Zhou Bangjun, Zeng Lirong
Center for Plant Science Innovation, Department of Plant Pathology, University of Nebraska, Lincoln, NE 68583 USA.
Southern Regional Collaborative Innovation Center for Grain and Oil Crops, Hunan Agricultural University, Changsha, 410128 China.
Plant Methods. 2017 Jul 21;13:59. doi: 10.1186/s13007-017-0210-6. eCollection 2017.
Virus-induced gene silencing (VIGS) has been used in many plant species as an attractive post transcriptional gene silencing (PTGS) method for studying gene function either individually or at large-scale in a high-throughput manner. However, the specificity and efficiency for knocking down members of a highly homologous gene family have remained to date a significant challenge in VIGS due to silencing of off-targets.
Here we present an improved method for the selection and evaluation of gene fragments used for VIGS to specifically and efficiently knock down members of a highly homologous gene family. Using this method, we knocked down twelve and four members, respectively of group III of the gene family encoding ubiquitin-conjugating enzymes (E2) in . Assays using these VIGS-treated plants revealed that the group III E2s are essential for plant development, plant immunity-associated reactive oxygen species (ROS) production, expression of the gene that is required for ROS production, and suppression of immunity-associated programmed cell death (PCD) by AvrPtoB, an effector protein of the bacterial pathogen . Moreover, functional redundancy for plant development and ROS production was found to exist among members of group III E2s.
We have found that employment of a gene fragment as short as approximately 70 base pairs (bp) that contains at least three mismatched nucleotides to other genes within any 21-bp sequences prevents silencing of off-target(s) in VIGS. This improved approach in the selection and evaluation of gene fragments allows for specific and efficient knocking down of highly homologous members of a gene family. Using this approach, we implicated group III E2s in plant development, immunity-associated ROS production, and suppression of multiple immunity-associated PCD by AvrPtoB. We also unraveled functional redundancy among group III members in their requirement for plant development and plant immunity-associated ROS production.
病毒诱导的基因沉默（VIGS）已在许多植物物种中用作一种有吸引力的转录后基因沉默（PTGS）方法，用于以高通量方式单独或大规模研究基因功能。然而，由于脱靶沉默，在VIGS中敲低高度同源基因家族成员的特异性和效率至今仍是一个重大挑战。
本文我们提出了一种改进的方法，用于选择和评估用于VIGS的基因片段，以特异性和高效地敲低高度同源基因家族的成员。使用该方法，我们分别敲低了编码泛素结合酶（E2）的基因家族第三组中的12个和4个成员。对这些经VIGS处理的植物进行的分析表明，第三组E2s对于植物发育、植物免疫相关的活性氧（ROS）产生、ROS产生所需基因的表达以及细菌病原体效应蛋白AvrPtoB对免疫相关程序性细胞死亡（PCD）的抑制至关重要。此外，发现第三组E2s成员之间在植物发育和ROS产生方面存在功能冗余。
我们发现，使用一个长度约为70个碱基对（bp）的基因片段，该片段在任何21bp序列中与其他基因至少有三个错配核苷酸，可防止VIGS中的脱靶沉默。这种在基因片段选择和评估方面的改进方法允许特异性和高效地敲低基因家族的高度同源成员。使用这种方法，我们确定了第三组E2s在植物发育、免疫相关ROS产生以及AvrPtoB对多种免疫相关PCD的抑制中的作用。我们还揭示了第三组成员在植物发育和植物免疫相关ROS产生需求方面的功能冗余。
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