# An Efficient Rice Virus-Induced Gene Silencing System Mediated by Wheat Dwarf Virus
## 1 Introduction  
Virus-induced gene silencing (VIGS) is a powerful technique that can down-regulating endogenous genes in plants, enabling rapid functional genomics studies. Its efficiency is determined by several key parameters, including infection efficiency, silencing efficacy, host range, speed of phenotypic expression, impact on plant growth, and ease of delivery [1]. Based on this approach, researchers can modify a plant's viral vector to achieve specific gene silencing [2, 3]. This technology does not require stable transformation and large mutant populations, and it is becoming an important tool to identify the heredity and function of plant genes [4]. Until now, there have been various plant viral vectors that have been developed, and an increasing number of plant species have been successfully utilized for gene function analysis using VIGS. _Tobarrius capsici_ (tobacco rattle virus, TRV), as one of the most well-established VIGS vectors, has been widely used in various solanaceous species, such as tomatoes, peppers, and tobacco [5]. Its widespread application in these plants could be attributed to its high silencing efficiency and technical feasibility [6, 7]. _Comovirus silique_ (bean pod mottle virus, BPMV) has been adapted as a VIGS vector in legumes, allowing for gene function studies in beans and other economically important legume species [8]. _Hordeivirus hordei_ (barley stripe mosaic virus, BSMV) has been used asa VIGS vector in monocot plants, particularly in maize, by targeting the endogenous host _Phytone desaturase_ (_PDS_) gene [9]. Rice (_Oryza sativa_), a critical staple crop feeding over half the global population, faces severe threats from _Magnaporthe oryzae_, the causative agent of rice blast disease. This pathogen accounts for annual yield losses of 10-30%, underscoring the urgency to identify resistance genes [10]. However, since rice blast resistance typically weakens within 3-5 years, the continuous updating of disease-resistant gene combinations is required to achieve sustainable rice production. VIGS enables rapid functional gene characterization, but most VIGS vectors preferentially infect dicotyledonous plants, with many viruses demonstrating low infection efficiency in monocotyledons. Another hurdle is the lack of a universal approach that can be applied to all plants. For example, we face hurdles around the incompatibility of VIGS vectors with the host and the inability to use VIGS for plant species which are not easily transformable [11]. Current reports indicate that only four RNA viruses and two DNA viruses have been successfully utilized as vectors for VIGS in monocot species, including BSMV-based in barley and wheat [12; 13]; _Bromovirus BMV_ in rice and barley [14]; _Potexvirus hambusae_ and its satellite RNA in _Brachtyodium distachyon_ [15]; _Tungrovirus oryzae_ (rice tangro bacilliform virus, RTBV) in rice [16; 17]; and _Mastrevirus hordei_ (wheat dwarf virus, WDV) in wheat and rice [18]. WDV, a member of the genus _Mastrevirus_ (family _Geminiviridae_), is a pathogen affecting cereal crops that is transmitted by the leafhopper _Psammettati alienus_ [19]. WDV consists of the following: Rep (LIR and SIR for replication), CP/V1 (silencing effector), and V2 (movement protein). For VIGS vectors, Rep is preserved for replication, while V2 is modified or deleted to insert target genes [20]. Recent studies have demonstrated WDV's potential as a VIGS vector in wheat and barley, achieving high-throughput silencing with minimal off-target effects. However, its application in rice remains underexplored, with only one report suggesting compatibility--a gap our study addresses [21]. Here, we optimize WDV-mediated VIGS in rice using two targets: _PDS_, a visual marker for silencing efficiency, and _Pi21_, a key blast resistance gene. We hypothesized that our VIGS protocol will efficiently silence endogenous genes in rice.