Virus-induced gene silencing (VIGS) is a versatile and attractive approach for functional gene characterization in plants. Although several VIGS vectors for maize (Zea mays) have been previously developed, their utilities are limited due to low viral infection efficiency, insert instability, short maintenance of silencing, inadequate inoculation method, or abnormal requirement of growth temperature. Here, we established a Cucumber mosaic virus (CMV)-based VIGS system for efficient maize gene silencing that overcomes many limitations of VIGS currently available for maize. Using two distinct strains, CMV-ZMBJ and CMV-Fny, we generated a pseudorecombinant-chimeric (Pr) CMV. Pr CMV showed high infection efficacy but mild viral symptoms in maize. We then constructed Pr CMV-based vectors for VIGS, dubbed Pr CMV VIGS. Pr CMV VIGS is simply performed by mechanical inoculation of young maize leaves with saps of Pr CMV-infected Nicotiana benthamiana under normal growth conditions. Indeed, suppression of isopentenyl/dimethylallyl diphosphate synthase (ZmIspH) expression by Pr CMV VIGS resulted in non-inoculated leaf bleaching as early as 5 d post-inoculation (dpi) and exhibited constant and efficient systemic silencing over the whole maize growth period up to 105 dpi. Furthermore, utilizing a ligation-independent cloning (LIC) strategy, we developed a modified Pr CMV-LIC VIGS vector, allowing easy gene cloning for high-throughput silencing in maize. Thus, our Pr CMV VIGS system provides a much-improved toolbox to facilitate efficient and long-duration gene silencing for large-scale functional genomics in maize, and our pseudorecombination-chimera combination strategy provides an approach to construct efficient VIGS systems in plants.

中文翻译：

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基于玉米花叶病毒的高效高通量伪重组嵌合黄瓜花叶病毒 VIGS


病毒诱导的基因沉默（VIGS）是一种多功能且有吸引力的植物功能基因表征方法。尽管之前已经开发了几种用于玉米（Zea mays）的VIGS载体，但由于病毒感染效率低、插入不稳定、沉默维持时间短、接种方法不当或生长温度要求异常，其实用性受到限制。在这里，我们建立了一种基于黄瓜花叶病毒（CMV）的 VIGS 系统，用于有效的玉米基因沉默，克服了目前可用于玉米的 VIGS 的许多限制。使用两种不同的毒株 CMV-ZMBJ 和 CMV-Fny，我们生成了假重组嵌合 (Pr) CMV。 Pr CMV 在玉米中表现出较高的感染效率，但病毒症状较轻。然后，我们构建了基于 Pr CMV 的 VIGS 载体，称为 Pr CMV VIGS。 Pr CMV VIGS 只需在正常生长条件下用感染 Pr CMV 的本塞姆氏烟草的汁液机械接种玉米幼叶即可进行。事实上，Pr CMV VIGS 对异戊烯基/二甲基烯丙基二磷酸合酶 (ZmIspH) 表达的抑制早在接种后 5 天 (dpi) 就导致未接种的叶片漂白，并在整个玉米生长期表现出持续有效的系统性沉默。 105 dpi。此外，利用不依赖于连接的克隆（LIC）策略，我们开发了一种改良的 Pr CMV-LIC VIGS 载体，可以轻松进行基因克隆，从而在玉米中实现高通量沉默。因此，我们的 Pr CMV VIGS 系统提供了一个大大改进的工具箱，以促进玉米大规模功能基因组学的高效和长期基因沉默，并且我们的假重组-嵌合体组合策略提供了一种在植物中构建高效 VIGS 系统的方法。