Verchot Jeanmarie, Thapa Aastha, Wijayasekara Dulanjani, Hoyt Peter R
Texas A&M Agrilife Center at Dallas;
Noble Research Center, Oklahoma State University.
J Vis Exp. 2018 Jul 27(137):57855. doi: 10.3791/57855.
This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.
这种宏基因组方法用于鉴定具有环状DNA基因组的植物病毒及其转录本。通常，在宿主中滴度较低或无法机械接种到另一个宿主的植物DNA病毒很难增殖以获得更高滴度的感染性物质。将感染的叶片在一种温和的缓冲液中研磨，该缓冲液具有推荐用于纯化大多数杆状副逆转录病毒的最佳pH值和离子组成。尿素用于分解捕获病毒粒子的包涵体并溶解细胞成分。差速离心可进一步将病毒粒子与植物污染物分离。然后用蛋白酶K处理去除衣壳。接着浓缩病毒DNA并用于下一代测序（NGS）。NGS数据用于组装重叠群，这些重叠群被提交到NCBI-BLASTn以识别生成数据集中的一部分病毒序列。在一个平行流程中，使用基于标准柱的RNA提取方法从感染的叶片中分离RNA。然后进行核糖体去除以富集一部分mRNA和病毒转录本。将RNA测序（RNA-seq）得到的组装序列提交到NCBI-BLASTn以识别该数据集中的一部分病毒序列。在我们的研究中，我们在这两个数据集中鉴定出了两个相关的全长杆状DNA病毒基因组。这种方法比另一种常见方法更可取，后者提取小RNA序列的总体群体以重建植物病毒基因组序列。后一种宏基因组流程可回收与病毒相关的序列，这些序列是插入植物基因组中的逆转录元件。这与生化或分子检测相结合以进一步识别活跃的感染因子。本研究中记录的方法可回收代表正在复制的病毒的序列，这些序列可能表明存在活跃的病毒感染。
