Optimization of a Virus-Induced Gene Silencing System with Soybean yellow common mosaic virus for Gene Function Studies in Soybeans.

The soybean (Glycine max (L.) Merr.) is an important legume crop. It is a major plant source of oil and provides protein-rich food for both human and animal consumption (Graham and Vance, 2003). Recently, a reference genome sequence of a cultivated soybean (G. max var. Williams 82) was released, and dozens of wild and cultivated soybeans have been resequenced (Kim et al., 2010; Lam et al., 2010; Schmutz et al., 2010). Furthermore, recent advances in next generation sequencing technologies have been applied to soybean research, leading to a large number of candidate genes. These genomic resources provide ample opportunities for elucidating gene functions involved in complex agronomical traits such as disease resistance, seed protein and oil content, and the number of seeds per pod in the soybean (Cook et al., 2012; Hwang et al., 2014; Pandy et al., 2011). Reverse genetics has been used for plant functional studies and typically involves various methods and tools, including gene silencing, overexpression, T-DNA tagging, transposon tagging, zinc-finger nucleases, homologous recombination, Deletagene, and the targeting of local lesions in the genome (Anai, 2012). Nevertheless, transformation represents the most invaluable tool for functional studies because it allows for the production of novel and genetically diverse genotypes. Stable genetic transformation is not yet considered routine in the soybean due to several limitations to soybean transformation. Only limited cultivars, such as Jack, Williams 82, Throne, and Bert can be transformed (Yamada et al., 2012). Moreover, transformation efficiency is low, and the construction of transformed soybeans requires long periods of time (over 1 year) (Mano et al., 2014). Virus-induced gene silencing (VIGS) is an attractive alternative for elucidating gene function in soybean without the need for stable transformation (Burch-Smith et al., 2004). VIGS has been widely used to silence target genes of interest through plant natural defense mechanisms (Lu et al., 2003). VIGS involves the delivery of a recombinant virus to plants containing a fragment of the plant gene that is intended to be silenced. The plant defense mechanism system then decreases not only the virus but also the targeted endogenous plant gene expression through post-transcriptional gene silencing (Robertson, 2004). Many viral vectors, both RNA and DNA, have been developed for gene silencing analysis. These viruses include Tobacco mosaic virus, Potato virus X, Tobacco rattle virus, Barley stripe mosaic virus, Pea early browning virus, Poplar mosaic virus, Brome mosaic virus, Cabbage leaf curl virus, African cassava mosaic virus, and Tomato yellow leaf curl China virus (Robertson, 2004; Unver and Budak, 2009). However, only a few VIGS vectors are now available for soybean research based on the Bean pod mottle virus (BPMV) (Zhang and Ghabrial, 2006), Cucumber mosaic virus (CMV) (Nagamatsu et al., 2007), and Apple latent spherical virus (ALSV) (Yamagishi and Yoshikawa, 2009). Based on USDA soybean germplasm collection, there exist more than 14,000 accessions including wild, landrace, cultivated, and introduced soybeans (Bandillo et al., 2015). These VIGS vectors may not be able to be introduced into some soybean accessions, because some cultivars might have resistant genes against these viruses. Furthermore, ALSV and BPMV are not found in Korea and cannot be used as VIGS vectors in this area, necessitating new and more appropriate virus vectors to be developed for VIGS-based functional analyses of the soybean. Recently, Lim et al. (2015) developed a new VIGS system based on the Soybean yellow common mosaic virus (SYCMV) (Nam et al., 2012). Unlike ALSV, BPMV, and CMV consisting of bi- or tri-partite RNA genomes, SYCMV has a single stranded RNA genome. SYCMV-derived VIGS system is more efficient than other systems for developing VIGS constructs easily and rapidly. For determining the effectiveness of VIGS, growth conditions, including photoperiods and the developmental stage of the plants at the time of inoculation, were cited as the most important factors (Burch-Smith et al., 2006). In addition, the VIGS vector, strain, culture concentration, inoculation method, photoperiod, plant age, and plant growth temperature have been highlighted as key parameters for VIGS effectiveness (Burch-Smith et al., 2004; Wang et al., 2006). Therefore, much effort has been invested to ascertain the optimal conditions for VIGS in plants (Pang et al., 2013; Sung et al., 2014; Wang et al., 2013). The aim of this study was to determine the optimal conditions for the SYCMV-based VIGS system through investigation of several factors that potentially influence VIGS efficiency. This study demonstrates that a new SYCMV-based VIGS system could be useful for a wide range of applications in soybean functional genomics.