Sakuta Kohei, Uchida Keiko, Fukuhara Toshiyuki, Komatsu Ken, Okada Ryo, Moriyama Hiromitsu
Laboratory of Molecular and Cellular Biology, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Japan.
Laboratory of Plant Pathology, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Japan.
Front Microbiol. 2023 Sep 12;14:1243068. doi: 10.3389/fmicb.2023.1243068. eCollection 2023.
Two endornaviruses, Phytophthora endornavirus 2 (PEV2) and Phytophthora endornavirus 3 (PEV3), have been discovered in pathogens targeting asparagus. In this study, we analyzed the nick structure in the RNA genomes of PEV2 and PEV3 in the host oomycetes. Northern blot hybridization using positive and negative strand-specific RNA probes targeting the 5' and 3' regions of PEV2 and PEV3 RNA genomes revealed approximately 1.0 kilobase (kb) RNA fragments located in the 5' regions of the two genomes. 3' RACE analysis determined that the size of the RNA fragments were 958 nucleotides (nt) for PEV2 and 968 nt for PEV3. We have successfully constructed full-length cDNA clones of the entire RNA genomes of PEV2 and PEV3 using a homologous recombination system in the yeast, . These full-length cDNA sequences were ligated downstream of a constitutive expression promoter () or a galactose-inducing promoter () in the shuttle vector to enable the production of the full-length RNA transcripts of PEV2 and PEV3 in yeast cells. Interestingly, a 1.0 kb RNA fragment from the PEV3 positive-strand transcript was also detected with a 5'-region RNA probe, indicating that site-specific cleavage also occurred in yeast cells. Further, when PEV2 or PEV3 mRNA was overexpressed under the promoter, yeast cell growth was suppressed. A fusion protein combining EGFP to the N-terminus of the full-length PEV2 ORF or C-terminus of the full-length PEV3 ORF was expressed, and allowed PEV2 and PEV3 ORFs to be successfully visualized in yeast cells. Expression of the fusion protein also revealed presence of heterogeneous bodies in the cells.
在针对芦笋的病原体中发现了两种内质网病毒，即疫霉内质网病毒2（PEV2）和疫霉内质网病毒3（PEV3）。在本研究中，我们分析了宿主卵菌中PEV2和PEV3 RNA基因组中的切口结构。使用针对PEV2和PEV3 RNA基因组5'和3'区域的正链和负链特异性RNA探针进行的Northern印迹杂交显示，在两个基因组的5'区域中存在约1.0千碱基（kb）的RNA片段。3' RACE分析确定，PEV2的RNA片段大小为958个核苷酸（nt），PEV3的为968 nt。我们利用酵母中的同源重组系统成功构建了PEV2和PEV3整个RNA基因组的全长cDNA克隆。这些全长cDNA序列被连接到穿梭载体中组成型表达启动子（）或半乳糖诱导型启动子（）的下游，以便在酵母细胞中产生PEV2和PEV3的全长RNA转录本。有趣的是，用5'区域RNA探针也检测到了来自PEV3正链转录本的1.0 kb RNA片段，这表明在酵母细胞中也发生了位点特异性切割。此外，当PEV2或PEV3 mRNA在启动子下过表达时，酵母细胞生长受到抑制。表达了一种将EGFP与全长PEV2 ORF的N端或全长PEV3 ORF的C端结合的融合蛋白，从而使PEV2和PEV3 ORF在酵母细胞中成功可视化。融合蛋白的表达还揭示了细胞中存在异质体。
