相互作用，以及这些相互作用如何扰乱生化过程从而导致PCD。这为研究基于蛋白质的生长抑制剂提供了一个平台，使用我们目前使用的方法来确定新型植物来源的抗真菌药物的作用模式。最后，我们将通过高通量的转录组测序，或细胞中所有的RNA分子的测序，来研究寄生线虫对异体识别的反应。我们还将研究自然产生的寄生虫病毒CHV1和抑制子突变dcl2-如何能够减弱不相容相关的PCD。这项工作将利用我们最近完成的寄生微孢子虫的基因组序列，寄生微孢子虫是导致板栗树枯萎病的病原体。这项研究将为这种植物病原菌和其他植物病原体的生物防治策略提供见解。总体而言，这项研究将帮助培养遗传学、微生物学和生物化学等高市场领域的6名博士、7名硕士和30多名本科生。这项以学生为中心的研究将促进我们在基础科学和应用科学领域的知识。

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.