# An Efficient Rice Virus-Induced Gene Silencing System Mediated by Wheat Dwarf Virus
## 4 Discussion
It relies on the viral replication protein (Rep) for rolling-circle replication, enabling transient high-level expression of the target gene fragment. The vector design can activate the host RNA interference (RNAi) pathway, leading to the specific silencing of the target gene. Unlike retrovirus such as RTBV, WDV does not integrate into the host genome, thereby avoiding risks associated with long-term genetic modifications and reducing growth abnormalities caused by insertional mutations [35; 36]. In this study, rice and its susceptibility _OsPi21_ gene were selected to evaluate the WDV vector system. To verify the feasibility of the WDV vector, firstly, pCambiia1300-WDV-_OsPDSi_ was used as a positive control; the results show that the leaves of rice plants treated with VIGS were obviously albino, and the expression of _OsPDS_ in rice was significantly down-regulated compared to the control group, which preliminarily determined the effectiveness of the vector. Then, the virus vector was used to construct pCambiia1300-WDV-_OsPi21_ to silence rice _OsPi21_, which further proved the feasibility of the tool vector constructed based on WDV. And further studies indicate that it will provide a new gene silencing tool vector for the study of rice blast resistant varieties. A previous study reported that, due to the lack of proofreading mechanisms and many replication cycles, the payload genes in the WDV vector may rapidly accumulate point mutations, leading to the inactivation of these non-essential insertions [37; 38]. This involves recombination events in the viral vector that may lead to the production of new functional viral genomes and cause the spread of unwanted pathogens [39]. While, in this study, it was found that the WDV fragment itself had no significant effect on the growth ability of rice, and there were few severe streak symptoms in wheat [40], and because _Geminiviridae_ has the characteristics of rapid infection, proliferation, transcription, and expression without integration into the plant genome, WDV-mediated VIGS typically causes milder symptoms with minimal impact on host growth and development, while its high silencing efficiency is confirmed through phenotypic and physiological comparisons with control groups. However, like other VIGS approaches, potential limitations should be acknowledged, including the transient nature of the silencing, possible variability in efficiency between individual plants or under different conditions, and the theoretical potential for off-target effects that warrant careful sequence selection. Despite these considerations, it has become a highly efficient plant expression vector with broad prospects for development. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app15115818/s1](https://www.mdpi.com/article/10.3390/app15115818/s1)]([https://www.mdpi.com/article/10.3390/app15115818/s1](https://www.mdpi.com/article/10.3390/app15115818/s1)), Figure S1: WDV-VIGS vector models. Conceived and designed the experiments: [PERSON], [PERSON] ([PERSON]), [PERSON] ([PERSON]) and [PERSON] Performed the cloning experiments: [PERSON], [PERSON] ([PERSON]) and [PERSON] Performed the rice transformation: [PERSON], [PERSON] and [PERSON]. Performed the phenotyping: [PERSON], [PERSON] ([PERSON]), [PERSON] and [PERSON]. Performed the DNA extraction and genotyping analysis: [PERSON], [PERSON] ([PERSON]), [PERSON], [PERSON] and [PERSON]. Drafted the manuscript: [PERSON], [PERSON], [PERSON] ([PERSON]) and [PERSON] Edited the manuscript: [PERSON], [PERSON], [PERSON], [PERSON] ([PERSON]) and [PERSON] All authors have read and agreed to the published version of the manuscript. This research was funded by Natural Science Foundation of Zhejiang province in China (LQ24C140003, LY20C140003) and Hangzhou Normal University Graduate Research Innovation Promotion Project, and the APC was funded by Hangzhou Normal University. Not applicable.