Department of Plant Pathology, Cornell University, Ithaca, NY 14853.
Proc Natl Acad Sci U S A. 1989 Jun;86(12):4496-500. doi: 10.1073/pnas.86.12.4496.
Several lines of evidence are presented to allow us to conclude that tobacco mosaic virus (TMV) RNA enters the chloroplast in vivo. Chloroplasts were prepared from either directly inoculated or systemically infected leaves of tobacco plants inoculated with one of several strains of the virus and from uninfected control plants. Intact chloroplasts were isolated on Percoll gradients and treated with pancreatic RNase and thermolysin to destroy potential TMV virions and RNA on the outside or bound to their surfaces. Northern blot analysis of RNA extracted from these chloroplasts demonstrated that full-length TMV RNA was present within the chloroplasts prepared from both directly inoculated and systemically invaded leaves. Only genomic length, but not subgenomic length, RNA was found in the chloroplast extracts, indicating a selectivity of the transport of the viral RNA into the chloroplast. A temperature-sensitive TMV mutant (Ts 38), in which no virions are formed at 35 degrees C, was used to demonstrate that at that restrictive temperature viral RNA is detected in the chloroplast, indicating that free viral RNA can enter the chloroplast rather than intact virions. To our knowledge, the transport of a foreign RNA species into chloroplasts has not been reported previously.
有几条证据表明，烟草花叶病毒（TMV）RNA 能够在体内进入叶绿体。叶绿体是从直接接种或系统感染烟草植株的几种病毒株系的叶片中分离出来的，也从未感染的对照植物中分离出来。完整的叶绿体在 Percoll 梯度上分离出来，并使用胰蛋白酶和热稳定蛋白酶处理，以破坏潜在的 TMV 病毒粒子和位于其表面或与之结合的 RNA。从这些叶绿体中提取的 RNA 的 Northern blot 分析表明，全长 TMV RNA 存在于直接接种和系统入侵叶片制备的叶绿体中。在叶绿体提取物中仅发现基因组长度的 RNA，而不是亚基因组长度的 RNA，表明病毒 RNA 进入叶绿体具有选择性。使用温度敏感型 TMV 突变体（Ts 38），在 35 摄氏度下不形成病毒粒子，证明在该限制温度下在叶绿体中检测到病毒 RNA，表明游离的病毒 RNA 可以进入叶绿体，而不是完整的病毒粒子。据我们所知，以前尚未报道过将外源 RNA 种类转运到叶绿体中。
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