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

该奖项由分子和细胞生物科学分部的细胞系统群和整合组织系统分部的过程、结构和完整性群共同资助。细胞信号转导机制传递外部刺激，从而管理该细胞对环境做出反应的能力。然而，这种反应可能会因感染病毒而中断。为了探索这种情况可能发生的机制，道博士将使用可以稳定地被RNA病毒(低病毒)感染的真菌栗疫病菌(栗疫病菌)。这个项目将集中在进化上保守的异三聚体G蛋白信号通路，该信号通路在所有真核生物中将细胞内级联与细胞外刺激偶联。在低病毒存在的情况下，三个G蛋白亚基的数量会减少，但发生这种情况的机制尚不清楚。BDM-1是一种被称为光导蛋白样蛋白的蛋白质家族的成员，似乎是稳定这一途径的必要成分。这种蛋白质是磷酸化的。道博士假设，这种修饰受到低度病毒的影响。这会导致G-蛋白复合体无法正确组装，并随后导致信号通路的失败。为了解决这个问题，道博士将尝试使用免疫共沉淀技术来确认预测的亚基之间的关系，这种技术可以监测两种蛋白质的联系。对磷酸化BDM-1的需求将通过BDM-1的定点突变体进行分析，在这些突变体中，预测的磷酸化位点被无法磷酸化的氨基酸取代，或者模拟磷酸化的存在。为了阐明病毒基因组中影响BDM-1的潜在区域，道博士将引入嵌合(混合)病毒结构，其中包含部分病毒序列，这些序列赋予宿主不同的表型，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。分析真菌毒力所需的信号成分将有助于更好地理解信号的控制以及与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保守的蛋白质家族，同时也有助于识别病毒介导的G蛋白信号调节机制。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防治策略等领域的长期贡献外，该项目还将扩大学生培训，为少数族裔服务机构的研究生和本科生提供宝贵的分子方法学经验。学生们还将通过科学会议和与日本宫城大学教职员工的互访，接触到新墨西哥州立大学以外的项目。此外，该项目的材料将用于加强PI的分子和细胞真菌学课程，并将通过出版物和网站传播数据。

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.