Role of Heterotrimeric G-Proteins and Related Components In Hypovirulence and Virulence of the Plant Pathogen Cryphonectria Parasitica

该奖项由分子细胞生物科学部的细胞系统组和综合有机体系统部的过程、结构完整性组共同资助。细胞信号转导机制传递外部刺激，从而控制细胞对环境的反应能力。然而，这种反应可能会被病毒感染中断。为了探索可能发生这种情况的机制，Dawe博士将使用可以被RNA病毒（hypoviruses）稳定感染的真菌Cryphonectria parasitica（栗疫病真菌）。这个项目将集中在进化上保守的异源三聚体G蛋白信号通路，在所有真核生物中将细胞内级联反应耦合到细胞外刺激。三个G蛋白亚基的量在低病毒存在下减少，但发生这种情况的机制尚不清楚。BDM-1是一种被称为类胰蛋白酶蛋白的蛋白质家族的成员，似乎是稳定该途径的重要组成部分。这种蛋白质是磷酸化的。Dawe博士推测这种改变是由Hypovirus破坏的。 这导致G蛋白复合物不能正确组装，以及随后的信号传导通路失败。为了解决这个问题，Dawe博士将尝试使用免疫共沉淀技术来确认亚基的预测关系，该技术允许监测两种蛋白质的缔合。将使用BDM-1的定点突变体分析磷酸化BDM-1的需求，其中预测的磷酸化位点被不能磷酸化或模拟磷酸化存在的氨基酸取代。为了阐明病毒基因组中影响BDM-1的潜在区域，Dawe博士将引入嵌合（混合）病毒构建体，该构建体包含赋予宿主不同表型的病毒序列部分，包括改变BDM-1的积累。通过监测BDM-1的修饰和产生的变化，可以确定病毒基因组中负责影响BDM-1行为的特定区域。对真菌毒力所需的信号组分的分析将导致对信号控制的更深入理解以及丝状真菌生长和发育的关系。确定BDM-1的作用将有助于了解一个保守的蛋白质家族，同时也有助于确定病毒介导的G蛋白信号转导调节机制。除了对真菌发育，植物病理学，病毒-宿主相互作用和潜在的基于真菌病毒的生物控制策略领域的长期贡献外，该项目还将扩大学生培训，为少数民族服务机构的研究生和本科生提供分子方法学方面的宝贵经验。学生们还将通过科学会议和与日本宫城大学教员的互访，接触到新墨西哥州州立大学以外的项目。此外，该项目的材料将用于提高PI的分子和细胞真菌学课程，数据将通过出版物和网站传播进行分发。

This award is funded jointly by the Cellular Systems Cluster in the Division of Molecular & Cellular Biosciences and the Processes, Structures & Integrity Cluster in the Division of Integrative Organismal Systems.Cellular signal transduction mechanisms transmit external stimuli and thereby govern the ability of that cell to respond to its environment. However, such responses may be interrupted by infection with viruses. To explore mechanisms by which this may occur, Dr. Dawe will use the fungus Cryphonectria parasitica (the chestnut blight fungus) that can be stably infected by RNA viruses (hypoviruses). This project will focus on the evolutionarily conserved heterotrimeric G-protein signaling pathway that couple intracellular cascades to extracellular stimuli in all eukaryotes. The amounts of the three G-protein subunits are reduced in the presence of hypovirus, but the mechanism by which this occurs is unknown. BDM-1, a member of a protein family called phosducin-like proteins, appears to be an essential component for stabilizing this pathway. This protein is phosphorylated. and Dr. Dawe hypothesizes that this modification is compromised by the hypovirus. This leads to failure of the G-protein complex to properly assemble, and a subsequent failure of the signaling pathway. To address this problem, Dr. Dawe will attempt to confirm the predicted relationship of subunits using co-immunoprecipitation techniques that permit monitoring of the association of two proteins. The requirement for phosphorylated BDM-1 will be analyzed with site directed mutants of BDM-1 in which predicted phosphorylation sites are replaced with amino acids that either cannot be phosphorylated, or mimic the presence of phosphorylation. To elucidate the potential regions in the viral genome that affect BDM-1, Dr. Dawe will introduce chimeric (mixed) virus constructs containing parts of viral sequences that confer different phenotypes on the host, including altered accumulation of BDM-1. By monitoring of the resulting changes to modification and production of BDM-1 the specific regions of the viral genome that are responsible for affecting BDM-1 behavior can be determined.The analysis of signaling components required for fungal virulence will lead to greater understanding of the control of signaling and the relationship to growth and development in filamentous fungi. Determining the role of BDM-1 will contribute to the knowledge of a conserved family of proteins, while also identifying mechanisms of virus-mediated modulation of G-protein signaling. In addition to long-term contributions to the fields of fungal development, plant pathology, virus-host interactions and potential mycovirus-based biological control strategies, this project will expand student training, providing valuable experience in molecular methodologies for graduate and undergraduate students at a minority-serving institution. Students will also be exposed to projects beyond New Mexico State University through scientific meetings and reciprocal visits with a faculty member at Miyagi University in Japan. In addition, material from this project will be used to enhance the PI's Molecular and Cellular Mycology class and data will be distributed through publication and web site dissemination.