Bipartite Geminivirus Movement Protein Function

该项目的目的是阐明植物病毒编码的运动蛋白(MPS)如何协调病毒基因组的复制及其在植物细胞内和之间的定向运动，并探索MPS在组织限制植物病毒感染中的作用。在这一层面上了解病毒与宿主的相互作用对于抗击病毒疾病非常重要，因为只有从这些知识中才能制定出合理的抗病毒策略。遗传学研究表明，MPS决定了植物病毒的寄主范围和致病特性。因此，了解它们的功能对于开发对广泛的双生病毒具有抗性的植物的工程策略是至关重要的。两部分双生病毒南瓜曲叶病毒(SqLCV)和甘蓝曲叶病毒(CLCV)编码两个MPS，NSP和MPB。它们协同作用，在细胞内和细胞之间移动病毒单链DNA基因组。NSP是一种核穿梭蛋白，它将病毒基因组在细胞核内的复制位置结合起来，并在细胞核和细胞质之间移动。MPB将NSP-基因组复合体捕获在细胞质中，并将它们沿着独特的内质网(ER)衍生的小管移动到发育中的韧皮部细胞的壁上。通过定点突变，已在每个MP中确定了功能结构域。NSP和MPB之间的相互作用受植物细胞特有的活性调节，突变研究表明这种调节可能涉及MPS的磷酸化。绿色荧光蛋白(GFP)的使用进一步表明SqLCV的侵染是韧皮部受限的。要了解NSP和MPB的功能，它们所通过的信号通路，以及MPB在限制病毒侵染韧皮部中的作用，现在需要进行生化和遗传学研究。我们还想了解并开发新的体内和体外方法来研究它们的功能。该项目的具体目标是研究MPB-ER的相互作用及其在病毒感染的韧皮部限制中的作用，研究AtNSI的功能，AtNSI是一种与NSP相互作用的未知功能拟南芥蛋白，并鉴定与MPB相互作用的宿主细胞蛋白。这些方法将利用葫芦科植物和拟南芥的不同特点。将对表达游离GFP的CLCV和CLCV-SqLCV重组体进行研究，以确定CLCV在拟南芥中的侵染是否受韧皮部限制，以及MPB在侵染拟南芥的能力中所起的作用。将MPB与由组织特异性启动子表达的GFP在转基因拟南芥中的融合将被用来确定MPB在限制病毒侵染韧皮部中的作用以及韧皮部发育对病毒运动的影响。确定AtNSI的亚细胞位置以及组织特异性和时间表达模式将提供有关其功能的信息。体外与NSP的直接结合试验和酵母反向双杂交筛选将被用于鉴定在AtNSI相互作用中存在缺陷的NSP突变体；这些突变将通过基于PCR的技术进行筛选。识别与MPB相互作用的宿主蛋白将确定其功能和如何靶向ER，这将通过使用酵母SOS招募系统、相互作用克隆和与MPB的共纯化来完成。

The goal of the project is to elucidate how plant virus-encoded movement proteins (MPs) act to coordinate replication of the viral genome with its directional movement through and between plant cells, and to explore the role of MPs in conferring tissue restriction to plant virus infection. Understanding virus-host interactions at this level is important for combating viral diseases, for only from such knowledge can rational anti-viral strategies be developed. Genetic studies show that MPs determine the host range and pathogenic properties of plant viruses. Thus, understanding their functions is essential for developing strategies to engineer plants that will be resistant to a broad range of geminiviruses.The bipartite geminiviruses squash leaf curl virus (SqLCV) and cabbage leaf curl virus (CLCV) encode two MPs, NSP and MPB. These act cooperatively to move the viral single strand DNA genome within and between cells. NSP is a nuclear shuttle protein that binds viral genomes at their site of replication in the nucleus, and moves these between the nucleus and the cytoplasm. MPB traps NSP-genome complexes in the cytoplasm and moves them along unique endoplasmic reticulum (ER)-derived tubules to and across the walls of developing phloem cells. By site-directed mutagenesis, functional domains have been identified within each MP. The interactions between NSP and MPB are regulated by an activity that is specific to plant cells, and mutational studies suggest that this regulation may involve phosphorylation of the MPs . Use of the green fluorescent protein (GFP) has further shown that SqLCV infection is phloem-limited. To understand the functions of NSP and MPB, the signaling pathways through which they act, and the role of MPB in the phloem restriction of virus infection, now requires biochemical and genetic studies. We also want to understand, and develop new in vivo and in vitro approaches to investigate their functions. The specific goals of the project are to investigate MPB-ER interactions and their roles in the phloem-restriction of virus infection, to investigate the function of AtNSI, an Arabidopsis protein of unknown function that interacts with NSP and to identify host cell proteins that interact with MPB. These approaches will take advantage of different features of cucurbits and Arabidopsis. CLCV expressing free GFP and CLCV-SqLCV recombinants will be studied to determine if CLCV infection is phloem-restricted in Arabidopsis and the role of MPB in the ability to infect Arabidopsis. Fusions of MPB with GFP expressed from tissue-specific promoters in transgenic Arabidopsis wll be used to determine the role of MPB in the phloem limitation to virus infection and the effects of phloem development on virus movement. Determining the subcellular location and tissue-specific and temporal expression patterns of AtNSI will provide information on its function. Direct binding assays with NSP in vitro and a yeast reverse two-hybrid screen will be used to to identify NSP mutants with defects in AtNSI interaction; these will be screened by a PCR-based technique. Identifying host proteins that interact with MPB will define its function and how it targets to ER, and will be accomplished by using the yeast SOS-recruitment system, interaction cloning, and co-purification with MPB.