芸薹黄化病毒（BrYV）P3a蛋白影响病毒系统侵染的机制研究

Plant viruses are obligate intracellular parasites, of which systemic infection proceeds through three main steps: intracellular replication, cell-to-cell movement, and long-distance movement. During the past decades, lots of efforts were devoted to determine the viral factors involved in systemic infection of virus and to elucidate the molecular mechanisms underlying this process. Polerovirus belongs to the Luteoviridae family. Viruses in this genus are worldwide distributed infecting many crops of economic importance and causing severe plant diseases and yield losses. Coat protein (CP) and readthrough protein (RTP) were shown to play roles in the systemic infection of poleroviruses. In addition to CP and RTP, a protein encoded by a newly found short open reading frame (ORF) in poleroviruses, termed P3a, was reported to participate in the systemic infection of poleroviruses recently. However, the function of P3a was not completely understood, and little is known about the mechanism for the P3a-dependent systemic infection. Brassica yellows virus (BrYV) is a novel polerovirus which was discovered and distributed widely in China. Intriguingly, we found that mutation of Pro18 residue in P3a abolished the systemic infection of BrYV in plants. Based on previous studies, this project will focus on the effect of P3a in systemic infection of BrYV and the mechanism underlying it, with emphasis on BrYV and its P3a mutant. Biochemical and molecular biological technologies were employed together with transcriptome analysis in this study. Spatio-temporal differences in systemic infection process between BrYV and its P3a mutant will be analyzed, in order to further investigate the role of P3a in systemic infection of BrYV. Using multiple approaches, host proteins which may interact with P3a and differential expression genes which may be involved in BrYV systemic infection will be identified, followed by reverse genetic analysis of their function. These results, together with the biochemical characterization of P3a protein and its mutant, should finally elucidate the molecular basis of P3a function in BrYV systemic infection. This study will provide new evidences in understanding the molecular mechanism of virus systemic infection, and offer theoretical guidance for development of virus resistance crops using genome editing in the future.

与系统侵染相关的病毒蛋白验证及分子机制探索一直是研究的重点之一。近期研究表明，马铃薯卷叶病毒属中新发现的小蛋白P3a为系统侵染所必需，但对其如何发挥作用的分子机制尚无深入研究。前期研究中我们发现该属芸薹黄化病毒（BrYV）P3a第18位氨基酸突变导致病毒无法系统侵染。以此为契机，基于该属病毒病害在生产上的重要性和国内外研究现状，本项目拟以BrYV为研究对象，通过分子生物学手段分析比较野生型与P3a突变体病毒侵染过程的时空差异，进一步明确P3a突变对系统侵染的影响；并通过生物化学、分子生物学结合转录组分析，筛选与P3a互作或在侵染过程中差异表达的寄主因子，并利用反向遗传学对其功能进行验证；结合P3a及其突变体的生化特性研究，以期在分子层面明确P3a在BrYV系统侵染中的作用机制。研究结果不仅能为阐明病毒系统侵染的分子机制提供理论依据，还可以为基于基因编辑技术创制抗病毒作物新种质提供参考。

与系统侵染过程相关的病毒蛋白验证及分子机制探索一直是研究的重点之一。在马铃薯卷叶病毒属中，除了外壳蛋白和通读蛋白以外，新发现的小蛋白P3a为系统侵染所必需的，但其发挥作用的分子机制尚不清楚。前期研究过程中，我们发现该属芸薹黄化病毒（Brassica yellows virus, BrYV）P3a第18位氨基酸的突变导致病毒丧失系统侵染植物的能力。以此为契机，本项目以BrYV为研究对象，对P3a在BrYV系统侵染过程中发挥的功能及背后的分子机制进行了探究。本项目首先分析和比较了野生型与P3a突变体病毒侵染过程的时空差异，验证了P3a的突变主要影响了病毒的系统侵染能力。随后，通过异源互补实验证明了P3a蛋白的功能对病毒建立系统侵染至关重要，并明确了第18位的脯氨酸为P3a蛋白发的关键氨基酸位点。进一步的分析表明，野生型病毒能够进入接种叶叶柄，而突变体病毒则不能，这暗示了P3a蛋白可能参与病毒进入韧皮组织的过程。另外，我们还发现马铃薯卷叶病毒属和黄症病毒属病毒编码的P3a蛋白均能异源互补突变体病毒BrYVP18L和BrYVAGC的系统侵染，暗示着这两个属的P3a从进化上和功能上都是保守的。有意思的是，豌豆耳突花叶病毒2号（PEMV 2）可以通过协生互作帮助突变体病毒BrYVP18L和BrYVAGC在本生烟上建立系统侵染，而这种协生互作和P3a的功能之间存在冗余。综合运用分子生物学和生物化学手段对P3a及其突变体蛋白P3aP18L进行比较分析，发现二者具有相似的亚细胞定位。值得注意的是，虽然P3a和P3aP18L在体内都能发生自身互作，但是P3aP18L比P3a的互作能力更强。对不同标签的P3a及P3aP18L蛋白进行检测时发现，突变体比野生型P3a产生更加稳定的二聚体。此外，本项目利用分裂泛素酵母双杂交系统分析了与可能与P3a互作的BrYV编码蛋白, 还以P3a为诱饵从寄主cDNA文库中筛选了与P3a互作的寄主蛋白，并对候选蛋白进行了初步的功能分析。本项目关于P3a作用机制的研究为阐明病毒系统侵染的分子机制提供了理论依据，还为基于基因编辑技术创制抗病毒作物新种质提供参考。本项目培养博士研究生1名、硕士研究生2名，研究结果发表SCI收录论文1篇。