Liu Yongliang, Lyu Ruiqing, Singleton Joshua J, Patra Barunava, Pattanaik Sitakanta, Yuan Ling
Department of Plant and Soil Sciences and Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington, KY, 40546, USA.
Plant Methods. 2024 Feb 12;20(1):26. doi: 10.1186/s13007-024-01154-x.
Virus-induced gene silencing (VIGS) is widely used in plant functional genomics. However, the efficiency of VIGS in young plantlets varies across plant species. Additionally, VIGS is not optimized for many plant species, especially medicinal plants that produce valuable specialized metabolites.
We evaluated the efficacy of five-day-old, etiolated seedlings of Catharanthus roseus (periwinkle) for VIGS. The seedlings were vacuum-infiltrated with Agrobacterium tumefaciens GV3101 cells carrying the tobacco rattle virus (TRV) vectors. The protoporphyrin IX magnesium chelatase subunit H (ChlH) gene, a key gene in chlorophyll biosynthesis, was used as the target for VIGS, and we observed yellow cotyledons 6 days after infiltration. As expected, the expression of CrChlH and the chlorophyll contents of the cotyledons were significantly decreased after VIGS. To validate the cotyledon based-VIGS method, we silenced the genes encoding several transcriptional regulators of the terpenoid indole alkaloid (TIA) biosynthesis in C. roseus, including two activators (CrGATA1 and CrMYC2) and two repressors (CrGBF1 and CrGBF2). Silencing CrGATA1 led to downregulation of the vindoline pathway genes (T3O, T3R, and DAT) and decreased vindoline contents in cotyledons. Silencing CrMYC2, followed by elicitation with methyl jasmonate (MeJA), resulted in the downregulation of ORCA2 and ORCA3. We also co-infiltrated C. roseus seedlings with TRV vectors that silence both CrGBF1 and CrGBF2 and overexpress CrMYC2, aiming to simultaneous silencing two repressors while overexpressing an activator. The simultaneous manipulation of repressors and activator resulted in significant upregulation of the TIA pathway genes. To demonstrate the broad application of the cotyledon-based VIGS method, we optimized the method for two other valuable medicinal plants, Glycyrrhiza inflata (licorice) and Artemisia annua (sweet wormwood). When TRV vectors carrying the fragments of the ChlH genes were infiltrated into the seedlings of these plants, we observed yellow cotyledons with decreased chlorophyll contents.
The widely applicable cotyledon-based VIGS method is faster, more efficient, and easily accessible to additional treatments than the traditional VIGS method. It can be combined with transient gene overexpression to achieve simultaneous up- and down-regulation of desired genes in non-model plants. This method provides a powerful tool for functional genomics of medicinal plants, facilitating the discovery and production of valuable therapeutic compounds.
病毒诱导的基因沉默（VIGS）在植物功能基因组学中被广泛应用。然而，VIGS在幼苗中的效率因植物物种而异。此外，VIGS尚未针对许多植物物种进行优化，尤其是那些能产生有价值的特殊代谢产物的药用植物。
我们评估了长春花（Catharanthus roseus）五天大的黄化幼苗用于VIGS的效果。用携带烟草脆裂病毒（TRV）载体的根癌农杆菌GV3101细胞对幼苗进行真空浸润。原卟啉IX镁螯合酶亚基H（ChlH）基因是叶绿素生物合成中的关键基因，被用作VIGS的靶标，浸润后6天我们观察到子叶变黄。正如预期的那样，VIGS处理后长春花ChlH的表达和子叶中的叶绿素含量显著降低。为了验证基于子叶的VIGS方法，我们沉默了长春花中编码萜类吲哚生物碱（TIA）生物合成的几种转录调节因子的基因，包括两种激活因子（CrGATA1和CrMYC2）和两种抑制因子（CrGBF1和CrGBF2）。沉默CrGATA1导致长春花碱途径基因（T3O、T3R和DAT）下调，子叶中长春花碱含量降低。沉默CrMYC2，随后用茉莉酸甲酯（MeJA）诱导，导致ORCA2和ORCA3下调。我们还用能沉默CrGBF1和CrGBF2并过表达CrMYC2的TRV载体共浸润长春花幼苗，旨在同时沉默两种抑制因子并过表达一种激活因子。对抑制因子和激活因子的同时操作导致TIA途径基因显著上调。为了证明基于子叶的VIGS方法的广泛适用性，我们对另外两种有价值的药用植物胀果甘草（Glycyrrhiza inflata）和青蒿（Artemisia annua）优化了该方法。当携带ChlH基因片段的TRV载体浸润到这些植物的幼苗中时，我们观察到子叶变黄，叶绿素含量降低。
与传统的VIGS方法相比，基于子叶的VIGS方法具有广泛的适用性，速度更快、效率更高，并且更容易进行额外处理。它可以与瞬时基因过表达相结合，以实现非模式植物中所需基因的同时上调和下调。该方法为药用植物功能基因组学提供了一个强大的工具，有助于发现和生产有价值的治疗性化合物。
