Is the cucumber mosaic virus 2b silencing suppressor protein regulated by direct interactions with the viral 1a protein?

博士论文申请题目：利用黄瓜花叶病毒2b沉默抑制蛋白和病毒1a蛋白的相互作用，确定沉默途径与植物抗虫机制之间的关系。黄瓜花叶病毒（CMV） 2b沉默抑制蛋白抑制短干扰（si）RNA介导的RNA沉默（一种有效的抗病毒机制）和微RNA调控的植物发育。2b蛋白的这两种活性是不同的：抗病毒沉默的抑制取决于2b结合双链siRNA的能力，而其对mirna调控的发育的影响主要取决于2b与Argonaut (AGO)1蛋白的相互作用。2b蛋白对AGO1控制的抗虫防御系统的作用被另一种CMV蛋白1a蛋白拮抗，1a蛋白是一种复制相关蛋白，以前没有发现与沉默有关。最近的证据表明，这种效果是通过2b和1a病毒蛋白之间的直接相互作用实现的。本博士的目的是利用1a和2b衍生的野生型和突变型构建物来表征1a和2b之间的相互作用，用于蛋白质结合研究，包括双分子荧光互补和FLAG下拉。这些技术，连同荧光显微镜技术，也将用于确定1a和2b病毒蛋白的定位和细胞生物学。本博士将试图确定1a是否也会影响2b的沉默抑制活性，或者仅仅是其对miRNA通路的影响。最后，本博士将在蚜虫实验中使用1a和2b衍生的野生型和突变型构建物来评估中断沉默途径对植物抗虫性的影响。

PhD project strategic theme: Bioscience for sustainable agriculture and foodProposed title for PhD: Using the cucumber mosaic virus 2b silencing suppressor protein and the viral 1a protein interaction to identify the relationship between silencing pathways and plant insect resistance mechanisms.The cucumber mosaic virus (CMV) 2b silencing suppressor protein inhibits RNA silencing mediated by short-interfering (si)RNAs (a potent antiviral mechanism) and micro(mi)RNA-regulated plant development. These two activities of the 2b protein are distinct: suppression of antiviral silencing depends on 2b's ability to bind double-stranded siRNA, while its effect on miRNA-regulated development depend largely on the interaction of 2b with Argonaut (AGO)1 protein. The effect of the 2b protein on an anti-insect defence system controlled by AGO1 are antagonized by another CMV protein the 1a protein, a replication-associated protein with no previously known connection to silencing. Recent evidence suggests that this effect is achieved through a direct interaction between the 2b and 1a viral proteins. The aim of this PhD is to characterise the interaction between 1a and 2b using 1a and 2b-derived wild-type and mutant constructs for protein binding studies including bimolecular fluorescence complementation and FLAG pull-downs. These techniques, along with fluorescent microscopy techniques, will also be used to identify the localisation and cell biology of the 1a and 2b viral proteins. This PhD will attempt to determine if 1a also affects the silencing suppressor activity of 2b or just its effects on miRNA pathways. Finally, this PhD will use 1a and 2b-derived wild-type and mutant constructs in aphid assays to assess the effects of disrupted silencing pathways on insect resistance in plants.