近日，云南農(nóng)業(yè)大學(xué)在線發(fā)表文獻(xiàn)《Establishment and application of a root wounding–immersion method for efficient virus-induced gene silencing in plants》，文獻(xiàn)中實(shí)驗(yàn)使用LUYOR-3410RB觀測GFP在煙草上的表達(dá)。

文獻(xiàn)摘要：

In the post-genomic era, virus-induced gene silencing (VIGS) has played an important role in research on reverse genetics in plants. Commonly used Agrobacterium-mediated VIGS inoculation methods include stem scratching, leaf infiltration, use of agrodrench, and air-brush spraying. In this study, we developed a root wounding–immersion method in which 1/3 of the plant root (length) was cut and immersed in a tobacco rattle virus (TRV)1:TRV2 mixed solution for 30 min. We optimized the procedure in Nicotiana benthamiana and successfully silenced N. benthamiana, tomato (Solanum lycopersicum), pepper (Capsicum annuum L.), eggplant (Solanum melongena), and Arabidopsis thaliana phytoene desaturase (PDS), and we observed the movement of green fluorescent protein (GFP) from the roots to the stem and leaves. The silencing rate of PDS in N. benthamiana and tomato was 95–**. In addition, we successfully silenced two disease-resistance genes, SITL5 and SITL6, to decrease disease resistance in tomatoes (CLN2037E). The root wounding–immersion method can be used to inoculate large batches of plants in a short time and with high efficiency, and fresh bacterial infusions can be reused several times. The most important aspect of the root wounding–immersion method is its application to plant species susceptible to root inoculation, as well as its ability to inoculate seedlings from early growth stages. This method offers a means to conduct large-scale functional genome screening in plants.

3.1 Optimization of the root wounding–immersion procedure

Current inoculation methods mainly include Agrobacterium-mediated injection infiltration and vacuum infiltration. However, injecting Agrobacterium cultures into the seedling leaves of plants with tough tissues, such as soya beans and maize, can be challenging. Additionally, the leaves of these plants need to be fully unfolded to ensure successful injection (Ratcliff et al., 2001). For roots that are susceptible to inoculation and early-growth seedlings, we developed an inoculation method known as root wounding–immersion (Figure 1B). To test the feasibility of the root wounding–immersion method, we chose PDS as a reporter gene because the leaves of plants in which PDS is silenced tend to show symptoms of photobleaching (Kumagai et al., 1995; Liu et al., 2002a). To visualize TRV viral transport from the roots to the above-ground parts of plants, we cloned tobacco NbPDS fragments and inserted them into the vector pTRV2-GFP. Uninjured tobacco was infected according to the method described in Section 2.3 and the silencing rate was found to be less than 1%, so we used uninoculated plants as controls. The frequency of VIGS was defined as the number of plants that show silencing phenotype (photobleaching) after inoculation with TRV2-GFP-NbPDS. In N. benthamiana, the ratio of positive silenced plants was 95.8%, and GFP insertion did not modify the gene silencing capacity of the TRV vector (Tian et al., 2014). Under illumination with the portable excitation light source (LUYOR-3410RB), pTRV2-GFP-NbPDS was transferred from the roots to the stem and leaves (Figure 2F). We designed two different infection methods for different temperatures (Supplementary Table S1): “concurrent inoculation” and “successive inoculation.” Photobleaching was observed in N. benthamiana plants as early as day 6 (Figure 2A), which was faster than the 7–10 days reported in the study of Ryu et al. (2004).

文獻(xiàn)地址：https://doi.org/10.3389/fpls.2024.1336726   

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