Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA.
Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, 12 Rue de Général Zimmer, 67084 Strasbourg, France.
J Virol Methods. 2018 Jul;257:16-21. doi: 10.1016/j.jviromet.2018.04.006. Epub 2018 Apr 6.
One of the greatest hindrances to the study of grapevine fanleaf virus (GFLV) is the dearth of robust protocols for reliable, scalable, and cost-effective inoculation of host plants, especially methods which allow for rapid and targeted manipulation of the virus genome. Agroinoculation fulfills these requirements: it is a relatively rapid, inexpensive, and reliable method for establishing infections, and enables genetic manipulation of viral sequences by modifying plasmids. We designed a system of binary plasmids based on the two genomic RNAs [RNA1 (1) and RNA2 (2)] of GFLV strains F13 (F) and GHu (G) and optimized parameters to maximize systemic infection frequency in Nicotiana benthamiana via agroinoculation. The genomic make-up of the inoculum (G1-G2 and reassortant F1-G2), the identity of the co-infiltrated silencing suppressor (grapevine leafroll associated virus 2 p24), and temperature at which plants were maintained (25 °C) significantly increased systemic infection, while high optical densities of infiltration cultures (OD of 1.0 or 2.0) increased the consistency of systemic infection frequency in N. benthamiana. In contrast, acetosyringone in the bacterial culture media, regardless of concentration, had no effect. Plasmids in this system are amenable to rapid and reliable manipulation by one-step site-directed mutagenesis, as shown by the creation of infectious RNA1 chimeras of the GFLV-F13 and GHu strains. The GFLV agroinoculation plasmids described here, together with the optimized protocol for bacterial culturing and plant maintenance, provide a robust system for the establishment of systemic GFLV infection in N. benthamiana and the rapid generation of GFLV mutants, granting a much-needed tool for investigations into GFLV-host interactions.
葡萄扇叶病毒（GFLV）研究的最大障碍之一是缺乏可靠、可扩展且具有成本效益的宿主植物接种方法，尤其是能够快速且有针对性地操纵病毒基因组的方法。农杆菌介导的基因转化（Agroinoculation）满足了这些要求：它是一种相对快速、廉价且可靠的建立感染的方法，并通过修饰质粒来实现病毒序列的遗传操作。我们设计了一个基于 GFLV 菌株 F13（F）和 GHu（G）的两个基因组 RNA[RNA1（1）和 RNA2（2）]的二元质粒系统，并优化了参数，以通过农杆菌介导的基因转化在本氏烟中最大限度地提高系统感染频率。接种物的基因组构成（G1-G2 和重组 F1-G2）、共浸润的沉默抑制子（葡萄卷叶伴随病毒 2 p24）的身份以及植物维持的温度（25°C）显著增加了系统感染，而高渗透培养物的光密度（OD 值为 1.0 或 2.0）增加了本氏烟中系统感染频率的一致性。相比之下，细菌培养基中的乙酰丁香酮，无论浓度如何，都没有影响。该系统中的质粒易于通过一步定点突变进行快速可靠的操作，正如 GFLV-F13 和 GHu 菌株的 RNA1 嵌合体的感染性 RNA 的创建所示。本文描述的 GFLV 农杆菌接种质粒，以及优化的细菌培养和植物维持方案，为在本氏烟中建立系统 GFLV 感染和快速产生 GFLV 突变体提供了一个强大的系统，为研究 GFLV-宿主相互作用提供了急需的工具。
