RNA指导的RNA聚合酶1基因在植物防御类病毒侵染中的作用及机制

RNA指导的RNA聚合酶1基因(RDR1)是RNA沉默途径中的重要基因，也是植物通过RNA沉默防御病毒侵染必需的。然而，其在植物防御类病毒侵染中的作用及机制仍不清楚。本项目采用马铃薯纺锤形块茎类病毒/番茄和啤酒花矮化类病毒/本氏烟的组合为实验体系，使用病毒诱导的基因沉默(VIGS)、RNA干扰(RNAi)、基因编辑和过表达转基因的方法，改变番茄和本氏烟中RDR1基因的表达水平。通过接种类病毒，分析RDR1基因表达的变化对类病毒侵染的影响，从而明确其在植物防御类病毒侵染中的作用。此外，利用小RNA测序技术，分析RDR1基因的表达与类病毒来源的小RNA(vd-sRNA)的合成之间的关系，并找出RDR1特异性扩增的vd-sRNA，利用表达人工miRNA的方法，验证其沉默效率，从而解析RDR1基因参与植物防御类病毒侵染的分子机制。本研究将深化对植物通过RNA沉默防御类病毒侵染的分子机制的理解。

RNA-directed RNA polymerase 1 gene (RDR1) is important for RNA silencing and is necessary for plant antiviral defense through RNA silencing mechanism. However, the role of RDR1 in plant defense response to viroid infection remains unknown. This project will use the combinations of potato spindle tuber viroid (PSTVd)/tomato and hop stunt viroid (HSVd)/Nicotiana benthamiana to identify the function and mechanism of RDR1 in plant resistance to viroid infection. Tomato RDR1 gene (LeRDR1) will be down-regulated by the technology of virus-induced gene silencing (VIGS), knocked down by RNA interference (RNAi), and knocked out using CRISPR/Cas9 system. RDR1 of N. tabacum (NtRDR1) and LeRDR1 will be overexpressed in N. benthamiana and tomato plants using transgene technology. The obtained plants with different expression levels of RDR1 will be inoculated with viroid followed with the detection of viroid and viroid-derived small RNA (vd-sRNA). The influence of altered RDR1 expressions on viroid infection can reveal the function of RDR1 in plant defense response to viroid infection. Next, small RNA sequencing technology will be used to verify vd-sRNA accumulation level in viroid-infected tomato and N. benthamiana plants and to identity RDR1 specifically amplified vd-sRNAs in HSVd-infected N. benthamiana plants with a high expression level of NtRDR1. RNA silencing efficiencies of these vd-sRNA will be determined in N. benthamiana using the method of artificial miRNA expression. Thus, how RDR1 regulates plant resistance to viroid will be revealed. The results will deepen the understanding of the mechanism of plant resistance to viroid.