# An Efficient Rice Virus-Induced Gene Silencing System Mediated by Wheat Dwarf Virus
## 4 Discussion  
The RTBV vector could be successfully introduced into rice via Agrobacteriumium-mediated transformation, effectively suppressing the _PDS_ gene in the treated rice leaves [16]. In each case, real-time PCR-based assessments indicated an approximately 40-80% reduction in the accumulation of _PDS_ transcripts. However, quartz sand treatment of leaves are required in the RTBV friction inoculation method, where the force must be controlled to avoid leaf damage [17]. In contrast, in this WDV-mediated VIGS system, transformation can be achieved by vacuum-soaking the germinated seeds in _Agrobacteriumium_ suspension, effectively addressing the drawback of the long growth cycle of rice. The VIGS vector constructed using pCamba1300-ubi and WDV has strong infectivity, good repeatability, and few adverse reactions. After Agrobacteriumium treatment with rice seeds infiltrated with _Agrobacteriumium_ carrying the WDV-_OsPDSi_ construct, a larger area of albino symptoms appears on the new leaves within 2-3 weeks, indicating a better silencing effect. Moreover, the vacuum-soaking method is simple to operate and does not require complex manipulation skills [28; 29]. This suggests that the WDV vector is more reliable in practical applications, providing consistent silencing effects. Moreover, in this experiment, the V2 movement protein gene of the WDV was deleted, thus the viral vector has less impact on the growth and development of rice, compared to RTBV, which may cause pathogenic effects on rice or affect its growth and development. The WDV vector exhibits am optimal silencing effect when the _OsPDS/OsPi21_ target gene fragment is 60 bp, indicating its better silencing efficiency relative to RTBV, which may require longer gene fragments to achieve effective gene silencing. Most target gene fragments inserted in reverse into the recombinant vector yield better silencing effects than those inserted in the same direction [30]. Early studies in BSMV-mediated VIGS systems demonstrate that inserting hairpin structures can enhance the silencing phenotype or down-regulate the expression of the target gene [31]. However, recent studies in the VIGS system mediated by BSMV and BMV vectors found that these short reverse repeated sequences are very unstable and that their silencing effectiveness is not as good as that of the fragments inserted in the same direction, suggesting that research on the silencing system in poaceous plants is needed to determine the universal effectiveness of reverse repeated sequences [12]. In this experiment, we adopted a strategy of inserting both forward and reverse sequences to construct the VIGS vector. Unfortunately, the sequencing results failed to obtain the expected sequence information, which may be due to the formation of hairpin structures during the sequencing of constructs containing simultaneously inserted forward and reverse sequences. Moreover, the experimental phenomena observations showed no significant difference from the single-direction insertion vector, which may imply that, in this experimental system, the direction of the inserted fragment has no significant effect on silencing efficiency or that the potential advantage of reverse insertion was not demonstrated under the conditions of this experiment. The key in VIGS technology is the selection and construction of viral vectors. Although there are many plant viruses, such as RNA viruses, DNA viruses, and satellite viruses, have been adapted as VIGS vectors, these three types of vectors have their own characteristics and application scope [32; 33]. The host range infected by different types of vectors is limited, leading to great differences in the efficiency of gene silencing. The silencing efficiency of VIGS is also affected by the interaction between the host plant and the virus, as well as environmental factors [34]. As a member of the Geminiviridae family, WDV possesses a single-stranded DNA genome that efficiently transfects host cells via _Agrobacterium_-mediated vacuum infiltration.