Phytochrome and pheromone signalling during the induction of a new cell type involved in sexual fusion in filamentous fungi

丝状子囊菌真菌产生的无性孢子称为分生孢子。子囊菌是真菌中最大的一类，含有大多数作物和人类的真菌病原体。分生孢子是这些生物的主要生殖细胞和传播媒介，但在有性生殖过程中也可以作为雄性受精的媒介。我们已经发现，模式子囊真菌神经孢子菌的分生孢子可以产生三种类型的丝状细胞：(a)参与集落建立的分生孢子芽管，(b)通过细胞融合参与形成复杂细胞网络的分生孢子吻合管，以及(c)最近发现的与异性细胞性融合的分生孢子性管。分生孢子性管的产生机制，特别是它们如何受性信息素和光的调节，是本研究的重点。一个有趣的方面是，分生孢子性管是在最靠近光源的一侧形成的（即它们表现出正向的向光性）。我们研究计划的第一个目标是分析信息素信号的作用。特别是，我们将确定合成性信息素是否能诱导分生孢子性管的形成。我们发现分生孢子性管是在红光下形成的，这涉及到两个被称为光敏色素的红光感受器。我们的第二个目标将是分析这些光敏色素受体如何对红光作出反应，并在此过程中调节分生孢子性管的形成。粗神经孢子虫是第一个完成基因组测序的丝状真菌，结果显示它拥有约10,000个基因。神经孢子菌的约1万个基因中的每一个都在发生突变，以产生“基因敲除突变体”。我们的第三个目标是筛选几百个这样的基因敲除突变体，以确定它们在分孢子性管的形成和/或向光性方面是否有缺陷。我们还获得了另外两种光感受器参与分生孢子性管形成的证据：一种称为隐色素的蓝光光感受器和一种属于视蛋白家族的绿光光感受器（其中还包括人眼中发现的视紫红质）。这些光感受器在真菌中的作用目前尚不清楚。我们的第四个目标将是分析隐色素和两种不同的视蛋白在分生孢子性管的形成及其可能的嗜光性生长中的作用。第五个目标将涉及可视化光敏色素、隐色素和视蛋白的光感受器，通过荧光标记，使它们能够在活细胞中用荧光显微镜成像。通过这种方式，我们将能够直接监测它们是否会改变它们的亚细胞位置以响应光照。我们的最终目标是确定不同的光感受器是否相互作用，并对光信号做出一致的反应，从而导致分生孢子性管的形成和向光性。

The asexual spore produced by filamentous ascomycete fungi is called a conidium. The ascomycetes are the largest group of fungi and contain the majority of crop and human fungal pathogens. Conidia are the main reproductive cells and dispersal agents of these organisms, but can also act as male fertilizing agents during sexual reproduction. We have discovered that conidia of the model ascomycete fungus, Neurospora crassa, can produce three types of filamentous cells: (a) the conidial germ tube which is involved in colony establishment, (b) the conidial anastomosis tube which is involved in forming complex cellular networks by undergoing cell fusion, and (c) the most recently discovered conidial sex tube which is involved in sexual fusion with cells of the opposite sex. The mechanism by which conidial sex tubes are produced, and particularly how they are regulated by sex pheromones and by light, is the focus of the research in the present proposal. One interesting aspect is that the conidial sex tubes are formed on the side closest to the light source (i.e. they exhibit a positive phototropism). The first objective of our research proposal will be to analyse the role of pheromone signalling. In particular, we will determine whether a synthetic sex pheromone can induce conidial sex tubes to form. We have found that conidial sex tubes are formed in response to red light and this involves two red light photoreceptors called phytochromes. Our second objective will be to analyse how these phytochrome receptors respond to red light, and in doing so, regulate the formation of conidial sex tubes. Neurospora crassa was the first filamentous fungus to have its genome completely sequenced and as a result has been shown to possess ~ 10,000 genes. Each of Neurospora's ~ 10,000 genes is being mutated to produce 'knockout mutants. Our third objective will be to screen several hundred of these knockout mutants to determine if they are defective in conidial sex tube formation and/or phototropism. We have also obtained evidence that two other photoreceptors are involved in the formation of conidial sex tubes: a blue light photoreceptor called cryptochrome, and a green light photoreceptor belonging to the opsin family of proteins (which also includes the photoreceptor rhodopsin which is found in the human eye). The roles of these photoreceptors in fungi are currently unknown. Our fourth objective will be to analyse the roles of cryptochrome and two different opsins in the formation of conidial sex tubes and possibly their phototropic growth. The fifth objective will involve visualizing the phytochrome, cryptochrome and opsin photoreceptors by tagging them with fluorescent labels which allow them to be imaged in living cells with a fluorescence microscope. In this way we will be able to directly monitor whether they change their subcellular location in response to light exposure. Our final objective will be to be to determine if the different photoreceptors interact and act in concert in response to the light signals that result in the formation and phototropism of conidial sex tubes.