Julius Kühn-Institut, Federal Research Centre for Cultivated Plants, Institute for Plant Protection in Fruit Crops and Viticulture, D-69221 Dossenheim, and Ruprecht-Karls-Universität Heidelberg, Centre for Organismal Studies, 69120 Heidelberg, Germany.
Julius Kühn-Institut, Federal Research Centre for Cultivated Plants, Institute for Plant Protection in Fruit Crops and Viticulture, D-69221 Dossenheim, Germany.
Plant Dis. 2017 Dec;101(12):2110-2115. doi: 10.1094/PDIS-04-17-0573-RE. Epub 2017 Sep 15.
Construction and agroinoculation of full-length infectious cDNA clones of plant RNA viruses have been used in plant virology to prove Koch's postulates and for development of viruses as vectors for expressing foreign genes in plants. Four full-length cDNA clones (pIF3-12, pIF3-14, pIF3-15, and pIF3-19) of Apple chlorotic leaf spot virus (ACLSV) isolate 38/85 were produced. Two of the four full-length cDNA clones (pIF3-15 and pIF3-19) proved to be infectious on Nicotiana occidentalis 37B test plants by agroinoculation and were then mechanically transmissible to healthy N. occidentalis 37B. The genomic cDNAs of ACLSV pIF3-15 and pIF3-19 shared nucleotide identity of 77.5%, demonstrating mixed infections of multiple strains of ACLSV in the source tree of isolate 38/85. The two full-length cDNA clones were agroinoculated to apple seedlings by a newly developed vacuum infiltration method. The success rate of agroinoculation was greater than 78%, defined as the number of PCR positive seedlings to the number of apple seedlings that survived. ACLSV was transmissible from agroinoculated seedlings by cleft grafting. The results of this study will be useful for construction of infectious cDNA clones of plant viruses from full-length PCR fragments and agroinoculating woody host plants using the vacuum infiltration method outlined here.
植物 RNA 病毒全长感染性 cDNA 克隆的构建和农杆菌接种已被用于植物病毒学，以证明科赫假设，并开发病毒作为载体，在植物中表达外源基因。我们构建了苹果褪绿叶斑病毒（ACLSV）分离株 38/85 的四个全长 cDNA 克隆（pIF3-12、pIF3-14、pIF3-15 和 pIF3-19）。这四个全长 cDNA 克隆中的两个（pIF3-15 和 pIF3-19）通过农杆菌接种证明对 Nicotiana occidentalis 37B 测试植物具有感染性，随后可以机械传播到健康的 N. occidentalis 37B。ACLSV pIF3-15 和 pIF3-19 的基因组 cDNA 共享 77.5%的核苷酸同一性，表明在分离株 38/85 的原始树木中存在 ACLSV 多种株系的混合感染。我们通过一种新开发的真空渗透方法将这两个全长 cDNA 克隆接种到苹果幼苗中。农杆菌接种的成功率大于 78%，定义为 PCR 阳性幼苗的数量与幸存的苹果幼苗的数量之比。ACLSV 可以通过劈接从接种的幼苗中传播。本研究的结果将有助于使用这里概述的真空渗透方法从全长 PCR 片段构建植物病毒的感染性 cDNA 克隆，并对木本宿主植物进行农杆菌接种。
