Discovering the xylan-sensing pathway in the filamentous fungus Neurospora crassa

 描述(由申请人提供)：以最有效的方式识别和利用可用的营养是所有生物体共同的挑战。在人类中，不准确或失败的营养感知可能导致包括糖尿病和肥胖症在内的各种疾病，而癌症的进展已被证明依赖于葡萄糖摄取的增加和营养感知的变化。许多营养感知途径从酵母到人类都是保守的，单细胞真核生物中的营养感知研究对于阐明人类的营养感知途径具有重要意义。然而，在单细胞真核生物中营养信号的大部分工作都是在发芽酵母中完成的，这种酵母的碳水化合物利用谱相当有限。与发芽酵母不同，模式丝状真菌粗脉孢子菌能够利用各种各样的碳水化合物：从单糖到植物细胞壁中发现的复杂糖链。为了有效地利用可利用的资源，海藻必须能够感知并对这些不同碳水化合物的存在做出反应。已鉴定出几种转录因子可激活植物细胞壁降解酶的转录。其中之一是XLR1，当植物细胞壁成分木聚糖存在时，它激活半纤维素酶的转录。然而，尽管XLR1显然必须被激活才能诱导半纤维素酶的表达，但实现这一点的方法仍然不清楚。该项目的目标是确定和表征XLR1的上游调控因子以及它们与粗枝藻中其他营养传感途径的相互作用，这将通过完成以下三个具体目标来实现。第一个是筛选XLR1激活是结构性或不可诱导性的突变体，以确定参与木聚糖传感途径的基因，并表征它们在木聚糖传感中的功能和作用。第二个目标是利用定向进化来探测木聚糖感应通路内以及木聚糖感应通路和其他营养感应通路之间更微妙的遗传相互作用，以确定使粗粗木聚糖快速而准确的感应和利用最优化的突变，以及了解木聚糖感应通路如何适应评估细胞代谢状态的更大的通路方案。第三个目标是利用合成生物学来重建木聚糖感知途径。 在不能利用木聚糖作为碳源的芽生酵母中，利用前两种方法中确定的优化基因，旨在验证木聚糖传感途径中涉及的基因的鉴定。这些目标的完成将有助于揭示粗毛拟青霉的木聚糖感应途径及其与其他细胞信号途径的相互作用。我们希望这将提高我们对整个真核领域细胞信号的整体理解，因为扫描信号通路所涉及的分子机制的多样性有助于阐明广泛的生物学范式。

 DESCRIPTION (provided by applicant): Identifying and utilizing the nutrients available in the most efficient manner is a challenge common to all organisms. In humans inaccurate or failed nutrient sensing can result in a variety of diseases including diabetes and obesity, and cancer progression has been shown to rely on increased glucose uptake and changes in nutrient sensing. Many of the nutrient sensing pathways are conserved from yeast to humans, and studies on nutrient sensing in unicellular eukaryotes have been instrumental in elucidating nutrient sensing pathways in humans. However, much of the work on nutrient signaling in unicellular eukaryotes has been done in budding yeast, which has a fairly limited carbohydrate utilization repertoire. Unlike budding yeast, the model filamentous fungus, Neurospora crassa, is capable of utilizing a wide variety of carbohydrates: from simple sugars to the complex sugar chains found in plant cell walls. In order to efficiently exploit the available resources, N. crass must be capable of sensing and responding to the presence of these different carbohydrates. Several transcription factors have been identified in N. crassa that activate the transcription of plant cell wall-degrading enzymes. One of these is XLR1, which activates the transcription of hemicellulases when in the presence of the plant cell wall component xylan. However, while it seems evident that XLR1 must be activated in order to induce expression of hemicellulases, the method by which this is achieved is still unclear. The goal of this project is to identify and characterize upstream regulators of XLR1 and their interactions with other nutrient sensing pathways in N. crassa, which will be accomplished through the completion of the following three specific aims. The first is to screen for mutants in which XLR1 activation is either constitutive o uninducible to identify the genes involved in the xylan-sensing pathway and characterize their function and role in xylan sensing. The second aim is to use directed evolution to probe more subtle genetic interactions both within the xylan-sensing pathway and between the xylan-sensing pathway and other nutrient sensing pathways to identify mutations which optimize N. crassa for rapid and accurate xylan sensing and utilization as well as to understand how the xylan-sensing pathway fits into the larger scheme of pathways that assess the metabolic state of the cell. And the third aim is to use synthetic biology to reconstruct the xylan-sensing pathway in the budding yeast, Saccharomyces cerevisiae, which is not able to use xylan as a carbon source, using the optimized genes identified in the first two aims to validate the identification o genes involved in the xylan-sensing pathway. The completion of these aims should shed light on the xylan-sensing pathway in N. crassa and its interaction with other cellular signaling pathways. We expect this to improve our overall understanding of cellular signaling throughout the eukaryotic realm , since scanning the diversity of molecular mechanisms involved in signaling pathways is helpful in elucidating broad biological paradigms.