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

Abstract

Virus-induced gene silencing (VIGS) is an effective tool for the study of soybean gene function. Successful VIGS depends on the interaction between virus spread and plant growth, which can be influenced by environmental conditions. Recently, we developed a new VIGS system derived from the Soybean yellow common mosaic virus (SYCMV). Here, we investigated several environmental and developmental factors to improve the efficiency of a SYCMV-based VIGS system to optimize the functional analysis of the soybean. Following SYCMV: Glycine max-phytoene desaturase (GmPDS) infiltration, we investigated the effect of photoperiod, inoculation time, concentration of Agrobacterium inoculm, and growth temperature on VIGS efficiency. In addition, the relative expression of GmPDS between non-silenced and silenced plants was measured by qRT-PCR. We found that gene silencing efficiency was highest at a photoperiod of 16/8 h (light/dark) at a growth temperature of approximately 27°C following syringe infiltration to unrolled unifoliolate leaves in cotyledon stage with a final SYCMV:GmPDS optimal density (OD)600 of 2.0. Using this optimized protocol, we achieved high efficiency of GmPDS-silencing in various soybean germplasms including cultivated and wild soybeans. We also confirmed that VIGS occurred in the entire plant, including the root, stem, leaves, and flowers, and could transmit GmPDS to other soybean germplasms via mechanical inoculation. This optimized protocol using a SYCMV-based VIGS system in the soybean should provide a fast and effective method to elucidate gene functions and for use in large-scale screening experiments.

Improved apple latent spherical virus-induced gene silencing in multiple soybean genotypes through direct inoculation of agro-infiltrated Nicotiana benthamiana extract

“…Nine soybean genotypes infected with GmPDS-VIGS displayed photo-bleaching indicative of successful silencing of GmPDS1 , whereas the other ten genotypes exhibited no GmPDS1 silencing. Our findings are similar to those of [ 33 ] in that, of the multiple soybean germplasm tested for efficiency to soybean yellow common mosaic virus based VIGS vector, not all germplasm was susceptible to SYCMV and GmPDS silencing was measured at variable rates between genotypes. Judging from our results and those of [ 33 ], more optimization may be needed to gauge the impact of environmental factors such as, growth temperatures and Agrobacterium inoculum concentration on the efficiency of VIGS.…”

Virus-induced gene silencing for functional analysis of eds1 gene in tomato infected with Ralstonia solanacearum

“…The efficiency of virus-induced gene silencing with Soybean yellow common mosaic virus (SYCMV) in soybeans has been investigated by using quantitative reverse transcriptasepolymerase chain reaction (qRT-PCR) method at different plant growth stages. These results showed a high VIGS efficiency at the primary leaves stage and low efficiency at closed cotyledon stage (Kim et al, 2016). We found that silencing of eds1 gene was more effective in the inoculated seedlings at fully-opened cotyledon stage, whereas at the eight-leaf stage, VIGS efficiency was significantly reduced.…”

“…Our observation showed that the optimal temperature is 20°C with a high VIGS efficiency and this temperature did not show any tomato growth limitation. In SYCMV-based VIGS system this parameter has been determined at 27°C (Kim et al, 2016). The high-efficiency VIGS at the low temperature is due to the high susceptibility of plants to viruses because at the high temperature, siRNAs is slowly accumulated and then RNA silencing will be displayed to lower level (Tuttle et al, 2008;Sung et al, 2014).…”

Functional Analysis of A Soybean Ferredoxin-NADP Reductase (FNR) Gene in Response to Soybean Mosaic Virus

“…Therefore, the virus-induced gene silencing (VIGS) system extensively used for the over-expression and downregulation of genes in various crops, including soybean. Thus far, many VIGS systems, including bean pod mottle virus (BPMV), tobacco rattle virus (TRV), apple latent spherical virus (ALSV), and soybean yellow common mosaic virus (SYCMV), used for the functional analysis of candidate genes in soybean [8][9][10][11]. The BPMV VIGS system is successfully applied to describe the candidate genes function for different traits, including SMV resistance in soybean [12,13].…”