How do Spore killers kill spores? Elucidating the mechanism of meiotic drive by spore killing in Neurospora fungi.

这个项目研究了真菌粗神经孢子菌的两个基因，一个杀死真菌孢子，另一个阻止孢子被杀死。虽然这两个基因可以被认为形成了一个毒药和解毒剂系统，但它们的目的尚不清楚。它们对真菌有益还是有害？它们似乎形成了一种自私的伙伴关系，这种关系的杀伤和抵抗特性使这两种基因能够传递给生物体的每一个后代，即使一对配偶中只有一方拥有这种基因。这个项目的主要目标是确定这些基因，毒药和解毒剂，是如何实现这一非凡的壮举的。此外，这两个基因似乎推动了它们所在染色体的重大重组。因此，该项目的第二个目标是研究自私基因是真核生物基因组重组的主要驱动因素的假设。主要目标将由伊利诺斯州中部伊利诺斯州立大学的科学家、研究生和本科生组成的团队来实现，而次要目标将由同一团队与瑞典的进化生物学家合作来实现。此外，来自伊利诺伊州中部的一名GK-12 STEM教师将协助该项目。这不仅将为教师提供宝贵的研究经验，而且PI和教师将为小学至12年级的教室设计探究性学习活动，包括对N. crassa和其他无害微生物的实验。本研究研究的两个基因分别是rfk （N.草孢子杀灭基因）和rsk （N.草孢子抗性基因）。虽然rfk杀伤基因的边界还没有被精确定义，但其杀伤功能已经被追踪到sk2菌株第三条染色体上的一个1500碱基对DNA片段。抗风险基因位于同一染色体上。自然界中存在着不同的风险等位基因，并不是所有的等位基因都能对rfk产生抗性。此外，rsk具有蛋白质编码基因的典型特征。例如，它具有清晰可识别的开始密码子、停止密码子和开放阅读框。这与杀手基因形成鲜明对比，杀手基因没有明显的蛋白质编码特征，因此可能产生有毒的非编码RNA或有毒的蛋白质。该项目的第一个目标是区分这两种可能性，并收集孢子杀死机制的证据。首先，将对六个先前分离的rfk突变体进行测序，以产生破坏杀伤的突变目录；其次，RNA测序将用于绘制功能性rfk位点的转录本，并确定与杀伤相关的全局转录变化；第三步，利用RACE技术克隆rfk全长转录物；第四，位点定向诱变将用于确定随机插入突变是否比随机点突变更具杀伤破坏性；第五，一组6个点突变将被修复，以确定6个点突变的哪个组合对杀死的丧失至关重要，当它们都在同一个RFK等位基因中被发现时，它们会破坏杀死；第六，将从抗性和非抗性的RSK版本中产生蛋白质嵌合体，以确定抗性蛋白的哪些部分对功能至关重要。该项目的第二个目标是在另一种名为Sk-3的菌株上进行同样的实验。总之，该提案的两个目标将定义杀伤基因的边界，确定杀伤基因产物是有毒的RNA还是蛋白质，提供破坏孢子杀伤的突变目录，确定与孢子杀伤机制相关的转录变化，并确定抗性所需的蛋白质的关键区域，所有这些都针对Sk-2和Sk-3，这两个自私基因功能和进化的模型。

This project investigates two genes in the fungus Neurospora crassa, one that kills fungal spores and another that prevents spore killing. While these two genes can be thought of as forming a poison and antidote system, their purpose is unclear. Are they beneficial for the fungus or are they detrimental? It seems they have formed a selfish partnership whose killing and resistance properties allow the two genes to be transmitted to every one of the organism's offspring, even when only one parent of a mating pair possesses the genes. The primary goal of this project is to determine how these genes, poison and antidote, achieve this remarkable feat. Additionally, the two genes appear to have driven a major reorganization of the chromosome in which they reside. Therefore, a secondary goal of the project is to investigate the hypothesis that selfish genes are major drivers of genome reorganization in eukaryotic organisms. The primary goal will be pursued by a team of scientists, graduate students, and undergraduate students at Illinois State University in Central Illinois, while the secondary goal will be pursued by the same team in collaboration with evolutionary biologists based in Sweden. In addition, a GK-12 STEM teacher from Central Illinois will assist with the project. Not only will this provide valuable research experience to the teacher, the PI and the teacher will design inquiry-based learning activities for GK-12 classrooms that involve experimentation with N. crassa and other harmless microorganisms. The two genes under investigation in this study are rfk, the N. crassa spore killing gene, and rsk, the resistance gene. Although the boundaries of the rfk killing gene have not been precisely defined, the killing function has been tracked to a 1500 base pair fragment of DNA on the third chromosome of a strain called Sk 2. The rsk resistance gene is found on the same chromosome. Different rsk alleles exist in nature, and not all of them provide resistance to rfk. Additionally, rsk possesses features typical of protein-coding genes. For example, it possesses a clearly identifiable start codon, stop codon, and open reading frame. This is in stark contrast to the killer gene, which does not have obvious protein-coding features, and thus could produce either a toxic non-coding RNA or a toxic protein. The first aim of this project is to differentiate between these two possibilities and gather evidence on the mechanism of spore killing. First, six previously isolated rfk mutants will be sequenced to produce a catalog of mutations that disrupt killing; second, RNA sequencing will be employed to map transcripts from a functional rfk locus and to determine the global transcriptional changes associated with killing; third, the full-length rfk transcript will be cloned with Rapid Amplification of cDNA End (RACE) technology; fourth, site-directed mutagenesis will be used to determine if random insertion mutations are more disruptive of killing than random point mutations; fifth, a group of six point mutations, which are known to disrupt killing when all are found within the same rfk allele, will be repaired to determine which combination of the six is critical for loss of killing; and sixth, protein chimeras will be produced from resistant and non-resistant versions of RSK to determine which parts of the resistance protein are critical for function. The second aim of this project is to perform the same set of experiments on a different strain called Sk-3. Together, the two aims of this proposal will define the borders of the killing gene, determine if the killer gene product is a toxic RNA or a protein, provide a catalog of mutations that disrupt spore killing, identify transcriptional changes associated with the spore killing mechanism, and identify critical regions of the protein required for resistance, all for Sk-2 and Sk-3, two models of selfish gene function and evolution.