Instituto Nacional de Tecnología Agropecuaria, 1686 Hurlingham, Argentina.
Plant Physiol. 2011 Jul;156(3):1278-91. doi: 10.1104/pp.111.177345. Epub 2011 Apr 29.
Since the advent of the postgenomic era, efforts have focused on the development of rapid strategies for annotating plant genes of unknown function. Given its simplicity and rapidity, virus-induced gene silencing (VIGS) has become one of the preeminent approaches for functional analyses. However, several problems remain intrinsic to the use of such a strategy in the study of both metabolic and developmental processes. The most prominent of these is the commonly observed phenomenon of "sectoring" the tissue regions that are not effectively targeted by VIGS. To better discriminate these sectors, an effective marker system displaying minimal secondary effects is a prerequisite. Utilizing a VIGS system based on the tobacco rattle virus vector, we here studied the effect of silencing the endogenous phytoene desaturase gene (pds) and the expression and subsequent silencing of the exogenous green fluorescence protein (gfp) on the metabolism of Arabidopsis (Arabidopsis thaliana) leaves and tomato (Solanum lycopersicum) fruits. In leaves, we observed dramatic effects on primary carbon and pigment metabolism associated with the photobleached phenotype following the silencing of the endogenous pds gene. However, relatively few pleiotropic effects on carbon metabolism were observed in tomato fruits when pds expression was inhibited. VIGS coupled to gfp constitutive expression revealed no significant metabolic alterations after triggering of silencing in Arabidopsis leaves and a mild effect in mature green tomato fruits. By contrast, a wider impact on metabolism was observed in ripe fruits. Silencing experiments with an endogenous target gene of interest clearly demonstrated the feasibility of cosilencing in this system; however, carefully constructed control experiments are a prerequisite to prevent erroneous interpretation.
自从后基因组时代以来，人们一直致力于开发快速策略来注释未知功能的植物基因。由于其简单性和快速性，病毒诱导的基因沉默（VIGS）已成为功能分析的主要方法之一。然而，在研究代谢和发育过程中，该策略的使用仍然存在一些内在问题。其中最突出的问题是组织区域的“分区”现象，这种现象不能被 VIGS 有效靶向。为了更好地区分这些区域，需要一个显示最小二次效应的有效标记系统。我们利用基于烟草脆裂病毒载体的 VIGS 系统，研究了内源类胡萝卜素脱饱和酶基因（pds）沉默以及外源绿色荧光蛋白（gfp）表达和随后沉默对拟南芥（Arabidopsis thaliana）叶片和番茄（Solanum lycopersicum）果实代谢的影响。在叶片中，我们观察到内源性 pds 基因沉默后与光漂白表型相关的初级碳和色素代谢的显著影响。然而，当 pds 表达受到抑制时，在番茄果实中观察到的碳代谢多效性影响相对较少。VIGS 与 gfp 组成型表达相结合，在触发沉默后，在拟南芥叶片中未发现明显的代谢变化，在成熟的绿色番茄果实中仅观察到轻微的影响。相比之下，在成熟的果实中观察到更广泛的代谢影响。用感兴趣的内源靶基因进行的沉默实验清楚地证明了该系统中共沉默的可行性；然而，仔细构建对照实验是防止错误解释的前提。
